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Arginyltransferases (ATE1s) are eukaryotic enzymes that catalyze the non-ribosomal, post-translational addition of the amino acid arginine to an acceptor protein. While understudied, post-translation arginylation and ATE1 have major impacts on eukaryotic cellular homeostasis through both degradative and non-degradative effects on the intracellular proteome. Consequently, ATE1-catalyzed arginylation impacts major eukaryotic biological processes including the stress response, cellular motility, cardiovascular maturation, and even neurological function. Despite this importance, there is a lack of information on the structural and biophysical characteristics of ATE1, prohibiting a comprehensive understanding of the mechanism of this post-translational modification, and hampering efforts to design ATE1-specific therapeutics. To that end, this chapter details a protocol designed for the expression and the purification of ATE1 from Saccharomyces cerevisiae, although the approaches described herein should be generally applicable to other eukaryotic ATE1s. The detailed procedures afford high amounts of pure, homogeneous, monodisperse ATE1 suitable for downstream biophysical analyses such as X-ray crystallography, small angle X-ray scattering (SAXS), and cryo-EM techniques., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Small non-coding microRNAs (miRNAs) can modulate the outcome of virus infection. Here we explore the role of miRNAs in insect-virus interactions, in vivo, using the natural Drosophila melanogaster-Drosophila C virus (DCV) model system. Comparison of the miRNA expression profiles in DCV-infected and uninfected flies showed altered miRNA levels due to DCV infection, with the largest change in abundance observed for miR-956-3p. Knockout of miR-956 resulted to delayed DCV-induced mortality and decreased viral accumulation compared to wild-type flies. A screen of 84 putative miR-956-3p target genes identified regulation of Ectoderm-expressed 4 (Ect4) in miR-956 knockout flies and, separately, DCV infection. In Ect4 knockdown flies DCV-induced mortality occurred more quickly and virus accumulation was increased. Taken together, results suggest that the host-protective and antiviral consequences of miR-956 suppression during in vivo infection of D. melanogaster with its natural pathogen DCV is conferred through miR-956-3p induction of its target Ect4., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Recent changes in recommendations for mammography screening for women in their forties could have an impact on organized screening programs, as these require clear definition of target and eligible populations., Current Status: Internationally, a majority of programs target women fifty and over, and so the recent USPSTF age recommendations for screening are quite consistent with this practice. However, there is a good deal of variability in the availability of population-based screening programs to women in their forties should they choose to participate., Future Impact: Several programs are reviewing recommendations regarding the eligibility of women in their forties; as guidelines indicate women in this age group should be eligible for screening, even if not targeted, there is discussion as to whether they should be allowed in programs so that they benefit from the same programmatic quality assurance afforded to the target population of women over 50. Clear communication of the evidentiary basis for the eligibility criteria and target populations is required, so that the public is aware that programs are designed to deliver maximal population impact, while minimizing population risk., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Fragile X syndrome (FXS) is a common cause of inherited mental retardation and the best characterized form of autistic spectrum disorders. FXS is caused by the loss of functional fragile X mental retardation protein (FMRP), which leads to abnormalities in the differentiation of neural progenitor cells (NPCs) and in the development of dendritic spines and neuronal circuits. Brain-derived neurotrophic factor (BDNF) and its TrkB receptors play a central role in neuronal maturation and plasticity. We studied BDNF/TrkB actions in the absence of FMRP and show that an increase in catalytic TrkB expression in undifferentiated NPCs of Fmr1-knockout (KO) mice, a mouse model for FXS, is associated with changes in the differentiation and migration of neurons expressing TrkB in neurosphere cultures and in the developing cortex. Aberrant intracellular calcium responses to BDNF and ATP in subpopulations of differentiating NPCs combined with changes in the expression of BDNF and TrkB suggest cell subtype-specific alterations during early neuronal maturation in the absence of FMRP. Furthermore, we show that dendritic targeting of Bdnf mRNA was increased under basal conditions and further enhanced in cortical layer V and hippocampal CA1 neurons of Fmr1-KO mice by pilocarpine-induced neuronal activity represented by convulsive seizures, suggesting that BDNF/TrkB-mediated feedback mechanisms for strengthening the synapses were compromised in the absence of FMRP. Pilocarpine-induced seizures caused an accumulation of Bdnf mRNA transcripts in the most proximal segments of dendrites in cortical but not in hippocampal neurons of Fmr1-KO mice. In addition, BDNF protein levels were increased in the hippocampus but reduced in the cortex of Fmr1-KO mice in line with regional differences of synaptic plasticity in the brain of Fmr1-KO mice. Altogether, the present data suggest that alterations in the BDNF/TrkB signaling modulate brain development and impair synaptic plasticity in FXS., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective: The purpose of this paper is to outline the short-, medium- and long-term requirements of a strategy to evaluate the impact of HPV immunization and to define a framework to facilitate planning and evaluation., Method: This strategy was developed in Ontario from January to August 2008. Literature review was completed to assess existing material relevant to vaccine evaluation, and HPV vaccine specifically. Scientists and epidemiologists within our organization attended meetings to brainstorm and identify key requirements for vaccine evaluation. Other selected internal and external experts were consulted to review preliminary lists of potential indicators and questions for inclusion in an evaluation strategy., Results: Results are reported in three sections--literature review, proposed evaluation framework and data requirements., Conclusion: The first vaccine evaluation strategy that integrates primary and secondary prevention of cervical cancer is presented. Among women who are neither screened nor immunized, customized interventions will be required to ensure that they are aware of potential risks and benefits. This evaluation strategy may serve as a useful outline for jurisdictions in Canada and elsewhere. This new paradigm of combined primary and secondary intervention will encourage cooperation for effective evaluation of an integrated approach for control of cervical cancer and other HPV-related disease.

The lack of fragile X mental retardation protein (FMRP) causes fragile X syndrome, a common form of inherited mental retardation. Our previous studies revealed alterations in the differentiation of FMRP-deficient neural progenitors. Here, we show abnormalities in neurogenesis in the mouse and human embryonic FMRP-deficient brain as well as after in utero transfection of I304N mutated FMRP, which acts in a dominant negative manner in the wild-type mouse brain. Progenitors accumulated abnormally in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) mouse neocortex. An increased density of cells expressing sequentially an intermediate progenitor marker, T-box transcription factor (Tbr2), and a postmitotic neuron marker, T-brain 1 (Tbr1), indicated that the differentiation to glutamatergic cell lineages was particularly disturbed. These abnormalities were associated with an increased density of pyramidal cells of the layer V in the early postnatal neocortex suggesting a role for FMRP in the regulation of the differentiation of neocortical glutamatergic neurons.

Objective: This study was designed to compare the initial efficacy of Motivational Interviewing (MI), Online Transtheoretical Model (TTM)-tailored communications and a brief Health Risk Intervention (HRI) on four health risk factors (inactivity, BMI, stress and smoking) in a worksite sample., Method: A randomized clinical trial assigned employees to one of three recruitment strategies and one of the three treatments. The treatment protocol included an HRI session for everyone and in addition either a recommended three TTM online sessions or three MI in person or telephone sessions over 6 months. At the initial post-treatment assessment at 6 months, groups were compared on the percentage who had progressed from at risk to taking effective action on each of the four risks., Results: Compared to the HRI only group, the MI and TTM groups had significantly more participants in the Action stage for exercise and effective stress management and significantly fewer risk behaviors at 6 months. MI and TTM group outcomes were not different., Conclusion: This was the first study to demonstrate that MI and online TTM could produce significant multiple behavior changes. Future research will examine the long-term impacts of each treatment, their cost effectiveness, effects on productivity and quality of life and process variables mediating outcomes.

HIV envelope glycoprotein (Env) is the target for inducing neutralizing antibodies. Env is present on the virus surface as a trimer, and, upon binding to CD4, a cascade of events leads to structural rearrangement exposing the co-receptor binding site and entry into the CD4+ host target cells. We have designed monomeric and trimeric Env constructs with and without deletion of the variable loop 2 (ΔV2) from SF162, a subtype B primary isolate, and performed biophysical, biochemical and immunological studies to establish a potential structure–functional relationship. We expressed these Envs in CHO cells, purified the proteins to homogeneity and performed biophysical studies to define the binding properties to CD4, structural characteristics and exposure of epitopes recognized by b12 and CD4i mAb (17B) on both full-length and mutant HIV Env proteins. Parameters evaluated include oligomerization state, number and affinity of CD4 binding sites, enthalpy and entropy of the Env–CD4 interaction and affinity for b12 and 17b mAbs. We observed one CD4 binding site per monomer and three active CD4 binding sites per trimer. A40-fold difference in affinity of the gp120 monomer vs. the o-gp140 trimer towards CD4 was observed (Kd = 58 nM and 1.5 nM, respectively),whereas only a 2-fold difference was observed for the V2 deleted Envs (Kd of gp120ΔV2 = 19 nM, Kd of o-gp140DV2 = 9.3 nM). Monomers had 3-fold higher affinity to the mAb 17b and at least 3-fold weaker affinity to b12 compared to trimers, with gp120DV2 having the weakest affinity for b12 (Kd = 446 nM). Affinity of CD4 binding correlated with proportion of the antibodies induced against the conformational epitopes by the corresponding Envs, and changes in mAb binding correlated with the induction of antibodies directed against linear epitopes. Furthermore,biophysical analysis reveals that the V2 deletion has broad structural implications in the monomer not shared by the trimer, and these changes are reflected in the quality of the immune responses induced in rabbits. These data suggest that biophysical characteristics of HIV Env, such as affinity for CD4, and exposure of important neutralizing epitopes, such as those recognized by b12 mAb, may be important predictors of its in vivo efficacy and may serve as important surrogate markers for screening Env structures as potential vaccine candidates., (©2006 Elsevier Inc. All rights reserved.)