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The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk for mesothelioma and melanocytic tumors. Here, we show that pancreatic intraepithelial neoplasia driven by oncogenic mutant KrasG12D progressed to pancreatic adenocarcinoma in the absence of BAP1. The Hippo pathway was deregulated in BAP1-deficient pancreatic tumors, with the tumor suppressor LATS exhibiting enhanced ubiquitin-dependent proteasomal degradation. Therefore, BAP1 may limit tumor progression by stabilizing LATS and thereby promoting activity of the Hippo tumor suppressor pathway. Significance: BAP1 is mutated in a broad spectrum of tumors. Pancreatic Bap1 deficiency causes acinar atrophy but combines with oncogenic Ras to produce pancreatic tumors. BAP1-deficient tumors exhibit deregulation of the Hippo pathway. See related commentary by Brekken, p. 1624

In a pilot study, heavy water labeling was used to determine hepatitis B surface antigen (HBsAg) turnover rates in chronic hepatitis B (CHB) patients. The mean (standard deviation) half-life of HBsAg in blood was 6.7 (5.5) days, which reflects recent production in the liver and supports strategies aimed at reducing HBsAg production in CHB patients.

Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody-siRNA complexes provide a possible solution. However, initial reports of antibody-siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody-siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.

SummaryLive attenuated vaccines remain the safest, most cost-effective intervention against viral infections. Because live vaccine strains are generated empirically and the basis for attenuation is usually ill defined, many important viruses lack an efficient live vaccine. Here, we present a general strategy for the rational design of safe and effective live vaccines that harnesses the microRNA-based gene-silencing machinery to control viral replication. Using poliovirus as a model, we demonstrate that insertion of small miRNA homology sequences into a viral genome can restrict its tissue tropism, thereby preventing pathogenicity and yielding an attenuated viral strain. Poliovirus strains engineered to become targets of neuronal-specific miRNAs lost their ability to replicate in the central nervous system, leading to significant attenuation of neurovirulence in infected animals. Importantly, these viruses retained the ability to replicate in nonneuronal tissues. As a result, these engineered miRNA-regulated viruses elicited strong protective immunity in mice without producing disease.

Expression of chloroplast genes is primarily regulated posttranscriptionally, and a number of RNA elements, found in either the 5'- or 3'-untranslated regions (UTRs) of plastid mRNAs, that impact gene expression have been identified. Complex regulatory and feedback mechanisms influence both translation and protein accumulation, making assignment of roles for specific RNA elements difficult. To identify specific contributions made by various UTRs on translation of plastid mRNAs, we used a heterologous gfp reporter gene that is fused combinatorially to chloroplast 5'- and 3'-UTRs. In general, the 5'-UTR, including the promoter, of the plastid atpA and psbD genes produced the highest levels of chimeric mRNA and protein accumulation, while the 5'-UTR of the rbcL and psbA genes produced less mRNA and protein. Varying the 3'-UTR had little impact on mRNA and protein accumulation, as long as a 3'-UTR was present. Overall, accumulation of chimeric mRNAs was proportional to protein accumulation, with a few notable exceptions. Light-regulated translation continues to operate in chimeric mRNAs containing the 5'-UTR of either the psbA or psbD mRNAs, despite translation of these two chimeric mRNAs at very different efficiencies, suggesting that translational efficiency and light-regulated translation are separate events. Translation of some chimeric mRNAs was much more efficient than others, suggesting that interactions between the untranslated and coding sequences can dramatically impact translational efficiency.

Biosynthesis of chloroplast proteins is to a large extent regulated post-transcriptionally, and a number of nuclear-encoded genes have been identified that are required for translation or stability of specific chloroplast mRNAs. A nuclear mutant of Chlamydomonas reinhardtii, hf261, deficient in the translation of the psbA mRNA, has reduced association of the psbA mRNA with ribosomes and is deficient in binding of the chloroplast localized poly (A) binding protein (cPAB1) to the psbA mRNA. Cloning of the hf261 locus and complementation of hf261 using a wt genomic clone has identified a novel gene, Tba1, for translational affector of psbA. Strains complemented with the wt Tba1 gene restore the ability of the psbA mRNA to associate with ribosomes, and restores RNA binding activity of cPAB1 for the psbA mRNA. Analysis of the Tba1 gene identified a protein with significant homology to oxidoreductases. The effect of Tba1 expression on the RNA binding activity of cPAB1, and on the association of psbA mRNA with ribosomes, implies that Tba1 functions as a redox regulator of cPAB1 RNA binding activity to indirectly promote psbA mRNA translation initiation. A model of chloroplast translation incorporating Tba1 and other members of the psbA mRNA binding complex is presented.

Binding of proteins to chloroplast-encoded mRNAs has been shown to be an essential part of chloroplast gene expression. Four nuclear-encoded proteins (38, 47, 55, and 60 kDa) have been identified that bind to the 5'-untranslated region of the Chlamydomonas reinhardtii psbA mRNA with high affinity and specificity. We have cloned a cDNA that represents the 38 kDa protein (RB38) and show that it encodes a novel RNA binding protein that is primarily localized within the chloroplast stroma. RB38 contains four 70 amino acid repeats with a high percentage of basic amino acids, as well as an amino-terminal extension predicted to act as a chloroplast import sequence. We demonstrate that the 38 kDa precursor protein is imported into isolated chloroplasts and interacts with high specificity to uridine-rich regions within the 5'-untranslated region of the psbA mRNA. While database searches have identified hypothetical proteins from several other eukaryotic species with high sequence similarity to the deduced amino acid sequence of RB38, no proteins with homology to RB38 have been biochemically characterized. Bioinformatic analysis of the RB38 sequence, together with structure analysis using circular dichroism and protein modeling, suggests that the 70 amino acid repeats within RB38 are similar in fold to previously identified RNA binding motifs, despite limited sequence homology.

A panel of catalytic antibodies which catalyze ester hydrolysis, transesterification, porphyrin metallation, Diels-Alder, and redox reactions has been selected for crystallographic study. While these examples are only a handful of the catalytic antibodies generated to date, they represent distinct and important aspects of antibody catalysis. Since the first reports of catalysis, a great deal of progress has been made with respect to the scope, selectivity, and efficiency of antibody catalysis and strategies for generating catalytic antibodies. However, it is clear that further progress in the field will benefit greatly from a detailed understanding of the molecular interactions occurring in the combining site. High-resolution crystallographic data should allow the importance of general base catalysis, entropy effects, electrophilic catalysis, and transition-state stabilization to be evaluated. Antibody and enzyme active sites have been shown to share considerable structural and mechanistic similarity, and ongoing structure-function studies of catalytic antibodies may enhance our understanding of the mechanisms and evolution of enzymatic catalysis. Structural studies of antibodies which perform a biological or highly selective reactions should enhance our ability to generate catalysts for important synthetic applications. Finally, the combination of high-resolution crystallographic analysis with rational mutagenesis should provide a basis for engineering antibodies with enhanced properties.

Beam driven plasma wakes show great promise for meter scale accelerators with high gradients. Plasma wakefield theory indicates that the achievable gradient is proportional to N/{sigma}{sub z}{sup 2}, and the bunches as short as 12 {micro}m {approx} 40 fsec in RMS length which are now possible at the Stanford Linear Accelerator Center (SLAC) are predicted to allow gradients in the tens to hundreds of GeV/m. We discuss the three stages of compression needed to achieve such short bunches. No technique currently available can measure these longitudinal profiles directly shot by shot, requiring an indirect method. We added a magnetic chicane near the end of SLAC's 3 km main accelerator to measure the energy spread of each bunch in a nondestructive manner. Additionally, we performed a series of detailed simulations of the main accelerator in LiTrack, a code developed at SLAC. By comparing each measured spectrum against the library of spectra from simulations, we can find the best match to determine the input conditions to the accelerator and the total longitudinal phase space of every shot in the machine. We discuss several methods employed to verify that the longitudinal profiles coming from simulations are accurate. We can use this information tomore » understand which particles are accelerated in each bunch, and by how much. Additionally, we use the longitudinal information to choose a subset of shots that always have the same incoming profiles to see the differing acceleration experienced by those shots as we vary the plasma density and length. This allows a more robust calculation of achieved gradient, as well as illuminating the effect of transverse deflections on that acceleration. Finally, we discuss other applications, as the technique for measuring the energy spectra and for matching to simulations is quite general.« less

Biosynthesis of chloroplast proteins is to a large extent regulated post-transcriptionally, and a number of nuclear-encoded genes have been identified that are required for translation or stability of specific chloroplast mRNAs. A nuclear mutant of Chlamydomonas reinhardtii, hf261, deficient in the translation of the psbA mRNA, has reduced association of the psbA mRNA with ribosomes and is deficient in binding of the chloroplast localized poly (A) binding protein (cPAB1) to the psbA mRNA. Cloning of the hf261 locus and complementation of hf261 using a wt genomic clone has identified a novel gene, Tba1, for translational affector of psbA. Strains complemented with the wt Tba1 gene restore the ability of the psbA mRNA to associate with ribosomes, and restores RNA binding activity of cPAB1 for the psbA mRNA. Analysis of the Tba1 gene identified a protein with significant homology to oxidoreductases. The effect of Tba1 expression on the RNA binding activity of cPAB1, and on the association of psbA mRNA with ribosomes, implies that Tba1 functions as a redox regulator of cPAB1 RNA binding activity to indirectly promote psbA mRNA translation initiation. A model of chloroplast translation incorporating Tba1 and other members of the psbA mRNA binding complex is presented.

The ability to couple photosynthetic electron transport and redox poise to plastid gene expression enables plants to respond to environmental conditions and coordinate nuclear and chloroplast activities in order to maintain photosynthetic efficiency. The plastid redox regulatory system serves as a paradigm for understanding redox-regulated gene expression. In this review, we will focus on posttranscriptional events of redox-regulated gene expression in the chloroplast. As redox regulation of enzymatic activities in the chloroplast will be covered in other reviews in this volume, as will discussions on the redox regulation of chloroplast transcription, we will concentrate on the available evidence for redox regulation of chloroplast translation, and mRNA splicing and turnover.

A classic hypothesis for enzyme catalysis is the induction of strain in the substrate. This notion was first expressed by Haldane with the lock and key analogy-"the key does not fit the lock perfectly but exercises a certain strain on it" (1). This mechanism has often been invoked to explain the catalytic efficiency of enzymes but has been difficult to establish conclusively (2-7). Here we describe X-ray crystallographic and mutational studies of an antibody metal chelatase which strongly support the notion that this antibody catalyzes metal ion insertion into the porphyrin ring by inducing strain. Analysis of the germline precursor suggests that this strain mechanism arose during the process of affinity maturation in response to a conformationally distorted N-alkylmesoporphyrin.

BACKGROUND: Much is written about the importance of follow-up in determining the effect of surgical treatment for obesity upon weight loss. When patients are lost to follow-up, it has been suggested that these patients should be considered as failures. This study was undertaken to determine the effect of incorporating patients not followed in a definition of success for weight loss at one year. METHODS: Data from 34 surgical practices were used to study the effect of using two different denominators, patients followed (Df) or patients eligible (De), to define success. The numerator used in both methods was the number of patients with