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Pharmaceutical litigation often begins when a generic drug company files an application to have its generic drug approved by the FDA. That application is received by the FDA in the District of Maryland. To \"submit\" it is a statutory act of patent infringement under the Hatch-Waxman Act. Establishing venue in subsequent Hatch-Waxman litigation can be complex because Hatch-Waxman litigation often involves simultaneous and independent lawsuits against many generic applicants. A Hatch-Waxman plaintiff might reasonably attempt to consolidate litigation in a single district court; Hatch-Waxman defendants might reasonably resist consolidation in the plaintiff's preferred venue. Recent Supreme Court and Federal Circuit case law has narrowed venue options for Hatch-Waxman plaintiffs. This Comment argues for an interpretation of Hatch-Waxman's statutory act of patent infringement and the patent venue rules that moves toward a centralized venue for Hatch-Waxman litigation in the District of Maryland.

Proteomic and genomic analysis of Polycomb group complexes in embryonic stem cells and neural progenitor cells identifies new PRC1 and PRC2 interaction partners and targets during neural lineage commitment. Although the core subunits of Polycomb group (PcG) complexes are well characterized, little is known about the dynamics of these protein complexes during cellular differentiation. We used quantitative interaction proteomics and genome-wide profiling to study PcG proteins in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We found that the stoichiometry and genome-wide binding of PRC1 and PRC2 were highly dynamic during neural differentiation. Intriguingly, we observed a downregulation and loss of PRC2 from chromatin marked with trimethylated histone H3 K27 (H3K27me3) during differentiation, whereas PRC1 was retained at these sites. Additionally, we found PRC1 at enhancer and promoter regions independently of PRC2 binding and H3K27me3. Finally, overexpression of NPC-specific PRC1 interactors in ESCs led to increased Ring1b binding to, and decreased expression of, NPC-enriched Ring1b-target genes. In summary, our integrative analyses uncovered dynamic PcG subcomplexes and their widespread colocalization with active chromatin marks during differentiation.

Interaction proteomics studies have provided fundamental insights into multimeric biomolecular assemblies and cell-scale molecular networks. Significant recent developments in mass spectrometry-based interaction proteomics have been fueled by rapid advances in label-free, isotopic, and isobaric quantitation workflows. Here, we report a quantitative protein–DNA and protein–nucleosome binding assay that uses affinity purifications from nuclear extracts coupled with isobaric chemical labeling and mass spectrometry to quantify apparent binding affinities proteome-wide. We use this assay with a variety of DNA and nucleosome baits to quantify apparent binding affinities of monomeric and multimeric transcription factors and chromatin remodeling complexes. Quantitative mass spectrometry enables the proteome-wide assessment of biomolecular binding affinities. While previous approaches mainly focused on protein–small molecule interactions, the authors here present a method to probe protein–DNA and protein–nucleosome binding affinities at proteome scale.

Genome-wide association studies (GWAS) have identified ~20 melanoma susceptibility loci, most of which are not functionally characterized. Here we report an approach integrating massively-parallel reporter assays (MPRA) with cell-type-specific epigenome and expression quantitative trait loci (eQTL) to identify susceptibility genes/variants from multiple GWAS loci. From 832 high-LD variants, we identify 39 candidate functional variants from 14 loci displaying allelic transcriptional activity, a subset of which corroborates four colocalizing melanocyte cis -eQTL genes. Among these, we further characterize the locus encompassing the HIV-1 restriction gene, MX2 (Chr21q22.3), and validate a functional intronic variant, rs398206. rs398206 mediates the binding of the transcription factor, YY1, to increase MX2 levels, consistent with the cis -eQTL of MX2 in primary human melanocytes. Melanocyte-specific expression of human MX2 in a zebrafish model demonstrates accelerated melanoma formation in a BRAF V600E background. Our integrative approach streamlines GWAS follow-up studies and highlights a pleiotropic function of MX2 in melanoma susceptibility. There are more than 20 known melanoma susceptibility genes. Here, using a massively parallel reporter assay, the authors identify risk-associated variants that alter gene transcription, and demonstrate that expression of one such gene, MX2 , leads to the promotion of melanoma in a zebrafish model.

David Adams, Julia Newton-Bishop, Timothy Bishop, Nicholas Hayward and colleagues identify loss-of-function variants in POT1 in several families with early onset multiple primary melanoma. They further show that these variants disrupt telomere binding by POT1 and are associated with increased telomere length. Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases 1 , and rare variants in CDK4 , BRCA2 , BAP1 and the promoter of TERT have also been linked to the disease 2 , 3 , 4 , 5 . Here we set out to identify new high-penetrance susceptibility genes by sequencing 184 melanoma cases from 105 pedigrees recruited in the UK, The Netherlands and Australia that were negative for variants in known predisposition genes. We identified families where melanoma cosegregates with loss-of-function variants in the protection of telomeres 1 gene ( POT1 ), with a proportion of family members presenting with an early age of onset and multiple primary tumors. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding and leading to increased telomere length. These findings suggest that POT1 variants predispose to melanoma formation via a direct effect on telomeres.

The nucleosome presents a formidable barrier to DNA-templated transcription by the RNA polymerase II machinery. Overcoming this transcriptional barrier in a locus-specific manner requires sequence-specific recognition of nucleosomal DNA by ‘pioneer’ transcription factors (TFs). Cell fate decisions, in turn, depend on the coordinated action of pioneer TFs at cell lineage-specific gene regulatory elements. Although it is already appreciated that pioneer factors play a critical role in cell differentiation, our understanding of the structural and biochemical mechanisms by which they act is still rapidly expanding. Recent research has revealed novel insight into modes of nucleosome-TF binding and uncovered kinetic principles by which nucleosomal DNA compaction affects both TF binding and residence time. Here, we review progress and argue that these structural and kinetic studies suggest new models of gene regulation by pioneer TFs.

Kevin Brown and colleagues functionally characterize a melanoma risk locus encompassing PARP1 , correlating the risk genotype to PARP1 gene expression levels in melanoma cells. They identify an intronic gene-regulatory variant in PARP1 and find that PARP1 can promote cell proliferation and rescue oncogene-induced senescence, likely through MITF . Previous genome-wide association studies have identified a melanoma-associated locus at 1q42.1 that encompasses a ∼100-kb region spanning the PARP1 gene. Expression quantitative trait locus (eQTL) analysis in multiple cell types of the melanocytic lineage consistently demonstrated that the 1q42.1 melanoma risk allele (rs3219090[G]) is correlated with higher PARP1 levels. In silico fine-mapping and functional validation identified a common intronic indel, rs144361550 (−/GGGCCC; r 2 = 0.947 with rs3219090), as displaying allele-specific transcriptional activity. A proteomic screen identified RECQL as binding to rs144361550 in an allele-preferential manner. In human primary melanocytes, PARP1 promoted cell proliferation and rescued BRAF V600E -induced senescence phenotypes in a PARylation-independent manner. PARP1 also transformed TERT -immortalized melanocytes expressing BRAF V600E . PARP1-mediated senescence rescue was accompanied by transcriptional activation of the melanocyte-lineage survival oncogene MITF , highlighting a new role for PARP1 in melanomagenesis.

Oxygen breathing at elevated partial pressures (PO 2 ’s) at or more than 3 atmospheres absolute (ATA) causes a reduction in brain γ-aminobutyric acid (GABA) levels that impacts the development of central nervous system oxygen toxicity (CNS-OT). Drugs that increase brain GABA content delay the onset of CNS-OT, but it is unknown if oxidant damage is lessened because brain tissue PO 2 remains elevated during hyperbaric oxygen (HBO 2 ) exposures. Experiments were performed in rats and mice to measure brain GABA levels with or without GABA transporter inhibitors (GATs) and its influence on cerebral blood flow, oxidant damage, and aspects of mitochondrial quality control signaling (mitophagy and biogenesis). In rats pretreated with tiagabine (GAT1 inhibitor), the tachycardia, secondary rise in mean arterial blood pressure, and cerebral hyperemia were prevented during HBO 2 at 5 and 6 ATA. Tiagabine and the nonselective GAT inhibitor nipecotic acid similarly extended HBO 2 seizure latencies. In mice pretreated with tiagabine and exposed to HBO 2 at 5 ATA, nuclear and mitochondrial DNA oxidation and astrocytosis was attenuated in the cerebellum and hippocampus. Less oxidant injury in these regions was accompanied by reduced conjugated microtubule-associated protein 1A/1B-light chain 3 (LC3-II), an index of mitophagy, and phosphorylated cAMP response element binding protein (pCREB), an initiator of mitochondrial biogenesis. We conclude that GABA prevents cerebral hyperemia and delays neuroexcitation under extreme HBO 2 , limiting oxidant damage in the cerebellum and hippocampus, and likely lowering mitophagy flux and initiation of pCREB-initiated mitochondrial biogenesis.

Transcription-blocking DNA lesions are removed by transcription-coupled nucleotide excision repair (TC-NER) to preserve cell viability. TC-NER is triggered by the stalling of RNA polymerase II at DNA lesions, leading to the recruitment of TC-NER-specific factors such as the CSA–DDB1–CUL4A–RBX1 cullin–RING ubiquitin ligase complex (CRL CSA ). Despite its vital role in TC-NER, little is known about the regulation of the CRL CSA complex during TC-NER. Using conventional and cross-linking immunoprecipitations coupled to mass spectrometry, we uncover a stable interaction between CSA and the TRiC chaperonin. TRiC’s binding to CSA ensures its stability and DDB1-dependent assembly into the CRL CSA complex. Consequently, loss of TRiC leads to mislocalization and depletion of CSA, as well as impaired transcription recovery following UV damage, suggesting defects in TC-NER. Furthermore, Cockayne syndrome (CS)-causing mutations in CSA lead to increased TRiC binding and a failure to compose the CRL CSA complex. Thus, we uncover CSA as a TRiC substrate and reveal that TRiC regulates CSA-dependent TC-NER and the development of CS. An integrated network of chaperones and protein degradation machineries called the proteostasis network (PN) is required to maintain protein homeostasis. Here the authors show that one of the components of the PN, the chaperonin TRiC, interacts with the core transcription-coupled nucleotide excision repair protein CSA to ensure its assembly into the CRLCSA complex.

Background Oxygen-rich breathing mixtures up to 100% are used in some underwater diving operations for several reasons. Breathing elevated oxygen partial pressures (PO 2 ) increases the risk of developing central nervous system oxygen toxicity (CNS-OT) which could impair performance or result in a seizure and subsequent drowning. We aimed to study the dynamics of the electrodermal activity (EDA) and heart rate (HR) while breathing elevated PO 2 in the hyperbaric environment (HBO 2 ) as a possible means to predict impending CNS-OT. Methods EDA is recorded during 50 subject exposures (26 subjects) to evaluate CNS-OT in immersed (head out of water) exercising divers in a hyperbaric chamber breathing 100% O 2 at 35 feet of seawater (FSW), (PO 2  = 2.06 ATA) for up to 120 min. Results 32 subject exposures exhibit symptoms “definitely” or “probably” due to CNS-OT before the end of the exposure, whereas 18 do not. We obtain traditional and time-varying spectral indices (TVSymp) of EDA to determine its utility as predictive physio markers. Variations in EDA and heart rate (HR) for the last 5 min of the experiment are compared to baseline values prior to breathing O 2 . In the subset of experiments where “definite” CNS-OT symptoms developed, we find a significant elevation in the mean ± standard deviation TVSymp value 57 ± 79 s and median of 10 s, prior to symptoms. Conclusions In this retrospective analysis, TVSymp may have predictive value for CNS-OT with high sensitivity (1.0) but lower specificity (0.48). Additional work is being undertaken to improve the detection algorithm. Plain Language Summary This study looked at the effects of breathing high levels of oxygen during underwater diving and the risk of central nervous system oxygen toxicity. This toxicity can cause problems with movement, seizures or even drowning. We wanted to see if changes in skin and heart activity could help predict the symptoms of toxicity. We tested 26 divers (50 dives) in a special chamber. They breathed pure oxygen at increased pressure (equivalent to being underwater at 35 feet). 32 dives showed signs of toxicity, while 18 did not. We looked at the electrodermal activity (a measurement of the skin conductance) and heart rate data to see if they could warn of an issue. We found that in dives where toxicity symptoms definitely developed, there were significant changes in electrodermal activity around 57 s before symptoms appeared. While this method was very sensitive, it wasn’t always specific. We are working on improving this prediction method. This may be used to warn divers of dangerous gases so they can switch breathing gases or move to a shallower depth, and can improve the chances of escaping a disabled submarine. Posada-Quintero et al. study the dynamics of the electrodermal activity and heart rate while breathing at elevated oxygen partial pressures in a hyperbaric environment. Electrodermal activitycan be used to predict the onset of central nervous system oxygen toxicity symptoms in divers resulting from prolonged exposure to a hyperbaric environment.