Altered gene expression and DNA damage in peripheral blood cells from Friedreich's ataxia patients: Cellular model of pathology

The neurodegenerative disease Friedreich's ataxia (FRDA) is the most common autosomal-recessively inherited ataxia and is caused by a GAA triplet repeat expansion in the first intron of the frataxin gene. In this disease, transcription of frataxin, a mitochondrial protein involved in iron homeostasis, is impaired, resulting in a significant reduction in mRNA and protein levels. Global gene expression analysis was performed in peripheral blood samples from FRDA patients as compared to controls, which suggested altered expression patterns pertaining to genotoxic stress. We then confirmed the presence of genotoxic DNA damage by using a gene-specific quantitative PCR assay and discovered an increase in both mitochondrial and nuclear DNA damage in the blood of these patients (p<0.0001, respectively). Additionally, frataxin mRNA levels correlated with age of onset of disease and displayed unique sets of gene alterations involved in immune response, oxidative phosphorylation, and protein synthesis. Many of the key pathways observed by transcription profiling were downregulated, and we believe these data suggest that patients with prolonged frataxin deficiency undergo a systemic survival response to chronic genotoxic stress and consequent DNA damage detectable in blood. In conclusion, our results yield insight into the nature and progression of FRDA, as well as possible therapeutic approaches. Furthermore, the identification of potential biomarkers, including the DNA damage found in peripheral blood, may have predictive value in future clinical trials

Rapid and Efficient One-Step Metabolic Pathway Integration in <i>E. coli</i>

Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in <i>Escherichia coli</i>. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the <i>E. coli</i> genome as a stable testing platform

Immunization with a heat-killed preparation of the environmental bacterium Mycobacterium vaccae promotes stress resilience in mice

The prevalence of inflammatory diseases is increasing in modern urban societies. Inflammation increases risk of stress-related pathology; consequently, immunoregulatory or antiinflammatory approaches may protect against negative stress-related outcomes. We show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Repeated immunization with a heat-killed preparation of Mycobacterium vaccae, an immunoregulatory environmental microorganism, reduced subordinate, flight, and avoiding behavioral responses to a dominant aggressor in a murine model of chronic psychosocial stress when tested 1-2wk following the final immunization. Furthermore, immunization with M. vaccae prevented stress-induced spontaneous colitis and, in stressed mice, induced anxiolytic or fear-reducing effects as measured on the elevated plus-maze, despite stress-induced gut microbiota changes characteristic of gut infection and colitis. Immunization with M. vaccae also prevented stress-induced aggravation of colitis in a model of inflammatory bowel disease. Depletion of regulatory T cells negated protective effects of immunization with M. vaccae on stress-induced colitis and anxiety-like or fear behaviors. These data provide a framework for developing microbiome- and immunoregulation-based strategies for prevention of stress-related pathologies