Project description:Inherited retinal diseases (IRDs) cause progressive loss of light-sensitive photoreceptors in the eye and can lead to blindness. Gene-based therapies for IRDs have shown remarkable progress in the past decade, but the vast majority of forms remain untreatable. In the era of personalised medicine, induced pluripotent stem cells (iPSCs) emerge as a valuable system for cell replacement and to model IRD because they retain the specific patient genome and can differentiate into any adult cell type. Three-dimensional (3D) iPSCs-derived retina-like tissue called retinal organoid contains all major retina-specific cell types: amacrine, bipolar, horizontal, retinal ganglion cells, Müller glia, as well as rod and cone photoreceptors. Here, we describe the main applications of retinal organoids and provide a comprehensive overview of the state-of-art analysis methods that apply to this model system. Finally, we will discuss the outlook for improvements that would bring the cellular model a step closer to become an established system in research and treatment development of IRDs.
Project description:Probiotics are live microorganisms that confer a health benefit to the host when administered in adequate amounts. This definition links probiotic efficacy to microbial viability. The current gold standard assay for probiotic potency is enumeration using classical microbiology plating-based procedures, yielding results in colony-forming units (CFU). One drawback to plating-based procedures is high variability due to intrinsic and extrinsic uncertainties. These uncertainties make comparison between analytical procedures challenging. In this article, we provide tools to reduce measurement uncertainty and strengthen the reliability of probiotic enumerations by using analytical procedure lifecycle management (APLM). APLM is a tool that uses a step-by-step process to define procedure performance based on the concept that the reportable value (final CFU result) must be fit for its intended use. Once the procedure performance is defined, the information gathered through APLM can be used to evaluate and compare procedures. Here, we discuss the theory behind applying APLM and give practical information about its application to CFU enumeration procedures for probiotics using a simulated example and data set. Data collected in a manufacturer's development laboratory is included to support application of the concept. Implementation of APLM can lead to reduced variability by identifying specific factors (e.g., the dilution step) with significant impact on the variability and providing insights to procedural modifications that lead to process improvement. Understanding and control of the analytical procedure is improved by using these tools. The probiotics industry can confidently apply the information and analytical results generated to make decisions about processes and formulation, including overage requirements. One benefit of this approach is that companies can reduce overage costs. More reliable procedures for viable cell count determinations will improve the quality evaluation of probiotic products, and hence manufacturing procedures, while ensuring that products deliver clinically demonstrated beneficial doses.
Project description:Vaccines are key in charting a path out of the COVID-19 pandemic. However, development of new vaccines is highly dependent on availability of analytical methods for their design and evaluation. This paper highlights the challenges presented in having to rapidly develop vaccine analytical tools during an ongoing pandemic, including the need to address progressive virus mutation and adaptation which can render initial assays unreliable or redundant. It also discusses the potential of new computational modeling techniques to model and analyze key viral proteins and their attributes to assist vaccine production and assay design. It then reviews the current range of analytical tools available for COVID-19 vaccine application, ranging from in vitro assays for immunogen characterization to assays to measure vaccine responses in vivo. Finally, it provides a future perspective for COVID-19 vaccine analytical tools and attempts to predict how the field might evolve over the next 5-10 years.
Project description:Dietary ingredient and nutrient composition may affect the efficacy of additives in broilers. Specific feed ingredients can represent dietary challenging conditions for broilers, resulting in impaired performances and health, which might be alleviated by dietary probiotics and postbiotics. We assessed the effects of a Lactobacilli probiotic (Pro) and postbiotic (Post) when added to a standard (SD) and challenge (CD) diet. A completely randomized block study with 2 diets (SD, CD) and 3 additive conditions (Control, Pro and Post) involving 1,368 one-day-old Ross male broilers, equally distributed among 36 pens, from d1 to d42 was conducted. Both diets were formulated to contain identical levels of nutrients, with CD formulated to be richer than SD in nonstarch polysaccharides using rye and barley as ingredients. Readout parameters included growth performance parameters, footpad lesions score, blood minerals and biochemical parameters, and tibia health, strength, and composition. Compared to SD, CD decreased BW (1,936 vs. 2,033 g; p = 0.001), increased FCR (p < 0.01) and impaired tibia health and strength (p < 0.05) at d35, thereby confirming the challenging effect of CD. Pro and Post increased BW in CD (+4.7 and +3.2%, respectively, at d35; P < 0.05) but not in the SD group, without affecting FCR. Independently of the diet, Pro increased plasma calcium, phosphorus and uric acid at d21 (+6.2, +7.4, and +15.5%, respectively) and d35 (+6.6, +6.2 and +21.0%, respectively) (P < 0.05) while Post increased plasma magnesium only at d21 (+11.3%; P = 0.037). Blood bile acids were affected by additives in an age- and diet-dependent manner, with some opposite effects between dietary conditions. Diet composition modulated Pro and Post effects on broiler growth performance. Additionally, Pro and Post affected animal metabolism and leg health diet-dependently for some but not all investigated parameters. Our findings show that the effects of pro- and postbiotics on the growth performance and physiology of broilers can be dependent on diet composition and thus possibly other factors affecting diet characteristics.
Project description:Plant small RNAs are a diverse and complex set of molecules, ranging in length from 21 to 24 nt, involved in a wide range of essential biological processes. Nowadays, high-throughput sequencing is the most commonly used method for the discovery and quantification of small RNAs. However, it is known that several biases can occur during the preparation of small RNA libraries, especially using low input RNA. We used two types of plant biological samples to evaluate the performance of seven commercially available methods for small RNA library construction, using different RNA input amounts. We show that when working with plant material, library construction methods have differing capabilities to capture small RNAs, and that different library construction methods provide better results when applied to the detection of microRNAs, phased small RNAs, or tRNA-derived fragments.
Project description:The use of flow cytometry to enumerate microorganisms is gaining traction over the traditional plate count technique on the basis of superior accuracy, precision and time-to-result. Here, we assessed the suitability of live/dead flow cytometry for the enumeration of mixed populations of probiotic bacteria (L. acidophilus, L. paracasei, L. plantarum, L. salivarius, B. lactis and B. bifidum) whilst comparing outcomes with plate counting. Using a novel gating strategy designed specifically for the enumeration of mixed populations, the application of flow cytometry resulted in the detection of higher numbers of viable bacteria with a greater level of repeatability than plate counting (RSD of 6.82 and 13.14% respectively). Across all multi-species blends tested, viable cell input was more accurately recovered by flow cytometry (101.8 ± 6.95%) than plate counts (81.37 ± 16.03%). However, when certain probiotic mixtures contained preparations with high numbers of non-viable cells in their total population, flow cytometry had the potential for overestimation of the viable population. Nevertheless, the comparative plate counts of these mixtures were low and variable, thus supporting the use of flow cytometry for the enumeration of viable bacteria in mixed populations. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-023-02792-2.
Project description:The oral cavity plays a crucial role in food digestion and immune protection. Thus, maintaining oral health is necessary. Postbiotic and heat-killed probiotic cells have shown increased antibacterial potential with stable viability compared with live strains. However, clinical evidence regarding their effect on oral health is insufficient. Therefore, in this study, we tested postbiotic lozenges of Lactobacillus salivarius subsp. salicinius AP-32, L. paracasei ET-66, and L. plantarum LPL28 and heat-killed probiotic lozenges of L. salivarius subsp. salicinius AP-32 and L. paracasei ET-66 for their effect on oral health. In total, 75 healthy individuals were blindly and randomly divided into placebo, postbiotic lozenge, and heat-killed probiotic lozenge groups and were administered the respective lozenge type for 4 weeks. Postbiotic and heat-killed probiotic lozenge groups demonstrated antibacterial activities with a considerable increase in L. salivarius in their oral cavity. Furthermore, their salivary immunoglobulin A, Lactobacillus, and Bifidobacterium increased. Subjective questionnaires completed by the participants indicated that participants in both the experimental groups developed better oral health and intestinal conditions than those in the placebo group. Overall, our study revealed that a food additive in the form of an oral postbiotic or heat-killed probiotic lozenge may effectively enhance oral immunity, inhibit the growth of oral pathogens, and increase the numbers of beneficial oral microbiota.
Project description:As the leading cause of acute gastroenteritis worldwide, human noroviruses (HuNoVs) have caused around 685 million cases of infection and nearly $60 billion in losses every year. Despite their highly contagious nature, an effective vaccine for HuNoVs has yet to become commercially available. Therefore, rapid detection and subtyping of noroviruses is crucial for preventing viral spread. Over the past half century, there has been monumental progress in the development of techniques for the detection and analysis of noroviruses. However, currently no rapid, portable assays are available to detect and subtype infectious HuNoVs. The purpose of this review is to survey and present different analytical techniques for the detection and characterization of noroviruses.
Project description:This study evaluates the potential of various yeast strains as probiotic and postbiotic agents for agglutinating enteric pathogens, offering a preventive approach to gastrointestinal infections. Different yeast species were tested in vitro against a range of pathogenic bacteria, including enterotoxigenic Escherichia coli ETEC, Shigella flexneri, Salmonella enterica serovar Typhimurium, and Salmonella enterica serovar Enteritidis, to assess their capacity for pathogen agglutination. Additionally, inactivated yeasts were obtained using a novel chemical treatment and employed to explore their efficacy as postbiotic agents. The results suggest that both live and inactivated yeasts are able to agglutinate the different pathogens, potentially limiting bacterial colonization. Notably, we also demonstrated that Wickerhamomyces anomalus, Saccharomyces cerevisiae, and Pichia fermentans, exhibiting agglutination activity, were capable of reducing bacterial adhesion to HeLa cells in vitro. This research highlights yeast's probiotic and postbiotic potential and supports the development of novel yeast-based products for preventing enteric infections.