Quantitative Study of the Chiral Organization of the Phage Genome Induced by the Packaging Motor.

Molecular motors that translocate DNA are ubiquitous in nature. During morphogenesis of double-stranded DNA bacteriophages, a molecular motor drives the viral genome inside a protein capsid. Several models have been proposed for the three-dimensional geometry of the packaged genome, but very little is known of the signature of the molecular packaging motor. For instance, biophysical experiments show that in some systems, DNA rotates during the packaging reaction, but most current biophysical models fail to incorporate this property. Furthermore, studies including rotation mechanisms have reached contradictory conclusions. In this study, we compare the geometrical signatures imposed by different possible mechanisms for the packaging motors: rotation, revolution, and rotation with revolution. We used a previously proposed kinetic Monte Carlo model of the motor, combined with Brownian dynamics simulations of DNA to simulate deterministic and stochastic motor models. We find that rotation is necessary for the accumulation of DNA writhe and for the chiral organization of the genome. We observe that although in the initial steps of the packaging reaction, the torsional strain of the genome is released by rotation of the molecule, in the later stages, it is released by the accumulation of writhe. We suggest that the molecular motor plays a key role in determining the final structure of the encapsidated genome in bacteriophages.

The Rabl configuration limits topological entanglement of chromosomes in budding yeast.

The three dimensional organization of genomes remains mostly unknown due to their high degree of condensation. Biophysical studies predict that condensation promotes the topological entanglement of chromatin fibers and the inhibition of function. How organisms balance between functionally active genomes and a high degree of condensation remains to be determined. Here we hypothesize that the Rabl configuration, characterized by the attachment of centromeres and telomeres to the nuclear envelope, helps to reduce the topological entanglement of chromosomes. To test this hypothesis we developed a novel method to quantify chromosome entanglement complexity in 3D reconstructions obtained from Chromosome Conformation Capture (CCC) data. Applying this method to published data of the yeast genome, we show that computational models implementing the attachment of telomeres or centromeres alone are not sufficient to obtain the reduced entanglement complexity observed in 3D reconstructions. It is only when the centromeres and telomeres are attached to the nuclear envelope (i.e. the Rabl configuration) that the complexity of entanglement of the genome is comparable to that of the 3D reconstructions. We therefore suggest that the Rabl configuration is an essential player in the simplification of the entanglement of chromatin fibers.

Resident-Led Palliative Care Education Project.

BACKGROUND AND OBJECTIVE: Despite the growth of palliative medicine, 39% of hospitals do not have palliative care teams for consultation or to provide resident education. We examined the impact of resident-led education in palliative care principles on attitudes toward and comfort with palliative medicine and end-of-life care among internal medicine residents. METHODS: An educational module designed by the authors was presented to other internal medicine residents in the program. Pre- and post-intervention survey data measuring residents' agreement with various statements regarding palliative medicine and end-of-life care were analyzed. Residents' agreement with various statements regarding palliative medicine and end-of-life care on a 5-point Likert scale was analyzed. RESULTS: Following the intervention, participants reported improved comfort with general knowledge of palliative medicine (p < 0.01), specific resources available to patients (p < 0.001), and explaining the difference between palliative care and end-of-life care (p < 0.001). In each of the seven specific domains of palliative medicine covered in the educational session, residents reported a statistically significant increase in comfort in all of the areas addressed (p < 0.05). CONCLUSION: This study demonstrates that a resident-led curriculum in palliative medicine can improve resident comfort within this still-under-represented area of medicine.

Use of prescription and nonprescription medications and supplements by cancer patients during chemotherapy: questionnaire validation.

BACKGROUND: Cancer patients take medications for coexisting disease and self-medicate with over-the-counter drugs (OTCs). A complete analysis of the use of prescription drugs, OTCs, and supplements during cancer treatment has never been done. METHODS: The study developed and validated a self-administered questionnaire on the use of concomitant medications by patients undergoing treatment with chemotherapy. The questionnaire listed 510 prescription medications, OTCs, and supplements (including vitamins, minerals, and herbs). Fifty-two subjects completed the questionnaire while visiting the infusion clinic to receive chemotherapy. On a subsequent visit the subjects brought their medications to the clinic and a pharmacist reviewed their completed questionnaire. RESULTS: Ninety-six percent of the subjects reported taking prescription medications within 3 days prior to chemotherapy, 71% reported taking OTCs and 69% reported use of supplements. The subjects took an average of 5.5 (range 0-13) prescription drugs, 2.2 (0-20) OTCs, and 1.9 (0-11) supplements. Twenty-one drugs were each taken by at least 10% of the subjects. Acetaminophen was taken by 59.6% of the subjects. One subject reported taking five acetaminophen-containing drugs. The questionnaire's sensitivity was 92.0%, specificity 99.9%. CONCLUSION: Within 3 days prior to chemotherapy, subjects took an average of 9.6 concomitant medications, many of which alter drug metabolism and or disposition. In clinical trials, multivariate analysis of all concomitant medications could add to clinically relevant data to identify drug interactions that negate or potentiate the efficacy of cancer treatment regimens. In some instances, apparent resistance of tumors to chemotherapy may be the result of drug interactions.

Thrombin-antithrombin appearance in out-of-hospital cardiac arrest.

OBJECTIVE: Intravascular thrombosis induced during out-of-hospital cardiac arrest (OOHCA) may contribute to the pathophysiology of cardiac arrest and complicate resuscitation. We characterized the prevalence of thrombogenesis during OOHCA by measuring plasma levels of thrombin-antithrombin complexes (TAT). METHODS: An observational cohort study of medical OOHCA patients in an urban emergency medical services (EMS) system. Subjects were patients>or=18 years suffering medical OOHCA. Citrated blood samples were drawn in the prehospital setting either directly from venous blood or during the placement of a central venous catheter and frozen (-70 degrees C). The EMS physician documented age, gender, time intervals, return of spontaneous circulation (ROSC), therapies administered and time of blood draw. TAT assays were performed by commercial ELISA. RESULTS: Eighty-eight patients (58% male) aged 63.4+/-15.9 years were enrolled in the study. Median [interquartile range IQR] TAT values in 80 samples (8 samples were grossly clotted and excluded) were 159.2 [38, 2, 522, 8] and ranged from 0.79 to 1,343.9. Patients transported to the hospital had lower TAT levels than those pronounced in the field (p=0.014). Of four EMS-witnessed arrests, three had return of pulses with TAT values of 0.79, 6.8, and 17.9. The fourth had a TAT over 525 after a long unsuccessful resuscitation. For subjects with TAT below 50 (n=23), all but three were witnessed arrests or received bystander CPR. CONCLUSIONS: Except for a single case witnessed by EMS and immediately defibrillated into a perfusing rhythm, all cases of OOHCA exhibited increased thrombotic state. Intravascular thrombosis may represent a global barrier to resuscitation and ultimately, end-organ perfusion.

The use of performance improvement methods to enhance emergency department patient satisfaction in the United States: a critical review of the literature and suggestions for future research.

OBJECTIVES: The authors reviewed the evidence on performance improvement methods for increasing emergency department (ED) patient satisfaction to provide evidence-based suggestions for clinical practice. METHODS: Data sources consisted of searches through MEDLINE, CINAHL, PSYCHINFO, Cochrane Library, and Emergency Medicine Abstracts and a manual search of references. Articles were included if they reported a performance improvement intervention targeting patient satisfaction in the ED setting. Articles on studies not conducted in the United States or that failed to provide enough details to allow critical evaluation of the study were excluded. Two authors used structured evaluation criteria to independently review each retained study. RESULTS: Nineteen articles met all selection criteria. Three studies found varying levels of support for multicomponent interventions, predominantly focused on implementation of clinical practice guidelines for specific presenting complaints and process redesign. Sixteen studies evaluated single-component interventions, with the following having at least one supportive study: using alternating patient assignment to provider teams rather than "zone"-based assignment, enhancing provider communication and customer service skills, incorporating information delivery interventions (e.g., pamphlets, video) that target patient expectations, using preformatted charts, and establishing ED-based observation units for specific conditions such as asthma and chest pain. CONCLUSIONS: There is modest evidence supporting a range of performance improvement interventions for improving ED patient satisfaction. Further work is needed before specific, evidence-based recommendations can be made regarding which process changes are most effective. Recommendations are made for improving the quality of performance improvement efforts in the ED setting.

Hypothermia and ERK activation after cardiac arrest.

Mild hypothermia improves survival and neurological outcome after cardiac arrest, as well as increasing activation of the extracellular-signal-regulated kinase (ERK) in hippocampus. ERK signaling is involved in neuronal growth and survival. We tested the hypothesis that the beneficial effects of hypothermia required ERK activation. ERK activation was measured by immunoblotting with phosphorylation-specific antibodies. Rats (n = 8 per group) underwent 8 min of asphyxial cardiac arrest and were resuscitated with chest compressions, ventilation, epinephrine and bicarbonate. At 30 min after resuscitation, vehicle (50% saline:50% DMSO) or the ERK kinase inhibitor U0126 (100 microg) was infused into the lateral ventricle. Cranial temperature was kept at either 33 degrees C (hypothermia) or 37 degrees C (normothermia) between 1 and 24 h. Neurological function was assessed daily for 14 days. Surviving neurons were counted in the hippocampus. A dose of 100 mug U0126 inhibited ERK bilaterally for 12 to 24 h and decreased phosphorylation of the ERK substrates ATF-2 and CREB. As in previous studies, hypothermia improved survival, neurological and histological outcome after cardiac arrest. However, survival, neurological score and histology did not differ between U0126 and vehicle-treated rats after cardiac arrest. Therefore, a dose of U0126 sufficient to inhibit biochemical markers of ERK signaling in hippocampus does not alter the beneficial effects of hypothermia induced after resuscitation in rats and did not affect recovery of normothermia-treated rats. These results suggest that hypothermia-induced improvement in outcomes does not require ERK activation.

An in vitro model of hepatitis C virion production.

The hepatitis C virus (HCV) is a major cause of liver disease worldwide. The understanding of the viral life cycle has been hampered by the lack of a satisfactory cell culture system. The development of the HCV replicon system has been a major advance, but the system does not produce virions. In this study, we constructed an infectious HCV genotype 1b cDNA between two ribozymes that are designed to generate the exact 5' and 3' ends of HCV. A second construct with a mutation in the active site of the viral RNA-dependent RNA polymerase (RdRp) was generated as a control. The HCV-ribozyme expression construct was transfected into Huh7 cells. Both HCV structural and nonstructural proteins were detected by immunofluorescence and Western blot. RNase protection assays showed positive- and negative-strand HCV RNA. Sequence analysis of the 5' and 3' ends provided further evidence of viral replication. Sucrose density gradient centrifugation of the culture medium revealed colocalization of HCV RNA and structural proteins in a fraction with the density of 1.16 g/ml, the putative density of HCV virions. Electron microscopy showed viral particles of approximately 50 nm in diameter. The level of HCV RNA in the culture medium was as high as 10 million copies per milliliter. The HCV-ribozyme construct with the inactivating mutation in the RdRp did not show evidence of viral replication, assembly, and release. This system supports the production and secretion of high-level HCV virions and extends the repertoire of tools available for the study of HCV biology.

Prevalence of troponin-T elevation during out-of-hospital cardiac arrest.

To determine the prevalence of myocardial ischemia before out-of-hospital cardiac arrest (OOHCA), we determined the prevalence of elevated cardiac troponin-T levels in subjects at the time of OOHCA. Plasma was collected from 63 subjects during resuscitation. Troponin levels were elevated (> or =0.03 ng/ml) in 25 subjects (39.7%; 95% confidence intervals [CI] 29% to 52%). Increasing age was associated with elevated troponin (OR 1.10; 95% CI 1.04 to 1.17). Elevated troponin levels did not reliably predict short-term outcome. Because troponin increases hours after the onset of ischemia, these data reveal that about 40% of OOHCA cases can undergo intervention before collapse.

Hypothermic reperfusion after cardiac arrest augments brain-derived neurotrophic factor activation.

Induction of mild hypothermia improves neurologic outcome after global cerebral ischemia. This study measured levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in hippocampal tissue of rats after resuscitation from 8 minutes of normothermic, asphyxial cardiac arrest. After resuscitation, rats were maintained either at normal temperature (37 degrees C) or cooled to mild hypothermia (33 degrees C, beginning 60 minutes after resuscitation). After 12 or 24 hours, neurotrophin levels in hippocampus were measured by immunoblotting. Ischemia and reperfusion increased hippocampal levels of BDNF. Induction of hypothermia during reperfusion potentiated the increase in BDNF after 24 hours, but not after 12 hours. Levels of NGF were not increased by postresuscitation hypothermia. Hypothermia also increased tissue levels and tyrosine phosphorylation of TrkB, the receptor for BDNF. Increased BDNF levels were correlated with activation of the extracellularly regulated kinase (ERK), a downstream element in the signal transduction cascade induced by BDNF. In contrast to the many deleterious processes during ischemia and reperfusion that are inhibited by induced hypothermia, increasing BDNF levels is a potentially restorative process that is augmented. Increased activation of BDNF signaling is a possible mechanism by which mild hypothermia is able to reduce the neuronal damage typically occurring after cardiac arrest.

Endotracheal drug administration during out-of-hospital resuscitation: where are the survivors?

BACKGROUND: Drugs administered endotracheally are effectively absorbed during normal spontaneous cardiac activity. However, animal cardiac arrest studies and limited clinical investigations do not support either the use of endotracheal (ET) drugs in doses currently recommended for adults or the method of direct endotracheal instillation. The purpose of this study was to compare the effect of intravenous (IV) and ET drug therapy on outcome from out-of-hospital cardiac arrest secondary to all cardiac arrest rhythms. DESIGN: Five and one-half year retrospective cohort study. SETTING: Municipal, university affiliated hospital. PATIENTS: Consecutive patients >18 years of age in nontraumatic out-of-hospital cardiac arrest who received advanced cardiac life support (ACLS) medications by only the ET or IV route were included. INTERVENTIONS: None. RESULTS: Five hundred and ninety-six patients met inclusion criteria (IV drugs=495, ET drugs=101). There was no difference between groups in the rate of witnessed arrest and the frequency of bystander cardiopulmonary resuscitation (CPR). In the ET drug group, a significantly greater number of patients had an initial documented arrest rhythm of asystole compared to the IV drug group (56 vs 37%, P=0.01). The rate of return of spontaneous circulation (27 vs 15%, P=0.01) and survival to hospital admission rate (20 vs 9%, P=0.01) were significantly greater in the IV drug group. No patient who received ET drugs survived to hospital discharge compared to 5% of those receiving IV drugs (P=0.01). CONCLUSION: For our out-of-hospital advanced rescuer system, ET drugs at recommended doses (twice the IV dose) injected into an ET tube during cardiac arrest and CPR were of no benefit.