The bioavailability of whole-grain rye-derived phytochemicals has not yet been comprehensively characterized, and different baking and manufacturing processes can modulate the phytochemical composition of breads and other rye products. The aim of our study was to find key differences in the phytochemical profile of plasma after the consumption of 3 breads containing rye bran when compared with a plain white wheat bread control. Plasma metabolite profiles of 12 healthy middle-aged men and women were analyzed using LC quadrupole time-of-flight mass spectrometry metabolomics analysis while fasting and at 60 min, 120 min, 240 min, and 24 h after consuming a meal that contained either 100% whole-grain sourdough rye bread or white wheat bread enriched with native unprocessed rye bran or bioprocessed rye bran. White wheat bread was used as the control. The meals were served in random order after a 12-h overnight fast, with at least 3 d between each occasion. Two sulfonated phenylacetamides, hydroxy-N-(2-hydroxyphenyl) acetamide and N-(2-hydroxyphenyl) acetamide, potentially derived from the benzoxazinoid metabolites, were among the most discriminant postprandial plasma biomarkers distinguishing intake of breads containing whole-meal rye or rye bran from the control white wheat bread. Furthermore, subsequent metabolite profiling analysis of the consumed breads indicated that different bioprocessing/baking techniques involving exposure to microbial metabolism (e.g., sourdough fermentation) have a central role in modulating the phytochemical content of the whole-grain and bran-rich breads.

Integrating viral vectors are efficient gene transfer tools, but their integration patterns have been associated with genotoxicity and oncogenicity. The recent development of highly specific designer nucleases has enabled target DNA modification and site-specific gene insertion at desired genomic loci. However, a lack of consensus exists regarding a perfect genomic safe harbour (GSH) that would allow transgenes to be stably and reliably expressed without adversely affecting endogenous gene structure and function. Ribosomal DNA (rDNA) has many advantages as a GSH, but efficient means to target integration to this locus are currently lacking. We tested whether lentivirus vector integration can be directed to rDNA by using fusion proteins consisting of the Human Immunodeficiency Virus 1 (HIV-1) integrase (IN) and the homing endonuclease I-PpoI, which has natural cleavage sites in the rDNA. A point mutation (N119A) was introduced into I-PpoI to abolish unwanted DNA cleavage by the endonuclease. The vector-incorporated IN-I-PpoIN119A fusion protein targeted integration into rDNA significantly more than unmodified lentivirus vectors, with an efficiency of 2.7%. Our findings show that IN-fusion proteins can be used to modify the integration pattern of lentivirus vectors, and to package site-specific DNA-recognizing proteins into vectors to obtain safer transgene integration.

Epilepsy is a highly heritable disorder affecting over 50 million people worldwide, of which about one-third are resistant to current treatments. Here we report a multi-ancestry genome-wide association study including 29,944 cases, stratified into three broad categories and seven subtypes of epilepsy, and 52,538 controls. We identify 26 genome-wide significant loci, 19 of which are specific to genetic generalized epilepsy (GGE). We implicate 29 likely causal genes underlying these 26 loci. SNP-based heritability analyses show that common variants explain between 39.6% and 90% of genetic risk for GGE and its subtypes. Subtype analysis revealed markedly different genetic architectures between focal and generalized epilepsies. Gene-set analyses of GGE signals implicate synaptic processes in both excitatory and inhibitory neurons in the brain. Prioritized candidate genes overlap with monogenic epilepsy genes and with targets of current antiseizure medications. Finally, we leverage our results to identify alternate drugs with predicted efficacy if repurposed for epilepsy treatment.

Rare-cutting endonucleases, such as the I-PpoI, can be used for the induction of double strand breaks (DSBs) in genome editing and targeted integration based on homologous recombination. For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques. For its applications, the endonuclease needs to be transported into the target cell nucleus, where the mechanism of transport may affect its function. Here, we have studied the lentiviral protein transduction of the integrase (IN)-PpoI fusion protein using the cis-packaging method. In genome-wide interaction studies, IN-fusion proteins were verified to bind their target sequence containing 28S ribosomal RNA (rRNA) genes with a 100-fold enrichment, despite the well-documented behavior of IN to be tethered into various genomic areas by host-cell factors. In addition, to estimate the applicability of the method, DSB-induced cytotoxic effects with different vector endonuclease configurations were studied in a panel of cells. Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA. In cell studies, certain cancerous cell lines were especially prone to DSBs in rRNA genes, which led us to test the protein transduction in a tumour environment in an in vivo study. In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases.