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Inhibitory activity of vaginal lactobacilli towards Candida spp.
No full textObjectives. Lactobacilli are the dominant bacteria of healthy vaginal microbiota and their principal function is to maintain an environment that restricts the growth of pathogenic and opportunistic microorganisms, as fungi belonging to the genus Candida. Lactobacilli form a critical line of defence against potential pathogens by lowering the environmental pH through lactic acid production and producing antimicrobial
compounds, or through competitive exclusion. Anyway, the mechanisms underlying antifungal activity against Candida spp. are still not fully understood. In this study, the potential activity against Candida spp. of different strains of vaginal lactobacilli was analysed, focusing on hydrogen peroxide generation, lactic acid production and antimicrobial supernatant fluids activity.
Methods. Seventeen strains of lactobacilli were isolated from vaginal swabs collected from pre-menopausal healthy women. They were taxonomically identified by sequencing the 16S ribosomal RNA gene.
Hydrogen peroxide generation was tested in a semi-quantitative assay on de Man, Rogosa, Sharpe (MRS) agar plates containing tetramethylbenzidine and horseradish peroxidase in anaerobic conditions. Isolates were scored as low, medium and high producing strains. Lactic acid production was measured in cell free supernatants of Lactobacillus cultures by 1H-nuclear magnetic resonance (NMR) analysis. Lactobacillus culture supernatants were tested for their fungistatic or fungicidal activity against 9 Candida strains isolated from vaginal swabs submitted to the Microbiology Laboratory of Sant’Orsola-Malpighi University Hospital of Bologna for routine diagnostic procedures, belonging to C. albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, and C. lusitaniae species. The in vitro activity of free-cell supernatants was determined by broth microdilution assay in accordance with EUCAST guidelines. To determine if Lactobacillus strains supernatants had a killing effect, samples from wells exhibiting less than 50% of growth were taken and spotted onto SD agar plates. Fungicidal activity was defined as a ≥3 log10 reduction from the starting inoculum.
Results. The Lactobacillus isolates were taxonomically identified as follows: 8 strains of L. crispatus (BC1-BC8), 6 strains of L. gasseri (BC9-BC14), and 3 strains of L. vaginalis (BC15-BC17). All Lactobacillus strains exhibited a good generation of hydrogen peroxide, while the production of lactic acid, even if recorded for all the strains tested, showed concentrations ranging from 4.8 to 50.9 mM. When the anti-fungal activity of Lactobacillus was assessed, L. crispatus supernatants were the most effective, especially versus C. albicans and C. lusitaniae. None of the Lactobacillus strains was able to interfere with C. krusei and C. parapsilosis. Detailed results of fungistatic or fungicidal activity are shown in figure 1.
Conclusion. A major potential application of this study concerns the identification of active Lactobacillus strains that could be administered as probiotics for prophylaxis and/or adjuvant therapy of vulvovaginal candidiasis. Further studies are ongoing to elucidate the mechanisms by which lactobacilli exert their protective functions against Candida
Approcci innovativi per l’identificazione di specie di lattobacilli: studio dei metaboliti mediante spettrometria di risonanza magnetica (1H-RMN)
No full textINTRODUZIONE. I lattobacilli rappresentano un ampio genere di microrganismi anaerobi Gram positivi di forma cocco-bacillare. Essi risiedono in differenti regioni anatomiche umane dove svolgono un ruolo chiave nel mantenere l’omeostasi microbica attraverso effetti diretti e capacità immuno-modulanti.
Scopo di questo lavoro è stato quello di utilizzare lo studio dei metaboliti batterici tramite spettroscopia di risonanza magnetica nucleare (1H-RMN), come approccio innovativo per l’identificazione di specie di lattobacilli.
MATERIALI E METODI. Un totale di 40 ceppi di lattobacilli di diversa origine sono stati inclusi nello studio. L’identificazione di specie, ottenuta mediante sequenziamento del gene 16s rRNA ha permesso di identificare 7 L. crispatus, 7 L. gasseri, 5 L. acidophilus, 5 L. delbrueckii, 2 L. vaginalis, 2 L. reuteri, 6 L. plantarum, 1 L. pentosus, 2 L. rhamnosus, 2 L. casei/paracasei e 1 L. brevis. L’analisi metabolica in 1H-RMN è stata eseguita con lo spettrometro AVANCE III (Bruker) sia sull’intero set di metaboliti intracellulari, dopo opportuna lisi batterica, sia sul gruppo di metaboliti extracellulari. Partendo dal dendrogramma basato sulle sequenze del gene 16s rRNA, i lattobacilli sono stati suddivisi in 7 gruppi e le differenze nella composizione del metaboloma sono state studiate con opportuni test statistici.
RISULTATI. L’analisi in 1H-RMN ha permesso di identificare 30 e 17 molecole nel metaboloma extracellulare e intracellulare, rispettivamente. Tali molecole erano rappresentate prevalentemente da aminoacidi, alcoli, monosaccaridi e acidi organici. I diversi gruppi di lattobacilli hanno mostrato complessivamente lo stesso tipo di metaboliti ma con significative differenze per quanto riguarda la concentrazione di singole molecole. In particolare, 4 molecole nel metaboloma extracellulare (acetoina, acetone, piruvato, glucosio) e 8 in quello intracellulare (AMP, lattato, lisina, NAD+, propionato, succinato, uracile e valina) hanno mostrato una differenza statisticamente significativa. Ad esempio, il gruppo dei L. crispatus ha mostrato il maggior consumo di glucosio (P=1x10-3) mentre la produzione di lattato è apparsa più significativa per le specie L. casei-L. paracasei-L. rhamnosus (P=1x10-3). CONCLUSIONI. L’analisi metabolomica ha permesso di identificare metaboliti marker caratteristici di alcuni gruppi di lattobacilli, che potrebbero chiarire i meccanismi che stanno alla base dell’effetto antimicrobico svolto da alcune specie
A peptidic hydrogel that may behave as a “Trojan Horse”
No full textA physical hydrogel prepared with the low-molecular-weight hydrogelator (LMWHG) CH2(C3H6CO-L-Phe-D-Oxd-OH)2 and water/ethanol mixture was applied as a potential “Trojan Horse” carrier into cells. By SEM and XRD analysis we could demonstrate that a fibrous structure is present in the xerogel, making a complex network. The gelator is derived from α-amino acids (Thr, Phe) and a fatty acid (azelaic acid) and is biocompatible: it was dosed to IGROV-1 cells, which internalized it, without significantly affecting the cell proliferation. To check the internalization process by confocal microscopy, fluorescent hydrogels were prepared, introducing the fluorescent dansyl moiety into the mixture
ENDOGENOUS LIPID PEROXIDATION PRODUCT 9-HSA REGULATES PROGENITOR FATE IN THE VERTEBRATE RETINA
No full textEfficacy and Safety of a Multistrain Probiotic Formulation Depends from Manufacturing
Full text linkBackgroundVariability in probiotics manufacturing may affect their properties, with potential implications for their efficacy and safety. This is of particular concern with probiotic products destined for use in patients with serious medical conditions, including human immunodeficiency virus (HIV) infection. The purpose of the study was to carry out a series of experiments comparing the properties of the US-made probiotic formulation originally commercialized under the brand name VSL#3®, with those of the Italian-made formulation now commercialized under the same name. The US-made formulation has previously shown beneficial effects at the intestinal and neurological levels in HIV-infected subjects as well as in patients with inflammatory bowel diseases and hepatic encephalopathy.MethodsEleven subjects receiving combined antiretroviral therapy for HIV-1 were treated for 6 months with the US-made VSL#3 formulation. At baseline and 6 months, T-cells were analyzed for phenotype and activation markers, and fecal samples were analyzed for bifidobacteria, lactobacilli, and their metabolites. The fecal metabolome was assessed using 1H-NMR spectroscopy. Production of metabolites of interest by bacteria obtained from sachets of the two formulations was compared in vitro and their effects on a rat intestinal epithelial cell line (IEC-6) were assessed. Particular attention was paid to the metabolite 1,3-dihydroxyacetone (DHA).ResultsAt 6 months, fecal samples showed a significant increase in the specific bacterial genera contained in the probiotic supplement. Immune activation was reduced as shown by a significant reduction in the percentage of CD4+CD38+HLA-DR+ T-cells at 6 months. Fecal concentrations of DHA decreased significantly. In vitro, significant differences in the production and metabolism of DHA were found between bacteria from the US-made and Italian-made formulations: the US-made formulation was able to metabolize DHA whereas the bacteria in the Italian-made formulation were producing DHA. DHA reduced the viability of Streptococcus thermophilus, reduced IEC-6 cell viability in a dose-dependent manner, and also led to a lower rate of repair to scratched IEC-6 cell monolayer.ConclusionOur data, in conjunction with previously published findings, confirm that the new Italian-made formulation of VSL#3® is different from the previous US-made VSL#3 and therefore its efficacy and safety in HIV-infected subjects is still unproven
Ruolo dell'Enantiomero (R)-9-HSA nel controllo della proliferazione in una linea di Adenocarcinoma del Colon umano
No full text9-hydroxystearic acid (9-HSA) is an endogenous lipoperoxidation product and its administration to HT29, a colon adenocarcinoma cell line, induced a proliferative arrest in G0/G1 phase mediated by a direct activation of the p21WAF1 gene, bypassing p53. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity, showing interesting features as a new anticancer drug.
The interaction of 9-HSA with the catalytic site of the 3D model has been tested with a docking procedure: noticeably, when interacting with the site, the (R)-9-enantiomer is more stable than the (S) one. Thus, in this study, (R)- and (S)-9-HSA were synthesized and their biological activity tested in HT29 cells. At the concentration of 50 M (R)-9-HSA showed a stronger antiproliferative effect than the (S) isomer, as indicated by the growth arrest in G0/G1. The inhibitory effect of (S)-9-HSA on HDAC1, HDAC2 and HDAC3 activity was less effective than that of the (R)-9-HSA in vitro, and the inhibitory activity of both the (R)- and the (S)-9-HSA isomer, was higher on HDAC1 compared to HDAC2 and HDAC3, thus demonstrating the stereospecific and selective interaction of 9-HSA with HDAC1. In addition, histone hyperacetylation caused by 9-HSA treatment was examined by an innovative HPLC/ESI/MS method. Analysis on histones isolated from control and treated HT29 confirmed the higher potency of (R)-9-HSA compared to (S)-9-HSA, severely affecting H2A-2 and H4 acetylation.
On the other side, it seemed of interest to determine whether the G0/G1 arrest of HT29 cell proliferation could be bypassed by the stimulation with the growth factor EGF. Our results showed that 9-HSA-treated cells were not only prevented from proliferating, but also showed a decreased [3H]thymidine incorporation after EGF stimulation. In this condition, HT29 cells expressed very low levels of cyclin D1, that didn’t colocalize with HDAC1. These results suggested that the cyclin D1/HDAC1 complex is required for proliferation. Furthermore, in the effort of understanding the
possible mechanisms of this effect, we have analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. EGF/EGFR/HDAC1 complex quantitatively increases in 9-HSA-treated cells but not in serum starved cells after EGF stimulation. Our data suggested that 9-HSA interaction with the catalytic site of the HDAC1 disrupts the HDAC1/cyclin D1 complex and favors EGF/EGFR recruitment by HDAC1, thus enhancing 9-HSA antiproliferative effects.
In conclusion 9-HSA is a promising HDAC inhibitor with high selectivity and specificity, capable of inducing cell cycle arrest and histone hyperacetylation, but also able to modulate HDAC1 protein interaction. All these aspects may contribute to the potency of this new antitumor agent
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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