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1.  Selective DNA demethylation by fusion of TDG with a sequence-specific DNA-binding domain 
Epigenetics  2012;7(4):344-349.
Our ability to selectively manipulate gene expression by epigenetic means is limited, as there is no approach for targeted reactivation of epigenetically silenced genes, in contrast to what is available for selective gene silencing. We aimed to develop a tool for selective transcriptional activation by DNA demethylation. Here we present evidence that direct targeting of thymine-DNA-glycosylase (TDG) to specific sequences in the DNA can result in local DNA demethylation at potential regulatory sequences and lead to enhanced gene induction. When TDG was fused to a well-characterized DNA-binding domain [the Rel-homology domain (RHD) of NFκB], we observed decreased DNA methylation and increased transcriptional response to unrelated stimulus of inducible nitric oxide synthase (NOS2). The effect was not seen for control genes lacking either RHD-binding sites or high levels of methylation, nor in control mock-transduced cells. Specific reactivation of epigenetically silenced genes may thus be achievable by this approach, which provides a broadly useful strategy to further our exploration of biological mechanisms and to improve control over the epigenome.
PMCID: PMC3368818  PMID: 22419066
demethylase; demethylation; DNA; gene function; TDG; transcriptional enhancement
2.  A Value-Based Analysis of Hemodynamic Support Strategies for High-Risk Heart Failure Patients Undergoing a Percutaneous Coronary Intervention 
The economic burden of heart disease is heavy and growing. As advanced technologies for treating heart disease become available, decision makers need to be able to assess the relative value of such options against existing standards of care.
To compare the clinical and economic benefits of a percutaneous ventricular assist device (pVAD) versus an intra-aortic balloon pump (IABP) observed during the 90-day duration of the PROTECT II clinical trial, and to supplement these findings with a simulation of the longer-term value of this technology through the use of a Markov model to estimate the incremental cost-effectiveness of a pVAD relative to an IABP, in terms of quality-adjusted life-years (QALYs).
Hospital bills were collected for patients enrolled in the PROTECT II trial who received hemodynamic support for high-risk percutaneous coronary intervention (PCI) provided by a pVAD (Impella 2.5) versus a conventional IABP during a 90-day episode of care (EOC). Length of stay, charges, and costs were analyzed for the index admissions, intensive care unit confinements, readmissions, and overall EOC. In addition, a probabilistic Markov model was used to project these parameters and their impact on a patient's quality of life for up to 10 years in relation to a pVAD versus an IABP.
Hospital costs for the index admission were lower for the IABP compared with the pVAD ($33,684 vs $47,667; P <.001), whereas readmission length of stay and costs were lower for the pVAD versus the IABP (5 days vs 7 days; and $11,007 vs $21,834, respectively; P <.001). The total 90-day hospital charges were similar for the pVAD and the IABP ($172,564 vs $172,758, respectively; P = .785); however, the total 90-day EOC cost was lower for the IABP than for the pVAD ($44,032 vs $53,171, respectively; P <.001). The median hospital days for the entire EOC were 7 days for the pVAD versus 9 days for the IABP (P = .008). Critical care stays were considerably shorter for a pVAD than for an IABP on readmissions (3.88 days vs 7.00 days; P = .145). Reduction in major adverse cardiovascular and cerebrovascular events resulted in a projected gain of 0.26 QALYs over 10 years, yielding an incremental cost-effectiveness ratio of $39,389/QALY.
For high-risk patients with advanced heart failure undergoing PCI, the new pVAD reduced major adverse events, critical care and readmission length of stay, and readmission cost over the 90-day EOC, and was determined to be cost-effective over the long-term. These findings can assist decision makers in forming value-based judgments with regard to new hemodynamic support strategies.
PMCID: PMC4031707  PMID: 24991349
3.  MARCO Regulates Early Inflammatory Responses against Influenza 
Lung macrophages use the scavenger receptor MARCO to bind and ingest bacteria, particulate matter, and post cellular debris. We investigated the role of MARCO in influenza A virus (IAV) pneumonia. In contrast to higher susceptibility to bacterial infection, MARCO−/− mice had lower morbidity and mortality from influenza pneumonia than wild-type (WT) mice. The early course of influenza in MARCO−/− lungs was marked by an enhanced but transient neutrophilic inflammatory response and significantly lower viral replication compared with the WT mice. At later time points, no significant differences in lung histopathology or absolute numbers of T lymphocyte influx were evident. Uptake of IAV by WT and MARCO−/− bronchoalveolar lavage macrophages in vitro was similar. By LPS coadministration, we demonstrated that rapid neutrophil and monocyte influx during the onset of influenza suppressed viral replication, indicating a protective role of early inflammation. We hypothesized that the presence of increased basal proinflammatory post cellular debris in the absence of scavenging function lowered the inflammatory response threshold to IAV in MARCO−/− mice. Indeed, MARCO−/− mice showed increased accumulation of proinflammatory oxidized lipoproteins in the bronchoalveolar lavage early in the infection process, which are the potential mediators of the observed enhanced inflammation. These results indicate that MARCO suppresses a protective early inflammatory response to influenza, which modulates viral clearance and delays recovery.
PMCID: PMC3262690  PMID: 21562316
inflammation; scavenger receptors; leukocytes; chemokines; pathology; oxidized lipoproteins
4.  Genome-Wide RNAi Screen in IFN-γ-Treated Human Macrophages Identifies Genes Mediating Resistance to the Intracellular Pathogen Francisella tularensis 
PLoS ONE  2012;7(2):e31752.
Interferon-gamma (IFN-γ) inhibits intracellular replication of Francisella tularensis in human monocyte-derived macrophages (HMDM) and in mice, but the mechanisms of this protective effect are poorly characterized. We used genome-wide RNA interference (RNAi) screening in the human macrophage cell line THP-1 to identify genes that mediate the beneficial effects of IFN-γ on F. tularensis infection. A primary screen identified ∼200 replicated candidate genes. These were prioritized according to mRNA expression in IFN-γ-primed and F. tularensis-challenged macrophages. A panel of 20 top hits was further assessed by re-testing using individual shRNAs or siRNAs in THP-1 cells, HMDMs and primary human lung macrophages. Six of eight validated genes tested were also found to confer resistance to Listeria monocytogenes infection, suggesting a broadly shared host gene program for intracellular pathogens. The F. tularensis-validated hits included ‘druggable’ targets such as TNFRSF9, which encodes CD137. Treating HMDM with a blocking antibody to CD137 confirmed a beneficial role of CD137 in macrophage clearance of F. tularensis. These studies reveal a number of important mediators of IFN-γ activated host defense against intracellular pathogens, and implicate CD137 as a potential therapeutic target and regulator of macrophage interactions with Francisella tularensis.
PMCID: PMC3281001  PMID: 22359626
5.  The Rationale and Design of the Pharmacist Intervention for Low Literacy in Cardiovascular Disease (PILL-CVD) Study 
Medication errors and adverse drug events are common after hospital discharge, due to changes in medication regimens, suboptimal discharge instructions, and prolonged time to follow-up. Pharmacist-based interventions may be effective in promoting the safe and effective use of medications, especially among high risk patients such as those with low health literacy.
Methods and Results
The Pharmacist Intervention for Low Literacy in Cardiovascular Disease (PILL-CVD) study is a randomized controlled trial conducted at 2 academic centers – Vanderbilt University Hospital and Brigham and Women’s Hospital. Patients admitted with acute coronary syndrome or acute decompensated heart failure were randomized to usual care or intervention. The intervention consisted of pharmacist-assisted medication reconciliation, inpatient pharmacist counseling, low-literacy adherence aids, and tailored telephone follow-up after discharge. The primary outcome is the occurrence of serious medication errors in the first 30 days after hospital discharge. Secondary outcomes are health care utilization, disease-specific quality of life, and cost effectiveness. Enrollment was completed September 2009. A total of 862 patients were enrolled, and 430 patients were randomized to receive the intervention. Analyses will determine whether the intervention was effective in reducing serious medication errors, particularly in patients with low health literacy.
The PILL-CVD study was designed to reduce serious medication errors after hospitalization through a pharmacist-based intervention. The intervention, if effective, will inform health care facilities on the use of pharmacist-assisted medication reconciliation, inpatient counseling, low-literacy adherence aids, and patient follow-up after discharge.
Clinical Trial Registration, NCT00632021
PMCID: PMC3021350  PMID: 20233982
coronary disease; heart failure; patients
6.  A pilot study assessing social support among cancer patients enrolled on clinical trials: a comparison of younger versus older adults 
This study tested the logistical feasibility of obtaining data on social support systems from cancer patients enrolled on clinical trials and compared the social support of older adults (age ≥65) and younger adults (<50 years of age) with cancer.
Patients had to be eligible for a phase II or phase III oncology clinical trial and enter the study prior to treatment. Patients filled out the Lubben Social Network Scale (LSNS) at baseline. The Symptom Distress Scale (SDS) and single-item overall quality of life (QOL) Uniscale were assessed at baseline and weekly for 4 weeks.
There was no significant difference in overall mean Lubben social support levels by age. Older patients had more relatives they felt close to (85% versus 53% with 5 or more relatives, P = 0.02), heard from more friends monthly (84% versus 53% with 3 or more friends, P = 0.02), less overall symptom distress (P = 0.03), less insomnia (P = 0.003), better concentration (P = 0.005), better outlook (P = 0.01), and less depression (P = 0.005) than younger patients.
Younger subjects reported worse symptoms, a smaller social support network, and fewer close friends and relatives than older subjects. Having someone to discuss decisions and seeing friends or relatives often was associated with longer survival.
PMCID: PMC3004576  PMID: 21188104
social support; Lubben scale; QOL; elderly
7.  Prediction of breast self-examination in a sample of Iranian women: an application of the Health Belief Model 
BMC Women's Health  2009;9:37.
Iranian women, many of whom live in small cities, have limited access to mammography and clinical breast examinations. Thus, breast self examination (BSE) becomes an important and necessary approach to detecting this disease in its early stages in order to limit its resultant morbidity and mortality. This study examined constructs arising from the Health Belief Model as predictors of breast self examination behavior in a sample of women living in Bandar Abbas, Iran.
This study was conducted in eight health centers located in Bandar Abbas, Iran. The sample consisted of 240 eligible women who were selected from referrals to the centers. The inclusion criteria were as follows: aged 30 years and over; and able to read and write Farsi. Women with breast cancer, who were pregnant, or breast feeding, were excluded from the study. Data were collected by using a self administered questionnaire which included demographic characteristics and Champion's Health Belief Model Scale. This instrument measures the concepts of disease susceptibility (3 items), seriousness (6 items), benefits (4 items), barriers (8 items) and self-efficacy (10 items).
The subjects' mean age was 37.2 (SD = 6.1) years. Just under a third of the subjects (31.7%) had performed BSE in the past and 7.1% of them performed it at least monthly. Perceived benefits and perceived self-efficacy of the women who performed BSE were significantly higher compared with women who did not practice BSE (p < 0.03). Furthermore, perceived barriers were lower among those who had performed BSE (p < 0.001). Logistic regression analysis indicated that women who perceived fewer barriers (OR: 0.70, 95% CI: 0.63-0.77, p < 0.001) and had higher self-efficacy (OR: 1.08, 95% CI: 1.02-1.13, p = 0.003) were more likely to perform BSE (R2 = 0.52).
Findings from this study indicated that perceived barriers and perceived self-efficacy could be predictors of BSE behavior among the sample of women. Therefore, BSE training programs that emphasize self-efficacy and address perceived barriers are recommended.
PMCID: PMC2806255  PMID: 20040093
8.  Comparison of the Effects of Leishmania major or Leishmania donovani Infection on Macrophage Gene Expression▿ †  
Infection and Immunity  2007;76(3):1186-1192.
The intracellular parasite Leishmania causes a wide spectrum of human disease, ranging from self-resolving cutaneous lesions to fatal visceral disease, depending on the species of Leishmania involved. The mechanisms by which different Leishmania species cause different pathologies are largely unknown. We have addressed this question by comparing the gene expression profiles of bone marrow-derived macrophages infected with either Leishmania donovani or L. major promastigotes. We found that the two species had very similar effects on macrophage gene expression. Both species caused a small (<2.5-fold) but statistically significant repression of several hundred genes. In addition, both species strongly induced and repressed about 60 genes. Comparing the effects of the two species showed that only 26 genes were regulated differently by L. major as opposed to L. donovani, including those for metallothioneins 1 and 2, HSP70 and -72, CCL4, Gadd45β, Dsp1, matrix metalloprotease 13, T-cell death-associated gene 51 (Tdag51), RhoB, spermine/spermidine N1-acyl transferase 1 (SSAT), and Cox2. L. donovani-infected macrophages were also found to express higher levels of Cox2 protein and prostaglandin E synthase mRNA than L. major-infected macrophages. While both species have previously been shown to trigger prostaglandin E synthesis by bystander cells, this study suggests that infected macrophages themselves express prostaglandin E-synthesizing genes only in response to L. donovani.
PMCID: PMC2258831  PMID: 18086813
9.  Role of Host Protein Tyrosine Phosphatase SHP-1 in Leishmania donovani-Induced Inhibition of Nitric Oxide Production  
Infection and Immunity  2006;74(11):6272-6279.
In order to survive within the macrophages of its host organism, the protozoan parasite Leishmania inhibits a number of critical, gamma interferon (IFN-γ)-inducible, macrophage functions, including the generation of nitric oxide. We have previously shown that the protein tyrosine phosphatase SHP-1 (Src-homology 2 domain containing phosphatase-1) is activated during Leishmania infection and plays an important role in both the survival of Leishmania within cultured macrophages and disease progression in vivo by inhibiting nitric oxide production. Here we use a SHP-1−/− macrophage cell line derived from motheaten mice to address the mechanisms by which SHP-1 prevents IFN-γ-dependent nitric oxide production during Leishmania donovani infection. We show that Leishmania inhibits nitric oxide production in response to IFN-γ poorly in SHP-1-deficient macrophages. This correlates with the inability of Leishmania to alter JAK2 and mitogen-activated protein kinase extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation and to prevent nuclear translocation of transcription factors NF-κB and AP-1, although the latter two to a lesser extent. Surprisingly, Leishmania inactivated the transcription factor STAT1 to a similar extent in SHP-1-deficient and wild-type macrophages, so STAT1 is not necessary for nitric oxide production by infected macrophages. Overall, this study demonstrates that induction of SHP-1 by Leishmania is vital for inhibition of nitric oxide generation and that this inhibition occurs through the inactivation of JAK2 and ERK1/2, and transcription factors NF-κB and AP-1.
PMCID: PMC1695482  PMID: 17057094
10.  Subversion Mechanisms by Which Leishmania Parasites Can Escape the Host Immune Response: a Signaling Point of View 
Clinical Microbiology Reviews  2005;18(2):293-305.
The obligate intracellular parasite Leishmania must survive the antimicrobial activities of its host cell, the macrophage, and prevent activation of an effective immune response. In order to do this, it has developed numerous highly successful strategies for manipulating activities, including antigen presentation, nitric oxide and oxygen radical generation, and cytokine production. This is generally the result of interactions between Leishmania cell surface molecules, particularly gp63 and LPG, and less well identified macrophage surface receptors, causing the distortion of specific intracellular signaling cascades. We describe some of the signaling pathways and intermediates that are repressed in infected cells, including JAK/STAT, Ca2+-dependent protein kinase C (PKC) isoforms, and mitogen-activated protein kinases (especially ERK1/2), and proteasome-mediated transcription factor degradation. We also discuss protein tyrosine phosphatases (particularly SHP-1), intracellular Ca2+, Ca2+-independent PKC, ceramide, and the suppressors of cytokine signaling family of repressors, which are all reported to be activated following infection, and the role of parasite-secreted cysteine proteases.
PMCID: PMC1082797  PMID: 15831826
11.  The p68 and p72 DEAD box RNA helicases interact with HDAC1 and repress transcription in a promoter-specific manner 
p68 (Ddx5) and p72 (Ddx17) are highly related members of the DEAD box family and are established RNA helicases. They have been implicated in growth regulation and have been shown to be involved in both pre-mRNA and pre-rRNA processing. More recently, however, these proteins have been reported to act as transcriptional co-activators for estrogen-receptor alpha (ERα). Furthermore these proteins were shown to interact with co-activators p300/CBP and the RNA polymerase II holoenzyme. Taken together these reports suggest a role for p68 and p72 in transcriptional activation.
In this report we show that p68 and p72 can, in some contexts, act as transcriptional repressors. Targeting of p68 or p72 to constitutive promoters leads to repression of transcription; this repression is promoter-specific. Moreover both p68 and p72 associate with histone deacetylase 1 (HDAC1), a well-established transcriptional repression protein.
It is therefore clear that p68 and p72 are important transcriptional regulators, functioning as co-activators and/or co-repressors depending on the context of the promoter and the transcriptional complex in which they exist.
PMCID: PMC514542  PMID: 15298701
13.  The Universal Transverse Mercator Code: A location code for disease reporting 
The Canadian Veterinary Journal  1988;29(10):825-829.
Since November 1987, all rabies specimen reports submitted by Agriculture Canada's District Veterinary Officers have required a new location code, the Universal Transverse Mercator Code (UTMC). In addition to the previously required entries for county, district, legal address and mailing addresses, the new code is set up for computer analysis and mapping. It is capable of pinpointing the origin of the specimen to within 100 meters anywhere in Canada that is covered by the National Topographic System 1:50,000 maps. Because of its 100 meter spatial resolution, the code is of great interest to those studying the occurrence and spread of rabies. The code will also be important in the detailed planning and evaluation of the Ontario rabies control scheme, scheduled for 1988. Agriculture Canada anticipates that the UTMC will also be used for reporting other animal diseases as well as for emergency disease reporting.
PMCID: PMC1680870  PMID: 17423142
18.  Cefazaflur, a New Parenteral Cephalosporin: In Vitro Studies 
Cefazaflur was tested in vitro against 262 strains of bacteria. Inhibitory and bactericidal concentrations were determined with two inoculum sizes of bacterial cells in Mueller-Hinton broth and nutrient broth. Agar dilution studies also were performed. When tested in agar, 5.0 μg or less of cefazaflur per ml inhibited almost all strains of Staphylococcus aureus, Escherichia coli, Klebsiella, and Proteus mirabilis. The drug was less active against Enterobacter and indole-positive Proteus, and 7.5 μg of antibiotic per ml inhibited approximately two-thirds to one-fourth of the strains. A concentration of 50 μg of cefazaflur per ml was required for inhibition of the enterococci. There was negligible activity against Pseudomonas. The drug demonstrated less activity in broth than in agar, and a major inoculum effect was seen with some strains. For example, with a lower inoculum, 2.5 μg of cefazaflur per ml killed all strains of E. coli, whereas with the higher inoculum, 7.5 μg of cefazaflur per ml, inhibited 64% and killed only 8% of strains. The activity of the drug for some strains was greater in Mueller-Hinton broth; for others, it was greater in nutrient broth. There were considerable differences in the results of the broth and agar studies for some species when the same medium was employed. Because of differences in activity found with different media, inocula, and method of testing, an evaluation of the eventual usefulness of cefazaflur must await the results of in vivo studies.
PMCID: PMC352055  PMID: 324399

Results 1-18 (18)