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In the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and 2,6-lutidine, α,α-disubstituted aldehydes condense with electron-rich aromatic aldehydes to yield β,β-disubstituted styrenes. More electron-rich aromatic aldehydes react more rapidly and in higher yield. Preliminary results suggest that the reaction may proceed via the ionization and formal deformylation of an aldol intermediate.

AAV gene transfer is a promising treatment for many patients with life-threatening genetic diseases. However, host immune response to the vector poses a significant challenge for the durability and safety of AAV-mediated gene therapy. Here, we characterize the innate immune response to AAV in human whole blood. We identified neutrophils, monocyte-related dendritic cells, and monocytes as the most prevalent cell subsets able to internalize AAV particles, while conventional dendritic cells were the most activated in terms of the CD86 co-stimulatory molecule upregulation. Although low titers (≤1:10) of AAV neutralizing antibodies (NAb) in blood did not have profound effects on the innate immune response to AAV, higher NAb titers (≥1:100) significantly increased pro-inflammatory cytokine/chemokine secretion, vector uptake by antigen presenting cells (APCs) and complement activation. Interestingly, both full and empty viral particles were equally potent in inducing complement activation and cytokine secretion. By using a compstatin-based C3 and C3b inhibitor, APL-9, we demonstrated that complement pathway inhibition lowered CD86 levels on APCs, AAV uptake, and cytokine/chemokine secretion in response to AAV. Together these results suggest that the pre-existing humoral immunity to AAV may contribute to trigger adverse immune responses observed in AAV-based gene therapy, and that blockade of complement pathway may warrant further investigation as a potential strategy for decreasing immunogenicity of AAV-based therapeutics.

To determine the effect of discontinuing routine oral contrast material on emergency department (ED) length of stay (LOS), time from order to CT completion, and preliminary report turnaround time (TAT). A HIPAA-compliant, IRB-waived, single-institution, retrospective cohort study was conducted on adult patients presenting with abdominal pain to the ED from October 2015 to April 2019. Routine oral contrast material was administered prior to July 2018 and discontinued thereafter. CT workflow (ED LOS, exam completion time, report TAT) data were analyzed in a univariate analysis before and after discontinuation of oral contrast. Pre- versus post-policy data were compared with 2-sided t tests. The primary outcome was ED LOS. Data were analyzed on a process control chart and confidence limits were adjusted using established criteria. There were 5020 included abdominopelvic CTs. After routine oral contrast material was discontinued, ED LOS (13.4 h vs 10.7 h, p

Germinal matrix hemorrhage-intraventricular hemorrhage is among the most common intracranial complications in premature infants. Early detection is important to guide clinical management for improved patient prognosis.The purpose of this study was to assess whether a convolutional neural network (CNN) can be trained via transfer learning to accurately diagnose germinal matrix hemorrhage on head ultrasound.Over a 10-year period, 400 head ultrasounds performed in patients ages 6 months or younger were reviewed. Key sagittal images at the level of the caudothalamic groove were obtained from 200 patients with germinal matrix hemorrhage and 200 patients without hemorrhage; all images were reviewed by a board-certified pediatric radiologist. One hundred cases were randomly allocated from the total for validation and an additional 100 for testing of a CNN binary classifier. Transfer learning and data augmentation were used to train the model.The median age of patients was 0 weeks old with a median gestational age of 30 weeks. The final trained CNN model had a receiver operating characteristic area under the curve of 0.92 on the validation set and accuracy of 0.875 on the test set, with 95% confidence intervals of [0.86, 0.98] and [0.81, 0.94], respectively.A CNN trained on a small set of images with data augmentation can detect germinal matrix hemorrhage on head ultrasounds with strong accuracy.

To determine the effect of discontinuing routine oral contrast material on emergency department (ED) length of stay (LOS), time from order to CT completion, and preliminary report turnaround time (TAT).A HIPAA-compliant, IRB-waived, single-institution, retrospective cohort study was conducted on adult patients presenting with abdominal pain to the ED from October 2015 to April 2019. Routine oral contrast material was administered prior to July 2018 and discontinued thereafter. CT workflow (ED LOS, exam completion time, report TAT) data were analyzed in a univariate analysis before and after discontinuation of oral contrast. Pre- versus post-policy data were compared with 2-sided t tests. The primary outcome was ED LOS. Data were analyzed on a process control chart and confidence limits were adjusted using established criteria.There were 5020 included abdominopelvic CTs. After routine oral contrast material was discontinued, ED LOS (13.4 h vs 10.7 h, p 0.001) and time from CT order to CT completion (2.7 h vs 2.1 h, p 0.001) declined. However, control chart analysis revealed improvement in overall LOS preceded the policy change by 9 months, while improvement in time to CT completion coincided with the policy change. Preliminary report TAT increased by 4 min after the policy change (29 min vs. 33 min, p 0.001).Discontinuation of routine oral contrast material in the ED accelerated time to CT completion but had a minor non-significant effect on overall ED LOS. Much of the reduction in overall LOS likely was due to unrelated process improvements.

The need for a malaria vaccine is indisputable. A single vaccine for Plasmodium pre-erythrocytic stages targeting the major sporozoite antigen circumsporozoite protein (CSP) has had partial success. Additionally, CD8+ T cells targeting liver-stage (LS) antigens induced by live attenuated sporozoite vaccines were associated with protection in human challenge experiments. To further evaluate protection mediated by LS antigens, we focused on exported pre-erythrocytic proteins (exported protein 1 (EXP1), profilin (PFN), exported protein 2 (EXP2), inhibitor of cysteine proteases (ICP), transmembrane protein 21 (TMP21), and upregulated in infective sporozoites-3 (UIS3)) expressed in all Plasmodium species and designed optimized, synthetic DNA (synDNA) immunogens. SynDNA antigen cocktails were tested with and without the molecular adjuvant plasmid IL-33. Immunized animals developed robust T cell responses including induction of antigen-specific liver-localized CD8+ T cells, which were enhanced by the co-delivery of plasmid IL-33. In total, 100% of mice in adjuvanted groups and 71%–88% in non-adjuvanted groups were protected from blood-stage disease following Plasmodium yoelii sporozoite challenge. This study supports the potential of synDNA LS antigens as vaccine components for malaria parasite infection.

This article examines U.S. Vice President Henry A. Wallace’s Cold War dissent as a window into racial geopolitics in a post–World War II era of decolonization and U.S. global power. Focused on Wallace and the United States, the article uses a wide range of published and archival sources to argue that Wallace and U.S. anticolonial liberal elites saw anti-racist egalitarian pressures in the post-1945 international system as not only a threat, as existing scholarship suggests, but also an opportunity for U.S. global expansion—particularly in the Pacific Rim. By the 1960s, Wallace and postwar anti-racist activists diminished in influence amid global Cold War pressures reviving racial restrictions and Cold War militarization after the Korean War. Nonetheless, Wallace’s anti-racist diplomacy, stemming from long-running U.S. and global liberal debates and political struggles over race and empire, suggests the wider role of anti-racist geopolitics and the paradoxical persistence of race as a global cultural concept in the postwar era.

The retinoblastoma protein (pRb/p105) tumor suppressor plays a pivotal role in cell cycle regulation by blockage of the G1-to-S-phase transition. pRb tumor suppressor activity is governed by a variety of posttranslational modifications, most notably phosphorylation by cyclin-dependent kinase (Cdk) complexes. Here we report a novel regulation of pRb through protein arginine methyltransferase 4 (PRMT4)-mediated arginine methylation, which parallels phosphorylation. PRMT4 specifically methylates pRb at the pRb C-terminal domain (pRb C(term)) on arginine (R) residues R775, R787, and R798 in vitro and R787 in vivo. Arginine methylation is important for efficient pRb C(term) phosphorylation, as manifested by the reduced phosphorylation of a methylation-impaired mutant, pRb (R3K). A methylmimetic form of pRb, pRb (R3F), disrupts the formation of the E2F-1/DP1-pRb complex in cells as well as in an isolated system. Finally, studies using a Gal4-E2F-1 reporter system show that pRb (R3F) expression reduces the ability of pRb to repress E2F-1 transcriptional activation, while pRb (R3K) expression further represses E2F-1 transcriptional activation relative to that for cells expressing wild-type pRb. Together, our results suggest that arginine methylation negatively regulates the tumor suppressor function of pRb during cell cycle control, in part by creating a better substrate for Cdk complex phosphorylation and disrupting the interaction of pRb with E2F-1.

Latent Kaposi's sarcoma-associated herpesvirus (KSHV) episomes are coated with viral latency-associated nuclear antigen (LANA). In contrast, LANA rapidly disassociates from episomes during reactivation. Lytic KSHV expresses polyadenylated nuclear RNA (PAN RNA), a long noncoding RNA (lncRNA). We report that PAN RNA promotes LANA-episome disassociation through an interaction with LANA which facilitates LANA sequestration away from KSHV episomes during reactivation. These findings suggest that KSHV may have evolved an RNA aptamer to regulate latent protein function.

Kaposi's sarcoma-associated herpesvirus (KSHV) latent genomes are tethered to host histones to form a minichromosome also known as an “episome.” Histones, which are core components of chromatin, are heavily modified by various histone-targeting enzymes. Posttranslational modifications of histones significantly influence accessibility of transcriptional factors and thus have profound effects on gene expression. Recent studies showed that epigenetic marks on the KSHV episome are well organized, exemplified by the absence of histone H3 lysine 9 (H3K9) methylation, a heterochromatic histone mark, from immediate early and latent gene promoters in naturally infected cells. The present study revealed a mechanistic insight into KSHV epigenome regulation via a complex consisting of LANA and the H3K9me1/2 histone demethylase JMJD1A/KDM3A. This complex was isolated from HeLa cell nuclear extracts stably expressing LANA and was verified by coimmunoprecipitation analyses and with purified proteins. LANA recruitment sites on the KSHV genome inversely correlated with H3K9me2 histone marks in naturally infected cells, and methylation of H3K9 significantly inhibited LANA binding to the histone H3 tail. Chromatin immunoprecipitation coupled with KSHV tiling arrays identified the recruitment sites of the complex, while depletion of LANA expression or overexpression of a KDM3A binding-deficient mutant decreased KDM3A recruitment to the KSHV genome. Finally, ablation of KDM3A expression from latently KSHV-infected cells significantly inhibited KSHV gene expression, leading to decreased KSHV replication during reactivation. Taken together, our results suggest that LANA may play a role in regulation of epigenetic marks on the KSHV genome, which is in part through association with the histone demethylase KDM3A.

New York City, New York: Oxford University Press, 2011. 223 pp. hardcover, $99; softcover, $21.95.

The Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a multifunctional protein with roles in gene regulation and maintenance of viral latency. Post-translational modification of LANA is important for functional diversification. Here, we report that LANA is subject to arginine methylation by protein arginine methyltransferase 1 in vitro and in vivo. The major arginine methylation site in LANA was mapped to arginine 20. This site was mutated to either phenylalanine (bulky hydrophobic, constitutive methylated mimetic) or lysine (positively charged, non-arginine methylatable) residues. The significance of the methylation in LANA function was examined in both the isolated form and in the context of the viral genome through the generation of recombinant KSHV. In addition, authentic LANA binding sites on the KSHV episome in naturally infected cells were identified using a whole genome KSHV tiling array. Although mutation of the methylation site resulted in no significant difference in KSHV LANA subcellular localization, we found that the methylation mimetic mutation resulted in augmented histone binding in vitro and increased LANA occupancy at identified LANA target promoters in vivo. Moreover, a cell line carrying the methylation mimetic mutant KSHV showed reduced viral gene expression relative to controls both in latency and in the course of reactivation. These results suggest that residue 20 is important for modulation of a subset of LANA functions and properties of this residue, including the hydrophobic character induced by arginine methylation, may contribute to the observed effects.

Accumulating evidence has suggested that epigenetic alternations are as important as genetic mutations in cancer development. It is proposed that tumors are arisen by “malignant reprogramming” driven by a combination of both genetic and epigenetic changes. It therefore comes as no surprise that histone demethylases, the newest members of the histone modifying enzymes, are found to be targets of mutations and dysregulation in cancer cells. In this review article, we provide an overview of the types of histone demethylases whose genetic structure or expression is altered in cancers, the action of histone demethylases in cancer development and their potential inhibitors. Special emphasis is placed on the roles of histone demethylases in prostate cancer progression.

Xcode has undergone another major revision. This time around, Xcode has one giant window with tabs. A ton of new features with this release make the developer’s job easier. Some of these new features include A single, unified window that brings everything together. The Jump Bar brings quicker navigation through a project as well as a single source file without taking up too much space. Interface Builder is fully integrated into Xcode allowing for even tighter integration between the nib and source. The Xcode Assistant is a two-pane editor that, when enabled, will pick an appropriate file to view next to the editor in which you are editing. LLVM 3.0 is fully integrated into the Xcode which means better syntax highlighting, code completion and many other features that LLVM has. Fix-it uses some features of LLVM to not only display compile errors but suggest quick fixes. Xcode has better integration with some common version control systems: Git and Subversion (SVN). A brand new debugger: LLDB is to GDB as LLVM is to GCC. Instruments has a new interface featuring a Jump Bar and other features borrowed from the Xcode interface.

There is nothing more annoying than a sluggish user interface. As developers, we have the job of ensuring applications are responsive and snappy. This is not always easy. Algorithms can become complicated and run for longer than expected. Designers design complicated views with heavy graphics. Combine the two, and you can easily run into a situation where a user interface becomes a bit choppy when scrolling. UITableViews are notorious for having this kind of issue.

In the previous chapter, we covered some highlights of how to use the new iOS developer tools. The chapters that follow will build on that base and show you some great techniques for turning your application into something that will bring your users back time and time again. In order to do this, we are going to spend the next several chapters walking through an application that has, shall we say, a few problems. Some will be obvious, and others will take some digging to find. The application we’re going to use is a virtual fruit stand. The requirements for the application are to display the product inventory including the product’s image, view a product’s details, add to a shopping cart, view the shopping cart, and check out. The application is in an alpha stage at this point; it has met the basic requirements but it needs some attention in regards to performance and has some crashing issues.

What a ride! We have a stable application with a good automated testing setup and static libraries too. We’ve used Instruments to help find bottlenecks in our application. Throughout this process, we’ve covered some nice features of Xcode and explored how to navigate through the tool set. Xcode has a lot of features that we haven’t covered though. That’s what this chapter is about. While the chapter is titled “One More Thing,” we’re going to cover more than that.

Development is over, the beta is out in the wild, and for the first time in weeks, you can sit back and relax. Right? Well, it’d be great if things were that easy, but they aren’t, and now you’re getting bug reports. Things are crashing, things are slow, and people are finding new and uncharted user journeys, which take them far away from the happy path you so carefully laid out before them.

So now we have an application that we think is ready to ship. Many a developer has been humbled by an application he or she thought was bulletproof, only to discover a litany of bugs once it was released. Often you, as the developer, will only exercise certain application paths or specific test cases. Sometimes, it takes fresh pair of eyes (and hands!) to truly put your application through its paces.

We’ve taken Super Checkout a long way since the beginning of this book. Testing has gone well, the feedback from the users is coming in, and they overwhelmingly want an iPad version of this application. So what do we do now? Do we separate out some of the common code and build two completely different applications, or do we build a universal binary? That decision depends on your goals for the application; it isn’t dictated by technology. It is generally a business decision, because multiple applications can mean more revenue. A universal application, on the other hand, is generally better for the end user, who can purchase one application that will run on the iPhone, iPod Touch, and iPad.

Now that your app is being thoroughly tested and your beta is underway, you’re starting to get requests for some new features. You’ve decided to bring a couple more people onto the project to help get it over the line. The new developers are good, but they aren’t as intimately familiar with the inner workings of your lovely app as you are, so you’re concerned that they might get in there and accidentally wreck the place. You’re very busy fixing bugs and adding features, so you can’t spare the time it takes to run all the tests and static analysis every time one of the new developers pushes an update to the project. Not only that, but adding developers always increases risk in integration bugs, especially on small projects.

So far, we’ve looked at how to remove memory leaks and make our interfaces scroll and animate without much lag. The application is starting to perform and act like a polished app that is ready for prime time. What is the next step in our process? Networking is the next step.

By now, you have written an iOS application or two. You have also learned that making a great app is hard work. From spontaneous crashes to memory leaks and bugs that create other bugs, the simplest of apps can quickly become a nightmare. Fortunately, these issues are easy to diagnose with the tools at our disposal.

In the previous chapter, we looked at an application that is basically feature complete but still needs a lot of work. We have our project set up with source control from Github, and we are ready to start fixing this application to make it ready for beta testing. The fun stuff starts in this chapter.

Antimicrobial peptides are distributed throughout the animal kingdom and are a key component of innate immunity. Salmonella typhimurium regulates mechanisms of resistance to cationic antimicrobial peptides through the two-component systems PhoP-PhoQ and PmrA-PmrB. Polymyxin resistance is encoded by the PmrA-PmrB regulon, whose products modify the lipopolysaccharide (LPS) core and lipid A regions with ethanolamine and add aminoarabinose to the 4' phosphate of lipid A. Two PmrA-PmrB-regulated S. typhimurium loci (pmrE and pmrF) have been identified that are necessary for resistance to polymyxin and for the addition of aminoarabinose to lipid A. One locus, pmrE, contains a single gene previously identified as pagA (or ugd) that is predicted to encode a UDP-glucose dehydrogenase. The second locus, pmrF, is the second gene of a putative operon predicted to encode seven proteins, some with similarity to glycosyltransferases and other complex carbohydrate biosynthetic enzymes. Genes immediately flanking this putative operon are also regulated by PmrA-PmrB and/or have been associated with S. typhimurium polymyxin resistance. This work represents the first identification of non-regulatory genes necessary for modification of lipid A and subsequent antimicrobial peptide resistance, and provides support for the hypothesis that lipid A aminoarabinose modification promotes resistance to cationic antimicrobial peptides.