Search

Supplemental Figures 1-5. Supplemental Figure 1. Kinase selectivity profile of ATRi. Supplemental Figure 2. Screening of inhibitors in cellular γ-H2AX assay in U2OS cells. Supplemental Figure 3. Flow cytometry profiles assessing γ-H2AX response to compare antimetabolite combinations with a fixed concentration of ATRi in U2OS cells. Supplemental Figure 4. γ-H2AX response and cell viability following ATRi treatment in U2OS cells. Supplemental Figure 5. In vivo pharmacokinetics and γ-H2AX pharmacodynamics of ATRi in GRANTA-519 (ATM mut) MCL tumor xenograft model.

Supplemental Table 2. SiRNA sequences (Supplemental data)

Supplemental Table 1. DiscoveRx ATR inhibitor profile

Supplemental Figure legends. Supplemental Figure 1. Kinase selectivity profile of ATRi. Supplemental Figure 2. Screening of inhibitors in cellular γ-H2AX assay in U2OS cells. Supplemental Figure 3. Flow cytometry profiles assessing γ-H2AX response to compare antimetabolite combinations with a fixed concentration of ATRi in U2OS cells. Supplemental Figure 4. γ-H2AX response and cell viability following ATRi treatment in U2OS cells. Supplemental Figure 5. In vivo pharmacokinetics and γ-H2AX pharmacodynamics of ATRi in GRANTA-519 (ATM mut) MCL tumor xenograft model.

Mechanisms to maintain genomic integrity are essential for cells to remain viable. Not surprisingly, disruption of key DNA damage response pathway factors, such as ataxia telangiectasia-mutated (ATM)/ataxia telangiectasia and RAD3-related (ATR) results in loss of genomic integrity. Here, a synthetic lethal siRNA-screening approach not only confirmed ATM but identified additional replication checkpoint proteins, when ablated, enhanced ATR inhibitor (ATRi) response in a high-content γ-H2AX assay. Cancers with inactivating ATM mutations exhibit impaired DNA double-stranded break (DSB) repair and rely on compensatory repair pathways for survival. Therefore, impairing ATR activity may selectively sensitize cancer cells to killing. ATR inhibition in an ATM-deficient context results in phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs) and leads to induction of γ-H2AX. Using both in vitro and in vivo models, ATR inhibition enhanced efficacy in ATM loss-of-function mantle cell lymphoma (MCL) compared with ATM wild-type cancer cells. In summary, single-agent ATR inhibitors have therapeutic utility in the treatment of cancers, like MCL, in which ATM function has been lost.Implications: These data suggest that single-agent ATR inhibitors have therapeutic utility and that ATR uses a complex and coordinated set of proteins to maintain genomic stability that could be further exploited. Mol Cancer Res; 13(1); 120–9. ©2014 AACR.

Material and Methods

This article evaluates a transition intervention project aimed at first-year undergraduate filmmaking students in the Manchester School of Art. In response to recently identified shifts in confidence and resilience linked to an increased rate of withdrawal, a Scholarship of Teaching and Learning research project was undertaken to address the issue and develop a transition strategy to promote engagement, confidence and progression. Research into studies of first-year transition and retention, and an analysis of both the pedagogical context of the course and wider ecology of the discipline, informed a remodelling of the autumn term curriculum with a focus on the first six weeks of study. The research revealed key themes of consequence to first-year curriculum design, including the impact of art and design’s ‘pedagogy of ambiguity’ and the significance of belongingness and community to filmmaking students.

Purpose To develop a reliable and efficient method for quantifying the area of preserved retinal pigment epithelium (RPE), facilitating the evaluation of disease progression or response to therapy in choroideremia (CHM). Methods The fundus autofluorescence images of CHM patients were captured at baseline and 1 year. A Photoshop-based method was developed to allow the reliable measurement of the RPE area. The results were compared with measurements generated by the Heidelberg Eye Explorer 2 (HEYEX2). The areas measured by two independent graders were compared to assess the test-retest reliability. Results By using the Photoshop-based method, the area of the RPE measured from 64 eyes was seen to decrease significantly (P < 0.001) at a rate of 2.57 ± 3.22 mm2 annually, and a percentage of 8.39% ± 5.24%. The average standard deviations for Photoshop were less than that for HEYEX2 (0.5-1.1 in grader 1; 0.4-1.6 in grader 2), indicating less intragrader variability. The RPE decrease as determined by the Photoshop-based method showed excellent reliability with an intraclass correlation coefficient of 0.944 (95% confidence interval, 0.907-0.966). In Bland-Altman plots, the Photoshop method also exhibited better intergrader agreement. Conclusions Photoshop-based quantification of preserved RPE area in patients with CHM is feasible and has better test-retest reliability compared with the HEYEX2 method. Translational Relevance An accurate quantification method for longitudinal RPE change in CHM patients is an important tool for the evaluation of efficacy in any therapeutic trials.

Cerebellar Purkinje cells (PCs) show conspicuous damages in many ataxic disorders. Targeted delivery of short nucleic acids, such as antisense oligonucleotides, to PCs may be a potential treatment for ataxic disorders, especially spinocerebellar ataxias (SCAs), which are mostly caused by a gain of toxic function of the mutant RNA or protein. However, oligonucleotides do not cross the blood-brain barrier (BBB), necessitating direct delivery into the central nervous system (CNS) through intra-thecal, intra-cisternal, intra-cerebral ventricular, or stereotactic parenchymal administration. We have developed a novel liposome (100 to 200 nm in diameter) formulation, DCL64, composed of dipalmitoyl-phosphatidylcholine, cholesterol, and poloxamer L64, which incorporates oligonucleotides efficiently (≥ 70%). Confocal microscopy showed that DCL64 was selectively taken up by brain microvascular endothelial cells by interacting with low-density lipoprotein receptor (LDLr) family members on cell surface, but not with other types of lipid receptors such as caveolin or scavenger receptor class B type 1. LDLr family members are implicated in brain microvascular endothelial cell endocytosis/transcytosis, and are abundantly localized on cerebellar PCs. Intravenous administration of DCL64 in normal mice showed distribution of oligonucleotides to the brain, preferentially in PCs. Mice that received DCL64 showed no adverse effect on hematological, hepatic, and renal functions in blood tests, and no histopathological abnormalities in major organs. These studies suggest that DCL64 delivers oligonucleotides to PCs across the BBB via intravenous injection with no detectable adverse effects. This property potentially makes DCL64 particularly attractive as a delivery vehicle in treatments of SCAs.

The travel books of Isabella Bird are noted for their accuracy and for their close attention to detail, measurements and statistics. In general, Unbeaten Tracks in Japan (1880) is no exception. However, when describing physical size, Bird vastly exaggerates the smallness of Japan and the Japanese while depicting herself as much larger in comparison – despite being roughly the same size as the average Japanese subject herself. In this article, I argue that Bird’s size distortions can be read as a sign of neurosis, and I demonstrate how subsequent writers such as Pierre Loti and Rudyard Kipling also used distortions in perspective and proportion to signify psychological discomfort. I describe how both Japanese and avant-garde Victorian art used perspective distortion to signal emotional distress, and suggest that art, as well as contemporary psychological theories, may have influenced the way these authors depicted physical size.

Mechanisms to maintain genomic integrity are essential for cells to remain viable. Not surprisingly, disruption of key DNA damage response pathway factors, such as ataxia telangiectasia-mutated (ATM)/ataxia telangiectasia and RAD3-related (ATR) results in loss of genomic integrity. Here, a synthetic lethal siRNA-screening approach not only confirmed ATM but identified additional replication checkpoint proteins, when ablated, enhanced ATR inhibitor (ATRi) response in a high-content γ-H2AX assay. Cancers with inactivating ATM mutations exhibit impaired DNA double-stranded break (DSB) repair and rely on compensatory repair pathways for survival. Therefore, impairing ATR activity may selectively sensitize cancer cells to killing. ATR inhibition in an ATM-deficient context results in phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs) and leads to induction of γ-H2AX. Using both in vitro and in vivo models, ATR inhibition enhanced efficacy in ATM loss-of-function mantle cell lymphoma (MCL) compared with ATM wild-type cancer cells. In summary, single-agent ATR inhibitors have therapeutic utility in the treatment of cancers, like MCL, in which ATM function has been lost. Implications: These data suggest that single-agent ATR inhibitors have therapeutic utility and that ATR uses a complex and coordinated set of proteins to maintain genomic stability that could be further exploited. Mol Cancer Res; 13(1); 120–9. ©2014 AACR.

The article as a whole seeks to explore new ways of looking at the literature of intercultural encounter using ideas of culture shock, cultural adaptation and narrative theory. It also demonstrates the importance of Anglophone literature about Japan to the understanding of how writers from the late nineteenth and early twentieth centuries sought to engage with people from other ethnic groups. From the 1850s, when Japan ended its policy of national isolation, until the beginning of World War II, the literary marketplace was flooded with numerous English-language texts which dealt with almost every aspect of Japanese life, covering subjects as diverse as art and design, botany, geology, women's rights, war, public health, theatre, linguistics and religion. However, despite the size of this Anglophone Japan corpus, only well-known texts such as Isabella Bird's Unbeaten Tracks in Japan and the works of Lafcadio Hearn have attracted substantial in-depth analysis. One reason for the neglect of the field is that previous decades of scholars regarded this literature as too ephemeral to be worth studying. Another may be that conventional Saidian frameworks for interpreting the literature of cultural encounter fail to provide a satisfactory understanding of relationships between westerners and the Japanese. However, this resistance to Saidian interpretation means that the Anglophone Japan corpus is ideal for testing alternative readings of cross-cultural literature. This, the first section of the article, presents a brief overview of Anglophone literature on Japan, and investigates some of the new strategies being put forward for interpreting these texts. The second part of the article will follow on by presenting a case study of one theme which dominated discussions of Japan in the early twentieth century: the figure of the samurai and the idea of ‘Bushidō’ (the ‘way of the samurai’). By undertaking this analysis, I demonstrate how ideas of culture shock and narrative psychology can be used to give a more nuanced understanding of the personal and social motives behind the literature of intercultural encounter.

Dealing with the abuses associated with widespread child labour is now considered to be a key task in any country's progress towards a full recognition of human rights. In Britain, the plight of th...

The aim of this paper is to investigate ways in which the dynamics of interprofessional work shaped older patients' "participation" in decision-making about discharge from acute hospital care in a medical directorate of a District General Hospital in Scotland. Twenty-two purposively selected older patients and their key professional hospital carers in three different ward environments participated in the study. An ethnographic approach was adopted, involving semi-structured interviews with patients and staff combined with rigorous observation of the practical context for staff and patient interactions during the discharge planning process over a 5-month period. Patients' and staff's understanding of "decision-making" and their priorities for discharge were different, but patients' perspectives fragmented and became invisible. Care routines, which centred around assessments and the decisions that flowed from these tended to exclude both staff and patients from active decision-making. Research and practice on patient involvement in discharge decision-making needs to focus on the organizational context, which shapes patients', unpaid carers' and staff's interactions and the dynamics by which some views are privileged and others excluded. Procedurally driven care routines and their impact on patients', carers' and staff's opportunity to actively engage in decision-making should be re-considered from an empowerment perspective.

A major obstacle in the treatment of glioblastoma (GBM) is the blood brain barrier (BBB), which restricts drug molecules in the bloodstream from reaching the tumor. The BBB may be disrupted at or near the GBM tumor core, but it is intact at the growing edge of the tumor, where invasive cells reside and can cause disease recurrence. To combat GBM more effectively, novel drug transport technology that crosses intact BBB is urgently needed. Brain microvascular endothelial cells (BMEC) found in the BBB are lined with low-density lipoprotein receptors (LDLr) and LDL-like receptor proteins (LRP), which are essential to the endocytosis/ transcytosis processes of BMEC. GBM cells are known to express higher levels of LDLr and LRP than normal brain cells. The objective of our research is to develop a novel lipid nanoparticle (NP) that targets LDLr and LRP to facilitate BMEC endocytosis/ transcytosis and GBM uptake. We focused on developing antisense oligonucleotides (oligos) as the drug cargo for our NP. Less than 20% of disease-associated genes are targetable by conventional drug molecules; however, we hypothesize that disease-associated genes, like EZH2 and ZEB1 transcriptional repressors, are amendable to antisense oligo inhibition. We engineered a novel NP which is composed of neutral lipids: phosphatidylcholine (PC), cholesterol (Chol) and a polymer called poloxamer (PXR). Toxicology studies indicated that NP composed of PC/Chol/PXR did not have adverse effects on mice. Flow cytometry and fluorescent microscopy revealed that PC/Chol/PXR NP was avidly taken up by BMEC, and that PC/Chol/PXR efficiently delivered antisense oligos to GBM cells. Additionally, intravenous injection of PC/Chol/PXR led to deposition of antisense oligos in intracranial GBM xenografts. Future studies will determine the efficacy of the innovative PC/Chol/PXR NP in suppressing target gene expression in intracranial GBM. (Supported by Florida Center for Brain Tumor Research and Accelerate Brain Cancer Cure).

Mutants of human adenovirus 5 (Ad5) with enhanced oncolytic activity were isolated by using a procedure termed bioselection. Two mutants, ONYX-201 and ONYX-203, were plaque purified from a pool of randomly mutagenized Ad5 that was repeatedly passaged in the human colorectal cancer cell line HT29, and they were subsequently characterized. ONYX-201 and ONYX-203 replicated more rapidly in HT29 cells than wild-type Ad5, and they lysed HT29 cells up to 1,000-fold more efficiently. The difference was most profound when cells were infected at a relatively low multiplicity of infection, presumably due to the compounding effects of multiple rounds of infection. This enhanced cytolytic activity was observed not only in HT29 cells but also in many other human cancer cell lines tested. In contrast, the cytotoxicity of the bioselected mutants in a number of normal primary human cells was similar to that of wild-type Ad5, thus enhancing the therapeutic index (cytotoxicity in tumor cells versus that in normal cells) of these oncolytic agents. Both ONYX-201 and -203 contain seven single-base-pair mutations when compared with Ad5, four of which were common between ONYX-201 and -203. The mutation at nucleotide 8350, shared by both mutant viruses, was shown to be essential for the observed phenotypes. This mutation was mapped to the i-leader region of the major late transcription unit, resulting in the truncation of 21 amino acids from the C terminus of the i-leader protein. This work demonstrates that bioselection is a powerful tool for developing novel tumor-selective oncolytic viruses. Other potential applications of this technology are discussed.

The use of genetically engineered, replication-selective viruses to treat cancer is being realized with viruses such as ONYX-015, a human adenovirus that selectively destroys p53 mutant cancer cells. To enhance further the clinical efficacy of ONYX-015 and viruses like it, we have developed a novel gene delivery system for replicating adenoviruses. This system has two unique features. First, it uses the endogenous adenoviral gene expression machinery (promoter, splicing, polyadenylation) to drive transgene expression. Second, a single region or gene in the multi-gene E3 transcription unit is selectively substituted for by the therapeutic transgene(s). Analyzing various transgene substitutions for the 6.7 K/gp19 K region of E3, we demonstrate the following: (1) transgene expression in this system is predictable and mimics the substituted endogenous gene expression pattern, (2) expression of surrounding E3 genes can be retained, (3) the insertion site choice can effect both the transgene expression level and the viral life cycle, and, (4) expression levels from this system are superior to those generated from a replication-defective virus using the HCMV enhancer-promoter and this is dependent on viral DNA replication. This unique methodology has broad application to the rapidly evolving field of replicating virus-based therapies.

in Hopkins' case, "the question" should really be "who is to be master?" I will here argue that, rather than merely dealing, as Humpty does, with bad-tempered adjectives and sulking verbs, Hopkins engages in a far wider contest for mastery of both language and textuality. In this, he embarks on a modified version of Hegel's Master-Slave dialectic, constantly testing power boundaries with both human and divine beings. The clinch of this struggle is the prerogative of Hopkins to inscribe himself as an authoritative figure within various social and spiritual power structures. The importance of this primarily linguistic negotiation includes Hopkins' ability to determine the nature of his own selfhood. E. Warwick Slinn noted that in recent structuralist and post-structuralist theory "consciousness is not some entity separate from language, but is inscribed in language [and is] itself 'written' in and through language."2 If we, like Jacques Derrida, acknowledge the instability of language systems, we must subsequently face the fragility of consciousness. If we are not "the master" of our language, we are not in control of our own being. This essay proposes that Hopkins anticipates the proposition that language and consciousness are interwoven, and that he enters into a dialogue where his poetic and ecclesiastical authority are jeopardized. I will demonstrate how a linguistic interpretation of the Master-Slave dialectic can be used to examine the power struggles

Pain can have a throbbing quality, especially when it is severe and disabling. It is widely held that this throbbing quality is a primary sensation of one's own arterial pulsations, arising directly from the activation of localized pain-sensory neurons by closely apposed blood vessels. We examined this presumption more closely by simultaneously recording the subjective report of the throbbing rhythm and the arterial pulse in human subjects of either sex with throbbing dental pain—a prevalent condition whose pulsatile quality is widely regarded a primary sensation. Contrary to the generally accepted view, which would predict a direct correspondence between the two, we found that the throbbing rate (44 bpm ± 3 SEM) was much slower than the arterial pulsation rate (73 bpm ± 2 SEM,p< 0.001), and that the two rhythms exhibited no underlying synchrony. Moreover, the beat-to-beat variation in arterial and throbbing events observed distinct fractal properties, indicating that the physiological mechanisms underlying these rhythmic events are distinct. Confirmation of the generality of this observation in other pain conditions would support an alternative hypothesis that the throbbing quality is not a primary sensation but rather an emergent property, or perception, whose “pacemaker” lies within the CNS. Future studies leading to an improved understanding of the neurobiological basis of clinically relevant pain qualities, such as throbbing, will also enhance our ability to measure and therapeutically target severe and disabling pain.

Objective: We recently reported that the heart rate and subjective throbbing rate in patients with migraine do not correspond, but were unable to establish the generality of this finding, nor were we able to rigorously test the possibility of a more subtle relationship between throbbing percepts and the peripheral arterial stimulus.Thus, in order to establish an independent neurobiological basis for this percept, we recorded subject reports of the throbbing rhythm while simultaneously recording the arterial pulse in subjects with acute dental pain, a pulsatile pain condition of indisputable peripheral origin. Background When severe and disabling, pain is often throbbing or pulsatile. The prevailing scientific view holds that this throbbing quality, such as in migraine, is a primary percept, arising directly from the mechanical activation of pain-sensory neurons closely apposed to blood vessels. Design/Methods: This was a cross-sectional study of the throbbing percept in a dental clinic. Results: We found that throbbing and arterial pulse rates had no correlation, and that the two rhythms showed no synchrony. Moreover, arterial and throbbing rate variability displayed distinct fractal scaling properties, implying that the mechanisms underlying these complex rhythms are unrelated. Conclusions: The present refutation of the “vascular theory” of throbbing percepts suggests an alternative possibility, that a CNS “pacemaker” underlies throbbing percepts, and could lead to novel treatment strategies for pain. Disclosure: Dr. Ahn has nothing to disclose. Dr. Mirza has nothing to disclose. Dr. Mo has nothing to disclose. Dr. Holt has nothing to disclose. Dr. Kairalla has nothing to disclose. Dr. Heft has nothing to disclose. Dr. Ding has nothing to disclose.