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Find video protocols related to scientific articles indexed in Pubmed.
Determining the Specificities of TALENs, Cas9, and Other Genome-Editing Enzymes.
Meth. Enzymol.
PUBLISHED: 11-16-2014
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The rapid development of programmable site-specific endonucleases has led to a dramatic increase in genome engineering activities for research and therapeutic purposes. Specific loci of interest in the genomes of a wide range of organisms including mammals can now be modified using zinc-finger nucleases, transcription activator-like effectornucleases, and CRISPR-associated Cas9 endonucleases in a site-specific manner, in some cases requiring relatively modest effort for endonuclease design, construction, and application. While these technologies have made genome engineering widely accessible, the ability of programmable nucleases to cleave off-target sequences can limit their applicability and raise concerns about therapeutic safety. In this chapter, we review methods to evaluate and improve the DNA cleavage activity of programmable site-specific endonucleases and describe a procedure for a comprehensive off-target profiling method based on the in vitro selection of very large (~10(12)-membered) libraries of potential nuclease substrates.
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Assessing Steatotic Liver Function after Ischemia-reperfusion Injury by In Vivo Multiphoton Imaging of Fluorescein Disposition.
Drug Metab. Dispos.
PUBLISHED: 11-09-2014
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Ischemia-reperfusion injury is a common complication during liver surgery, where steatotic livers are more prone to the injury and may become more prevalent in the growing obese population. This study aimed to characterize liver morphology and understand changes in subcellular function in steatotic livers exposed to ischemia-reperfusion injury through quantitative description of fluorescein distribution obtained by minimally-invasive in vivo multiphoton microscopy using a physiological pharmacokinetic model. Rats were fed a high fat diet for 7 days to induce liver steatosis. Partial ischemia was induced, following reperfusion for 4 hours, when fluorescein (10 mg/kg) was injected intravenously. Liver images, bile and blood were collected up to 180 min following injection. Ischemia-reperfusion injury was associated with an increase in alanine transaminase levels and apoptosis. In addition, steatosis had the presence of lipid droplets and an increase in the fluorescein associated fluorescence observed in the hepatocytes by multiphoton imaging. Analysis of the hepatic concentration-time profiles suggests that the steatosis induced increase in fluorescein associated fluorescence mainly arises by inducing the hepatic fluorescein metabolism. The combination of ischemia-reperfusion with steatosis exacerbates these effects further. This was confirmed by fluorescence lifetime imaging microscopy showing a decreased average fluorescence lifetime of the liver, indicative of increased production of the metabolite. Our results show the potential of non-invasive imaging of a dye to further improve our understanding of liver disease induced subcellular changes in vivo by also providing further quantitative measures of metabolic and biliary liver function and, hence, extending the qualitative liver function tests now available.
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Enhanced Delivery of Nano- and Submicron Particles using Elongated Microparticles.
Curr Drug Deliv
PUBLISHED: 09-04-2014
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Nanodermatology is a rapidly emerging field of study receiving significant interest because of its potential application in the prevention and treatment of skin diseases. However, nanoparticulate penetration into and through the skin is not feasible through topical application alone. Many physical and chemical approaches have been developed to enhance particulate penetration into skin. The most successful have been physical penetration enhancers. We have found that elongated microparticles can significantly improve topical nano- and microspheredelivery in an in vivo porcine model. The delivery efficiency was inversely related to the diameter of the payload. These data support a role for elongated microparticle enhanced delivery of nano- and submicron particulatecosmeceutical or therapeutic applications.
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Reprogramming the specificity of sortase enzymes.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-03-2014
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Staphylococcus aureus sortase A catalyzes the transpeptidation of an LPXTG peptide acceptor and a glycine-linked peptide donor and has proven to be a powerful tool for site-specific protein modification. The substrate specificity of sortase A is stringent, limiting its broader utility. Here we report the laboratory evolution of two orthogonal sortase A variants that recognize each of two altered substrates, LAXTG and LPXSG, with high activity and specificity. Following nine rounds of yeast display screening integrated with negative selection, the evolved sortases exhibit specificity changes of up to 51,000-fold, relative to the starting sortase without substantial loss of catalytic activity, and with up to 24-fold specificity for their target substrates, relative to their next most active peptide substrate. The specificities of these altered sortases are sufficiently orthogonal to enable the simultaneous conjugation of multiple peptide substrates to their respective targets in a single solution. We demonstrated the utility of these evolved sortases by using them to effect the site-specific modification of endogenous fetuin A in human plasma, the synthesis of tandem fluorophore-protein-PEG conjugates for two therapeutically relevant fibroblast growth factor proteins (FGF1 and FGF2), and the orthogonal conjugation of fluorescent peptides onto surfaces.
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Dynamism of the mitral annulus: a spatial and temporal analysis.
J. Cardiothorac. Vasc. Anesth.
PUBLISHED: 08-15-2014
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In this study, the authors sought to investigate the extent and timing of changes in mitral annular area during the cardiac cycle. Particularly, the authors assessed whether these changes were limited to the posterior part of the annulus or were more global in nature.
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Expanding the National Drug Abuse Treatment Clinical Trials Network to address the management of substance use disorders in general medical settings.
Subst Abuse Rehabil
PUBLISHED: 07-23-2014
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The Patient Protection and Affordable Care Act (2010) and the Mental Health Parity and Addiction Equity Act (2008) expand substance use disorder (SUD) care services in the USA into general medical settings. Care offered in these settings will engage substance-using patients in an integrated and patient-centered environment that addresses physical and mental health comorbidities and follows a chronic care model. This expansion of SUD services presents a great need for evidence-based practices useful in general medical settings, and reveals several research gaps to be addressed. The National Drug Abuse Treatment Clinical Trials Network of the National Institute on Drug Abuse can serve an important role in this endeavor. High-priority research gaps are highlighted in this commentary. A discussion follows on how the National Drug Abuse Treatment Clinical Trials Network can transform to address changing patterns in SUD care to efficiently generate evidence to guide SUD treatment practice within the context of recent US health care legislation.
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Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.
Nat. Biotechnol.
PUBLISHED: 07-22-2014
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Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.
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The effect of doxorubicin loading on response and toxicity with drug-eluting embolization in resectable hepatoma: a dose escalation study.
Anticancer Res.
PUBLISHED: 07-02-2014
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The dose-response relationship between doxorubicin and superabsorbent drug-eluting microspheres has not been established. In this study, we investigated the relationships between dose and delivery parameters as they pertain to toxicity and response in surgically resectable hepatocellular carcinoma (HCC).
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Videotaped behavior as a predictor of clinical outcome in rhesus macaques (Macaca mulatta).
Comp. Med.
PUBLISHED: 06-24-2014
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Understanding the behavior of laboratory NHP facilitates health assessment and clinical care. We sought to characterize the behavior of critically ill rhesus macaques (Macaca mulatta) and determine whether specific behaviors or behavioral changes might facilitate the determination of prognosis and clinical endpoints. Twenty-two critically-ill subjects were videorecorded after they were removed from the outdoor breeding colony for diagnostic work-up and treatment. Subjects were categorized as survivors (n = 15) and those that were euthanized according to existing clinical endpoints (n = 7). Behavior before, during, and after cageside examination was compared between these groups with regard to the presence or absence of direct observation. This approach allowed us to determine whether these settings revealed differences between groups or masking of behaviors during direct observation. Before cageside examination, several behaviors (for example, self-grooming and anxiety behaviors) were significantly more common in surviving subjects than in euthanized subjects. Few significant differences in behavior were detectable during or after the examination. Subjects that were eventually euthanized showed more illness-related behaviors; however, not all animals requiring euthanasia showed these signs when an observer was present. Furthermore, euthanized animals spent more time in an alert posture during direct observation than at other times. Therefore, direct observation of critically ill rhesus macaques may not yield the most accurate assessment of illness severity, and using video to assess behavior may be helpful for prognosis.
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Pulmonary ablation: a primer.
Can Assoc Radiol J
PUBLISHED: 06-20-2014
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Percutaneous image-guided thermal ablation is safe and efficacious in achieving local control and improving outcome in the treatment of both early stage non-small-cell lung cancer and pulmonary metastatic disease, in which surgical treatment is precluded by comorbidity, poor cardiorespiratory reserve, or unfavorable disease distribution. Radiofrequency ablation is the most established technology, but new thermal ablation technologies such as microwave ablation and cryoablation may offer some advantages. The use of advanced techniques, such as induced pneumothorax and the popsicle stick technique, or combining thermal ablation with radiotherapy, widens the treatment options available to the multidisciplinary team. The intent of this article is to provide the reader with a practical knowledge base of pulmonary ablation by concentrating on indications, techniques, and follow-up.
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Visceral adiposity is not associated with abdominal aortic aneurysm presence and growth.
Vasc Med
PUBLISHED: 06-19-2014
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Previous studies in rodent models and patients suggest that visceral adipose could play a direct role in the development and progression of abdominal aortic aneurysm (AAA). This study aimed to assess the association of visceral adiposity with AAA presence and growth. This study was a case-control investigation of patients that did (n=196) and did not (n=181) have an AAA who presented to The Townsville Hospital vascular clinic between 2003 and 2012. Cases were patients with AAA (infra-renal aortic diameter >30 mm) and controls were patients with intermittent claudication but no AAA (infra-renal aortic diameter <30 mm). All patients underwent computed tomography angiography (CTA). The visceral to total abdominal adipose volume ratio was estimated from CTAs by assessing total and visceral adipose deposits using an imaging software program. Measurements were assessed for reproducibility by repeat assessments on 15 patients. AAA risk factors were recorded at entry. Forty-five cases underwent two CTAs more than 6 months apart to assess AAA expansion. The association of visceral adiposity with AAA presence and growth was examined using logistic regression. Visceral adipose assessment by CTA was highly reproducible (mean coefficient of variation 1.0%). AAA was positively associated with older age and negatively associated with diabetes. The visceral to total abdominal adipose volume ratio was not significantly associated with AAA after adjustment for other risk factors. Patients with a visceral to total abdominal adipose volume ratio in quartile four had a 1.63-fold increased risk of AAA but with wide confidence intervals (95% CI 0.71-3.70; p=0.248). Visceral adiposity was not associated with AAA growth. In conclusion, this study suggests that visceral adiposity is not specifically associated with AAA presence or growth although larger studies are required to confirm these findings.
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A system for the continuous directed evolution of proteases rapidly reveals drug-resistance mutations.
Nat Commun
PUBLISHED: 06-14-2014
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The laboratory evolution of protease enzymes has the potential to generate proteases with therapeutically relevant specificities and to assess the vulnerability of protease inhibitor drug candidates to the evolution of drug resistance. Here we describe a system for the continuous directed evolution of proteases using phage-assisted continuous evolution (PACE) that links the proteolysis of a target peptide to phage propagation through a protease-activated RNA polymerase (PA-RNAP). We use protease PACE in the presence of danoprevir or asunaprevir, two hepatitis C virus (HCV) protease inhibitor drug candidates in clinical trials, to continuously evolve HCV protease variants that exhibit up to 30-fold drug resistance in only 1 to 3 days of PACE. The predominant mutations evolved during PACE are mutations observed to arise in human patients treated with danoprevir or asunaprevir, demonstrating that protease PACE can rapidly identify the vulnerabilities of drug candidates to the evolution of clinically relevant drug resistance.
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A naturally occurring, noncanonical GTP aptamer made of simple tandem repeats.
RNA Biol
PUBLISHED: 05-15-2014
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Recently, we used in vitro selection to identify a new class of naturally occurring GTP aptamer called the G motif. Here we report the discovery and characterization of a second class of naturally occurring GTP aptamer, the "CA motif." The primary sequence of this aptamer is unusual in that it consists entirely of tandem repeats of CA-rich motifs as short as three nucleotides. Several active variants of the CA motif aptamer lack the ability to form consecutive Watson-Crick base pairs in any register, while others consist of repeats containing only cytidine and adenosine residues, indicating that noncanonical interactions play important roles in its structure. The circular dichroism spectrum of the CA motif aptamer is distinct from that of A-form RNA and other major classes of nucleic acid structures. Bioinformatic searches indicate that the CA motif is absent from most archaeal and bacterial genomes, but occurs in at least 70 percent of approximately 400 eukaryotic genomes examined. These searches also uncovered several phylogenetically conserved examples of the CA motif in rodent (mouse and rat) genomes. Together, these results reveal the existence of a second class of naturally occurring GTP aptamer whose sequence requirements, like that of the G motif, are not consistent with those of a canonical secondary structure. They also indicate a new and unexpected potential biochemical activity of certain naturally occurring tandem repeats.
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Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling.
Nat. Chem. Biol.
PUBLISHED: 05-14-2014
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Probes that form covalent bonds with RNA molecules on the basis of their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with unusually nucleophilic RNAs. We used these probes to identify reactive genome-encoded RNAs, resulting in the discovery of a 42-nt catalytic RNA from an archaebacterium that reacts with a 2,3-disubstituted epoxide at N7 of a specific guanosine. Detailed characterization of the catalytic RNA revealed the structural requirements for reactivity. We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest. This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate. We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind the ASH1 mRNA.
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CT-guided percutaneous cryoablation for osteoid osteoma: initial experience in adults.
AJR Am J Roentgenol
PUBLISHED: 04-25-2014
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The purpose of this study was to investigate the safety and efficacy of CT-guided percutaneous cryoablation for the treatment of osteoid osteoma in adults.
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A DNA-based molecular probe for optically reporting cellular traction forces.
Nat. Methods
PUBLISHED: 04-24-2014
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We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can in principle be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.
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Development of serum antibodies during early infancy in rhesus macaques: implications for humoral immune responses to vaccination at birth.
Vaccine
PUBLISHED: 04-22-2014
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A better understanding of immune responses in human infants could lead to more effective immunization and vaccination strategies in early life.
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Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones.
Nature
PUBLISHED: 03-27-2014
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Despite decades of speculation that inhibiting endogenous insulin degradation might treat type-2 diabetes, and the identification of IDE (insulin-degrading enzyme) as a diabetes susceptibility gene, the relationship between the activity of the zinc metalloprotein IDE and glucose homeostasis remains unclear. Although Ide(-/-) mice have elevated insulin levels, they exhibit impaired, rather than improved, glucose tolerance that may arise from compensatory insulin signalling dysfunction. IDE inhibitors that are active in vivo are therefore needed to elucidate IDE's physiological roles and to determine its potential to serve as a target for the treatment of diabetes. Here we report the discovery of a physiologically active IDE inhibitor identified from a DNA-templated macrocycle library. An X-ray structure of the macrocycle bound to IDE reveals that it engages a binding pocket away from the catalytic site, which explains its remarkable selectivity. Treatment of lean and obese mice with this inhibitor shows that IDE regulates the abundance and signalling of glucagon and amylin, in addition to that of insulin. Under physiological conditions that augment insulin and amylin levels, such as oral glucose administration, acute IDE inhibition leads to substantially improved glucose tolerance and slower gastric emptying. These findings demonstrate the feasibility of modulating IDE activity as a new therapeutic strategy to treat type-2 diabetes and expand our understanding of the roles of IDE in glucose and hormone regulation.
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Ceramide modulates pre-mRNA splicing to restore the expression of wild-type tumor suppressor p53 in deletion-mutant cancer cells.
Biochim. Biophys. Acta
PUBLISHED: 02-26-2014
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Mutants of tumor suppressor p53 not only lose the activity in genome stabilizing and in tumor suppression, but also exhibit oncogenic function in cancer cells. Most efforts in restoring p53 biological activity focus on either altering mutant-protein conformation or introducing an exogenous p53 gene into cells to eliminate p53-mutant cancer cells. Being different from these, we report that ceramide can restore the expression of wild-type p53 and induce p53-dependent apoptosis in deletion-mutant cancer cells. We show that endogenous long-carbon chain ceramide species (C16- to C24-ceramides) and exogenous C6-ceramide, rather than other sphingolipids, restore wild-type mRNA (intact exon-5), phosphorylated protein (Ser15 in exon-5) of p53, and p53-responsive proteins, including p21 and Bax, in ovarian cancer cells, which predominantly express a deleted exon-5 of p53 mutant before treatments. Consequently, the restored p53 sensitizes these p53-mutant cancer cells to DNA damage-induced growth arrest and apoptosis. Furthermore, we elucidate that ceramide activates protein phosphatase-1, and then the dephosphorylated serine/arginine-rich splicing-factor 1 (SRSF1) is translocated to the nucleus, thus promoting pre-mRNA splicing preferentially to wild-type p53 expression. These findings disclose an unrecognized mechanism that pre-mRNA splicing dysfunction can result in p53 deletion-mutants. Ceramide through SRSF1 restores wild-type p53 expression versus deletion-mutant and leads cancer cells to apoptosis. This suggests that heterozygous deletion-mutants of p53 can be restored in posttranscriptional level by using epigenetic approaches.
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Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.
Nat. Biotechnol.
PUBLISHED: 02-23-2014
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Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ?15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 'nickases', recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least fourfold higher than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the versatility of this approach for highly specific genome-wide editing.
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Identification of ligand-target pairs from combined libraries of small molecules and unpurified protein targets in cell lysates.
J. Am. Chem. Soc.
PUBLISHED: 02-17-2014
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We describe the development and validation of interaction determination using unpurified proteins (IDUP), a method that selectively amplifies DNA sequences identifying ligand+target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. By operating in cell lysates, IDUP preserves native post-translational modifications and interactions with endogenous binding partners, thereby enabling the study of difficult-to-purify targets and increasing the potential biological relevance of detected interactions compared with methods that require purified proteins. In IDUP, target proteins are associated with DNA oligonucleotide tags either non-covalently using a DNA-linked antibody or covalently using a SNAP-tag. Ligand-target binding promotes hybridization of a self-priming hairpin that is extended by a DNA polymerase to create a DNA strand that contains sequences identifying both the target and its ligand. These sequences encoding ligand+target pairs are selectively amplified by PCR and revealed by high-throughput DNA sequencing. IDUP can respond to the effect of affinity-modulating adaptor proteins in cell lysates that would be absent in ligand screening or selection methods using a purified protein target. This capability was exemplified by the 100-fold amplification of DNA sequences encoding FRB+rapamycin or FKBP+rapamycin in samples overexpressing both FRB and FKBP (FRB·rapamycin+FKBP, Kd ? 100 fM; FKBP·rapamycin+FRB, Kd = 12 nM). In contrast, these sequences were amplified 10-fold less efficiently in samples overexpressing either FRB or FKBP alone (rapamycin+FKBP, Kd ? 0.2 nM; rapamcyin+FRB, Kd = 26 ?M). Finally, IDUP was used to process a model library of DNA-linked small molecules and a model library of cell lysates expressing SNAP-target fusions combined in a single sample. In this library×library experiment, IDUP resulted in enrichment of sequences corresponding to five known ligand+target pairs ranging in binding affinity from Kd = 0.2 nM to 3.2 ?M out of 67,858 possible combinations, with no false positive signals enriched to the same extent as that of any of the bona fide ligand+target pairs.
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Retrospective study of the impact of fellowship training on two quality and safety measures in uterine artery embolization.
J Am Coll Radiol
PUBLISHED: 02-12-2014
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To measure the impact of 1-year interventional fellowship training on fluoroscopic time and contrast media utilization in uterine artery embolization (UAE).
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Negative selection and stringency modulation in phage-assisted continuous evolution.
Nat. Chem. Biol.
PUBLISHED: 02-02-2014
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Phage-assisted continuous evolution (PACE) uses a modified filamentous bacteriophage life cycle to substantially accelerate laboratory evolution experiments. In this work, we expand the scope and capabilities of the PACE method with two key advances that enable the evolution of biomolecules with radically altered or highly specific new activities. First, we implemented small molecule-controlled modulation of selection stringency that enables otherwise inaccessible activities to be evolved directly from inactive starting libraries through a period of evolutionary drift. Second, we developed a general negative selection that enables continuous counterselection against undesired activities. We integrated these developments to continuously evolve mutant T7 RNA polymerase enzymes with ?10,000-fold altered, rather than merely broadened, substrate specificities during a single three-day PACE experiment. The evolved enzymes exhibit specificity for their target substrate that exceeds that of wild-type RNA polymerases for their cognate substrates while maintaining wild type-like levels of activity.
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Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene.
PLoS Pathog.
PUBLISHED: 02-01-2014
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Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo), which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s) via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S) genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting.
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A practical guide to magnetic resonance vascular imaging: techniques and applications.
Ann Vasc Surg
PUBLISHED: 01-31-2014
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Magnetic resonance angiography is a technique used to image both central and peripheral arteries using contrast and noncontrast techniques. These techniques are similar in that a bright signal, which appears white within blood vessels, is generated and the background tissues, veins, and stationary tissues are dark. This allows for assessment of anatomy and vascular disease. Extracellular gadolinium-based contrast agents allow for excellent visualization of both central and peripheral arteries. Acquiring images during first pass is required for high-contrast images within arteries, thereby limiting contamination with contrast enhancement of veins and soft tissue. Contrast-enhanced techniques using time-resolved angiography and blood pool contrast agents minimize this temporal limitation. Noncontrast techniques eliminate the uncommon but potentially fatal complications associated with gadolinium contrast agents, such as nephrogenic systemic fibrosis. These techniques including phase contrast and time-of-flight sequences have inferior contrast resolution compared with contrast-enhanced techniques and are susceptible to artifacts, which can limit interpretation. The advantage, however, is the ability to assess vascular disease in patients with severe renal failure without the added risks of gadolinium contrast media. The aim of this review is to outline the different techniques available for imaging both the arterial and venous systems, their advantages and disadvantages, and the indications in vascular disease.
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Aliskiren limits abdominal aortic aneurysm, ventricular hypertrophy and atherosclerosis in an apolipoprotein-E-deficient mouse model.
Clin. Sci.
PUBLISHED: 01-31-2014
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Aliskiren is a direct renin inhibitor developed to treat hypertension. Several clinical studies have suggested that aliskiren has beneficial effects on cardiovascular diseases beyond its antihypertensive effect. In the present study, we examined whether aliskiren limits the progression of AAA (abdominal aortic aneurysm), VH (ventricular hypertrophy) and atherosclerosis in an AngII (angiotensin II)-infused mouse model. ApoE-/- (apolipoprotein-E-deficient) mice were infused subcutaneously with AngII (1000 ng/kg of body weight per day; 4 weeks) to induce AAA and VH. At the completion of the AngII infusion, mice were randomly allocated to three groups to receive vehicle control, low-dose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day) for 4 weeks. Suprarenal aortic diameter assessed by ultrasound was significantly smaller in mice administered aliskiren at days 42 and 56. Aliskiren also significantly reduced the normalized heart weight, ventricular myocyte cell width and aortic arch atherosclerosis. Aliskiren lowered PRR (pro-renin receptor) expression and MAPK (mitogen-activated protein kinase) activity in the suprarenal aorta and heart. Aortic infiltration of T-lymphocytes and macrophages was reduced by aliskiren. In conclusion, aliskiren limits the progression of AAA, VH and atherosclerosis in an AngII-infused mouse model.
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In the absence of conflicting testimony young children trust inaccurate informants.
Dev Sci
PUBLISHED: 01-20-2014
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The present research investigated the nature of the inferences and decisions young children make about informants with a prior history of inaccuracies. Across three experiments, 3- and 4-year-olds (total N = 182) reacted to previously inaccurate informants who offered testimony in an object-labeling task. Of central interest was children's willingness to accept information provided by an inaccurate informant in different contexts of being alone, paired with an accurate informant, or paired with a novel (neutral) informant. Experiments 1 and 2 showed that when a previously inaccurate informant was alone and provided testimony that was not in conflict with the testimony of another informant, children systematically accepted the testimony of that informant. Experiment 3 showed that children accepted testimony from a neutral informant over an inaccurate informant when both provided information, but accepted testimony from an inaccurate informant rather than seeking information from an available neutral informant who did not automatically offer information. These results suggest that even though young children use prior history of accuracy to determine the relative reliability of informants, they are quite willing to trust the testimony of a single informant alone, regardless of whether that informant had previously been reliable.
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Persistent babesiosis in a Rhesus macaque (Macaca mulatta) infected with a simian-human immunodeficiency virus.
J. Med. Primatol.
PUBLISHED: 01-16-2014
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A rhesus macaque developed chronic anemia, lymphocytic leukocytopenia, fever, and anorexia while immunodeficient following inoculation with a simian-human immunodeficiency virus.
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Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity.
Nat. Methods
PUBLISHED: 01-11-2014
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Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have activity against related off-target sequences. To better understand TALEN specificity, we profiled 30 unique TALENs with different target sites, array length and domain sequences for their abilities to cleave any of 10(12) potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant that exhibits equal on-target cleavage activity but tenfold lower average off-target activity in human cells.
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MSH1-Induced Non-Genetic Variation Provides a Source of Phenotypic Diversity in Sorghum bicolor.
PLoS ONE
PUBLISHED: 01-01-2014
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MutS Homolog 1 (MSH1) encodes a plant-specific protein that functions in mitochondria and chloroplasts. We showed previously that disruption or suppression of the MSH1 gene results in a process of developmental reprogramming that is heritable and non-genetic in subsequent generations. In Arabidopsis, this developmental reprogramming process is accompanied by striking changes in gene expression of organellar and stress response genes. This developmentally reprogrammed state, when used in crossing, results in a range of variation for plant growth potential. Here we investigate the implications of MSH1 modulation in a crop species. We found that MSH1-mediated phenotypic variation in Sorghum bicolor is heritable and potentially valuable for crop breeding. We observed phenotypic variation for grain yield, plant height, flowering time, panicle architecture, and above-ground biomass. Focusing on grain yield and plant height, we found some lines that appeared to respond to selection. Based on amenability of this system to implementation in a range of crops, and the scope of phenotypic variation that is derived, our results suggest that MSH1 suppression provides a novel approach for breeding in crops.
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Septic Arthritis Due to Moraxella osloensis in a Rhesus Macaque (Macaca mulatta).
Comp. Med.
PUBLISHED: 12-12-2013
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A 5.5-y-old Chinese-origin female rhesus macaque (Macaca mulatta) presented for bilateral hindlimb lameness. The primate had been group-reared in an SPF breeding colony and was seronegative for Macacine herpesvirus 1, SIV, simian retrovirus type D, and simian T-lymphotropic virus. The macaques previous medical history included multiple occasions of swelling in the left tarsus, and trauma to the right arm and bilateral hands. In addition, the macaque had experienced osteomyelitis of the left distal tibia and rupture of the right cranial cruciate ligament that had been surgically repaired. Abnormal physical examination findings on presentation included a thin body condition, mild dehydration, and bilaterally swollen stifles that were warm to the touch, with the right stifle more severely affected. Mild instability in the left stifle was noted, and decreased range of motion and muscle atrophy were present bilaterally. Hematologic findings included marked neutrophilia and lymphopenia and moderate anemia. Arthrocentesis and culture of joint fluid revealed Moraxella-like organisms. Treatment with enrofloxacin was initiated empirically and subsequently switched to cephalexin, which over time alleviated the joint swelling and inflammation. Definitive diagnosis of Moraxella osloensis septic arthritis was made through isolation of the organism and sequencing of the 16S rDNA region. To our knowledge, this report is the first description of Moraxella osloensis septic arthritis in a rhesus macaque.
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Cryoablation and cementoplasty of a pathologic fracture in the sternum.
Singapore Med J
PUBLISHED: 10-25-2013
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A 49-year-old man with metastatic melanoma and pathologic fracture of the sternum was deemed to be a poor candidate for general anaesthesia. He suffered severe pain and range of motion limitation that did not respond to narcotic therapy. Ultimately, the lesion was managed with computed tomography-guided cryoablation and subsequent cementoplasty, and saw good initial clinical results.
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Diagnosis and therapy of acute hemorrhage in patients with pelvic fractures.
Semin Musculoskelet Radiol
PUBLISHED: 10-07-2013
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Within the past 15 years, significant advances in the imaging of multiorgan and complex trauma primarily due to the improvement of cross-sectional imaging have resulted in the optimization of the expedient diagnosis and management of the polytrauma patient. At the forefront, multidetector computed tomography (MDCT) has become the cornerstone of modern emergency departments and trauma centers. In many institutions, MDCT is the de facto diagnostic tool upon trauma activation. In the setting of pelvic imaging, MDCT (with its high spatial resolution and sensitivity as well as short acquisition times) allows for rapid identification and assessment of pelvic hemorrhage leading to faster triage and definitive management. In trauma centers throughout the world, angiography and minimally invasive catheter-based embolization techniques performed by interventional radiologists have become the standard of care for patients with acute pelvic trauma and related multiorgan hemorrhage. In an interdisciplinary setting, embolization may be performed either alone or as an adjunct procedure with open or closed reduction and stabilization techniques. A team-based approach involving multiple disciplines (e.g., radiology, traumatology, orthopedic surgery, intensive care medicine) is crucial to monitor and treat the actively bleeding patient appropriately.
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Brivanib in patients with advanced hepatocellular carcinoma who were intolerant to sorafenib or for whom sorafenib failed: results from the randomized phase III BRISK-PS study.
J. Clin. Oncol.
PUBLISHED: 08-26-2013
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Brivanib is a selective dual inhibitor of vascular endothelial growth factor and fibroblast growth factor receptors implicated in tumorigenesis and angiogenesis in hepatocellular carcinoma (HCC). An unmet medical need persists for patients with HCC whose tumors do not respond to sorafenib or who cannot tolerate it. This multicenter, double-blind, randomized, placebo-controlled trial assessed brivanib in patients with HCC who had been treated with sorafenib.
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Brivanib versus sorafenib as first-line therapy in patients with unresectable, advanced hepatocellular carcinoma: results from the randomized phase III BRISK-FL study.
J. Clin. Oncol.
PUBLISHED: 08-26-2013
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Brivanib is a dual inhibitor of vascular-endothelial growth factor and fibroblast growth factor receptors that are implicated in the pathogenesis of hepatocellular carcinoma (HCC). Our multinational, randomized, double-blind, phase III trial compared brivanib with sorafenib as first-line treatment for HCC.
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Do SmartSite antireflux valves limit the flow rate of 0.9% normal saline through intravenous cannulas?
Eur J Emerg Med
PUBLISHED: 08-24-2013
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The aim of the study was to determine whether the placement of a SmartSite antireflux valve between a bag of fluid with line giving set and an intravenous cannula decreases the flow rate. Fluid flow rates were compared in paired experiments with and without an antireflux valve using a pressure infuser or allowing fluid to flow by gravity only for four different sizes of intravenous cannulas (20-14 G). Antireflux valves significantly decreased the fluid flow rates for all cannula sizes, irrespective of whether a pressure infuser was used. The decrease in the flow rate was most marked for larger cannulas [gravity only: 14 G: 52% (95% confidence interval (CI): 37-68), 16 G: 39% (95% CI: 34-43); pressure bag: 14 G: 74% (95% CI: 70-78), 16 G: 56% (95% CI: 54-57); all P<0.001]. Where maximum fluid flow rates are desirable, clinicians should consider not using an antireflux valve.
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In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites.
Nucleic Acids Res.
PUBLISHED: 08-14-2013
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Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described-one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.
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Sequence-controlled polymers.
Science
PUBLISHED: 08-10-2013
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Sequence-controlled polymers are macromolecules in which monomer units of different chemical nature are arranged in an ordered fashion. The most prominent examples are biological and have been studied and used primarily by molecular biologists and biochemists. However, recent progress in protein- and DNA-based nanotechnologies has shown the relevance of sequence-controlled polymers to nonbiological applications, including data storage, nanoelectronics, and catalysis. In addition, synthetic polymer chemistry has provided interesting routes for preparing nonnatural sequence-controlled polymers. Although these synthetic macromolecules do not yet compare in functional scope with their natural counterparts, they open up opportunities for controlling the structure, self-assembly, and macroscopic properties of polymer materials.
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Aberrant celio-mesenteric supply of the splenic flexure: provoking a bleed.
World J. Gastroenterol.
PUBLISHED: 08-03-2013
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Lower gastrointestinal hemorrhage presents a common indication for hospitalization and account for over 300,000 admissions per year in the United States. Multimodality imaging is often required to aid in localization of the hemorrhage prior to therapeutic intervention if endoscopic treatment fails. Imaging includes computer tomography angiography, red blood cell tagged scintigraphy and conventional angiography, with scintigraphy being the most sensitive followed by computer tomography angiography. Aberrant celio-mesenteric supply occurs in 2% of the population; however failure to identify this may result in failed endovascular therapy. Computer tomography angiography is sensitive for arterial hemorrhage and delineates the anatomy, allowing the treating physician to plan an endovascular approach. If at the time of conventional angiography, the active bleed is not visualized, but the site of bleeding has been identified on computer tomography angiography, provocative angiography can be utilized in order to stimulate bleeding and subsequent targeted treatment. We describe a case of lower gastrointestinal hemorrhage at the splenic flexure supplied by a celio-mesenteric branch in a patient and provocative angiography with tissue plasminogen activator utilized at the time of treatment to illicit the site of hemorrhage and subsequent treatment.
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Pomegranate extract inhibits the bone metastatic growth of human prostate cancer cells and enhances the in vivo efficacy of docetaxel chemotherapy.
Prostate
PUBLISHED: 07-22-2013
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Docetaxel treatment is the only first-line chemotherapy with a survival benefit in metastatic castration-resistant prostate cancer (PCa). Nonetheless, most patients become docetaxel resistant and inevitably progress with no cure. In this study, we investigated the potential of pomegranate extract (PE) in targeting metastatic castration-resistant PCa and improving docetaxel chemotherapy.
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Neoadjuvant doxorubicin/cyclophosphamide followed by ixabepilone or paclitaxel in early stage breast cancer and evaluation of ?III-tubulin expression as a predictive marker.
Oncologist
PUBLISHED: 07-12-2013
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This randomized phase II trial was designed to compare the rate of pathologic complete response (pCR) induced by neoadjuvant cyclophosphamide plus doxorubicin (AC) followed by ixabepilone or paclitaxel in women with early stage breast cancer (BC). Expression of ?III-tubulin as a predictive marker was also evaluated.
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Transformation inverse design.
Opt Express
PUBLISHED: 06-22-2013
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We present a new technique for the design of transformation-optics devices based on large-scale optimization to achieve the optimal effective isotropic dielectric materials within prescribed index bounds, which is computationally cheap because transformation optics circumvents the need to solve Maxwells equations at each step. We apply this technique to the design of multimode waveguide bends (realized experimentally in a previous paper) and mode squeezers, in which all modes are transported equally without scattering. In addition to the optimization, a key point is the identification of the correct boundary conditions to ensure reflectionless coupling to untransformed regions while allowing maximum flexibility in the optimization. Many previous authors in transformation optics used a certain kind of quasiconformal map which overconstrained the problem by requiring that the entire boundary shape be specified a priori while at the same time underconstraining the problem by employing "slipping" boundary conditions that permit unwanted interface reflections.
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Experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-14-2013
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To what extent are evolutionary outcomes determined by a populations recent environment, and to what extent do they depend on historical contingency and random chance? Here we apply a unique experimental system to investigate evolutionary reproducibility and path dependence at the protein level. We combined phage-assisted continuous evolution with high-throughput sequencing to analyze evolving protein populations as they adapted to divergent and then convergent selection pressures over hundreds of generations. Independent populations of T7 RNA polymerase genes were subjected to one of two selection histories ("pathways") demanding recognition of distinct intermediate promoters followed by a common final promoter. We observed distinct classes of solutions with unequal phenotypic activity and evolutionary potential evolve from the two pathways, as well as from replicate populations exposed to identical selection conditions. Mutational analysis revealed specific epistatic interactions that explained the observed path dependence and irreproducibility. Our results reveal in molecular detail how protein adaptation to different environments, as well as stochasticity among populations evolved in the same environment, can both generate evolutionary outcomes that preclude subsequent convergence.
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Flow-injection MS/MS for gas-phase chiral recognition and enantiomeric quantitation of a novel boron-containing antibiotic (GSK2251052A) by the mass spectrometric kinetic method.
Anal. Chem.
PUBLISHED: 05-07-2013
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The present work demonstrates, for the first time, the application of the mass spectrometric kinetic method for quantitative chiral purity determination by automatic flow-injection MS/MS. The particular compound analyzed is GSK2251052A, a novel boron-containing systemic antibiotic for the treatment of multidrug-resistant Gram-negative bacterial infections. Chiral recognition and quantitation of GSK2251052A was achieved based on the competitive dissociation kinetics of the Cu(II)-bound trimeric complex [Cu(II)(A)(ref*)2-H](+) (A = GSK2251052A or its R-enantiomer, ref* = L-tryptophan) that gives rise to Cu(II)-bound dimeric complexes. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, characteristic of the kinetic method, allow chiral purity determination of pharmaceutical compounds during enantioselective synthesis. By using flow-injection MS/MS, enantiomeric quantitation of GSK2251052A by the kinetic method proved to be fast (2 min for analysis of each sample) and to have accuracy comparable to chiral LC-MS/MS and LC-UV methods as well as the method using chiral derivatization followed by LC-MS/MS analysis. This flow-injection MS/MS method represents an alternative approach to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful for rapid screening of chiral drugs during pharmaceutical development.
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Prevalence and risk factors of posterior vitreous detachment in a Chinese adult population: the Handan eye study.
BMC Ophthalmol
PUBLISHED: 05-01-2013
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To describe the prevalence and associations of posterior vitreous detachment (PVD) in a rural adult Chinese population.
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High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.
Nat. Biotechnol.
PUBLISHED: 03-30-2013
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The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.
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Immobilization of Actively Thromboresistant Assemblies on Sterile Blood-Contacting Surfaces.
Adv Healthc Mater
PUBLISHED: 03-23-2013
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Rapid one-step modification of thrombomodulin with alkylamine derivatives such as azide, biotin, and PEG is achieved using an evolved sortase (eSrtA) mutant. The feasibility of a point-of-care scheme is demonstrated herein to site-specifically immobilize azido-thrombomodulin on sterilized commercial ePTFE vascular grafts, which exhibit superior thromboresistance compared with commercial heparin-coated grafts in a primate model of acute graft thrombosis.
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A randomized, phase II, three-arm study of two schedules of ixabepilone or paclitaxel plus bevacizumab as first-line therapy for metastatic breast cancer.
Breast Cancer Res. Treat.
PUBLISHED: 02-28-2013
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The aim of this phase II trial was to estimate the objective response rate (ORR) of two different schedules of ixabepilone [weekly or every 3 weeks (Q3W)] combined with bevacizumab, relative to a reference arm of weekly paclitaxel and bevacizumab. Patients with human epidermal growth factor receptor 2-normal, chemotherapy-naïve metastatic breast cancer (MBC) were randomized 3:3:2 to ixabepilone 16 mg/m(2) weekly plus bevacizumab 10 mg/kg Q2W (Arm A: n = 46); ixabepilone 40 mg/m(2) Q3W (reduced to 32 mg/m(2) after four cycles of treatment) plus bevacizumab 15 mg/kg Q3W (Arm B: n = 45); or paclitaxel 90 mg/m(2) weekly plus bevacizumab 10 mg/kg intravenous infusion Q2W (Arm C: n = 32). Of 123 randomized patients, 122 were treated. All were followed for ?19 months; 5 % of patients remained on study treatment at the time of this analysis. Grade 3 or 4 neutropenia was more common in Arm B (60 %) than Arms A (16 %) or C (22 %); other adverse events were similar. The investigator-assessed ORR was 48, 71, and 63 % for Arms A, B, and C, respectively. Median progression-free survival (randomized patients) was 9.6 months in Arm A, 11.9 months in Arm B, and 13.5 months in Arm C. In conclusion, ixabepilone Q3W plus bevacizumab has clinical activity as first-line therapy for MBC relative to paclitaxel plus bevacizumab, but with significantly greater risk of grade 3 or 4 neutropenia. In addition, these data suggest that weekly dosing of ixabepilone may be less active than Q3W dosing, but with less neutropenia.
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A population-based experimental model for protein evolution: effects of mutation rate and selection stringency on evolutionary outcomes.
Biochemistry
PUBLISHED: 02-14-2013
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Protein evolution is a critical component of organismal evolution and a valuable method for the generation of useful molecules in the laboratory. Few studies, however, have experimentally characterized how fundamental parameters influence protein evolution outcomes over long evolutionary trajectories or multiple replicates. In this work, we applied phage-assisted continuous evolution (PACE) as an experimental platform to study evolving protein populations over hundreds of rounds of evolution. We varied evolutionary conditions as T7 RNA polymerase evolved to recognize the T3 promoter DNA sequence and characterized how specific combinations of both mutation rate and selection stringency reproducibly result in different evolutionary outcomes. We observed significant and dramatic increases in the activity of the evolved RNA polymerase variants on the desired target promoter after selection for 96 h, confirming positive selection occurred under all conditions. We used high-throughput sequencing to quantitatively define convergent genetic solutions, including mutational "signatures" and nonsignature mutations that map to specific regions of protein sequence. These findings illuminate key determinants of evolutionary outcomes, inform the design of future protein evolution experiments, and demonstrate the value of PACE as a method for studying protein evolution.
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Impaired acetylcholine-induced endothelium-dependent aortic relaxation by caveolin-1 in angiotensin II-infused apolipoprotein-E (ApoE-/-) knockout mice.
PLoS ONE
PUBLISHED: 02-05-2013
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The angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE(-/-)) mouse model is widely used to study atherosclerosis and abdominal aortic aneurysm. An increase in blood pressure has been reported in this model however the underlying mechanism has not been fully explored. In this study, we investigated whether vasomotor dysfunction develops in AngII-infused ApoE(-/-) mice and the underlying mechanism involved.
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Gluten-sensitive enteropathy coincides with decreased capability of intestinal T cells to secrete IL-17 and IL-22 in a macaque model for celiac disease.
Clin. Immunol.
PUBLISHED: 01-29-2013
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Celiac disease (CD) is an autoimmune disorder caused by intolerance to dietary gluten. The interleukin (IL)-17 and IL-22 function as innate regulators of mucosal integrity. Impaired but not well-understood kinetics of the IL-17/22 secretion was described in celiac patients. Here, the IL-17 and IL-22-producing intestinal cells were studied upon their in vitro stimulation with mitogens in class II major histocompatibility complex-defined, gluten-sensitive rhesus macaques. Pediatric biopsies were collected from distal duodenum during the stages of disease remission and relapse. Regardless of dietary gluten content, IL-17 and IL-22-producing cells consisted of CD4+ and CD8+ T lymphocytes as well as of lineage-negative (Lin-) cells. Upon introduction of dietary gluten, capability of intestinal T cells to secrete IL-17/22 started to decline (p<0.05), which was paralleled with gradual disruption of epithelial integrity. These data indicate that IL-17/22-producing cells play an important role in maintenance of intestinal mucosa in gluten-sensitive primates.
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Evaluation of the effect of information integration in displays for ICU nurses on situation awareness and task completion time: A prospective randomized controlled study.
Int J Med Inform
PUBLISHED: 01-26-2013
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The study measured whether nurses situation awareness would increase and task completion time decrease when they used an integrated information display compared to traditional displays for medication management, patient awareness and team communication.
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Interleukin-10 prevents epithelial cell apoptosis by regulating IFN? and TNF? expression in rhesus macaque colon explants.
Cytokine
PUBLISHED: 01-23-2013
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Interleukin-10 (IL-10) is an important immunomodulatory cytokine that plays an obligate role in regulating inflammatory responses. Here we demonstrated the role of IL-10 in regulating crypts length and breadth as well as maintaining the survival of epithelial cells using rhesus colon explant cultures. Anti-IL-10 antibody treatment of colon explant cultures induced increased production of inflammatory cytokines/molecules like IFN?, TNF?, CD107a and perforin as well as increased epithelial cell apoptosis compared to media controls tested. Our results suggest that IL-10 plays a crucial role in maintaining mucosal homeostasis by regulating mucosal IFN? and TNF? cytokine production.
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Discovery of widespread GTP-binding motifs in genomic DNA and RNA.
Chem. Biol.
PUBLISHED: 01-22-2013
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Biological RNAs that bind small molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genome-encoded RNA fragments for naturally occurring GTP aptamers. Several aptamer classes were identified, including one (the "G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ~75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (~300 ?M). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding.
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Biomarker analysis of neoadjuvant doxorubicin/cyclophosphamide followed by ixabepilone or Paclitaxel in early-stage breast cancer.
Clin. Cancer Res.
PUBLISHED: 01-22-2013
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Predictive biomarkers offer the potential to improve the benefit:risk ratio of a therapeutic agent. Ixabepilone achieves comparable pathologic complete response (pCR) rates to other active drugs in the neoadjuvant setting. This phase II trial was designed to investigate potential biomarkers that differentiate response to this agent.
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Enzyme-free translation of DNA into sequence-defined synthetic polymers structurally unrelated to nucleic acids.
Nat Chem
PUBLISHED: 01-21-2013
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The translation of DNA sequences into corresponding biopolymers enables the production, function and evolution of the macromolecules of life. In contrast, methods to generate sequence-defined synthetic polymers with similar levels of control have remained elusive. Here, we report the development of a DNA-templated translation system that enables the enzyme-free translation of DNA templates into sequence-defined synthetic polymers that have no necessary structural relationship with nucleic acids. We demonstrate the efficiency, sequence-specificity and generality of this translation system by oligomerizing building blocks including polyethylene glycol, ?-(D)-peptides, and ?-peptides in a DNA-programmed manner. Sequence-defined synthetic polymers with molecular weights of 26 kDa containing 16 consecutively coupled building blocks and 90 densely functionalized ?-amino acid residues were translated from DNA templates using this strategy. We integrated the DNA-templated translation system developed here into a complete cycle of translation, coding sequence replication, template regeneration and re-translation suitable for the iterated in vitro selection of functional sequence-defined synthetic polymers unrelated in structure to nucleic acids.
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Selective trust: childrens use of intention and outcome of past testimony.
Dev Psychol
PUBLISHED: 01-21-2013
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Childrens epistemic vigilance was examined for their reasoning about the intentions and outcomes of informants past testimony. In a 2 × 2 factorial design, 5- and 6-year-olds witnessed informants offering advice based on the intent to help or deceive others about the location of hidden prizes, with the advice leading to positive or negative outcomes. Informants then suggested to the children where to search for hidden prizes. Children trusted informants who had previously tried to help others more than informants who had previously tried to deceive others, regardless of past outcome. In addition, children trusted informants with positive past outcomes more than informants with negative past outcomes, regardless of intention. By varying intention and outcome independently, this study revealed that when children are deciding whether to trust testimony, they take into account the informants mental states but also give slightly greater weight to the informants past outputs.
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The effect of formulation on the penetration of coated and uncoated zinc oxide nanoparticles into the viable epidermis of human skin in vivo.
Eur J Pharm Biopharm
PUBLISHED: 01-09-2013
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The use of nanoparticulate zinc oxide (ZnO-NP) in sunscreens and other cosmetic products has raised public health concerns. The two key issues are the extent of exposure to ZnO-NP and the likely hazard after the application of ZnO-NP in sunscreen and cosmetic products to humans in vivo. Our aims were to assess exposure by the extent of ZnO-NP penetration into the viable epidermis and hazard by changes in the viable epidermal redox state for a number of topical products. Of particular interest is the role of the particle coating, formulation used, and the presence of any enhancers. Multiphoton tomography with fluorescence lifetime imaging microscopy (MPT-FLIM) was used to simultaneously observe ZnO-NP penetration and potential metabolic changes within the viable epidermis of human volunteers after topical application of various ZnO-NP products. Coated and uncoated ZnO-NP remained in the superficial layers of the SC and in the skin furrows. We observed limited penetration of coated ZnO-NP dispersed in a water-in-oil emulsion formulation, which was predominantly localized adjacent to the skin furrow. However, the presence of ZnO-NP in the viable epidermis did not alter the metabolic state or morphology of the cells. In summary, our data suggest that some limited penetration of coated and uncoated ZnO-NP may occur into viable stratum granulosum epidermis adjacent to furrows, but that the extent is not sufficient to affect the redox state of those viable cells.
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Chromobacterium violaceum infections in 13 non-human primates.
J. Med. Primatol.
PUBLISHED: 12-29-2011
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Recently, an Indian-origin macaque was found dead and Chromobacterium violaceum was isolated from the skin wound, and hepatic and pulmonary abscesses.
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University of Queensland vital signs dataset: development of an accessible repository of anesthesia patient monitoring data for research.
Anesth. Analg.
PUBLISHED: 12-20-2011
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Data recorded from the devices used to monitor a patients vital signs are often used in the development of displays, alarms, and information systems, but high-resolution, multiple-parameter datasets of anesthesia monitoring data from patients during anesthesia are often difficult to obtain. Existing databases have typically been collected from patients in intensive care units. However, the physical state of intensive care patients is dissimilar to those undergoing surgery, more frequent and marked changes to cardiovascular and respiratory variables are seen in operating room patients, and additional and highly relevant information to anesthesia (e.g., end-tidal agent monitoring, etc.) is omitted from these intensive care databases. We collected a set of high-quality, high-resolution, multiple-parameter monitoring data suitable for anesthesia monitoring research.
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Double-outlet right ventricle and double septal defects in a Rhesus macaque (Macaca mulatta).
J. Vet. Diagn. Invest.
PUBLISHED: 12-06-2011
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A 6-year-old male India-origin Rhesus macaque (Macaca mulatta) presented with thin body condition and muscular atrophy. Thoracic auscultation revealed a grade VI/VI pansystolic murmur bilaterally. Radiographs showed cardiomegaly with significant left atrial and biventricular enlargement, a dilated pulmonary artery, and hepatomegaly. Electrocardiogram revealed a normal sinus rhythm interspersed with ventricular bigeminy. Hematology showed mild polycythemia and prerenal azotemia. Necropsy demonstrated double-outlet right ventricle with a large subaortic ventricular septal defect, subpulmonary stenosis, small atrial septal defect, and right ventricular hypertrophy. The major histological finding was severe chronic passive hepatic congestion. Double-outlet right ventricle is a rare congenital heart disease, both in human beings and animals.
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DNA-enabled self-assembly of plasmonic nanoclusters.
Nano Lett.
PUBLISHED: 10-25-2011
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DNA nanotechnology provides a versatile foundation for the chemical assembly of nanostructures. Plasmonic nanoparticle assemblies are of particular interest because they can be tailored to exhibit a broad range of electromagnetic phenomena. In this Letter, we report the assembly of DNA-functionalized nanoparticles into heteropentamer clusters, which consist of a smaller gold sphere surrounded by a ring of four larger spheres. Magnetic and Fano-like resonances are observed in individual clusters. The DNA plays a dual role: it selectively assembles the clusters in solution and functions as an insulating spacer between the conductive nanoparticles. These particle assemblies can be generalized to a new class of DNA-enabled plasmonic heterostructures that comprise various active and passive materials and other forms of DNA scaffolding.
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Pericarditis - clinical features and management.
Aust Fam Physician
PUBLISHED: 10-18-2011
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Pericarditis is an important diagnosis to consider in a patient presenting with chest pain. It is diagnosed in 5% of patients presenting to hospital emergency departments with chest pain in the absence of a myocardial infarction.
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Automatic detection and segmentation of lymph nodes from CT data.
IEEE Trans Med Imaging
PUBLISHED: 10-03-2011
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Lymph nodes are assessed routinely in clinical practice and their size is followed throughout radiation or chemotherapy to monitor the effectiveness of cancer treatment. This paper presents a robust learning-based method for automatic detection and segmentation of solid lymph nodes from CT data, with the following contributions. First, it presents a learning based approach to solid lymph node detection that relies on marginal space learning to achieve great speedup with virtually no loss in accuracy. Second, it presents a computationally efficient segmentation method for solid lymph nodes (LN). Third, it introduces two new sets of features that are effective for LN detection, one that self-aligns to high gradients and another set obtained from the segmentation result. The method is evaluated for axillary LN detection on 131 volumes containing 371 LN, yielding a 83.0% detection rate with 1.0 false positive per volume. It is further evaluated for pelvic and abdominal LN detection on 54 volumes containing 569 LN, yielding a 80.0% detection rate with 3.2 false positives per volume. The running time is 5-20 s per volume for axillary areas and 15-40 s for pelvic. An added benefit of the method is the capability to detect and segment conglomerated lymph nodes.
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Ras-induced and extracellular signal-regulated kinase 1 and 2 phosphorylation-dependent isomerization of protein tyrosine phosphatase (PTP)-PEST by PIN1 promotes FAK dephosphorylation by PTP-PEST.
Mol. Cell. Biol.
PUBLISHED: 08-29-2011
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Protein tyrosine phosphatase (PTP)-PEST is a critical regulator of cell adhesion and migration. However, the mechanism by which PTP-PEST is regulated in response to oncogenic signaling to dephosphorylate its substrates remains unclear. Here, we demonstrate that activated Ras induces extracellular signal-regulated kinase 1 and 2-dependent phosphorylation of PTP-PEST at S571, which recruits PIN1 to bind to PTP-PEST. Isomerization of the phosphorylated PTP-PEST by PIN1 increases the interaction between PTP-PEST and FAK, which leads to the dephosphorylation of FAK Y397 and the promotion of migration, invasion, and metastasis of v-H-Ras-transformed cells. These findings uncover an important mechanism for the regulation of PTP-PEST in activated Ras-induced tumor progression.
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The development of distrust.
Child Dev
PUBLISHED: 08-08-2011
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Preschool-age childrens reasoning about the reliability of deceptive sources was investigated. Ninety 3- to 5-year-olds watched several trials in which an informant gave advice about the location of a hidden sticker. Informants were either helpers who were happy to give correct advice, or trickers who were happy to give incorrect advice. Three-year-olds tended to accept all advice from both helpers and trickers. Four-year-olds were more skeptical but showed no preference for advice from helpers over trickers, even though they differentiated between helpers and trickers on metacognitive measures. Five-year-olds systematically preferred advice from helpers. Selective trust was associated with childrens ability to make mental state inferences.
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p300-Dependent ATF5 acetylation is essential for Egr-1 gene activation and cell proliferation and survival.
Mol. Cell. Biol.
PUBLISHED: 07-26-2011
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ATF5 has been shown to be a critical regulator of cell proliferation and survival; however, the underlying mechanism remains largely unknown. We demonstrate here that ATF5 interacts with the transcriptional coactivator p300, which acetylates ATF5 at lysine-29 (K29), which in turn enhances the interaction between ATF5 and p300 and binding of the ATF5/p300 complex to the ATF5 response element (ARE) region of the Egr-1 promoter. ARE-bound ATF5/p300 acetylates lysine-14 (K14) of nucleosomal histone H3 at both the ARE and serum response element (SRE) of the Egr-1 promoter, which facilitates binding of extracellular signal-regulated kinase (ERK)-phosphorylated Elk-1 to the SRE, activating the Egr-1 promoter. Interference of p300-dependent acetylation of ATF5 or nucleosomal histone H3 or blockade of ERK-dependent Elk-1 phosphorylation abrogates ATF5-dependent Egr-1 activation and cell proliferation and survival. These findings assign a central role for the ATF5/p300 complex in ATF5 function and suggest that coordinated actions by ATF5, p300, Elk-1, and ERK/mitogen-activated protein kinase (MAPK) are essential for ATF5-dependent Egr-1 activation and cell proliferation and survival.
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Gas-phase derivatization via the Meerwein reaction for selective and sensitive LC-MS analysis of epoxides in active pharmaceutical ingredients.
J Pharm Biomed Anal
PUBLISHED: 06-28-2011
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A gas-phase derivatization strategy is reported by using the gas-phase Meerwein reaction for rapid and direct LC-MS analysis of epoxides, which are potential genotoxic impurities (GTIs) in active pharmaceutical ingredients (APIs). This class-selective ion/molecule reaction occurs between epoxides and the ethylnitrilium ion (CH(3)-C?NH?CH(3)-C=NH) that is generated by atmospheric pressure ionizations (when acetonitrile is used as the mobile phase). Density functional theory (DFT) calculations at the B3LYP/6-311+G(d,p) level show that the gas-phase Meerwein reaction is thermodynamically favorable. Commonly used atmospheric pressure ionization techniques including ESI, APCI and APPI were evaluated for optimal formation of the Meerwein reaction products. APCI appears to be the method of choice since it offers better sensitivity and more robust detection under typical LC-MS instrumentation conditions. Quantitative analysis of epoxides can be achieved by either single ion monitoring (SIM) or multiple reaction monitoring (MRM) of the Meerwein reaction products. We demonstrate herein quantitative analysis of two potential GTIs of SB797313 and SB719133 in APIs. The validated methods afford excellent linearity (r(2)?0.999), sensitivity (LOD?1 ppm by w/w in 10 mg/mL APIs) and recovery (ranging from 92% to 102%), as well as accuracy (?2.8% difference) and precision (?2.2% RSD) based on injections of six prepared standards. This novel strategy is particularly useful when a target analyte is difficult to be directly analyzed by LC-MS (e.g. due to poor ionization) or unstable in the course of solution-phase derivatization.
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