Developmental and epileptic encephalopathies (DEEs) are a group of severe epilepsies characterized by refractory seizures and developmental impairment. Sequencing approaches have identified causal genetic variants in only about 50% of individuals with DEEs. [1][2][3] This suggests that unknown genetic etiologies exist, potentially in the $98% of human genomes not covered by exome sequencing (ES). Here we describe seven likely pathogenic variants in regions outside of the annotated coding exons of the most frequently implicated epilepsy gene, SCN1A, encoding the alpha-1 sodium channel subunit. We provide evidence that five of these variants promote inclusion of a ''poison'' exon that leads to reduced amounts of full-length SCN1A protein. This mechanism is likely to be broadly relevant to human disease; transcriptome studies have revealed hundreds of poison exons, 4,5 including some present within genes encoding other sodium channels and in genes involved in neurodevelopment more broadly. 6 Future research on the mechanisms that govern neuronal-specific splicing behavior might allow researchers to co-opt this system for RNA therapeutics.

Despite significant advances in combinations of radiotherapy and chemotherapy, altered fractionation schedules and image-guided radiotherapy, many cancer patients fail to benefit from radiation. A prevailing hypothesis is that targeting repair of DNA double strand breaks (DSBs) can enhance radiation effects in the tumor and overcome therapeutic resistance without incurring off-target toxicities. Unrepaired DSBs can block cancer cell proliferation, promote cancer cell death and induce cellular senescence. Given the slow progress to date translating novel DSB repair inhibitors as radiosensitizers, we have explored drug repurposing, a proven route to improving speed, costs and success rates of drug development. In a prior screen where we tracked resolution of ionizing radiation-induced foci (IRIF) as a proxy for DSB repair, we had identified pitavastatin (Livalo), an HMG-CoA reductase inhibitor commonly used for lipid lowering, as a candidate radiosensitizer. Here we report that pitavastatin and other lipophilic statins are potent inhibitors of DSB repair in breast and melanoma models both in vitro and in vivo. When combined with ionizing radiation, pitavastatin increased persistent DSBs, induced senescence and enhanced acute effects of radiation on radioresistant melanoma tumors. shRNA knockdown implicated HMG-CoA reductase, farnesyl diphosphate synthase, and protein farnesyl transferase in IRIF resolution, DSB repair and senescence. These data confirm on-target activity of statins, though via inhibition of protein prenylation rather than cholesterol biosynthesis. In light of prior studies demonstrating enhanced efficacy of radiotherapy in patients taking statins, this work argues for clinical evaluation of lipophilic statins as non-toxic radiosensitizers to enhance the benefits of image-guided radiotherapy.

The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapyinduced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma in vitro and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence in vivo. Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. Implications: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.

Neem (Azadirachta indica A. Juss) is one of the most versatile tropical evergreen tree species known in India since the Vedic period (1500 BC–600 BC). Neem tree is a rich source of limonoids, having a wide spectrum of activity against insect pests and microbial pathogens. Complex tetranortriterpenoids such as azadirachtin, salanin and nimbin are the major active principles isolated from neem seed. Absolutely nothing is known about the biochemical pathways of these metabolites in neem tree. To identify genes and pathways in neem, we sequenced neem genomes and transcriptomes using next generation sequencing technologies. Assembly of Illumina and 454 sequencing reads resulted in 267 Mb, which accounts for 70% of estimated size of neem genome. We predicted 44,495 genes in the neem genome, of which 32,278 genes were expressed in neem tissues. Neem genome consists about 32.5% (87 Mb) of repetitive DNA elements. Neem tree is phylogenetically related to citrus, Citrus sinensis. Comparative analysis anchored 62% (161 Mb) of assembled neem genomic contigs onto citrus chromomes. Ultrahigh performance liquid chromatography-mass spectrometry-selected reaction monitoring (UHPLC-MS/SRM) method was used to quantify azadirachtin, nimbin, and salanin from neem tissues. Weighted Correlation Network Analysis (WCGNA) of expressed genes and metabolites resulted in identification of possible candidate genes involved in azadirachtin biosynthesis pathway. This study provides genomic, transcriptomic and quantity of top three neem metabolites resource, which will accelerate basic research in neem to understand biochemical pathways.

Archaeal proteasomes share many features with their eukaryotic counterparts and serve as important models for assembly. Proteasomes are also found in certain bacterial lineages yet their assembly mechanism is thought to be fundamentally different. Here we investigate α-ring formation using recombinant proteasomes from the archaeon Methanococcus maripaludis. Through an engineered disulfide cross-linking strategy, we demonstrate that double α-rings are structurally analogous to half-proteasomes and can form independently of single α-rings. More importantly, via targeted mutagenesis, we show that single α-rings are not required for the efficient assembly of 20S proteasomes. Our data support updating the currently held “α-ring first” view of assembly, initially proposed in studies of archaeal proteasomes, and present a way to reconcile the seemingly separate bacterial assembly mechanism with the rest of the proteasome realm. We suggest that a common assembly network underpins the absolutely conserved architecture of proteasomes across all domains of life.