PCR-based analytics of gene therapies using adeno-associated virus vectors: Considerations for cGMP method development.

The field of gene therapy has evolved and improved so that today the treatment of thousands of genetic diseases is now possible. An integral aspect of the drug development process is generating analytical methods to be used throughout clinical and commercial manufacturing. Enumeration and identification assays using genetic testing are critical to ensure the safety, efficacy, and stability of many active pharmaceutical ingredients.

The Effect of subsp. Bl-04 on Influenza A Virus Infection in Mice.

Influenza A virus infection is a major global disease requiring annual vaccination. Clinical studies indicate that certain probiotics may support immune function against influenza and other respiratory viruses, but direct molecular evidence is scarce. Here, mice were treated with a placebo or subsp.

An in vitro model of the chicken gastrointestinal tract with special emphasis to the cecal microbiota.

An in vitro model of the upper gastrointestinal tract as well as the chicken cecum was developed to have a predictive tool for estimating the production performance of animals by analyzing the feeding value of a certain diet. The upper gastrointestinal tract consists of a batch type model, whereas the cecal model is comprised of 4 semi-continuous connected vessels inoculated with cecal or fecal microbes. The upper gastrointestinal tract and cecal simulations were both run with a corn- and a wheat-based diet to simulate 2 typical feed types.

Genomic and epigenetic landscapes drive CRISPR-based genome editing in .

is a commensal bacterial genus ubiquitous in the human gastrointestinal tract, which is associated with a range of health benefits. The advent of CRISPR-based genome editing technologies provides opportunities to investigate the genetics of important bacteria and transcend the lack of genetic tools in bifidobacteria to study the basis for their health-promoting attributes. Here, we repurpose the endogenous type I-G CRISPR-Cas system and adopt an exogenous CRISPR base editor for genome engineering in subsp.

Locked Nucleic Acid Hydrolysis Probes for the Specific Identification of Probiotic Strains subsp. DSM 15954 and Bi-07™.

Probiotic health benefits are now well-recognized to be strain specific. Probiotic strain characterization and identification is thus important in clinical research and in the probiotic industry. This is becoming especially important with reports of probiotic products failing to meet the declared strain content, potentially compromising their efficacy.

Transcriptomics reveal different metabolic strategies for acid resistance and gamma-aminobutyric acid (GABA) production in select Levilactobacillus brevis strains.

Background: Of the many neurotransmitters in humans, gamma-aminobutyric acid (GABA) shows potential for improving several mental health indications such as stress and anxiety. The microbiota-gut-brain axis is an important pathway for GABAergic effects, as microbially-secreted GABA within the gut can affect host mental health outcomes. Understanding the molecular characteristics of GABA production by microbes within the gut can offer insight to novel therapies for mental health.

Epigenetics: A New Frontier in Probiotic Research.

Research into the benefits of probiotics has progressed beyond interventional studies to identifying the underlying molecular mechanisms. Health-promoting effector molecules produced by probiotics are well documented and have been linked to specific genes and even individual nucleotides. However, the factors controlling the expression of these molecules are poorly understood and we argue that epigenetic influences likely play an important role in mediating the health-promoting attributes of probiotics.

Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains.

Traditional microbiological enumeration methods have long been employed as the standard evaluation procedure for probiotic microorganisms. These methods are labor intensive, have long-time to results and inherently have a high degree of variability - up to 35%. As clinical probiotic and microbiome science continues to grow and develop, it is increasingly important that researchers thoroughly define and deliver the targeted probiotic dose.

Novel Genes and Metabolite Trends in Bifidobacterium longum subsp. infantis Bi-26 Metabolism of Human Milk Oligosaccharide 2'-fucosyllactose.

Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between bifidobacteria utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2'-fucosyllactose (2'-FL) in Bifidobacterium longum subsp. infantis Bi-26.

Transcriptional and Functional Analysis of Bifidobacterium animalis subsp. lactis Exposure to Tetracycline.

Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority recommends testing resistance using microdilution culture techniques previously used to establish inhibitory thresholds for the genus. Many subsp.

Absolute Enumeration of Probiotic Strains NCFM and subsp. Bl-04 via Chip-Based Digital PCR.

The current standard for enumeration of probiotics to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability and the inability to discern between bacterial strains. Accurate probiotic cell counts are important to confirm the delivery of a clinically documented dose for its associated health benefits. A method is described using chip-based digital PCR (cdPCR) to enumerate subsp.

Metabolic Fate of C-Labeled Polydextrose and Impact on the Gut Microbiome: A Triple-Phase Study in a Colon Simulator.

The present study introduces a novel triple-phase (liquids, solids, and gases) approach, which employed uniformly labeled [U-C] polydextrose (PDX) for the selective profiling of metabolites generated from dietary fiber fermentation in an in vitro colon simulator using human fecal inocula. Employing C NMR spectroscopy, [U-C] PDX metabolism was observed from colonic digest samples. The major C-labeled metabolites generated were acetate, butyrate, propionate, and valerate.

Safety evaluation of HOWARU Restore (Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp. lactis Bl-04 and B. lactis Bi-07) for antibiotic resistance, genomic risk factors, and acute toxicity.

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp.

Genotyping by PCR and High-Throughput Sequencing of Commercial Probiotic Products Reveals Composition Biases.

Recent advances in microbiome research have brought renewed focus on beneficial bacteria, many of which are available in food and dietary supplements. Although probiotics have historically been defined as microorganisms that convey health benefits when ingested in sufficient viable amounts, this description now includes the stipulation "well defined strains," encompassing definitive taxonomy for consumer consideration and regulatory oversight. Here, we evaluated 52 commercial dietary supplements covering a range of labeled species using plate counting and targeted genotyping.

CRISPR immunity drives rapid phage genome evolution in Streptococcus thermophilus.

Unlabelled: Many bacteria rely on CRISPR-Cas systems to provide adaptive immunity against phages, predation by which can shape the ecology and functioning of microbial communities. To characterize the impact of CRISPR immunization on phage genome evolution, we performed long-term bacterium-phage (Streptococcus thermophilus-phage 2972) coevolution experiments. We found that in this species, CRISPR immunity drives fixation of single nucleotide polymorphisms that accumulate exclusively in phage genome regions targeted by CRISPR.

Strong bias in the bacterial CRISPR elements that confer immunity to phage.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide adaptive immunity against phage via spacer-encoded CRISPR RNAs that are complementary to invasive nucleic acids. Here, we challenge Streptococcus thermophilus with a bacteriophage, and used PCR-based metagenomics to monitor phage-derived spacers daily for 15 days in two experiments. Spacers that target the host chromosome are infrequent and strongly selected against, suggesting autoimmunity is lethal.

Analysis of the Lactobacillus casei supragenome and its influence in species evolution and lifestyle adaptation.

Background: The broad ecological distribution of L. casei makes it an insightful subject for research on genome evolution and lifestyle adaptation. To explore evolutionary mechanisms that determine genomic diversity of L.