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Uveal melanomas are molecularly distinct from cutaneous melanomas and lack mutations in BRAF, NRAS, KIT, and NF1. Instead, they are characterized by activating mutations in GNAQ and GNA11, two highly homologous alpha subunits of G(alpha q/11) heterotrimeric G proteins, and in PLCB4 (phospholipase C beta 4), the downstream effector of G(alpha q) signaling(1-3). We analyzed genomics data from 136 uveal melanoma samples and found a recurrent mutation in CYSLTR2 (cysteinyl leukotriene receptor 2) encoding a p.Leu129Gln substitution in 4 of 9 samples that lacked mutations in GNAQ, GNA11, and PLCB4 but in 0 of 127 samples that harbored mutations in these genes. The Leu129Gln CysLT(2)R mutant protein constitutively activates endogenous G(alpha q) and is unresponsive to stimulation by leukotriene. Expression of Leu129Gln CysLT(2)R in melanocytes enforces expression of a melanocyte-lineage signature, drives phorbol ester-independent growth in vitro, and promotes tumorigenesis in vivo. Our findings implicate CYSLTR2 as a uveal melanoma oncogene and highlight the critical role of G(alpha q) signaling in uveal melanoma pathogenesis.

Here, we present the first description of an isolation syndrome in a patient who suffered prolonged cardiac arrest and underwent a standard therapeutic hypothermia protocol. Two years after the arrest, the patient demonstrated no motor responses to commands, communication capabilities, or visual tracking at the bedside. However, resting neuronal metabolism and electrical activity across the entire anterior forebrain was found to be normal despite severe structural injuries to primary motor, parietal, and occipital cortices. In addition, using quantitative electroencephalography, the patient showed evidence for willful modulation of brain activity in response to auditory commands revealing covert conscious awareness. A possible explanation for this striking dissociation in this patient is that altered neuronal recovery patterns following therapeutic hypothermia may lead to a disproportionate preservation of anterior forebrain cortico-thalamic circuits even in the setting of severe hypoxic injury to other brain areas. Compared to recent reports of other severely brain-injured subjects with such dissociation of clinically observable (overt) and covert behaviors, we propose that this case represents a potentially generalizable mechanism producing an isolation syndrome of blindness, motor paralysis, and retained cognition as a sequela of cardiac arrest and therapeutic hypothermia. Our findings further support that highly-preserved anterior cortico-thalamic integrity is associated with the presence of conscious awareness independent from the degree of injury to other brain areas.

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes-UtpA and UtpB-interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 50 end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 50 ETS and U3 snoRNA as well as the 30 boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.

Developmental specification of germ cells lies at the heart of inheritance, as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs)(1,2), precursors of sex-specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own 'latent' form of pluripotency. For example, PGCs express a number of transcription factors in common with embryonic stem (ES) cells, including OCT4 (encoded by Pou5f1), SOX2, NANOG and PRDM14 (refs 2-4). A biochemical mechanism by which these transcription factors converge on chromatin to produce the dramatic rearrangements underlying ES-cell-and PGC-specific transcriptional programs remains poorly understood. Here we identify a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification in mice. Cbfa2t2(-/-) mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional 'passenger' role of a co-repressor, CBFA2T2 functions synergistically with transcription factors at the crossroads of the fundamental developmental plasticity between uni- and pluripotency.

Sofosbuvir displays a high phenotypic barrier to resistance, and it is a component of several combination therapies for hepatitis C virus (HCV) infections. HCV fitness can be a determinant of decreased sensitivity to direct-acting antiviral agents such as telaprevir or daclatasvir, but fitness-dependent decreased drug sensitivity has not been established for drugs with a high phenotypic barrier to resistance. Low-and high-fitness HCV populations and biological clones derived from them were used to infect Huh-7.5 hepatoma cells. Sofosbuvir efficacy was analyzed by measuring virus progeny production during several passages and by selection of possible sofosbuvir resistance mutations determined by sequencing the NS5B-coding region of the resulting populations. Sofosbuvir exhibited reduced efficacy against high-fitness HCV populations, without the acquisition of sofosbuvir-specific resistance mutations. A reduced sofosbuvir efficacy, similar to that observed with the parental populations, was seen for high-fitness individual biological clones. In independently derived high-fitness HCV populations or clones passaged in the presence of sofosbuvir, M289L was selected as the only substitution in the viral polymerase NS5B. In no case was the sofosbuvir-specific resistance substitution S282T observed. High HCV fitness can lead to decreased sensitivity to sofosbuvir, without the acquisition of specific sofosbuvir resistance mutations. Thus, fitness-dependent drug sensitivity can operate with HCV inhibitors that display a high barrier to resistance. This mechanism may underlie treatment failures not associated with selection of sofosbuvir-specific resistance mutations, linked to in vivo fitness of pretreatment viral populations.

While engagement of the inhibitory Fc gamma-receptor (Fc gamma R) IIB is an absolute requirement for in vivo antitumor activity of agonistic mouse anti-CD40 monoclonal antibodies (mAbs), a similar requirement for human mAbs has been disputed. By using a mouse model humanized for its Fc gamma Rs and CD40, we revealed that Fc gamma RIIB engagement is essential for the activity of human CD40 mAbs, while engagement of the activating Fc gamma RIIA inhibits this activity. By engineering Fc variants with selective enhanced binding to Fc gamma RIIB, but not to Fc gamma RIIA, significantly improved antitumor immunity was observed. These findings highlight the necessity of optimizing the Fc domain for this class of therapeutic antibodies by using appropriate preclinical models that accurately reflect the unique affinities and cellular expression of human Fc gamma R.

Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (similar to 3-times, similar to 8-times, and similar to 80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.