Development of a Python-based electron ionization mass spectrometry amino acid and peptide fragment prediction model.

The increased fragmentation caused by harsher ionization methods used during mass spectrometry such as electron ionization can make interpreting the mass spectra of peptides difficult. Therefore, the development of tools to aid in this spectral analysis is important in utilizing these harsher ionization methods to study peptides, as these tools may be more accessible to some researchers. We have compiled fragmentation mechanisms described in the literature, confirmed them experimentally, and used them to create a Python-based fragment prediction model for peptides analyzed under direct exposure probe electron ionization mass spectrometry. This initial model has been tested using single amino acids as well as targeted libraries of short peptides. It was found that the model does well in predicting fragments of peptides composed of amino acids for which the model is well-defined, but several cases where additional mechanistic information needs to be incorporated have been identified.

G6PD Orchestrates Genome-Wide DNA Methylation and Gene Expression in the Vascular Wall.

Recent advances have revealed the importance of epigenetic modifications to gene regulation and transcriptional activity. DNA methylation, a determinant of genetic imprinting and the de novo silencing of genes genome-wide, is known to be controlled by DNA methyltransferases (DNMT) and demethylases (TET) under disease conditions. However, the mechanism(s)/factor(s) influencing the expression and activity of epigenetic writers and erasers, and thus DNA methylation, in healthy vascular tissue is incompletely understood. Based on our recent studies, we hypothesized that glucose-6-phosphate dehydrogenase (G6PD) is a modifier of DNMT and TET expression and activity and an enabler of gene expression. In the aorta of CRISPR-edited rats with the Mediterranean G6PD variant, we determined DNA methylation by whole-genome bisulfite sequencing, gene expression by RNA sequencing, and large artery stiffness by echocardiography. Here, we documented higher expression of Dnmt1, Dnmt3a, Tet2, and Tet3 in aortas from Mediterranean G6PDS188F variant (a loss-of-function single nucleotide polymorphism) rats than their wild-type littermates. Concomitantly, we identified 17,618 differentially methylated loci genome-wide (5787 hypermethylated loci, including down-regulated genes encoding inflammation- and vasoconstriction-causing proteins, and 11,827 hypomethylated loci, including up-regulated genes encoding smooth muscle cell differentiation- and fatty acid metabolism-promoting proteins) in aortas from G6PDS188F as compared to wild-type rats. Our results demonstrated that nitric oxide, which is generated in a G6PD-derived NADPH-dependent manner, increases TET and decreases DNMT activity. Further, we observed less large artery (aorta) stiffness in G6PDS188F as compared to wild-type rats. These results establish a noncanonical function of the wild-type G6PD and G6PDS188F variant in the regulation of DNA methylation and gene expression in healthy vascular tissue and reveal that the G6PDS188F variant contributes to reducing large artery stiffness.

Studies in CRISPR-generated Mediterranean G6PD variant rats reveal G6PD orchestrates genome-wide DNA methylation and gene expression in vascular wall.

Background: Recent advances have revealed the importance of epigenetic modifications to gene regulation and transcriptional activity. DNA methylation, a determinant of genetic imprinting and de novo silencing of genes genome-wide, is known to be controlled by DNA methyltransferases (DNMT) and demethylases (TET) under disease conditions. However, the mechanism(s)/factor(s) influencing the expression and activity of DNMTs and TETs, and thus DNA methylation, in healthy vascular tissue is incompletely understood. Based on our recent studies, we hypothesized that glucose-6-phosphate dehydrogenase (G6PD) is a modifier of DNMT and TET expression and activity and an enabler of gene expression. Methods: In aorta of CRISPR-edited rats with the Mediterranean G6PD variant we determined DNA methylation by whole-genome bisulfite sequencing, gene expression by RNA sequencing, and large artery stiffness by echocardiography. Results: Here, we documented higher expression of Dnmt3a, Tet2, and Tet3 in aortas from Mediterranean G6PDS188F variant (a loss-of-function single nucleotide polymorphism) rats than their wild-type littermates. Concomitantly, we identified 17,618 differentially methylated loci genome-wide (5,787 hypermethylated loci, including down-regulated genes encoding inflammation- and vasoconstriction-causing proteins, and 11,827 hypomethylated loci, including up-regulated genes encoding smooth muscle cell differentiation- and fatty acid metabolism-promoting proteins) in aorta from G6PDS188F as compared to wild-type rats. Further, we observed less large artery (aorta) stiffness in G6PDS188F as compared to wild-type rats. Conclusions: These results establish a noncanonical function of the wild-type G6PD and G6PDS188F variant in the regulation of DNA methylation and gene expression in healthy vascular tissue and reveals G6PDS188F variant contributes to reduce large artery stiffness.

Human cortical interneurons optimized for grafting specifically integrate, abort seizures, and display prolonged efficacy without over-inhibition.

Previously, we demonstrated the efficacy of human pluripotent stem cell (hPSC)-derived GABAergic cortical interneuron (cIN) grafts in ameliorating seizures. However, a safe and reliable clinical translation requires a mechanistic understanding of graft function, as well as the assurance of long-term efficacy and safety. By employing hPSC-derived chemically matured migratory cINs in two models of epilepsy, we demonstrate lasting efficacy in treating seizures and comorbid deficits, as well as safety without uncontrolled growth. Host inhibition does not increase with increasing grafted cIN densities, assuring their safety without the risk of over-inhibition. Furthermore, their closed-loop optogenetic activation aborted seizure activity, revealing mechanisms of graft-mediated seizure control and allowing graft modulation for optimal translation. Monosynaptic tracing shows their extensive and specific synaptic connections with host neurons, resembling developmental connection specificity. These results offer confidence in stem cell-based therapy for epilepsy as a safe and reliable treatment for patients suffering from intractable epilepsy.

Mediterranean G6PD variant mitigates expression of DNA methyltransferases and right heart pressure in experimental model of pulmonary hypertension.

DNA methylation potentially contributes to the pathogenesis of pulmonary hypertension (PH). However, the role of DNA methyltransferases (DNMTs: 1, 3a, and 3b), the epigenetic writers, in modulating DNA methylation observed in PH remains elusive. Our objective was to determine DNMT activity and expression in the lungs of experimental rat models of PH. Because the activity of DNMTs is metabolically driven, another objective was to determine the role of glucose-6-phosphate dehydrogenase (G6PD) in regulating DNMT expression and activity in the lungs of novel loss-of-function Mediterranean G6PD variant (G6PDS188F) rats. As outlined for modeling PH, rats injected with sugen5416 (SU) were placed in a hypoxia (Hx) chamber set at 10% oxygen for 3 weeks and then returned to normoxia (Nx) for 5 weeks (SU/Hx/Nx). Rats kept in atmospheric oxygen and treated with SU were used as controls. We assessed the activity and expression of DNMTs in the lungs of rats exposed to SU/Hx/Nx. WT rats exposed to SU/Hx/Nx developed hypertension and exhibited increased DNMT activity and Dnmt1 and Dnmt3b expression. In G6PDS188F rats, which developed less of a SU/Hx/Nx-induced increase in right ventricle pressure and hypertrophy than WT rats, we observed a diminished increase in expression and activity of DNMTs, DNA hypomethylation, increased histone acetylation and methylation, and increased expression of genes encoding NOS3 and SOD2-vascular-protective proteins. Collectively, increased DNMTs contribute to reduced expression of protective genes and to the pathogenesis of SU/Hx/Nx-induced experimental PH. Notably, G6PD regulates the expression of DNMTs and protective proteins in the lungs of hypertensive rats.