α-Lipoic acid induces Endoplasmic Reticulum stress-mediated apoptosis in hepatoma cells

Hepatocellular carcinoma (HCC) is the most common liver cancer and a major cause of adult death. The current treatments for HCC suffer from drug resistance and poor prognosis; therefore, novel therapeutic agents are urgently needed. Phytochemicals have been proposed to treat a range of cancers. Among them, α-lipoic acid (α-LA), a naturally synthesized antioxidant found in various dietary animal and plant sources, prevents oxidant-mediated cell death in normal cells while inducing apoptosis in several cancer cell lines. Previously, we demonstrated that the treatment of hepatoma cells with α-LA induced apoptosis, which was preceded by the generation of reactive oxygen species (ROS) and activation of the p53 protein, a known inducer of mitochondria-mediated apoptosis. Several studies have shown that ROS-induced apoptosis is associated with endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) activation. Herein, we investigated if α-LA-induced apoptosis in hepatoma cell lines was ER stress- and UPR-mediated by gene expression profiling analyses. UPR and ER stress pathways were the most up-regulated after treatment with α-LA. This finding, which has been confirmed by expression analyses of ER- and UPR-associated proteins, provides a better understanding of the molecular mechanisms behind the anti-tumoral action of α-LA on hepatoma cells

1H NMR spectroscopy-based metabolomics analysis for the diagnosis of symptomatic E. coli-associated urinary tract infection (UTI).

BACKGROUND: Urinary tract infection (UTI) is one of the most common diagnoses in girls and women, and to a lesser extent in boys and men younger than 50 years. Escherichia coli, followed by Klebsiella spp. and Proteus spp., cause 75-90% of all infections. Infection of the urinary tract is identified by growth of a significant number of a single species in the urine, in the presence of symptoms. Urinary culture is an accurate diagnostic method but takes several hours or days to be carried out. Metabolomics analysis aims to identify biomarkers that are capable of speeding up diagnosis. METHODS: Urine samples from 51 patients with a prior diagnosis of Escherichia coli-associated UTI, from 21 patients with UTI caused by other pathogens (bacteria and fungi), and from 61 healthy controls were analyzed. The 1H-NMR spectra were acquired and processed. Multivariate statistical models were applied and their performance was validated using permutation test and ROC curve. RESULTS: Orthogonal Partial Least Squares-discriminant Analysis (OPLS-DA) showed good separation (R2Y = 0.76, Q2=0.45, p < 0.001) between UTI caused by Escherichia coli and healthy controls. Acetate and trimethylamine were identified as discriminant metabolites. The concentrations of both metabolites were calculated and used to build the ROC curves. The discriminant metabolites identified were also evaluated in urine samples from patients with other pathogens infections to test their specificity. CONCLUSIONS: Acetate and trimethylamine were identified as optimal candidates for biomarkers for UTI diagnosis. The conclusions support the possibility of a fast diagnostic test for Escherichia coli-associated UTI using acetate and trimethylamine concentrations

A Deep Look at the Vaginal Environment During Pregnancy and Puerperium

A deep comprehension of the vaginal ecosystem may hold promise for unraveling the pathophysiology of pregnancy and may provide novel biomarkers to identify subjects at risk of maternal-fetal complications. In this prospective study, we assessed the characteristics of the vaginal environment in a cohort of pregnant women throughout their different gestational ages and puerperium. Both the vaginal bacterial composition and the vaginal metabolic profiles were analyzed. A total of 63 Caucasian women with a successful pregnancy and 9 subjects who had a first trimester miscarriage were enrolled. For the study, obstetric examinations were scheduled along the three trimester phases (9-13, 20-24, 32-34 gestation weeks) and puerperium (40-55 days after delivery). Two vaginal swabs were collected at each time point, to assess the vaginal microbiome profiling (by Nugent score and 16S rRNA gene sequencing) and the vaginal metabolic composition (1H-NMR spectroscopy). During pregnancy, the vaginal microbiome underwent marked changes, with a significant decrease in overall diversity, and increased stability. Over time, we found a significant increase of Lactobacillus and a decrease of several genera related to bacterial vaginosis (BV), such as Prevotella, Atopobium and Sneathia. It is worth noting that the levels of Bifidobacterium spp. tended to decrease at the end of pregnancy. At the puerperium, a significantly lower content of Lactobacillus and higher levels of Gardnerella, Prevotella, Atopobium, and Streptococcus were observed. Women receiving an intrapartum antibiotic prophylaxis for Group B Streptococcus (GBS) were characterized by a vaginal abundance of Prevotella compared to untreated women. Analysis of bacterial relative abundances highlighted an increased abundance of Fusobacterium in women suffering a first trimester abortion, at all taxonomic levels. Lactobacillus abundance was strongly correlated with higher levels of lactate, sarcosine, and many amino acids (i.e., isoleucine, leucine, phenylalanine, valine, threonine, tryptophan). Conversely, BV-associated genera, such as Gardnerella, Atopobium, and Sneathia, were related to amines (e.g., putrescine, methylamine), formate, acetate, alcohols, and short-chain fatty-acids (i.e., butyrate, propionate)

Gardnerella vaginalis clades in pregnancy: New insights into the interactions with the vaginal microbiome

Gardnerella vaginalis (GV) is an anaerobic bacterial species involved in the pathogenesis of bacterial vaginosis (BV), a condition of vaginal dysbiosis associated with adverse pregnancy outcomes. GV strains are categorized into four clades, characterized by a different ability to produce virulence factors, such as sialidase. We investigated the distribution of GV clades and sialidase genes in the vaginal ecosystem of a cohort of pregnant women, assessing the correlations between GV clades and the whole vaginal microbiome. A total of 61 Caucasian pregnant women were enrolled. Their vaginal swabs, collected both at the first and third trimester of pregnancy, were used for (i) evaluation of the vaginal status by Nugent score, (ii) vaginal microbiome profiling by 16S rRNA sequencing, (iii) detection and quantification of GV clades and sialidase A gene by qPCR assays. DNA of at least one GV clade was detected in most vaginal swabs, with clade 4 being the most common one. GV clade 2, together with the presence of multiple clades (&gt;2 simultaneously), were significantly associated with a BV condition. Significantly higher GV loads and sialidase gene levels were found in BV cases, compared to the healthy status. Clade 2 was related to the major shifts in the vaginal microbial composition, with a decrease in Lactobacillus and an increase in several BV-related taxa. As the number of GV clades detected simultaneously increased, a group of BV-associated bacteria tended to increase as well, while Bifidobacterium tended to decrease. A negative correlation between sialidase gene levels and Lactobacillus, and a positive correlation with Gardnerella, Atopobium, Prevotella, Megasphaera, and Sneathia were observed. Our results added knowledge about the interactions of GV clades with the inhabitants of the vaginal microbiome, possibly helping to predict the severity of BV and opening new perspectives for the prevention of pregnancy-related complications

Torquetenovirus in pregnancy: Correlation with vaginal microbiome, metabolome and pro-inflammatory cytokines

Torquetenovirus (TTV) is a negative sense, single-stranded DNA virus present in many body fluids of apparently healthy individuals. At present, it is considered a non-pathogenic endogenous virus. TTV can be detected in the vagina of pregnant women, its abundance being modulated with the extent of immune system activation. Until now, there is only scarce information regarding the association between TTV and the composition of the vaginal environment. Therefore, this study aimed to assess the presence of TTV in the vaginal ecosystem of a cohort of white women with a normal pregnancy (n = 60) at different gestational stages (first, second and third trimester) and in 9 subjects suffering a first trimester miscarriage. For each woman, we determined (i) the presence and titer of TTV, (ii) the vaginal bacterial composition by means of Nugent score and 16S rRNA gene sequencing, (iii) the vaginal metabolic profiles through H-1-NMR spectroscopy, and (iv) the vaginal concentration of two pro-inflammatory cytokines (IL-6 and IL-8). More than one third of women were found negative for TTV at all gestational stages. Although not statistically significant, the positivity for TTV dropped from 53.3% in the first to 36.6% in the third trimester. TTV loads varied greatly among vaginal samples, ranging between 2 x 10(1) and 2 x 10(5) copies/reaction. No difference in TTV prevalence and loads was observed between women with normal pregnancies and miscarriages. The presence of TTV was more common in women with a higher vaginal leucocyte count (p = 0.02). The levels of IL-6 (p = 0.02), IL-8 (p = 0.03), propionate (p = 0.001) and cadaverine (p = 0.006) were significantly higher in TTV-positive samples. TTV titer was positively correlated with the concentrations of 4-hydroxyphenyllactate (p &lt; 0.0001), isoleucine (p = 0.01) and phenylalanine (p = 0.04). TTV-positive samples were characterized by a higher relative abundance of Sneathia (p = 0.04) and Shuttleworthia (p = 0.0009). In addition, a trend toward a decrease of Lactobacillus crispatus and Lactobacillus jensenii, and an increase of Lactobacillus iners was observed for TTV-positive samples. In conclusion, we found that TTV is quite common in women with normal pregnancy outcomes, representing a possible predictor of local immune status

Cross sectional evaluation of the gut-microbiome metabolome axis in an Italian cohort of IBD patients.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract of uncertain origin, which includes ulcerative colitis (UC) and Crohn's disease (CD). The composition of gut microbiota may change in IBD affected individuals, but whether dysbiosis is the cause or the consequence of inflammatory processes in the intestinal tissue is still unclear. Here, the composition of the microbiota and the metabolites in stool of 183 subjects (82 UC, 50 CD, and 51 healthy controls) were determined. The metabolites content and the microbiological profiles were significantly different between IBD and healthy subjects. In the IBD group, Firmicutes, Proteobacteria, Verrucomicrobia, and Fusobacteria were significantly increased, whereas Bacteroidetes and Cyanobacteria were decreased. At genus level Escherichia, Faecalibacterium, Streptococcus, Sutterella and Veillonella were increased, whereas Bacteroides, Flavobacterium, and Oscillospira decreased. Various metabolites including biogenic amines, amino acids, lipids, were significantly increased in IBD, while others, such as two B group vitamins, were decreased in IBD compared to healthy subjects. This study underlines the potential role of an inter-omics approach in understanding the metabolic pathways involved in IBD. The combined evaluation of metabolites and fecal microbiome can be useful to discriminate between healthy subjects and patients with IBD

Comparison of tetraacetylethylendiamine + sodium perborate and sodium hypochlorite cytotoxicity on L929 fibroblasts

Introduction: Tetraacetylethylenediamine in association with sodium perborate (TAED+P) can be suggested for its use as an endodontic disinfectant because of its antimicrobial activity against different bacterial species when used at low concentrations. The purpose of this study was to measure the cytotoxicity of TAED+P on L929 fibroblasts and to compare it with that of sodium hypochlorite (NaOCl). Methods: L929 fibroblasts were grown in Dulbecco Modified Eagle Medium containing 10% fetal calf serum (FCS) at 37°C and 5% CO 2. At confluence, cells were split, plated in a 96-well plate, and incubated for 24 hours to allow attachment. The two disinfectants TAED+P and NaOCI were tested at various concentrations. The neutral red uptake and the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assays were used to evaluate the cell viability. The 50% inhibitory dose values for both disinfectants were calculated and statistically analyzed. The effect of both disinfectants on fibroblast viability was also determined in the presence of various concentrations of FCS. One-way analysis of variance with post hoc analysis using Tukey multiple comparison test was used for parametric data. Results: Both disinfectants induced a dose-related loss of cell viability; TAED+P resulted less cytotoxic than NaOCI in all the examined experimental conditions. Conclusions: These data support the possible use of TAED+P as an endodontic irrigant. Further studies are required to analyze its antibacterial activity against endodontic pathogens

ALTERAZIONI DEL MICROBIOMA FARINGEO IN CORSO DI INFEZIONI DA NEISSERIA GONORRHOEAE

Introduzione. Le faringiti da Neisseria gonorrhoeae sono in aumento tra gli omosessuali maschi (MSM). Queste infezioni decorrono generalmente in maniera asintomatica e possono fungere da serbatoio per la diffusione di casi di gonorrea, dal momento che spesso sono asintomatiche e possono persistere nel tempo. Inoltre, per capire l\u2019importanza di tali quadri, \ue8 importante sottolineare come a livello faringeo ci siano varie neisserie commensali, che possono trasferire geni di resistenza alle cefalosporine a N. gonorrhoeae. Lo scopo di questo studio \ue8 stato quello di valutare i profili della comunit\ue0 batterica del microbioma faringeo in corso di infezione da gonorrea, in soggetti MSM. Materiali e metodi. Sono stati analizzati 70 tamponi faringei provenienti da pazienti afferenti all\u2019ambulatorio MTS del Policlinico S. Orsola Malpighi di Bologna. Sulla base delle indagini molecolari ottenute mediante una Real-Time PCR (VERSANT\uae CT/GC 1.0 Assay, Siemens), attualmente utilizzata nella routine diagnostica della U.O. di Microbiologia, i pazienti sono stati divisi in due gruppi: soggetti non infetti (n=45) and pazienti con gonorrea faringea (n=25). Partendo dal DNA genomico estratto, sono state amplificate e sequenziate le regioni V3-V4 del gene 16S, per analizzare la composizione del microbioma faringeo (MiSeq Illumina). Per valutare la distribuzione e la bio-diversit\ue0 dei microrganismi sono state eseguite valutazioni di \u3b1- e \u3b2-diversit\ue0. Le differenze di abbondanza tassonomica tra i gruppi in studio sono state analizzate mediante il Mann-Whitney t-test. Un p-values &lt; 0.05 \ue8 stato considerato significativo. Lo studio ha ricevuto parere favorevole da parte del comitato etico del Policlinico (78/2017/U/Tess). Risultati. L\u2019analisi di \u3b1-diversit\ue0 non ha evidenziato differenze significative nei soggetti infetti. Seppure il microbioma faringeo di tutti i soggetti fosse dominato dalle famiglie Prevotellaceae, Veillonellaceae e Streptococcaceae, \ue8 stato possibile mettere in evidenza una differenza significativa nell\u2019abbondanza relativa di batteri anaerobi, pi\uf9 elevata in soggetti con gonorrea. Il loro microbioma, infatti, era pi\uf9 ricco in batteri dei generi Treponema, Parvimonas, Peptococcus e della famiglia Prevotellaceae, con una contemporanea riduzione di generi aerobi come Pseudomonas and Escherichia. Conclusioni. Il nostro studio \ue8 il primo a far luce sulla composizione del microbioma faringeo in corso di gonorrea. I dati ottenuti, sebbene preliminari, possono contribuire a comprendere la patogenesi delle infezioni faringee da gonorrea e a sviluppare strategie innovative per limitare la loro diffusione