Project description:We have recently described antitumor properties of Lactobacillus casei BL23 strain in both a mouse allograft model of human papilloma virus (HPV)-induced cancer and dimethylhydrazine-associated colorectal cancer. However, the mechanisms underlying these beneficial effects are still unknown. Interestingly, in vitro cellular models show that this bacterium is able to stimulate the production of high levels of IL-2. Because this cytokine has well-known antitumor properties, we decided to explore its role in the anti-cancer effects of BL23 using the HPV-induced cancer model. We found a negative correlation between IL-2 and tumor size confirming the necessity of IL-2 to protect from tumor development. Then, we blocked IL-2 synthesis using neutralizing monoclonal antibodies in mice that were challenged with lethal levels of tumor cells; this led to a significant reduction in the protective abilities of BL23. Next, we used a genetically modified strain of Lactococcus lactis to deliver exogenous IL-2 to the system, and in doing so, we were able to partially mimic the antitumor properties of BL23. Additionally, we showed the systemic role of T-cells in tumor protection through a negative correlation between tumor size and T-cells subpopulations and an increasement of BL23-specific local Foxp3 levels in tumor-bearing mice. Finally, we observed a negative correlation between tumor size and NK+ cells, but local recruitment of NK cells and cytotoxic activity appeared specific to BL23 treatment. Taken together, our data suggest that IL-2 signaling pathway plays an important role in the anti-tumoral effects of probiotic strain L. casei BL23. These results encourage further investigation in the use of probiotic strains for potential therapeutic applications to clinical practice, in particular for the treatment of colorectal cancer. Furthermore, our approach could be extended and applied to other potential beneficial microorganisms, such as gut microbiota, in order to better understand the crosstalk between microbes and the host.
Project description:The short-chain fatty acid butyrate plays critical roles in human gut health, affecting immunomodulation, cell differentiation, and apoptosis, while also serving as the preferred carbon source for colon cells. In this work, we have engineered a model probiotic organism, Escherichia coli Nissle 1917 (EcN, serotype O6:K5:H1), to produce butyrate from genomic loci up to approximately 1 g/L (11 mM). Then, for real-time monitoring of butyrate production in cultures, we developed a high-throughput biosensor that responds to intracellular butyrate concentrations, with green fluorescent protein as the reporter. This work provides a foundation for studies of butyrate for therapeutic applications.
Project description:Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a three-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate, and photobleaching rate of mEos3.2 sensitive to the buffer conditions likely by permeabilizing the yeast cell membrane. Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that 405-nm irradiation drove some red-state mEos3.2 molecules to enter an intermediate dark state, which can be converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to quantitatively compare conditions for imaging mEos3.2-tagged molecules in yeast cells. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells.
Project description:The aim of the study was to determine differences in the proteome and peptidome and zinc concentrations in the serum and tissues of chickens supplemented with a multi-strain probiotic and/or zinc glycine chelate in ovo. A total of 1400 fertilized broiler eggs (Ross × Ross 708) were divided into four groups: a control and experimental groups injected with a multi-strain probiotic, with zinc glycine chelate, and with the multi-strain probiotic and zinc glycine chelate. The proteome and peptidome were analyzed using SDS-PAGE and MALDI-TOF MS, and the zinc concentration was determined by flame atomic absorption spectrometry. We showed that in ovo supplementation with zinc glycine chelate increased the Zn concentration in the serum and yolk sac at 12 h post-hatch. The results of SDS-PAGE and western blot confirmed the presence of Cu/Zn SOD in the liver and in the small and large intestines at 12 h and at 7 days after hatching in all groups. Analysis of the MALDI-TOF MS spectra of chicken tissues showed in all experimental groups the expression of proteins and peptides that regulate immune response, metabolic processes, growth, development, and reproduction.
Project description:Appropriate semen processing and assessment are critical for successful infertility treatment. We investigated whether laboratory procedures including semen preparation and incubation affect sperm DNA integrity. A total of 153 infertile men were involved. Conventional semen parameters and sperm chromatin structure assay (SCSA) parameters, that is, DNA fragmentation index (%DFI) and high DNA stainability (%HDS), were assessed on the fresh ejaculated semen samples, which were treated and incubated under different conditions. Negative correlations were identified between the %DFI and sperm concentration, motility, progressive motility and morphology. A lower percentage of DFI was detected in spermatozoa when density gradient centrifugation (DGC) was followed by swim-up treatment in comparison with DGC alone (P < 0.01). Although the %DFI increased in a time-dependent manner with incubation both at room temperature (RT) and at 37 degrees C in air, the %DFI after 24 h at RT was significantly lower than that at 37 degrees C (P < 0.05). Incubation with 5% CO2 was effective in maintaining sperm motility (P < 0.01); however, it induced further elevation of %DFI (P < 0.001). Thus, sperm DNA damage was associated with longer incubation periods. Interestingly, common culture conditions, such as maintaining pH and temperature, compromised the sperm DNA integrity.
Project description:The objective of this study is to explore the probiotic properties and optimal growth conditions of Lactiplantibacillus plantarum BG24. L. plantarum BG24 exhibited a remarkable ability to utilize lactose, and to grow under acidic conditions and in the presence of high levels of bile salts. The strain showed the highest antibacterial activity against L. monocytogenes Scott A (zone of inhibition: 26 mm). L. plantarum BG24 was found to be resistant to 8 of the tested 19 antibiotics using the disc diffusion method.and its multiple antibiotic resistance (MAR) index was calculated as 0.421. The adhesion rate to human intestinal epithelial Caco-2 cells was determined as 37.51%. The enzyme profile of L. plantarum BG24 was investigated using API ZYM test kit and the highest enzymatic activities were found for Leucine arylamidase, β-glucosidase, Valine arylamidase, β-galactosidase and N-acetyl-β-glucosaminidase. L. plantarum BG24 strain showed higher microbial growth under static conditions (6.60 OD600) compared to 100 rpm (5.73 OD600) and 200 rpm (5.02 OD600) shaking speed due to its facultative anaerobic characteristic. However, different inoculation rates and glucose addition did not make a statistically significant difference on biomass formation (p > 0.05). The specific growth rate of L. plantarum BG24 was 0.416 h-1, the doubling time was 1.67 h, and the biomass productivity value was 0.14 gL-1 h-1 in the original MRS broth (pH 5.7) while higher values were found as 0.483 h-1, 1.43 h and 0.17 gL-1 h-1, respectively, in MRS broth (pH 6.5) medium enriched with 5 g/L yeast extract. The stirred tank bioreactor was used to optimise the growth of BG24 strain. The process variables was optimized at 0.05 vvm of aeration rate, 479 rpm of agitation speed, 3% of inoculation rate and 18 h of incubation time. The maximum biomass (g/L) production was obtained as 3.84 g/L at the optimized conditions.
Project description:A probiotic formulation combining Lactobacillus helveticus Rosell®-52, Bifidobacterium infantis Rosell®-33, and Bifidobacterium bifidum Rosell®-71 with fructooligosaccharides, first commercialized in China, has been sold in over 28 countries since 2002. Clinical studies with this blend of strains were conducted mainly in pediatric populations, and most were published in non-English journals. This comprehensive review summarizes the clinical studies in infants and children to evaluate the efficacy of this probiotic for pediatric indications. Literature searches for pediatric studies on Biostime® or Probiokid® (non-commercial name) in 6 international and Chinese databases identified 28 studies, which were classified by indications. Twelve studies show that the probiotic significantly increases the efficacy of standard diarrhea treatment regardless of etiology, reducing the risk of unresolved diarrhea (RR 0.31 [0.23; 0.42]; p < 0.0001) by 69%. In eight studies, the probiotic enhanced immune defenses, assessed by levels of various immune competence and mucosal immunity markers (six studies), and reduced the incidence of common infections (two studies). The probiotic improved iron deficiency anemia treatment efficacy (three studies), reducing the risk of unresolved anemia by 49% (RR 0.51 [0.28; 0.92]; p = 0.0263) and significantly reducing treatment side effects by 47% (RR 0.53 [0.37; 0.77]; p = 0.0009). Other studies support further investigation into this probiotic for oral candidiasis, eczema, feeding intolerance in premature babies, or hyperbilirubinemia in newborns.