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Background

Post-translational modification of ribosomal subunit proteins (RPs) is emerging as an important means of regulating gene expression. Recently, regulatory ubiquitination of small RPs RPS10 and RPS20 by the ubiquitin ligase ZNF598 was found to function in ribosome sensing and stalling on internally polyadenylated mRNAs during ribosome quality control (RQC). Here, we reveal that ZNF598 and RPS10 negatively regulate interferon-stimulated gene (ISG) expression in primary cells, depletion of which induced ISG expression and a broad antiviral state. However, cell lines lacking interferon responses revealed that ZNF598 E3 ligase activity and ubiquitination of RPS20, but not RPS10, were specifically required for poxvirus replication and synthesis of poxvirus proteins whose encoding mRNAs contain unusual 5′ poly(A) leaders. Our findings reveal distinct functions for ZNF598 and its downstream RPS targets, one that negatively regulates ISG expression and infection by a range of viruses while the other is positively exploited by poxviruses.

The integration of digital technologies at institutions of higher education are profoundly influencing formal learning on a global scale. Social-constructivist models of fullyonline learning are well-positioned to address the demands of government, and economic and social-development organizations for civically-engaged individuals with strong problem-solving, critical-thinking and collaboration competencies. With an established record of performance at the University of Ontario Institute of Technology (UOIT), Canada, the Fully Online Learning Community(FOLC) is one such model. This paper theorizes FOLC as a response to several problematics, including (a) the aforementioned demand for greater educational focus on higher-order competency development, (b) the deficiencies of distance education and MOOCs as learningmodels, and (c) a quest for new learning models that strengthen deliberation skills and deepen democratic experience. As a divergent fork of the Community of Inquiry model, FOLC describes collaborative learning as a symbiosis of social and cognitive interactions amplified through effective use of synchronous and asynchronous digital affordances. Furthermore, it models democratized learning communities that reduce transactional distance between learners and educators, incorporates authentic assessment, and encourages negotiated technology affordances and cognitive outcomes while distributing responsibility for constructive criticality. Having positioned FOLC conceptually, and addressed current limitations, a research agenda for extending its empirical foundations, and leveraging UOIT’s EILAB affordances, is presented. The underlying argument is that self-regulating and transformative learning communities can be established and sustained in fully online environments, and that such communities (a) produce a diversity of beneficial learning outcomes, and (b) deepen the democratic functioning of learners and their social contexts.

Clonal cytogenetic evolution with additional chromosomal abnormalities (ACAs) in chronic myelogenous leukemia (CML) is generally associated with decreased response to tyrosine kinase inhibitor (TKI) therapy and adverse survival. Although ACAs are considered as a sign of disease progression and have been used as one of the criteria for accelerated phase, the differential prognostic impact of individual ACAs in CML is unknown, and a classification system to reflect such prognostic impact is lacking. In this study, we aimed to address these questions using a large cohort of CML patients treated in the era of TKIs. We focused on cases with single chromosomal changes at the time of ACA emergence and stratified the 6 most common ACAs into 2 groups: group 1 with a relatively good prognosis including trisomy 8, −Y, and an extra copy of Philadelphia chromosome; and group 2 with a relatively poor prognosis including i(17)(q10), −7/del7q, and 3q26.2 rearrangements. Patients in group 1 showed much better treatment response and survival than patients in group 2. When compared with cases with no ACAs, ACAs in group 2 conferred a worse survival irrelevant to the emergence phase and time. In contrast, ACAs in group 1 had no adverse impact on survival when they emerged from chronic phase or at the time of CML diagnosis. The concurrent presence of 2 or more ACAs conferred an inferior survival and can be categorized into the poor prognostic group.

ABSTRACTMycobacterium tuberculosismust sense and adapt to host environmental cues to establish and maintain an infection. The two-component regulatory system PhoPR plays a central role in sensing and responding to acidic pH within the macrophage and is required for M. tuberculosisintracellular replication and growth in vivo. Therefore, the isolation of compounds that inhibit PhoPR-dependent adaptation may identify new antivirulence therapies to treat tuberculosis. Here, we report that the carbonic anhydrase inhibitor ethoxzolamide inhibits the PhoPR regulon and reduces pathogen virulence. We show that treatment of M. tuberculosiswith ethoxzolamide recapitulates phoPRmutant phenotypes, including downregulation of the core PhoPR regulon, altered accumulation of virulence-associated lipids, and inhibition of Esx-1 protein secretion. Quantitative single-cell imaging of a PhoPR-dependent fluorescent reporter strain demonstrates that ethoxzolamide inhibits PhoPR-regulated genes in infected macrophages and mouse lungs. Moreover, ethoxzolamide reduces M. tuberculosisgrowth in both macrophages and infected mice. Ethoxzolamide inhibits M. tuberculosiscarbonic anhydrase activity, supporting a previously unrecognized link between carbonic anhydrase activity and PhoPR signaling. We propose that ethoxzolamide may be pursued as a new class of antivirulence therapy that functions by modulating expression of the PhoPR regulon and Esx-1-dependent virulence.

ABSTRACTThe mycobacterial Esx-1 (ESAT-6 system 1) exporter translocates virulence factors across the cytoplasmic membrane to the cell wall, cell surface, and the bacteriological medium in vitro. The mechanisms underlying substrate targeting to distinct locations are unknown. Several Esx-1 substrates are N-α-terminally acetylated. The role of this rare modification in bacteria is unclear. We sought to identify genes required for Esx-1 substrate modification, transport, and localization. Pathogenic mycobacteria lyse Acanthamoeba castellaniiin an Esx-1-dependent manner. We conducted a genetic screen to identify Mycobacterium marinumstrains which failed to lyse amoebae. We identified a noncytotoxic M. marinumstrain with a transposon insertion in a predicted N-α-terminal acetyltransferase not previously linked to mycobacterial pathogenesis. Disruption of this gene led to attenuation of virulence, failure to induce a type I interferon response during macrophage infection, and loss of hemolytic activity. The major Esx-1 substrates, EsxA and EsxB, were exported to the cell surface, but only low levels were released into the bacteriological medium. The balance of EsxA N-α-terminal acetylation was disrupted, resulting in a mycobacterial strain in which surface-associated EsxA was hyperacetylated. Genetic complementation completely restored Esx-1 function and the levels of N-α-terminally acetylated EsxA on the surface but restored only low levels of Esx-1 substrates in the bacteriological medium. Our results reveal a novel gene required for mycobacterial Esx-1 export. Our findings indicate that maintaining the homeostasis of Esx-1 substrate N-α-terminal acetylation is essential for Esx-1-mediated virulence. We propose an inverse correlation between EsxA acetylation and virulence.

Background:Children born to mothers with gestational diabetes mellitus (GDM) experience increased risk of developing hypertension, type 2 diabetes mellitus, and obesity. Disrupted function of endothelial colony-forming cells (ECFCs) may contribute to this enhanced risk. The goal of this study was to determine whether cord blood ECFCs from GDM pregnancies exhibit altered functionality.Methods:ECFCs isolated from the cord blood of control and GDM pregnancies were assessed for proliferation, senescence, and Matrigel network formation. The requirement for p38MAPK in hyperglycemia-induced senescence was determined using inhibition and overexpression studies.Results:GDM-exposed ECFCs were more proliferative than control ECFCs. However, GDM-exposed ECFCs exhibited decreased network-forming ability in Matrigel. Aging of ECFCs by serial passaging led to increased senescence and reduced proliferation of GDM-exposed ECFCs. ECFCs from GDM pregnancies were resistant to hyperglycemia-induced senescence compared with those from controls. In response to hyperglycemia, control ECFCs activated p38MAPK, which was required for hyperglycemia-induced senescence. In contrast, GDM-exposed ECFCs showed no change in p38MAPK activation under equivalent conditions.Conclusion:Intrauterine exposure of ECFCs to GDM induces unique phenotypic alterations. The resistance of GDM-exposed ECFCs to hyperglycemia-induced senescence and decreased p38MAPK activation suggest that these progenitor cells have undergone changes that induce tolerance to a hyperglycemic environment.

Bacteria use a variety of secretion systems to transport proteins beyond their cell membrane to interact with their environment. For bacterial pathogens, these systems are key virulence determinants that transport bacterial proteins into host cells. Genetic screens to identify bacterial genes required for export have relied on enzymatic or fluorescent reporters fused to known substrates to monitor secretion. However, they cannot be used in analysis of all secretion systems, limiting the implementation across bacteria. Here, we introduce the first application of a modified form of whole colony MALDI-TOF MS to directly detect protein secretion from intact bacterial colonies. We show that this method is able to specifically monitor the ESX-1 system protein secretion system, a major virulence determinant in both mycobacterial and Gram-positive pathogens that is refractory to reporter analysis. We validate the use of this technology as a high throughput screening tool by identifying an ESAT-6 system 1-deficient mutant from a Mycobacterium marinumtransposon insertion library. Furthermore, we also demonstrate detection of secreted proteins of the prevalent type III secretion system from the Gram-negative pathogen, Pseudomonas aeruginosa.This method will be broadly applicable to study other bacterial protein export systems and for the identification of compounds that inhibit bacterial protein secretion.

Anger is a frequently experienced negative emotion that occurs identified in all ages and across all cultures (DiGiuseppe & Tafrate, 2007). Parent anger is concerning because it has been associated with dysfunctional discipline strategies, child abuse, child noncompliance, and various emotional problems during childhood (see Smith Slep & O’Leary, 2001). The Parent Anger Scale is a new measure for parent anger developed based on new data in the field. The purpose of this study was to psychometrically validate the Parent Anger Scale (PAS). Results show adequate properties of the scale based on a 1-factor solution.

Irrational and rational cognitions/beliefs are evaluative cognitive structures. Irrational thinking has been consistently associated with psychopathology; in contrast, rational thinking is considered important for resiliency. We conducted two studies to construct and validate the Parent Rational and Irrational Beliefs Scale (P-RIBS), a new self-report measure of parent irrational and rational cognitions/beliefs. Results show a three-factor solution for P-RIBS. The P-RIBS displayed strong psychometric, support evidencing adequate internal consistencies and concurrent validity.

Gamma/delta T cell lymphomas (γ/δ TCL) represent rare, often aggressive types of T cell malignancy that are clinically and pathologically diverse. Most γ/δ TCL occur as a hepatosplenic or subcutaneous type. To date, analysis of the T cell receptor δ (TCRδ) gene repertoire of hepatosplenic γ/δ TCL (γ/δ HSTCL) and subcutaneous panniculitis-like γ/δ TCL (γ/δ SPTCL) has been reported only in a limited number of cases. In this study we analyzed 11 γ/δ HSTCL and 4 γ/δ SPTCL by polymerase chain reaction and immunostaining to determine their usage of the Vδ subtypes (Vδ1–6). It is noteworthy that 10 of 11 γ/δ HSTCL expressed the Vδ1 gene. The remaining case also expressed T cell receptor δ (TCRδ) as determined by flow cytometry and TCRδ rearrangement in Southern blot. However, the Vδ gene expressed by this lymphoma could not be determined, which suggests usage of an as yet unidentified Vδ gene. In striking contrast to the γ/δ HSTCL, all 4 γ/δ SPTCL expressed the Vδ2 gene. Our data demonstrate that γ/δ HSTCL are preferentially derived from the Vδ1 subset of γ/δ T lymphocytes, whereas γ/δ SPTCL are preferentially derived from the Vδ2 subset. The pattern of Vδ gene expression in HSTCL and SPTCL corresponds to the respective, predominant γ/δ T cell subsets normally found in the spleen and skin. This finding suggests that γ/δ TCL are derived from normal γ/δ T lymphocytes which reside in the affected tissues. Furthermore, the selective, lymphoma type-specific Vδ gene segment usage may provide a molecular tool to distinguish better among various types of γ/δ TCL lymphoma particularly in the clinically advanced, widely disseminated cases.

To assess the association between race and insurance and Cesarean delivery rates after adjusting for clinical risk factors that increase the likelihood of cesarean delivery.

Nuclear oncoproteins are among the most rapidly degraded intracellular proteins. Previous work has implicated the ubiquitin-mediated proteolytic system in the turnover of short-lived intracellular proteins. In the present study, we have evaluated the potential role of the ubiquitin system in the degradation of the specific nuclear oncoproteins encoded by the N-myc, c-myc, c-fos, p53 and E1A genes. Each of these nuclear oncoproteins was synthesized in vitro by transcription of the appropriate cDNA and translation of the resulting mRNA in the presence of [35S]methionine. Degradation of labeled proteins was monitored in the ubiquitin cell-free system. ATP stimulated the degradation of all the proteins between 3- and 10-fold. The degradation was completely inhibited by neutralizing antibody directed against the ubiquitin-activating enzyme, E1, the first enzyme in the ubiquitin-mediated proteolytic cascade. Moreover, degradation in E1-depleted lysates could be restored in each case by the addition of affinity-purified E1. These data suggest that the ubiquitin system mediates the degradation of these oncoproteins in vitro. Degradation of other proteins, such as superoxide dismutase, cytochrome c, enolase, RNase A, and ornithine decarboxylase, is not mediated by the ubiquitin cell-free system. This suggests that the nuclear oncoproteins studied here possess specific signals that target them for rapid turnover by this proteolytic pathway. Furthermore, the relative sensitivity to degradation of various E1A mutants in vivo is also maintained in the cell-free system, suggesting that the ubiquitin pathway may play a role in the cellular degradation of these proteins as well.

This paper reports the study of an anode-supported SOFC cell containing an LSCF-SDC composite cathode. The SOFC cell was tested at different temperatures and reactant flow rates. After testing, the cell was sectioned and characterized using SEM/EDS. Such analysis indicated that no structural damage and no significant interdiffusion of elements among the layers occurred. The measured electrochemical performance data at different temperatures indicate an Arrhenius behavior or temperature activated processes. The low-porosity anode functional layer appears to be very sensitive to low hydrogen contents. The electrochemical performance is also affected by changing air flow rates.

Wood: Pfizer: Honoraria, Other: Laboratory Services Agreement; Juno: Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Seattle Genetics: Honoraria, Other: Laboratory Services Agreement. Lozanski:Stemline Therapeutics Inc.: Research Funding; Beckman Coulter: Research Funding; Boehringer Ingelheim: Research Funding; Genentech: Research Funding. Mukundan:CCS Associates: Employment. Higley:CCS Associates: Employment. Sigman:CCS Associates: Equity Ownership. Borowitz:BD Biosciences: Research Funding; Medimmune: Research Funding; Bristol Myers Squibb: Research Funding; HTG Molecular: Consultancy.

We have identified and characterized a distinct non-linearity in the time course of the reaction of mammalian DNA polymerase β with synthetic polynucleotides. Nucleotide incorporation is biphasic; an initial burst of activity decays exponentially to a lower steady-state velocity. This slow transition in polymerase activity is not due to substrate depletion, abortive complex formation, or enzyme inactivation. The data are consistent with description of the β-polymerase as a hysteretic enzyme, a finding which provides a potential explanation for the non-hyperbolic kinetics which have been reported previously for this polymerase. We have also found, in contrast to some previous data, that the nucleotide analogue, N2-(p-n-butylphenyl)-2′-deoxyguanosine-5′-triphosphate (BuPdGTP), is an inhibitor of the β-polymerase. When poly(dC) oligo(dG) is used as template primer,inhibition of the initial velocity is competitive with dGTP with a K1 of 1.25μM. On activated DNA, however,β-polymerase displays sensitivity to BuPdGTP which overlaps with that previously reported for DNA polymerase δ; 100μM BuPdGTP is required to inhibit the initial velocity of a dGTP-deficient, truncated assay. Finally, we demonstrate that, in addition to its inhibition of initial velocity, BuPdGTP also modulates both the rate constant of the slow transition in polymerase activity, and the steady-state velocity of the β-polymerase.