Quantitative recovery of viable Lactobacillus paracasei CNCM I-1572 (L. casei DG®) after gastrointestinal passage in healthy adults

Probiotics are live microorganisms, and viability after transit through the gastrointestinal tract (GIT) is considered an inherent property of the health benefits of probiotics. The aim of the present study was to quantify the viable and total loads of Lactobacillus paracasei DG cells after passage through the GIT following the consumption of the probiotic product Enterolactis (L. casei DG\uae; L. paracasei CNCM I-1572; L. paracasei DG) from drinkable vials by healthy adults. We developed a novel method for discriminating and enumerating culturable L. paracasei DG cells based on the unique sticky, filamentous phenotype of this strain on MRS agar containing vancomycin and kanamycin. The identity of DG was also confirmed with strain-specific primers by colony PCR. This method was used for a recovery study of the DG strain to quantify viable cells in the fecal samples of 20 volunteers during a 1-week probiotic consumption period and a 1-week follow-up. We isolated L. paracasei DG from at least one fecal sample from all the volunteers. The highest concentration of viable DG cells [ranging from 3.6 to 6.7 log10colony-forming unit (CFU) per gram of feces] in the feces was observed between 4 and 8 days from the beginning of Enterolactis intake and for up to 5 days after cessation of intake. As expected, the total DG count determined by real-time quantitative PCR (qPCR) was mostly higher than the viable DG cells recovered. Viable count experiments, carried out by combining ad hoc culture-based discriminative conditions and strain-specific molecular biological protocols, unambiguously demonstrated that L. paracasei DG can survive gastrointestinal transit in healthy adults when ingested as Enterolactis in drinkable vials containing no less than one billion CFU at the end of shelf life

Impact of a multistrain probiotic formulation with high bifidobacterial content on the fecal bacterial community and short-chain fatty acid levels of healthy adults

The consumption of probiotic products is continually increasing, supported by growing scientific evidence of their efficacy. Considering that probiotics may primarily affect health (either positively or negatively) through gut microbiota modulation, the first aspect that should be evaluated is their impact on the intestinal microbial ecosystem. In this study, we longitudinally analyzed the bacterial taxonomic composition and organic acid levels in four fecal samples collected over the course of four weeks from 19 healthy adults who ingested one capsule a day for two weeks of a formulation containing at least 70 billion colony-forming units, consisting of 25% lactobacilli and 75% Bifidobacterium animalis subsp. lactis. We found that 16S rRNA gene profiling showed that probiotic intake only induced an increase in a single operational taxonomic unit ascribed to B. animalis, plausibly corresponding to the ingested bifidobacterial strain. Furthermore, liquid chromatography/mass spectrometry revealed a significant increase in the lactate and acetate/butyrate ratio and a trend toward a decrease in succinate following probiotic administration. The presented results indicate that the investigated probiotic formulation did not alter the intestinal bacterial ecosystem of healthy adults and suggest its potential ability to promote colonization resistance in the gut through a transient increase in fecal bifidobacteria, lactic acid, and the acetate/butyrate ratio

Association between aortic calcification, cardiovascular events, and mortality in kidney and pancreas-kidney transplant recipients

BACKGROUND: Cardiovascular (CV) disease is the leading cause of death in kidney and simultaneous pancreas-kidney (SPK) transplant recipients. Assessing abdominal aortic calcification (AAC), using lateral spine x-rays and the Kaupilla 24-point AAC (0-24) score, may identify transplant recipients at higher CV risk. METHODS: Between the years 2000 and 2015, 413 kidney and 213 SPK first transplant recipients were scored for AAC at time of transplant and then followed for CV events (coronary heart, cerebrovascular, or peripheral vascular disease), graft-loss, and all-cause mortality. RESULTS: The mean age was 44 ± 12 years (SD) with 275 (44%) having AAC (26% moderate: 1-7 and 18% high: ≥8). After a median of 65 months (IQR 29-107 months), 46 recipients experienced CV events, 59 died, and 80 suffered graft loss. For each point increase in AAC, the unadjusted hazard ratios (HR) for CV events and mortality were 1.11 (95% CI 1.07-1.15) and 1.11 (1.08-1.15). These were similar after adjusting for age, gender, smoking, transplant type, dialysis vintage, and diabetes: aHR 1.07 (95% CI 1.02-1.12) and 1.09 (1.04-1.13). For recipients with high versus no AAC, the unadjusted and fully-adjusted HRs for CV events were 5.90 (2.90-12.02) and 3.51 (1.54-8.00), for deaths 5.39 (3.00-9.68) and 3.38 (1.71-6.70), and for graft loss 1.30 (0.75-2.28) and 1.94 (1.04-3.27) in age and smoking history-adjusted analyses. CONCLUSION: Kidney and SPK transplant recipients with high AAC have 3-fold higher CV and mortality risk and poorer graft outcomes than recipients without AAC. AAC scoring may be useful in assessing and targeted risk-lowering strategies

Effect of oral consumption of capsules containing Lactobacillus paracasei LPC-S01 on the vaginal microbiota of healthy adult women: a randomized, placebo-controlled, double-blind crossover study

Oral consumption of probiotics is practical and can be an effective solution to preserve vaginal eubiosis. Here, we studied the ability of orally administered Lactobacillus paracasei LPC-S01 (DSM 26760) to affect the composition of the vaginal microbiota and colonize the vaginal mucosa in nondiseased adult women. A total of 40 volunteers took oral probiotic (24 billion CFU) or placebo capsules daily for 4 weeks, and after a 4-week washout, they switched to placebo or probiotic capsules according to the crossover design. A total of 23 volunteers completed the study according to the protocol. Before and after capsule ingestion, vaginal swabs were collected for qPCR quantification to detect L. paracasei LPC-S01 and for 16S rRNA gene sequencing. Vaginal swabs were grouped according to their bacterial taxonomic structure into nine community state types (CSTs), four of which were dominated by lactobacilli. Lactobacillus paracasei LPC-S01 was detected in the vagina of two participants. Statistical modeling (including linear mixed-effects model analysis) demonstrated that daily intake of probiotic capsules reduced the relative abundance of Gardnerella spp. Quantitative PCR with Gardnerella vaginalis primers confirmed this result. Considering the pathogenic nature of G. vaginalis, these results suggest a potential positive effect of this probiotic capsule on the vaginal microbial ecosystem

191pd_pr9 weeks versus 1 year adjuvant trastuzumab for her2 early breast cancer subgroup analysis of the shorther trial allows to identify patients for whom a shorter trastuzumab administration may have a favourable risk benefit ratio

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RESEARCHERS' PYRAMID. A NEW OPPORTUNITY FOR ITALIAN ONCOLOGY RESEARCH INFRASTRUCTURE?

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Cutaneous barrier leakage and gut inflammation drive skin disease in Omenn syndrome

Background: Severe early-onset erythroderma and gut inflammation, with massive tissue infiltration of oligoclonal activated T cells are the hallmark of Omenn syndrome (OS). Objective: The impact of altered gut homeostasis in the cutaneous manifestations of OS remains to be clarified. Methods: We analyzed a cohort of 15 patients with OS and the 129Sv/C57BL/6 knock-in Rag2R229Q/R229Q (Rag2R229Q) mouse model. Homing phenotypes of circulating lymphocytes were analyzed by flow cytometry. Inflammatory cytokines and chemokines were examined in the sera by ELISA and in skin biopsies by immunohistochemistry and in situ RNA hybridization. Experimental colitis was induced in mice by dextran sulfate sodium salt. Results: We show that memory/activated T cells from patients with OS and from the Rag2R229Q mouse model of OS abundantly express the skin homing receptors cutaneous lymphocyte associated antigen and CCR4 (Ccr4), associated with high levels of chemokine C-C motif ligands 17 and 22. Serum levels of LPS are also elevated. A broad TH1/TH2/TH17 inflammatory signature is detected in the periphery and in the skin. Increased Tlr4 expression in the skin of Rag2R229Q mice is associated with enhanced cutaneous inflammation on local and systemic administration of LPS. Likewise, boosting colitis in Rag2R229Q mice results in increased frequency of Ccr4+ splenic T cells and worsening of skin inflammation, as indicated by epidermal thickening, enhanced epithelial cell activation, and dermal infiltration by TH1 effector T cells. Conclusions: These results support the existence of an interplay between gut and skin that can sustain skin inflammation in OS

The C-terminal domain from S. cerevisiae Pat1 displays two conserved regions involved in decapping factor recruitment

Eukaryotic mRNA decay is a highly regulated process allowing cells to rapidly modulate protein production in response to internal and environmental cues. Mature translatable eukaryotic mRNAs are protected from fast and uncontrolled degradation in the cytoplasm by two cis-acting stability determinants: a methylguanosine (m(7)G) cap and a poly(A) tail at their 5' and 3' extremities, respectively. The hydrolysis of the m(7)G cap structure, known as decapping, is performed by the complex composed of the Dcp2 catalytic subunit and its partner Dcp1. The Dcp1-Dcp2 decapping complex has a low intrinsic activity and requires accessory factors to be fully active. Among these factors, Pat1 is considered to be a central scaffolding protein involved in Dcp2 activation but also in inhibition of translation initiation. Here, we present the structural and functional study of the C-terminal domain from S. cerevisiae Pat1 protein. We have identified two conserved and functionally important regions located at both extremities of the domain. The first region is involved in binding to Lsm1-7 complex. The second patch is specific for fungal proteins and is responsible for Pat1 interaction with Edc3. These observations support the plasticity of the protein interaction network involved in mRNA decay and show that evolution has extended the C-terminal alpha-helical domain from fungal Pat1 proteins to generate a new binding platform for protein partners

Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis

Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis

Probiotic supplementation influences the diversity of the intestinal microbiota during early stages of farmed Senegalese sole (Solea senegalensis, Kaup, 1858)

Ingestion of bacteria at early stages results in establishment of a primary intestinal microbiota which likely undergoes several stages along fish life. The role of this intestinal microbiota regulating body functions is crucial for larval development. Probiotics have been proved to modulate this microbiota and exert antagonistic effects against fish pathogens. In the present study, we aimed to determine bacterial diversity along different developmental stages of farmed Senegalese sole (Solea senegalensis) after feeding probiotic (Shewanella putrefaciens Pdp11) supplemented diet for a short period (10–30 days after hatching, DAH). Intestinal lumen contents of sole larvae fed control and probiotic diets were collected at 23, 56, 87, and 119 DAH and DNA was amplified using 16S rDNA bacterial domain-specific primers. Amplicons obtained were separated by denaturing gradient gel electrophoresis (DGGE), cloned, and resulting sequences compared to sequences in GenBank. Results suggest that Shewanella putrefaciens Pdp11 induces a modulation of the dominant bacterial taxa of the intestinal microbiota from 23 DAH. DGGE patterns of larvae fed the probiotic diet showed a core of bands related to Lactobacillus helveticus, Pseudomonas acephalitica, Vibrio parahaemolyticus,and Shewanella genus, together with increased Vibri o genus presence. In addition, decreased number of clones related to Photobacterium damselae subsp piscicida at 23 and 56 DAH was observed in probiotic-fed larvae. A band corresponding to Shewanella putrefaciens Pdp11 was sequenced as predominant from 23 to 119 DAH samples, confirming the colonization by the probiotics. Microbiota modulation obtained via probiotics addition emerges as an effective tool to improve Solea senegalensis larviculture.En prens