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Find video protocols related to scientific articles indexed in Pubmed.
A four-month gatifloxacin-containing regimen for treating tuberculosis.
N. Engl. J. Med.
PUBLISHED: 10-23-2014
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Shortening the course of treatment for tuberculosis would be a major improvement for case management and disease control. This phase 3 trial assessed the efficacy and safety of a 4-month gatifloxacin-containing regimen for treating rifampin-sensitive pulmonary tuberculosis.
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Protein-DNA binding in the absence of specific base-pair recognition.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-15-2014
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Until now, it has been reasonably assumed that specific base-pair recognition is the only mechanism controlling the specificity of transcription factor (TF)-DNA binding. Contrary to this assumption, here we show that nonspecific DNA sequences possessing certain repeat symmetries, when present outside of specific TF binding sites (TFBSs), statistically control TF-DNA binding preferences. We used high-throughput protein-DNA binding assays to measure the binding levels and free energies of binding for several human TFs to tens of thousands of short DNA sequences with varying repeat symmetries. Based on statistical mechanics modeling, we identify a new protein-DNA binding mechanism induced by DNA sequence symmetry in the absence of specific base-pair recognition, and experimentally demonstrate that this mechanism indeed governs protein-DNA binding preferences.
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Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix.
Nucleic Acids Res.
PUBLISHED: 09-27-2014
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MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.
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Comparison of safety of sotalol versus amiodarone in patients with atrial fibrillation and coronary artery disease.
Am. J. Cardiol.
PUBLISHED: 06-18-2014
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Sotalol is a commonly prescribed antiarrhythmic drug (AAD) used for maintaining sinus rhythm in patients with atrial fibrillation (AF). Although randomized studies have found that sotalol can significantly delay time to AF recurrence, its association with mortality is less clear, particularly among those with coronary artery disease. We examined outcomes of 2,838 patients with coronary artery disease and AF. Using Cox proportional hazards modeling, landmark analysis, and time-dependent covariates for drug therapy, we compared cumulative survival among patients treated with sotalol (n = 226), amiodarone (n = 856), or no AAD (n = 1,756). Median follow-up was 4.2 years (interquartile range [IQR] 2.0-7.4). The median age was 68 years (IQR 60-75). Compared with those treated with amiodarone or no AAD, patients treated with sotalol were less likely to be black (6% vs 13% vs 13%) and have a previous myocardial infarction (35% vs 51% vs 48%) or a left ventricular ejection fraction <40% (13% vs 26% vs 21%). In follow-up, persistence of sotalol was limited; 97% of patients treated with sotalol were treated for <25% of the follow-up period. In adjusted analysis accounting for time on therapy, sotalol use was associated with an increased risk of all-cause death compared with no drug (hazard ratio 1.53, 95% confidence interval 1.19 to 1.96, p = 0.0009), but a decreased risk of death compared with amiodarone (hazard ratio 0.72, 95% confidence interval 0.55 to 0.91, p = 0.0141). In conclusion, sotalol therapy was more frequently used in patients with fewer co-morbidities, often discontinued early in follow-up, and was associated with increased mortality compared with no AAD but decreased mortality relative to amiodarone.
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Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA.
Nucleic Acids Res.
PUBLISHED: 06-03-2014
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AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (DNA) containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ?70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ?22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.
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Morbidity and mortality associated with liver resections for primary malignancies in children.
Pediatr. Surg. Int.
PUBLISHED: 02-26-2014
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Liver resection (LR) is a high-risk procedure with limited data in the pediatric surgical literature regarding short-term outcomes. Our aim was to characterize the patient population and short-term outcomes for children undergoing LR for malignancy.
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Obesity has minimal impact on clinical outcomes in children with inflammatory bowel disease.
J. Pediatr. Surg.
PUBLISHED: 02-18-2014
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Childhood obesity is an increasing problem in affluent societies throughout the world. We sought to identify the impact of obesity on the outcome of inflammatory bowel disease (IBD) and determine differences (if any) between ulcerative colitis (UC) and Crohn's disease (CD).
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Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI.
Sci Rep
PUBLISHED: 01-28-2014
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The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3' to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5' to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.
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A cross-sectional analysis of HIV and hepatitis C clinical trials 2007 to 2010: the relationship between industry sponsorship and randomized study design.
Trials
PUBLISHED: 01-16-2014
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The proportion of clinical research sponsored by industry will likely continue to expand as federal funds for academic research decreases, particularly in the fields of HIV/AIDS and hepatitis C (HCV). While HIV and HCV continue to burden the US population, insufficient data exists as to how industry sponsorship affects clinical trials involving these infectious diseases. Debate exists about whether pharmaceutical companies undertake more market-driven research practices to promote therapeutics, or instead conduct more rigorous trials than their non-industry counterparts because of increased resources and scrutiny. The ClinicalTrials.gov registry, which allows investigators to fulfill a federal mandate for public trial registration, provides an opportunity for critical evaluation of study designs for industry-sponsored trials, independent of publication status. As part of a large public policy effort, the Clinical Trials Transformation Initiative (CTTI) recently transformed the ClinicalTrials.gov registry into a searchable dataset to facilitate research on clinical trials themselves.
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A single-center, retrospective analysis evaluating the utilization of the opioid risk tool in opioid-treated cancer patients.
J Pain Palliat Care Pharmacother
PUBLISHED: 01-13-2014
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The Opioid Risk Tool (ORT) is a screening tool used to assess risk of opioid misuse by stratifying aberrant drug-seeking behaviors and/or identifying known risk factors for drug abuse. The objectives of this study were to risk stratify opioid misuse in a cancer pain population and determine the most common patient risk factors associated with misuse utilizing the ORT. This was a retrospective analysis conducted at an academic comprehensive cancer center. Patients were referred by an oncologist or hematologist to an outpatient palliative care clinic. One-hundred and fourteen patients with cancer (n = 107) or sickle cell disease (n = 7) were evaluated from July 2012 to July 2013. During the clinical interview, patients responded to a clinician administered ORT. Based on the ORT score, patients were stratified into low, moderate, or high risk for opioid misuse. Sample size included 57 men and 57 women. Sixty-five, 21, and 28 patients were deemed low, moderate, and high risk based on the ORT, respectively. The most common risk factors for opioid misuse were a history of depression (women = 32; men = 22) and family history of alcohol abuse (women = 26; men = 22). There was no difference between men and women in the prevalence of depression (P = .17) or family history of alcohol abuse (P = .57). The least common risk factor was a personal history of prescription drug abuse (n = 1) in women and history of preadolescent sexual abuse in men (n = 0). Twenty-five percent (n = 28) of the sample population were deemed high risk based on the ORT. Screening of cancer patients in the palliative care setting suggests that risk factors for opioid misuse exist. Stratifying patients based on a routine screening tool may help identify cancer patients at risk for aberrant drug behaviors.
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The increasing incidence of adolescent bariatric surgery.
J. Pediatr. Surg.
PUBLISHED: 08-16-2013
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Morbid obesity continues to be a significant problem within the United States, as overweight/obesity rates are nearing 33%. Bariatric surgery has had success in treating obesity in adults and is becoming a viable treatment option for obese adolescents.
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Mechanisms of functional mitral regurgitation in ischemic cardiomyopathy determined by transesophageal echocardiography (from the Surgical Treatment for Ischemic Heart Failure Trial).
Am. J. Cardiol.
PUBLISHED: 07-15-2013
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The mechanisms underlying functional mitral regurgitation (MR) and the relation between mechanism and severity of MR have not been evaluated in a large, multicenter, randomized controlled trial. Transesophageal echocardiography (TEE) was performed in 215 patients at 17 centers in the Surgical Treatment for Ischemic Heart Failure (STICH) trial. Both 2-dimensional (n = 215) and 3-dimensional (n = 81) TEEs were used to assess multiple quantitative measurements of the mechanism and severity of MR. By 2-dimensional TEE, leaflet tenting area, anterior and posterior leaflet angles, mitral annulus diameter, left ventricular (LV) end-systolic volume index, LV ejection fraction (LVEF), and sphericity index (p <0.05 for all) were significantly different across MR grades. By 3-dimensional TEE, mitral annulus area, leaflet tenting area, LV end-systolic volume index, LVEF, and sphericity index (p <0.05 for all) were significantly different across MR grades. A multivariate analysis showed a trend for annulus area (p = 0.069) and LV end-systolic volume index (p = 0.071) to predict effective regurgitant orifice area and for annulus area (p = 0.018) and LV end-systolic volume index (p = 0.073) to predict vena contracta area. In the STICH trial, multiple quantitative parameters of the mechanism of functional MR are related to MR severity. The mechanism of functional MR in ischemic cardiomyopathy is heterogeneous, but no single variable stands out as a strong predictor of quantitative severity of MR.
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Stability selection for regression-based models of transcription factor-DNA binding specificity.
Bioinformatics
PUBLISHED: 07-02-2013
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The DNA binding specificity of a transcription factor (TF) is typically represented using a position weight matrix model, which implicitly assumes that individual bases in a TF binding site contribute independently to the binding affinity, an assumption that does not always hold. For this reason, more complex models of binding specificity have been developed. However, these models have their own caveats: they typically have a large number of parameters, which makes them hard to learn and interpret.
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Evaluation of initial and steady-state gatifloxacin pharmacokinetics and dose in pulmonary tuberculosis patients by using monte carlo simulations.
Antimicrob. Agents Chemother.
PUBLISHED: 06-17-2013
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A 4-month regimen of gatifloxacin with rifampin, isoniazid, and pyrazinamide is being evaluated for the treatment of tuberculosis in a phase 3 randomized controlled trial (OFLOTUB). A prior single-dose study found that gatifloxacin exposure increased by 14% in the combination. The aims of the study are to evaluate the initial and steady-state pharmacokinetics of gatifloxacin when daily doses are given to patients with newly diagnosed drug-sensitive pulmonary tuberculosis as part of a combination regimen and to evaluate the gatifloxacin dose with respect to the probability of attaining a pharmacokinetic/pharmacodynamic target. We describe the population pharmacokinetics of gatifloxacin from the first dose to a median of 28 days in 169 adults enrolled in the OFLOTUB trial in Benin, Guinea, Senegal, and South Africa. The probability of achieving a ratio of ?125 for the area under the concentration time curve to infinity (AUC0-?) for the free fraction of gatifloxacin over the MIC (fAUC/MIC) was investigated using Monte Carlo simulations. The median AUC0-? of 41.2 ?g · h/ml decreased on average by 14.3% (90% confidence interval [CI], -90.5% to +61.5%) following multiple 400-mg daily doses. At steady state, 90% of patients achieved an fAUC/MIC of ?125 only when the MIC was <0.125 ?g/ml. We conclude that systemic exposure to gatifloxacin declines with repeated daily 400-mg doses when used together with rifampin, isoniazid, and pyrazinamide, thus compensating for any initial increase in gatifloxacin levels due to a drug interaction. (The OFLOTUB study has been registered at ClinicalTrials.gov under registration no. NCT00216385.).
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Characteristics of oncology clinical trials: insights from a systematic analysis of ClinicalTrials.gov.
JAMA Intern Med
PUBLISHED: 05-24-2013
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Clinical trials are essential to cancer care, and data about the current state of research in oncology are needed to develop benchmarks and set the stage for improvement.
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A cluster randomized study of the safety of integrated treatment of trachoma and lymphatic filariasis in children and adults in Sikasso, Mali.
PLoS Negl Trop Dis
PUBLISHED: 05-01-2013
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Neglected tropical diseases are co-endemic in many areas of the world, including sub Saharan Africa. Currently lymphatic filariasis (albendazole/ivermectin) and trachoma (azithromycin) are treated separately. Consequently, financial and logistical benefit can be gained from integration of preventive chemotherapy programs in such areas.
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Stroke after primary percutaneous coronary intervention in patients with ST-segment elevation myocardial infarction: timing, characteristics, and clinical outcomes.
Circ Cardiovasc Interv
PUBLISHED: 04-02-2013
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Stroke is a rare but potentially devastating complication of acute myocardial infarction. Little is known about stroke timing, characteristics, and clinical outcomes in patients with ST-segment elevation myocardial infarction treated with primary percutaneous coronary intervention (PCI).
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The natural history of uterine leiomyomas: light and electron microscopic studies of fibroid phases, interstitial ischemia, inanosis, and reclamation.
Obstet Gynecol Int
PUBLISHED: 03-05-2013
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We propose, and offer evidence to support, the concept that many uterine leiomyomas pursue a self-limited life cycle. This cycle can be arbitrarily divided on the basis of morphologic assessment of the collagen content into 4 phases: (1) proliferation, (2) proliferation and synthesis of collagen, (3) proliferation, synthesis of collagen, and early senescence, and (4) involution. Involution occurs as a result of both vascular and interstitial ischemia. Interstitial ischemia is the consequence of the excessive elaboration of collagen, resulting in reduced microvascular density, increased distance between myocytes and capillaries, nutritional deprivation, and myocyte atrophy. The end stage of this process is an involuted tumor with a predominance of collagen, little to no proliferative activity, myocyte atrophy, and myocyte cell death. Since many of the dying cells exhibit light microscopic and ultrastructural features that appear distinct from either necrosis or apoptosis, we refer to this process as inanosis, because it appears that nutritional deprivation, or inanition, is the underlying cause of cell death. The disposal of myocytes dying by inanosis also differs in that there is no phagocytic reaction, but rather an apparent dissolution of the cell, which might be viewed as a process of reclamation as the molecular contents are reclaimed and recycled.
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Genomic regions flanking E-box binding sites influence DNA binding specificity of bHLH transcription factors through DNA shape.
Cell Rep
PUBLISHED: 02-12-2013
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DNA sequence is a major determinant of the binding specificity of transcription factors (TFs) for their genomic targets. However, eukaryotic cells often express, at the same time, TFs with highly similar DNA binding motifs but distinct in vivo targets. Currently, it is not well understood how TFs with seemingly identical DNA motifs achieve unique specificities in vivo. Here, we used custom protein-binding microarrays to analyze TF specificity for putative binding sites in their genomic sequence context. Using yeast TFs Cbf1 and Tye7 as our case studies, we found that binding sites of these bHLH TFs (i.e., E-boxes) are bound differently in vitro and in vivo, depending on their genomic context. Computational analyses suggest that nucleotides outside E-box binding sites contribute to specificity by influencing the three-dimensional structure of DNA binding sites. Thus, the local shape of target sites might play a widespread role in achieving regulatory specificity within TF families.
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Dialysate calcium concentration and the risk of sudden cardiac arrest in hemodialysis patients.
Clin J Am Soc Nephrol
PUBLISHED: 01-31-2013
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The optimal dialysate calcium concentration to maintain normal mineralization and reduce risk of cardiovascular events in hemodialysis patients is debated. Guidelines suggest that dialysate Ca concentration should be lowered to avoid vascular calcification, but cardiac arrhythmias may be more likely to occur at lower dialysate Ca. Concurrent use of QT-prolonging medications may also exacerbate arrhythmic risk. This study examined the influence of serum Ca, dialysate Ca, and QT interval-prolonging medications on the risk of sudden cardiac arrest in a cohort of hemodialysis patients.
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The State of Infectious Diseases Clinical Trials: A Systematic Review of ClinicalTrials.gov.
PLoS ONE
PUBLISHED: 01-01-2013
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There is a paucity of clinical trials informing specific questions faced by infectious diseases (ID) specialists. The ClinicalTrials.gov registry offers an opportunity to evaluate the ID clinical trials portfolio.
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Extrauterine listeriosis in the gravid mouse influences embryonic growth and development.
PLoS ONE
PUBLISHED: 01-01-2013
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Gravid mice and other rodents inoculated with Listeria monocytogenes typically fail to clear an intrauterine infection and either succumb or expel their intrauterine contents. We took advantage of this property to investigate the effects of an extrauterine infection on parameters of pregnancy success. Pregnant mice were selected for our study if they showed no clinical signs of listeriosis following oral inoculation at 7.5 gestational days (gd), and had no detectable intrauterine colony forming units (cfu) at near term (18.5 gd). The range of oral doses employed was 10?-10? cfu per mouse for two listerial serotype strains (4nonb and 1/2a). At all doses, inoculation resulted in a decrease in average near-term (18.5 gd) fetal weight per litter compared to sham inoculated controls. Additionally, embryonic death (indicated by intrauterine resorptions) was exhibited by some inoculated mice but was absent in all sham inoculated animals. In parallel experiments designed to detect possible loss of placental function, gravid uteruses were examined histopathologically and microbiologically 96 h after oral inoculation. Placental lesions were associated with high (> 10?), but not low (< 10²) or absent intrauterine cfu. In vitro, mouse embryonic trophoblasts were indistinguishable from mouse enterocytes in terms of their sensitivity to listerial exposure. A model consistent with our observations is one in which products (host or bacterial) generated during an acute infection enter embryos transplacentally and influences embryonic survival and slows normal growth in utero.
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Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-09-2011
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The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kD(a) and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3? in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer.
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MPP8 mediates the interactions between DNA methyltransferase Dnmt3a and H3K9 methyltransferase GLP/G9a.
Nat Commun
PUBLISHED: 07-29-2011
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DNA CpG methylation and histone H3 lysine 9 (H3K9) methylation are two major repressive epigenetic modifications, and these methylations are positively correlated with one another in chromatin. Here we show that G9a or G9a-like protein (GLP) dimethylate the amino-terminal lysine 44 (K44) of mouse Dnmt3a (equivalent to K47 of human DNMT3A) in vitro and in cells overexpressing G9a or GLP. The chromodomain of MPP8 recognizes the dimethylated Dnmt3aK44me2. MPP8 also interacts with self-methylated GLP in a methylation-dependent manner. The MPP8 chromodomain forms a dimer in solution and in crystals, suggesting that a dimeric MPP8 molecule could bridge the methylated Dnmt3a and GLP, resulting in a silencing complex of Dnmt3a-MPP8-GLP/G9a on chromatin templates. Together, these findings provide a molecular explanation, at least in part, for the co-occurrence of DNA methylation and H3K9 methylation in chromatin.
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Admission, discharge, or change in B-type natriuretic peptide and long-term outcomes: data from Organized Program to Initiate Lifesaving Treatment in Hospitalized Patients with Heart Failure (OPTIMIZE-HF) linked to Medicare claims.
Circ Heart Fail
PUBLISHED: 07-08-2011
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B-type natriuretic peptide (BNP) has been associated with short- and long-term postdischarge prognosis among hospitalized patients with heart failure. It is unknown if admission, discharge, or change from admission to discharge BNP measure is the most important predictor of long-term outcomes.
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Coordinated methyl-lysine erasure: structural and functional linkage of a Jumonji demethylase domain and a reader domain.
Curr. Opin. Struct. Biol.
PUBLISHED: 06-06-2011
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Both components of chromatin (DNA and histones) are subjected to dynamic postsynthetic covalent modifications. Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers), and readers of the epigenetic code. Known histone lysine demethylases include flavin-dependent monoamine oxidase lysine-specific demethylase 1 and ?-ketoglutarate-Fe(II)-dependent dioxygenases containing Jumonji domains. Importantly, the Jumonji domain often associates with at least one additional recognizable domain (reader) within the same polypeptide that detects the methylation status of histones and/or DNA. Here, we summarize recent developments in characterizing structural and functional properties of various histone lysine demethylases, with emphasis on a mechanism of crosstalk between a Jumonji domain and its associated reader module(s). We further discuss the role of recently identified Tet1 enzyme in oxidizing 5-methylcytosine to 5-hydroxymethylcytosine in DNA.
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Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling.
Nucleic Acids Res.
PUBLISHED: 04-22-2011
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SET domain containing 6 (SETD6) monomethylates the RelA subunit of nuclear factor kappa B (NF-?B). The ankyrin repeats of G9a-like protein (GLP) recognizes RelA monomethylated at Lys310. Adjacent to Lys310 is Ser311, a known phosphorylation site of RelA. Ser311 phosphorylation inhibits Lys310 methylation by SETD6 as well as binding of Lys310me1 by GLP. The structure of SETD6 in complex with RelA peptide containing the methylation site, in the presence of S-adenosyl-L-methionine, reveals a V-like protein structure and suggests a model for NF-?B binding to SETD6. In addition, structural modeling of the GLP ankyrin repeats bound to Lys310me1 peptide provides insight into the molecular basis for inhibition of Lys310me1 binding by Ser311 phosphorylation. Together, these findings provide a structural explanation for a key cellular signaling pathway centered on RelA Lys310 methylation, which is generated by SETD6 and recognized by GLP, and incorporate a methylation-phosphorylation switch of adjacent lysine and serine residues. Finally, SETD6 is structurally similar to the Rubisco large subunit methyltransferase. Given the restriction of Rubisco to plant species, this particular appearance of the protein lysine methyltransferase has been evolutionarily well conserved.
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Polymorphisms associated with in vitro aspirin resistance are not associated with clinical outcomes in patients with coronary artery disease who report regular aspirin use.
Am. Heart J.
PUBLISHED: 03-17-2011
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We hypothesized that single-nucleotide polymorphisms (SNPs) associated with heightened in vitro platelet function during aspirin exposure (which we define as "laboratory aspirin resistance") would be associated with greater risk for death, myocardial infarction (MI) or stroke among patients with coronary artery disease regularly using aspirin.
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Pharmacokinetics of combined treatment with praziquantel and albendazole in neurocysticercosis.
Br J Clin Pharmacol
PUBLISHED: 02-22-2011
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Neurocysticercosis is the most common cause of acquired epilepsy in the world. Antiparasitic treatment of viable brain cysts is of clinical benefit, but current antiparasitic regimes provide incomplete parasiticidal efficacy. Combined use of two antiparasitic drugs may improve clearance of brain parasites. Albendazole (ABZ) has been used together with praziquantel (PZQ) before for geohelminths, echinococcosis and cysticercosis, but their combined use is not yet formally recommended and only scarce, discrepant data exist on their pharmacokinetics when given together. We assessed the pharmacokinetics of their combined use for the treatment of neurocysticercosis.
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Potential drug development candidates for human soil-transmitted helminthiases.
PLoS Negl Trop Dis
PUBLISHED: 02-19-2011
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Few drugs are available for soil-transmitted helminthiasis (STH); the benzimidazoles albendazole and mebendazole are the only drugs being used for preventive chemotherapy as they can be given in one single dose with no weight adjustment. While generally safe and effective in reducing intensity of infection, they are contra-indicated in first-trimester pregnancy and have suboptimal efficacy against Trichuris trichiura. In addition, drug resistance is a threat. It is therefore important to find alternatives.
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Structural insights for MPP8 chromodomain interaction with histone H3 lysine 9: potential effect of phosphorylation on methyl-lysine binding.
J. Mol. Biol.
PUBLISHED: 02-09-2011
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M-phase phosphoprotein 8 (MPP8) harbors an N-terminal chromodomain and a C-terminal ankyrin repeat domain. MPP8, via its chromodomain, binds histone H3 peptide tri- or di-methylated at lysine 9 (H3K9me3/H3K9me2) in submicromolar affinity. We determined the crystal structure of MPP8 chromodomain in complex with H3K9me3 peptide. MPP8 interacts with at least six histone H3 residues from glutamine 5 to serine 10, enabling its ability to distinguish lysine-9-containing peptide (QTARKS) from that of lysine 27 (KAARKS), both sharing the ARKS sequence. A partial hydrophobic cage with three aromatic residues (Phe59, Trp80 and Tyr83) and one aspartate (Asp87) encloses the methylated lysine 9. MPP8 has been reported to be phosphorylated in vivo, including the cage residue Tyr83 and the succeeding Thr84 and Ser85. Modeling a phosphate group onto the side-chain hydroxyl oxygen of Tyr83 suggests that the negatively charged phosphate group could enhance the binding of positively charged methyl-lysine or create a regulatory signal by allowing or inhibiting binding of other protein(s).
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Medical and psychosocial complications associated with method of bladder management after traumatic spinal cord injury.
Arch Phys Med Rehabil
PUBLISHED: 01-31-2011
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To determine the relationships between bladder management method and medical complications (renal calculi or decubitus ulcers), number of hospital days, and psychosocial factors. We hypothesized that indwelling catheterization would be associated with more complications, more hospitalizations, and worse psychosocial outcomes compared with other bladder management methods.
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Structural basis for human PHF2 Jumonji domain interaction with metal ions.
J. Mol. Biol.
PUBLISHED: 10-25-2010
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PHF2 belongs to a class of ?-ketoglutarate-Fe(2)(+)-dependent dioxygenases. PHF2 harbors a plant homeodomain (PHD) and a Jumonji domain. PHF2, via its PHD, binds Lys4-trimethylated histone 3 in submicromolar affinity and has been reported to have the demethylase activity of monomethylated lysine 9 of histone 3 in vivo. However, we did not detect demethylase activity for PHF2 Jumonji domain (with and without its linked PHD) in the context of histone peptides. We determined the crystal structures of PHF2 Jumonji domain in the absence and presence of additional exogenous metal ions. When Fe(2+) or Ni(2+) was added at a high concentration (50 mM) and allowed to soak in the preformed crystals, Fe(2+) or Ni(2+) was bound by six ligands in an octahedral coordination. The side chains of H249 and D251 and the two oxygen atoms of N-oxalylglycine (an analog of ?-ketoglutarate) provide four coordinations in the equatorial plane, while the hydroxyl oxygen atom of Y321 and one water molecule provide the two axial coordinations as the fifth and sixth ligands, respectively. The metal binding site in PHF2 closely resembles the Fe(2+) sites in other Jumonji domains examined, with one important difference-a tyrosine (Y321 of PHF2) replaces histidine as the fifth ligand. However, neither Y321H mutation nor high metal concentration renders PHF2 an active demethylase on histone peptides. Wild type and Y321H mutant bind Ni(2+) with an approximately equal affinity of 50 ?M. We propose that there must be other regulatory factors required for the enzymatic activity of PHF2 in vivo or that perhaps PHF2 acts on non-histone substrates. Furthermore, PHF2 shares significant sequence homology throughout the entire region, including the above-mentioned tyrosine at the corresponding iron-binding position, with that of Schizosaccharomyces pombe Epe1, which plays an essential role in heterochromatin function but has no known enzymatic activity.
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Diagnostics are central for a truly holistic approach against intestinal parasitic diseases.
Int. J. Infect. Dis.
PUBLISHED: 10-15-2010
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This is a perspectives piece on the central role of diagnostics for a truly holistic approach against gastrointestinal (GI) parasitic diseases. This article was motivated by a recent review in the International Journal of Infectious Diseases, where Absar Alum and colleagues (September 2010) reviewed the global burden, key transmission pathways, current tools and strategies, and provided their vision of a holistic approach to control GI protozoan and helminthic infections in humans. We argue that, as the success of multiple rounds of national deworming campaigns are actualized in various parts of the world, diagnostics become vital to achieve successful elimination and to aid pharmacovigilance against emerging pathogen resistance to the limited deworming pharmacopoeia.
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Adding a lysine mimic in the design of potent inhibitors of histone lysine methyltransferases.
J. Mol. Biol.
PUBLISHED: 03-29-2010
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Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers), and readers of the histone code. One common feature of these activities is the recognition of lysines in methylated and unmethylated states, whether they are substrates, reaction products, or binding partners. We applied the concept of adding a lysine mimic to an established inhibitor (BIX-01294) of histone H3 lysine 9 methyltransferases G9a and G9a-like protein by including a 5-aminopentyloxy moiety, which is inserted into the target lysine-binding channel and becomes methylated by G9a-like protein, albeit slowly. The compound enhances its potency in vitro and reduces cell toxicity in vivo. We suggest that adding a lysine or methyl-lysine mimic should be considered in the design of small-molecule inhibitors for other methyl-lysine writers, erasers, and readers.
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Identification and characterization of two Bordetella avium gene products required for hemagglutination.
Infect. Immun.
PUBLISHED: 03-29-2010
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Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.
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A novel classification system for traumatic central cord syndrome: the central cord injury scale (CCIS).
Spine
PUBLISHED: 03-16-2010
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Retrospective study of 37 patients with traumatic central cord syndrome.
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A methylation and phosphorylation switch between an adjacent lysine and serine determines human DNMT1 stability.
Nat. Struct. Mol. Biol.
PUBLISHED: 02-24-2010
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The protein lysine methyltransferase SET7 regulates DNA methyltransferase-1 (DNMT1) activity in mammalian cells by promoting degradation of DNMT1 and thus allows epigenetic changes via DNA demethylation. Here we reveal an interplay between monomethylation of DNMT1 Lys142 by SET7 and phosphorylation of DNMT1 Ser143 by AKT1 kinase. These two modifications are mutually exclusive, and structural analysis suggests that Ser143 phosphorylation interferes with Lys142 monomethylation. AKT1 kinase colocalizes and directly interacts with DNMT1 and phosphorylates Ser143. Phosphorylated DNMT1 peaks during DNA synthesis, before DNMT1 methylation. Depletion of AKT1 or overexpression of dominant-negative AKT1 increases methylated DNMT1, resulting in a decrease in DNMT1 abundance. In mammalian cells, phosphorylated DNMT1 is more stable than methylated DNMT1. These results reveal cross-talk on DNMT1, through modifications mediated by AKT1 and SET7, that affects cellular DNMT1 levels.
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Epidemiology and control of human gastrointestinal parasites in children.
Expert Rev Anti Infect Ther
PUBLISHED: 01-30-2010
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Parasites found in the human gastrointestinal tract can be largely categorized into two groups, protozoa and helminths. The soil-transmitted helminths (Ascaris lumbricoides, hookworm and Trichuris trichiura) are the most prevalent, infecting an estimated one-sixth of the global population. Infection rates are highest in children living in sub-Saharan Africa, followed by Asia and then Latin America and the Caribbean. The current momentum towards global drug delivery for their control is at a historical high through the efforts of numerous initiatives increasingly acting in coordination with donors, governments and local communities. Together, they have delivered enormous quantities of drugs, especially anthelmintics to children through nationwide annual or biannual mass drug administration largely coordinated through schools. However, a much larger and rapidly growing childhood population in these regions remains untreated and suffering from more than one parasite. Mass drug administration has profound potential for control but is not without considerable challenges and concerns. A principal barrier is funding. Stimulating a research and development pipeline, supporting the necessary clinical trials to refine treatment, in addition to procuring and deploying drugs (and sustaining these supply chains), requires substantial funding and resources that do not presently exist. Limited options for chemotherapy raise concerns about drug resistance developing through overuse, however, satisfactory pharmaco-epidemiology and monitoring for drug resistance requires more developed health infrastructures than are generally available. Further, the limited pharmacopeia does not include any effective second-line options if resistance emerges, and the research and development pipeline is severely depressed. Herein, we discuss the major gastrointestinal protozoa and helminths reviewing their impact on child health, changing epidemiology and how this relates to their control.
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Single-photon emission computed tomography myocardial perfusion imaging and the risk of sudden cardiac death in patients with coronary disease and left ventricular ejection fraction>35%.
J. Am. Coll. Cardiol.
PUBLISHED: 01-25-2010
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The aim of this study was to determine whether single-photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) is an effective method of risk stratification for sudden cardiac death (SCD) in patients with coronary artery disease (CAD) and left ventricular ejection fraction (LVEF)>35%.
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Albendazole and its metabolites in the breast milk of lactating women following a single oral dose of albendazole.
Br J Clin Pharmacol
PUBLISHED: 11-18-2009
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Albendazole (ABZ) is used in several anthelminthic drug programs. ABZ side-effects are generally mild, but ABZ-induced pancytopenia may be serious. In filariasis programmes, it may be necessary to administer ABZ to breastfeeding women. Few data are available on safety of ABZ for breastfed infants. In addition, the pharmacokinetics of ABZ and its metabolites in human milk is insufficiently investigated. The aim was to study pharmacokinetics of ABZ and its metabolites [ABZ sulphoxide (ABSX) and ABZ sulphone] in the breast milk lactating women after one single oral dose of ABZ.
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Enzymatic and structural insights for substrate specificity of a family of jumonji histone lysine demethylases.
Nat. Struct. Mol. Biol.
PUBLISHED: 10-29-2009
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Combinatorial readout of multiple covalent histone modifications is poorly understood. We provide insights into how an activating histone mark, in combination with linked repressive marks, is differentially read by two related human demethylases, PHF8 and KIAA1718 (also known as JHDM1D). Both enzymes harbor a plant homeodomain (PHD) that binds Lys4-trimethylated histone 3 (H3K4me3) and a jumonji domain that demethylates either H3K9me2 or H3K27me2. The presence of H3K4me3 on the same peptide as H3K9me2 makes the doubly methylated peptide a markedly better substrate of PHF8, whereas the presence of H3K4me3 has the opposite effect, diminishing the H3K9me2 demethylase activity of KIAA1718 without adversely affecting its H3K27me2 activity. The difference in substrate specificity between the two is explained by PHF8 adopting a bent conformation, allowing each of its domains to engage its respective target, whereas KIAA1718 adopts an extended conformation, which prevents its access to H3K9me2 by its jumonji domain when its PHD engages H3K4me3.
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Monte Carlo model for a prototype CT-compatible, anatomically adaptive, shielded intracavitary brachytherapy applicator for the treatment of cervical cancer.
Med Phys
PUBLISHED: 10-09-2009
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Current, clinically applicable intracavitary brachytherapy applicators that utilize shielded ovoids contain a pair of tungsten-alloy shields which serve to reduce dose delivered to the rectum and bladder during source afterloading. After applicator insertion, these fixed shields are not necessarily positioned to provide optimal shielding of these critical structures due to variations in patient anatomies. The authors present a dosimetric evaluation of a novel prototype intracavitary brachytherapy ovoid [anatomically adaptive applicator (A3)], featuring a single shield whose position can be adjusted with two degrees of freedom: Rotation about and translation along the long axis of the ovoid.
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Unresolved issues in anthelmintic pharmacology for helminthiases of humans.
Int. J. Parasitol.
PUBLISHED: 08-28-2009
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Helminth infections are an important constraint on the health and development of poor children and adults. Anthelmintic treatment programmes provide a safe and effective response, and increasing numbers of people are benefitting from these public health initiatives. Despite decades of clinical experience with anthelmintics for the treatment of human infections, relatively little is known about their clinical pharmacology. All of the drugs were developed initially in response to the considerable market for veterinary anthelmintics in high- and middle-income countries. In contrast, the greatest burden caused by these infections in humans is in resource-poor settings and as a result there has been insufficient commercial incentive to support studies on how these drugs work in humans, and how they should best be used in control programmes. The advent of mass drug administration programmes for the control of schistosomiasis, lymphatic filariasis, onchocerciasis and soil-transmitted helminthiases in humans increases the urgency to better understand and better monitor drug resistance, and to broaden the currently very narrow range of available anthelmintics. This provides fresh impetus for developing a comprehensive research platform designed to improve our understanding of these important drugs, in order to bring the scientific knowledge base supporting their use to a standard equivalent to that of drugs commonly used in developed countries. Furthermore, a better understanding of their clinical pharmacology will enable improved therapy and could contribute to the discovery of new products.
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Perirectal abscess infections related to MRSA: a prevalent and underrecognized pathogen.
J Surg Educ
PUBLISHED: 06-30-2009
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Methicillin-resistant Staphylococcus aureus (MRSA) is not a commonly recognized pathogen isolated from perirectal abscesses. Complex perirectal abscesses of MRSA origin may present a significant challenge to the physician and result in treatment failure. The aim of our study was to determine the prevalence of MRSA in our patient population with perirectal abscesses and whether antibiotics coverage, if given, was adequate.
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Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation.
J. Mol. Biol.
PUBLISHED: 06-19-2009
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Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp283, one of the most active hydrolase variants. Our initial investigation has focused on the role of an extended surface loop, created by linking the native N- and C-termini, on protein integrity. Incremental truncation of the loop partially compensates for observed losses in secondary structure and the permutants temperature of unfolding. Unexpectedly, the improvements are accompanied by quaternary-structure changes from monomer to dimer. The crystal structures of one truncated variant (cp283 Delta 7) in the apo-form determined at 1.49 A resolution and with a bound phosphonate inhibitor at 1.69 A resolution confirmed the formation of a homodimer by swapping of the enzymes 35-residue N-terminal region. Separately, the new protein termini at amino acid positions 282/283 convert the narrow access tunnel to the catalytic triad into a broad crevice for accelerated substrate entry and product exit while preserving the native active-site topology for optimal catalytic turnover.
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Comparison of a 3-D multi-group SN particle transport code with Monte Carlo for intracavitary brachytherapy of the cervix uteri.
J Appl Clin Med Phys
PUBLISHED: 05-11-2009
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A patient dose distribution was calculated by a 3D multi-group S N particle transport code for intracavitary brachytherapy of the cervix uteri and compared to previously published Monte Carlo results. A Cs-137 LDR intracavitary brachytherapy CT data set was chosen from our clinical database. MCNPX version 2.5.c, was used to calculate the dose distribution. A 3D multi-group S N particle transport code, Attila version 6.1.1 was used to simulate the same patient. Each patient applicator was built in SolidWorks, a mechanical design package, and then assembled with a coordinate transformation and rotation for the patient. The SolidWorks exported applicator geometry was imported into Attila for calculation. Dose matrices were overlaid on the patient CT data set. Dose volume histograms and point doses were compared. The MCNPX calculation required 14.8 hours, whereas the Attila calculation required 22.2 minutes on a 1.8 GHz AMD Opteron CPU. Agreement between Attila and MCNPX dose calculations at the ICRU 38 points was within +/- 3%. Calculated doses to the 2 cc and 5 cc volumes of highest dose differed by not more than +/- 1.1% between the two codes. Dose and DVH overlays agreed well qualitatively. Attila can calculate dose accurately and efficiently for this Cs-137 CT-based patient geometry. Our data showed that a three-group cross-section set is adequate for Cs-137 computations. Future work is aimed at implementing an optimized version of Attila for radiotherapy calculations.
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Relationship between anemia and health care costs in heart failure.
J. Card. Fail.
PUBLISHED: 04-30-2009
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Anemia is associated with higher morbidity and mortality in patients with heart failure (HF), but its implications for heath care costs are not well described.
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Elective inguinal hernia repair during Operation Iraqi Freedom.
Mil Med
PUBLISHED: 04-10-2009
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While there have been many publications regarding trauma care in the deployed environment, there is little discussion on the management of the more mundane maladies. This article examines the role of elective surgical intervention for inguinal hernia repairs within theater. Current U.S. policy transports service members out of theater for elective repair and convalescence. In these times of limited man power, this can represent a significant loss of the fighting strength.
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Continuous albendazole therapy in alveolar echinococcosis: long-term follow-up observation of 20 cases.
Trans. R. Soc. Trop. Med. Hyg.
PUBLISHED: 04-02-2009
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Twenty patients with primary hepatic alveolar echinococcosis were treated with continuous long-term albendazole at a dose of 20 mg/kg/d for an average of 5.7 years (1.5-13.5 years) and were followed up for an average of 12.7 years (4.1-13.5 years). The therapeutic effects were evaluated by clinical, laboratory (haemogram, liver function tests), ultrasonography and computed tomography (CT) examinations. The CT pattern of hepatic lesions was classified into three types: solid form (5 cases), pseudocystic form (6 cases) and geographic map (mixed) form (9 cases). Short-term results were encouraging. Jaundice and haemoptysis disappeared within 1 month of starting treatment, and hemiparesis in a patient with cerebral metastases also gradually improved. Treatment was less effective in two patients with advanced disease. On long-term follow-up, three patients were apparently cured, and the remaining 17 patients showed a good initial response, but recurrence occurred in 13 patients (65%). Therapeutic outcome was favourable with the solid form, but poor with the pseudocystic form. The mixed form was a transitional phase and slowly progressive. Involvement of the porta hepatis was the cause of various complications with high morbidity and mortality. Five patients (25%) died during the period of observation, the 10-year survival rate being, therefore, 75%.
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An international survey of the diagnosis, management, and treatment of hepatitis C in patients with end-stage renal disease.
Exp Clin Transplant
PUBLISHED: 03-02-2009
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Hepatitis C is one of the leading causes of death from liver disease in the United States, and is frequently associated with renal disease. Two major organizations-the American Association for the Study of Liver Disease and the National Kidney Foundation-have published recommendations regarding the treatment of hepatitis C in the presence of chronic kidney disease; however, these guidelines do not always provide the same recommendations. Given the paucity of data on adherence to the current guidelines, a survey was conducted to provide information about the current practices of physicians in comparison to the published guidelines.
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Survival trends in children with hepatoblastoma.
Pediatr. Surg. Int.
PUBLISHED: 02-27-2009
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Hepatoblastoma (HB) is a relatively rare pediatric malignancy. In this study, we present demographic data and a survival analysis from the largest patient cohort with HB reported to date.
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Mitral gradients and frequency of recurrence of mitral regurgitation after ring annuloplasty for ischemic mitral regurgitation.
Ann. Thorac. Surg.
PUBLISHED: 01-28-2009
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Undersized ring annuloplasty and surgical revascularization are commonly used to correct ischemic mitral regurgitation (MR), but published series have failed to demonstrate a benefit compared with revascularization alone. We hypothesized that surgical revascularization and annuloplasty lead to a durable repair, but may also lead to increased mitral gradients that could limit the benefit of the repair technique.
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Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294.
Nat. Struct. Mol. Biol.
PUBLISHED: 01-14-2009
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Histone lysine methylation is an important epigenetic mark that regulates gene expression and chromatin organization. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by methylating histone H3 Lys9. BIX-01294 was originally identified as a G9a inhibitor during a chemical library screen of small molecules and has previously been used in the generation of induced pluripotent stem cells. Here we present the crystal structure of the catalytic SET domain of GLP in complex with BIX-01294 and S-adenosyl-L-homocysteine. The inhibitor is bound in the substrate peptide groove at the location where the histone H3 residues N-terminal to the target lysine lie in the previously solved structure of the complex with histone peptide. The inhibitor resembles the bound conformation of histone H3 Lys4 to Arg8, and is positioned in place by residues specific for G9a and GLP through specific interactions.
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UHRF1, a modular multi-domain protein, regulates replication-coupled crosstalk between DNA methylation and histone modifications.
Epigenetics
PUBLISHED: 01-10-2009
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Cytosine methylation in DNA is a major epigenetic signal, and plays a central role in propagating chromatin status during cell division. However the mechanistic links between DNA methylation and histone methylation are poorly understood. A multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for DNA CpG maintenance methylation at replication forks, and mouse UHRF1-null cells show enhanced susceptibility to DNA replication arrest and DNA damaging agents. Recent data demonstrated that the SET and RING associated (SRA) domain of UHRF1 binds hemimethylated CpG and flips 5-methylcytosine out of the DNA helix, whereas its tandom tudor domain and PHD domain bind the tail of histone H3 in a highly methylation sensitive manner. We hypothesize that UHRF1 brings the two components (histones and DNA) carrying appropriate markers (on the tails of H3 and hemimethylated CpG sites) ready to be assembled into a nucleosome after replication.
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Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease.
Nucleic Acids Res.
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The MspJI modification-dependent restriction endonuclease recognizes 5-methylcytosine or 5-hydroxymethylcytosine in the context of CNN(G/A) and cleaves both strands at fixed distances (N(12)/N(16)) away from the modified cytosine at the 3-side. We determined the crystal structure of MspJI of Mycobacterium sp. JLS at 2.05-Å resolution. Each protein monomer harbors two domains: an N-terminal DNA-binding domain and a C-terminal endonuclease. The N-terminal domain is structurally similar to that of the eukaryotic SET and RING-associated domain, which is known to bind to a hemi-methylated CpG dinucleotide. Four protein monomers are found in the crystallographic asymmetric unit. Analytical gel-filtration and ultracentrifugation measurements confirm that the protein exists as a tetramer in solution. Two monomers form a back-to-back dimer mediated by their C-terminal endonuclease domains. Two back-to-back dimers interact to generate a tetramer with two double-stranded DNA cleavage modules. Each cleavage module contains two active sites facing each other, enabling double-strand DNA cuts. Biochemical, mutagenesis and structural characterization suggest three different monomers of the tetramer may be involved respectively in binding the modified cytosine, making the first proximal N(12) cleavage in the same strand and then the second distal N(16) cleavage in the opposite strand. Both cleavage events require binding of at least a second recognition site either in cis or in trans.
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Modest increase in peak VO2 is related to better clinical outcomes in chronic heart failure patients: results from heart failure and a controlled trial to investigate outcomes of exercise training.
Circ Heart Fail
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The prognostic ability of a single measurement of peak oxygen uptake (VO(2)) is well established in patients with chronic heart failure. The relation between a change in peak VO(2) and clinical outcomes is not well defined.
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Temporal and spatial distributions of ammonia-oxidizing archaea and bacteria and their ratio as an indicator of oligotrophic conditions in natural wetlands.
Water Res.
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Ammonia-oxidizing organisms play an important role in wetland water purification and nitrogen cycling. We determined soil nitrification rates and investigated the seasonal and spatial distributions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in three freshwater wetlands by using specific primers targeting the amoA genes of AOA and AOB and real-time quantitative polymerase chain reaction (qPCR). The nitrifying potentials of wetland soils ranged from 1.4 to 4.0 ?g g(-1) day(-1). The specific rates of ammonia oxidation activity by AOA and AOB at the Bee Hollow wetlands were 1.9 fmol NH(3) cell(-1) day(-1) and 36.8 fmol NH(3) cell(-1) day(-1), respectively. Soil nitrification potential was positively correlated with both archaeal and bacterial amoA abundance. However, the gene copies of AOA amoA were higher than those of AOB amoA by at least an order of magnitude in wetland soils and water in both summer and winter over a three year study period. AOB were more sensitive to low temperature than AOA. The amoA gene copy ratios of AOA to AOB in top soils (0-10 cm) ranged from 19 ± 4 to 100 ± 11 among the wetland sites. In contrast, the ratio of the wetland boundary soil was 10 ± 2, which was significantly lower than that of the wetland soils (P < 0.001). The NH(4)(+)-N concentrations in wetland water were lower than 2 mg/L throughout the study. The results suggest that ammonium concentration is a major factor influencing AOA and AOB population in wetlands, although other factors such as temperature, dissolved oxygen, and soil organic matter are involved. AOA are more persistent and more abundant than AOB in the nutrient-depleted oligotrophic wetlands. Therefore, ratio of AOA amoA gene copies to AOB amoA gene copies may serve as a new biological indicator for wetland condition assessment and wetland restoration applications.
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A pivotal registration phase III, multicenter, randomized tuberculosis controlled trial: design issues and lessons learnt from the Gatifloxacin for TB (OFLOTUB) project.
Trials
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There have been no major advances in tuberculosis (TB) drug development since the first East African/British Medical Research Council short course chemotherapy trial 35 years ago. Since then, the landscape for conducting TB clinical trials has profoundly changed with the emergence of HIV infection, the spread of resistant TB bacilli strains, recent advances in mycobacteriological capacity, and drug discovery. As a consequence questions have arisen on the most appropriate approach to design and conduct current TB trials. To highlight key issues discussed: Is a superiority, equivalence, or non-inferiority design most appropriate? What should be the primary efficacy outcome? How to consider re-infections in the definition of the outcome? What is the optimal length of patient follow-up? Is blinding appropriate when treatment duration in test arm is shorter? What are the appropriate assumptions for sample size calculation?
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A semimechanistic pharmacokinetic-enzyme turnover model for rifampin autoinduction in adult tuberculosis patients.
Antimicrob. Agents Chemother.
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The currently recommended doses of rifampin are believed to be at the lower end of the dose-response curve. Rifampin induces its own metabolism, although the effect of dose on the extent of autoinduction is not known. This study aimed to investigate rifampin autoinduction using a semimechanistic pharmacokinetic-enzyme turnover model. Four different structural basic models were explored to assess whether different scaling methods affected the final covariate selection procedure. Covariates were selected by using a linearized approach. The final model included the allometric scaling of oral clearance and apparent volume of distribution. Although HIV infection was associated with a 30% increase in the apparent volume of distribution, simulations demonstrated that the effect of HIV on rifampin exposure was slight. Model-based simulations showed close-to-maximum induction achieved after 450-mg daily dosing, since negligible increases in oral clearance were observed following the 600-mg/day regimen. Thus, dosing above 600 mg/day is unlikely to result in higher magnitudes of autoinduction. In a typical 55-kg male without HIV infection, the oral clearance, which was 7.76 liters · h?¹ at the first dose, increased 1.82- and 1.85-fold at steady state after daily dosing with 450 and 600 mg, respectively. Corresponding reductions of 41 and 42%, respectively, in the area under the concentration-versus-time curve from 0 to 24 h were estimated. The turnover of the inducible process was estimated to have a half-life of approximately 8 days in a typical patient. Assuming 5 half-lives to steady state, this corresponds to a duration of approximately 40 days to reach the induced state for rifampin autoinduction.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.