Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp

BACKGROUND: NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. RESULTS: We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. CONCLUSIONS: We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins

MicroRNA-143 activation regulates smooth muscle and endothelial cell crosstalk in pulmonary arterial hypertension

Rationale: The pathogenesis of PAH remains unclear. The four microRNAs representing the miR-143 and miR-145 stem loops are genomically clustered. Objective: To elucidate the transcriptional regulation of the miR-143/145 cluster, and the role of miR-143 in PAH. Methods and Results: We identified the promoter region that regulates miR-143/145 miRNA expression in pulmonary artery smooth muscle cells (PASMCs). We mapped PAH-related signalling pathways, including estrogens receptor (ER), liver X factor/retinoic X receptor (LXR/RXR), TGF-β (Smads), and hypoxia (HRE) that regulated levels of all pri-miR stem loop transcription and resulting miRNA expression. We observed that miR-143-3p is selectively upregulated compared to miR-143-5p during PASMC migration. Modulation of miR-143 in PASMCs significantly altered cell migration and apoptosis. In addition, we found high abundance of miR-143-3p in PASMCs-derived exosomes. Using assays with pulmonary arterial endothelial cells (PAECs) we demonstrated a paracrine pro-migratory and pro-angiogenic effect of miR-143-3p enriched exosomes from PASMC. Quantitative PCR and in situ hybridisation showed elevated expression of miR-143 in calf models of PAH as well as in samples from PAH patients. Moreover, in contrast to our previous findings that had not supported a therapeutic role in vivo, we now demonstrate a protective role for miR-143 in experimental PH in vivo in miR-143-/- and antimiR143-3p-treated mice exposed to chronic hypoxia in both preventative and reversal settings. Conclusions: MiR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while inhibition of miR-143-3p blocked experimental PH. Taken together these findings confirm an important role for the miR-143/145 cluster in PAH pathobiology

Intention in Investing Digital Gold Through E-Commerce Platforms

Gold can now be invested digitally through e-commerce platforms. However, Indonesian people generally invest in gold traditionally in physical form. This study aims to identify the intentions and behavior of investors in investing in gold on e-commerce platforms by proving the effect of Attitude, Subjective Norms, Perceived Behavioral Control, Perceived Usefulness, and Perceived Ease of Use variables on Intention to invest. The research uses quantitative methods with data collected through online surveys. The sample was selected using a purposive sampling method, with 261 respondents contained. The study’s results prove that Attitude, Perceived Behavioral Control, and Perceived Usefulness are significantly and positively related to the Intention to Invest. Perceived Usefulness is also demonstrated to have a significant and positive relationship with Attitude. On the other hand, Subjective Norms and Perceived Ease of Use are not significant to Intention to Invest. Perceived Ease of Use also does not demonstrate a substantial Attitude. This study reveals that variables of attitude, perceptions of behavioral control, and perceptions of use value are proven to increase investor intentions in investing in gold digitally. This research can also add to the knowledge in the study of gold investment, especially those carried out digitally through e-commerce platforms

Cobomarsen, an oligonucleotide inhibitor of miR-155, slows DLBCL tumor cell growth in vitro and in vivo

MicroRNA-155, is an oncogenic miRNA, highly expressed in B-cell malignancies, particularly in the non-Germinal Center B-cell or activated B-cell subtype of Diffuse Large B-cell Lymphoma (non-GCB/ABC-DLBCL), where it is considered a potential diagnostic and prognostic biomarker. Thus, miR-155 inhibition represents an important therapeutic strategy for B-cell lymphomas. In this study, we tested the efficacy and pharmacodynamic activity of an oligonucleotide inhibitor of miR-155, cobomarsen, in ABC-DLBCL cell lines and in corresponding xenograft mouse models. In addition, we assessed the therapeutic efficacy and safety of cobomarsen in a patient diagnosed with aggressive ABC-DLBCL

Optimizing Preprocessing and Analysis Pipelines for Single-Subject fMRI: 2. Interactions with ICA, PCA, Task Contrast and Inter-Subject Heterogeneity

A variety of preprocessing techniques are available to correct subject-dependant artifacts in fMRI, caused by head motion and physiological noise. Although it has been established that the chosen preprocessing steps (or “pipeline”) may significantly affect fMRI results, it is not well understood how preprocessing choices interact with other parts of the fMRI experimental design. In this study, we examine how two experimental factors interact with preprocessing: between-subject heterogeneity, and strength of task contrast. Two levels of cognitive contrast were examined in an fMRI adaptation of the Trail-Making Test, with data from young, healthy adults. The importance of standard preprocessing with motion correction, physiological noise correction, motion parameter regression and temporal detrending were examined for the two task contrasts. We also tested subspace estimation using Principal Component Analysis (PCA), and Independent Component Analysis (ICA). Results were obtained for Penalized Discriminant Analysis, and model performance quantified with reproducibility (R) and prediction metrics (P). Simulation methods were also used to test for potential biases from individual-subject optimization. Our results demonstrate that (1) individual pipeline optimization is not significantly more biased than fixed preprocessing. In addition, (2) when applying a fixed pipeline across all subjects, the task contrast significantly affects pipeline performance; in particular, the effects of PCA and ICA models vary with contrast, and are not by themselves optimal preprocessing steps. Also, (3) selecting the optimal pipeline for each subject improves within-subject (P,R) and between-subject overlap, with the weaker cognitive contrast being more sensitive to pipeline optimization. These results demonstrate that sensitivity of fMRI results is influenced not only by preprocessing choices, but also by interactions with other experimental design factors. This paper outlines a quantitative procedure to denoise data that would otherwise be discarded due to artifact; this is particularly relevant for weak signal contrasts in single-subject, small-sample and clinical datasets

Development of the Preferred Components for Co-Design in Research Guideline and Checklist: Protocol for a Scoping Review and a Modified Delphi Process

Background: There is increasing evidence that co-design can lead to more engaging, acceptable, relevant, feasible, and even effective interventions. However, no guidance is provided on the specific designs and associated methods or methodologies involved in the process. We propose the development of the Preferred Components for Co-design in Research (PRECISE) guideline to enhance the consistency, transparency, and quality of reporting co-design studies used to develop complex health interventions. Objective: The aim is to develop the first iteration of the PRECISE guideline. The purpose of the PRECISE guideline is to improve the consistency, transparency, and quality of reporting on studies that use co-design to develop complex health interventions. Methods: The aim will be achieved by addressing the following objectives: to review and synthesize the literature on the models, theories, and frameworks used in the co-design of complex health interventions to identify their common elements (components, values or principles, associated methods and methodologies, and outcomes); and by using the results of the scoping review, prioritize the co-design components, values or principles, associated methods and methodologies, and outcomes to be included in the PRECISE guideline. Results: The project has been funded by the Canadian Institutes of Health Research. Conclusions: The collective results of this project will lead to a ready-to-implement PRECISE guideline that outlines a minimum set of items to include when reporting the co-design of complex health interventions. The PRECISE guideline will improve the consistency, transparency, and quality of reports of studies. Additionally, it will include guidance on how to enact or enable the values or principles of co-design for meaningful and collaborative solutions (interventions). PRECISE might also be used by peer reviewers and editors to improve the review of manuscripts involving co-design. Ultimately, the PRECISE guideline will facilitate more efficient use of new results about complex health intervention development and bring better returns on research investments

Management of Hospital-Acquired Pneumonia at a Tertiary-Care Teaching Hospital

ABSTRACTBackground: The rising resistance of pathogens commonly implicated in hospital-acquired pneumonia to currently recommended empiric therapy may necessitate a change in the management of this condition.Objective: To determine the current therapeutic approach to the management of hospital-acquired pneumonia at an urban tertiary care hospital and to determine the need for a change to the institution’s guidelines, according to patterns of bacterial culture and sensitivity.Methods: A chart review was performed to identify patients in whom hospital-acquired pneumonia was diagnosed between January 1 and December 31, 2003.Results: The charts of 50 patients (15% of the 325 patients with a diagnosis of hospital-aquired pneumonia) were reviewed. Most patients (43 or 86%) had stayed on a ward, and the overall mean age was 77 years. The initial choice of antimicrobial regimen was selected empirically for 41 (82%) of the patients; levofloxacin 500 mg daily was the most commonly chosen single agent (15/41 or 37%). Ceftriaxone was also chosen frequently (10/41 or 24%) for the empiric management of hospital-acquired pneumonia. For patients whose therapy was culture-directed, the most commonly chosen agent was ciprofloxacin (5/9 or 56%). Sputum samples were obtained from 19 of the patients, and 3 species of bacteria were each cultured in more than 15% of these 19 samples: methicillin-sensitive Staphylococcus aureus (MSSA) (3/19 or 16%), Hemophilus influenzae (4/19 or 21%), and Serratia marcescens (3/19 or 16%).Conclusions: MSSA, Hemophilus influenzae, and Serratia marcescens were the most common causes of hospital-acquired pneumonia at this institution, but multidrug-resistant strains of these problematic organisms were not a concern. Therefore, broad-spectrum antibiotics, such as carbapenems, ceftazidime, and ciprofloxacin may be reserved for targeted use in appropriate patients. These agents should not be used in the routine empiric management of hospital-acquired pneumonia in the authors’ institution at this time.RÉSUMÉHistorique : La résistance croissante des agents pathogènes généralement mis en cause dans la pneumonie nosocomiale au traitement empirique actuellement recommandé pourrait commander un changement dans la prise en charge de cette affection.Objectif : Déterminer l’approche thérapeutique actuelle dans la prise en charge de la pneumonie nosocomiale au sein d’un hôpital urbain de soins tertiaires ainsi que la nécessité de modifier les lignes directrices de l’établissement selon les antibiogrammes.Méthodes : Une analyse des dossiers médicaux de patients chez qui l’on a diagnostiqué une pneumonie nosocomiale entre le 1er janvier et le 31 décembre 2003 a été effectuée.Résultats : Les dossiers de 50 patients ont été examinés (15 % des 325 patients avec un diagnostic de pneumonie nosocomiale). L’âge moyen des patients était de 77 ans et la plupart d’entre eux (43 ou 86 %) avaient séjourné dans un service hospitalier. L’antibiothérapie a été sélectionnée de façon empirique chez 41 (82 %) des patients; la lévofloxacine administrée à raison de 500 mg par jour était l’antibiotique le plus souvent choisi (15 patients sur 41 ou 37 %). La ceftriaxone était également choisie souvent (10 patients sur 41 ou 24 %) pour le traitement empirique de la pneumonie nosocomiale. Chez les patients dont le traitement était fondé sur les résultats de la culture, l’antibiotique le plus souvent retenu était la ciprofloxacine (5 patients sur 9 ou 56 %). Des échantillons de crachat ont été prélevés chez 19 des patients et trois souches de bactéries ont été chacune cultivées dans plus de 15 % des 19 échantillons : Staphylococcus aureus sensible à la méthicilline (3 échantillons sur 19 ou 16 %), Haemophilus influenzae (4 échantillons sur 19 ou 21 %) et Serratia marcescens (3 échantillons sur 19 ou 16 %).Conclusions : S. aureus sensible à la méthicilline, Haemophilus influenzae et S. marcescens étaient les principaux agents pathogènes responsables de la pneumonie nosocomiale dans cet établissement, et les souches de ces microorganismes résistantes à de multiples antibiotiques ne posaient pas problème. Par conséquent, les antibiotiques à large spectre, comme les carbapénèmes, la ceftazidime et la ciprofloxacine peuvent être réservés pour un usage ciblé chez des patients choisis. Ces agents ne devraient pas être utilisés comme traitement empirique systématique de la pneumonie nosocomiale dans cet établissement pour le moment

Angiogenesis in Ischemic Stroke and Angiogenic Effects of Chinese Herbal Medicine

Stroke is one of the major causes of death and adult disability worldwide. The underlying pathophysiology of stroke is highly complicated, consisting of impairments of multiple signalling pathways, and numerous pathological processes such as acidosis, glutamate excitotoxicity, calcium overload, cerebral inflammation and reactive oxygen species (ROS) generation. The current treatment for ischemic stroke is limited to thromolytics such as recombinant tissue plasminogen activator (tPA). tPA has a very narrow therapeutic window, making it suitable to only a minority of stroke patients. Hence, there is great urgency to develop new therapies that can protect brain tissue from ischemic damage. Recent studies have shown that new vessel formation after stroke not only replenishes blood flow to the ischemic area of the brain, but also promotes neurogenesis and improves neurological functions in both animal models and patients. Therefore, drugs that can promote angiogenesis after ischemic stroke can provide therapeutic benefits in stroke management. In this regard, Chinese herbal medicine (CHM) has a long history in treating stroke and the associated diseases. A number of studies have demonstrated the pro-angiogenic effects of various Chinese herbs and herbal formulations in both in vitro and in vivo settings. In this article, we present a comprehensive review of the current knowledge on angiogenesis in the context of ischemic stroke and discuss the potential use of CHM in stroke management through modulation of angiogenesis