New function of a well-known promoter: Enhancer activity of minimal CMV promoter enables efficient dual-cassette transgene expression.

BACKGROUND: Co-expression of multiple genes in single vectors has achieved varying degrees of success by employing two promoters and/or application of viral 2A-peptide or the internal ribosome entry-site (IRES). However, promoter interference, potential functional-interruption of expressed-proteins by 2A-generated residual peptides or weaker translation of IRES-mediated downstream genes has curtailed their utilization. Thus, there is the need for single vectors that robustly express multiple proteins for enhanced gene therapy applications. METHODS: We engineered lentiviral-vectors for dual-cassette expression of green fluorescent protein and mCherry in uni- or bidirectional architectures using the short-version (Es) of elongation factor 1α (EF) promoter and simian virus 40 promoter (Sv). The regulatory function of a core fragment (cC) from human cytomegalovirus promoter was investigated with cell-lineage specificity in NIH3T3 (fibroblast) and hematopoietic cell lines U937 (monocyte/macrophage), LCL (lymphoid), DAMI (megakaryocyte) and MEL (erythroid). RESULTS: The cC element in reverse-orientation not only boosted upstream Es promoter to levels comparable to full-length EF in DAMI, U937 and 3T3 cells, but also blocked the suppression of downstream Sv promoter by Es in U937 and 3T3 cells with further improved Sv activity in DAMI cells. Such lineage-restricted up-regulation is likely attributed to two protein-binding domains of cC and diverse expression of related factors in different cell types for enhancer and terminator activities, but not spacing function. CONCLUSIONS: Such a newly developed dual-cassette vector could be advantageous, particularly in hematopoietic cell-mediated gene/cancer therapy, by allowing for independent and robust co-expression of therapeutic gene(s) and/or a selectable gene or imaging marker in the same cells.

Standardized programming to reduce the burden of inappropriate therapies in implantable cardioverter defibrillators - Single centre follow up results.

BACKGROUND: Current algorithms and device morphology templates have been proposed in current Implantable Cardioverter-Defibrillators (ICDs) to minimize inappropriate therapies (ITS), but this has not been completely successful. AIM: Assess the impact of a deliberate strategy of using an atrial lead implant with standardized parameters; based on all current ICD discriminators and technologies, on the burden of ITS. METHOD: A retrospective single-centre analysis of 250 patients with either dual chamber (DR) ICDs or biventricular ICDs (CRTDs) over a (41.9 ± 27.3) month period was performed. The incidence of ITS on all ICD and CRTD patients was chronicled after the implementation of standardized programming. RESULTS: 39 events of anti-tachycardial pacing (ATP) and/or shocks were identified in 20 patients (8% incidence rate among patients). The total number of individual therapies was 120, of which 34% were inappropriate ATP, and 36% were inappropriate shocks. 11 patients of the 250 patients received ITS (4.4%). Of the 20 patients, four had ICDs for primary prevention and 16 for a secondary prevention. All the episodes in the primary indication group were inappropriate, while seven patients (43%) of the secondary indication group experienced inappropriate therapies. CONCLUSIONS: The burden of ITS in the population of patients receiving ICDs was 4.4% in the presence of atrial leads. The proposed rationalized programming criteria seems an effective strategy to minimize the burden of inappropriate therapies and will require further validation.

Advances in the treatment of atrial fibrillation.

Management of atrial fibrillation (AF) has changed greatly in the past 10 years. The advent of a greater understanding of the pathophysiology of AF has resulted in major therapeutic breakthroughs, both in invasive and non-invasive strategies. New antiarrhythmic agents with fewer side effects, new anticoagulants and technical advances in ablation have changed the treatment of this condition. Molecular modification of the highly effective amiodarone, to improve safety and tolerability, has produced promising analogues such as Dronedarone. Although this drug seems less effective than amiodarone in preventing AF recurrence, the drug presented an interesting data on reduction of stroke and cardiovascular death, a novel effect that needs further investigation. New antiarrhythmics with atria selectiveness such Vernakalant, might be useful for cardioversion in AF without ventricular proarrhythmia. Dabigatran, a prodrug that directly inhibits thrombin, represents an alternative to warfarin for anticoagulant treatment in selected patients. In AF ablation, technological advances are sure to result in the necessary improvements in the safety and procedures efficacy. These technologies include ablation catheters designed to electrically isolate the pulmonary veins with improved safety, efficacy, speed, and precision and improved imaging and electrical mapping systems. Although pulmonary vein isolation remains essential for most ablation procedures, the role of substrate modification has taken on increasing importance. In this article, we review the advances in the treatment of AF, focus on the new medications and advances in invasive procedures.

Before and After: Comparison of Legacy and Harmonized TCGA Genomic Data Commons' Data.

We present a systematic analysis of the effects of synchronizing a large-scale, deeply characterized, multi-omic dataset to the current human reference genome, using updated software, pipelines, and annotations. For each of 5 molecular data platforms in The Cancer Genome Atlas (TCGA)-mRNA and miRNA expression, single nucleotide variants, DNA methylation and copy number alterations-comprehensive sample, gene, and probe-level studies were performed, towards quantifying the degree of similarity between the 'legacy' GRCh37 (hg19) TCGA data and its GRCh38 (hg38) version as 'harmonized' by the Genomic Data Commons. We offer gene lists to elucidate differences that remained after controlling for confounders, and strategies to mitigate their impact on biological interpretation. Our results demonstrate that the hg19 and hg38 TCGA datasets are very highly concordant, promote informed use of either legacy or harmonized omics data, and provide a rubric that encourages similar comparisons as new data emerge and reference data evolve.

Critical pathways: the time is here for pharmacist involvement. American College of Clinical Pharmacy.

Successful development, implementation, and assessment of the effectiveness of critical pathways involves many processes and tools. Numerous pathways have been developed and the value of this tool in improving patient care has been demonstrated in some patient groups.27,29 Pharmacists are becoming more involved, but the window of opportunity is small. Critical pathways are routinely being utilized to optimally sequence time-appropriate interventions of the interdisciplinary plan of care set forth to achieve patient satisfaction and desired outcomes. Pharmacists must seize the chance to provide pharmaceutical care and assure their participation in the development and implementation of critical pathways.

Polyethylene glycol electrolyte lavage solution (PEG-ELS). A rapid, safe mechanical bowel preparation for colorectal surgery.

The use of polyethylene glycol electrolyte lavage solution (PEG-ELS) as a whole-bowel irrigation for colorectal surgery is retrospectively evaluated in 65 consecutive patients. Sixty-three (97%) of the patients were able to complete the lavage. Fifty-five (85%) of these tolerated the preparation without event. Unpleasant reactions of nausea, vomiting, or fullness resolved in most patients by decreasing the rate of ingestion. Twenty-seven patients had associated medical illnesses that may have been aggravated by volume overload, but none of these experienced any adverse effects from the lavage. Most patients completed the total lavage ingestion of 4 L in 4 hours, which resulted in a 1-day preparation prior to operation. There were no wound or septic complications in any of the lavage patients. This clinical review indicates that PEG-ELS is a safe, rapid, and effective mechanical bowel preparation that should be able to reduce in-hospital preparation time for most patients undergoing colorectal surgery.

Establishing an institution-specific therapeutic range for heparin.

The relationship between heparin concentration and activated partial thromboplastin time (aPTT) in pooled plasma was compared with that in patient samples to assess the feasibility of using heparin-spiked pooled plasma in determining a therapeutic range for aPTT. Blood samples were taken from 32 patients who had been receiving intravenous unfractionated heparin sodium for more than 24 hours. The samples were stored at -70 degrees C until anti-Xa assay within three months of collection. Pooled normal plasma was spiked with unfractionated heparin sodium to produce nominal anti-Xa concentrations of 0, 0.05, 0.1, 0.2, and 0.5 unit/mL. Heparin concentrations and a aPTT values were measured, and the relationship between the two was determined by linear regression. For the ex vivo samples, the range of aPTT values corresponding to therapeutic heparin concentrations of 0.3-0.7 anti-Xa unit/mL was 64-106 seconds, which corresponds to an aPTT range of 2.3-3.9 times the mean of the normal range (compared with the traditionally defined therapeutic range of 1.5-2.5 times the control value). For the in vitro samples, the aPTT range corresponding to heparin concentrations of 0.3-0.7 unit/mL was 121-256 seconds, which corresponds to an aPTT range of 4.4-9.4 times the mean of the normal range. Each institution should establish a therapeutic aPTT range by calibrating aPTT values against heparin concentrations from blood samples of patients receiving intravenous heparin.

Impact of a pharmacist/physician cooperative target drug monitoring program on prophylactic antibiotic prescribing in obstetrics and gynecology.

The pharmacist's role in promoting rational, cost-effective use of drugs has been described in the literature. In a target drug monitoring program (TDMP), a single agent or group of agents becomes targeted for review. Antibiotics have been the primary focus of TDMP because of their therapeutic impact and cost considerations. The objectives of this project were to assess the prophylactic antibiotic prescribing habits of OB/GYN physicians and to evaluate the impact of a pharmacist/physician cooperative TDMP on prophylactic antibiotic prescribing and cost. The study was conducted in three phases: 1) a retrospective chart review of 150 patients, 2) an in-service education session, and 3) a concurrent chart review of 107 patients. Patient selection, timing of preoperative dose, and use of single dose prophylaxis were according to criteria in greater than 90% of patients both before and after the in-service training. Compliance with recommended regimens increased from 45 to 73% after the in-service training. A cost savings was not realized because the physicians wished to use a regimen with anti-anaerobic coverage (i.e., cefotetan) rather than a less expensive agent. However, the cost of selection of resistant organisms must be considered when discouraging the use of multiple broad spectrum agents. Active involvement of the medical staff in a pharmacy-based TDMP produces a cooperative atmosphere in which to educate clinicians and promote rational prescribing habits.

A multidisciplinary approach to patient controlled analgesia.

Patient controlled analgesia (PCA) is an innovative method for delivering intravenous analgesia, which requires therapeutic and technical expertise from various health care professionals. This article describes a multi-disciplinary process of implementing a PCA program including approaches to solving problems encountered. A pharmacy and therapeutics subcommittee was established with various aspects of the program assigned to the medical, nursing and pharmacy staffs, intravenous (IV) therapy team, and clinical equipment support. A detailed comparison of PCA pumps was prepared to aid in selection. A pharmacy-based protocol describes the role of each health care professional. Usage guidelines are presented and evaluated. Physician order sheets and narcotic disposition forms were designed specifically for PCA. Problems encountered include dedicated IV access, PCA use in specialized hospital units, and use in pediatric patients. A multi-disciplinary approach was successful in implementing and maintaining a quality PCA program. Similar approaches should be used for other sophisticated drug-delivery systems.

Left subclavian vein occlusion after pacemaker insertion.

Subclavian vein thrombosis after pacemaker lead insertion is a well-described complication of pacemaker insertion. It is usually asymptomatic due to the presence of collateral venous flow but the clinical presentation is varied. This index case illustrates a typical clinical scenario and the value of computerised tomography (CT) in providing adequate delineation of the venous pathology.

Innovations in the pharmacologic management of heart failure.

Improved understanding of the pathophysiologic course of heart failure has led to many advances in pharmacologic therapy. Angiotensin-converting enzyme inhibitors represent the first effort at targeting neurohormonal activation in chronic heart failure. More recently, beta-adrenergic receptor antagonists have been shown effective in blocking chronic sympathetic nervous system activation. The roles of digoxin and the newer, vasoselective calcium channel blockers in heart failure have been better defined. Other agents targeting the neurohormonal system are under investigation. These include angiotensin-receptor antagonists, aldosterone inhibitors, and endothelin antagonists. Experience with phosphodiesterase inhibitors and adrenergic agents has confirmed the importance of neurohormonal activation in progression of heart failure. Despite angiotensin-converting enzyme inhibitor, diuretic, and digoxin therapy, mortality in heart failure remains high. Careful manipulation of the neurohormonal response to heart failure holds promise for altering the course of the disease.

Effect of analgesic treatment on the physiological consequences of acute pain.

Physiological responses to acute pain are described, and the effects of different analgesic techniques on these responses are discussed. The body's response to acute pain can cause adverse physiological effects. Pain can impede the return of normal pulmonary function, modify certain aspects of the stress response to injury, and alter hemodynamic values and cardiovascular function. It can produce immobility and contribute to thromboembolic complications. In addition, pain can slow a patient's recovery from surgery and contribute to increased morbidity. Fewer pulmonary complications occur when adequate analgesia is provided through the use of epidural narcotics and local anesthetics, particularly if the injury or surgery involves the lower part of the body. Continuous morphine infusions, intercostal nerve blocks, and transcutaneous electrical stimulation do not alter the frequency of pulmonary complications. The effectiveness of patient-controlled analgesia in reducing postoperative pulmonary complications is still not known. Epidural local anesthetic therapy inhibits the stress response, particularly in operations involving the lower abdomen or extremities; this technique is less effective during major abdominal procedures. Suppression of endocrine-metabolic changes following lower abdominal surgery requires neural block to the fourth thoracic segment. Epidural narcotics partially inhibit the stress response after lower abdominal or extremity surgery but not after upper abdominal or thoracic surgery. Local anesthetics applied to the surgical site, intercostal nerve blocks, and intrapleural and intraperitoneal administration also do not modify the stress response. Adequate analgesia through the use of local anesthetics and narcotics postoperatively generally results in improved cardiovascular function, decreased pulmonary morbidity and mortality, earlier ambulation, and decreased likelihood of deep vein thrombosis. Some data suggest that improved patient outcome occurs with adequate analgesia. Block of afferent and efferent neural pathways by local anesthetics seems to be the most effective analgesic modality in lessening the physiologic response to pain and injury.

Whole-bowel irrigation for mechanical colon cleansing.

The physiology, solution composition, indications, efficacy, and safety of whole-bowel irrigation (WBI) for mechanical bowel cleansing are reviewed. WBI with isotonic electrolyte solutions produces diarrhea when the infusion rate exceeds the capacity of the intestine to distend and absorb the solution. A number of solutions are used for WBI, including 0.9% sodium chloride, balanced electrolyte solutions, lactated Ringer's, mannitol, and electrolyte solutions containing polyethylene glycol 3350 (PEG). WBI solution administration rates vary from 15-90 mL/min, by oral ingestion or nasogastric tube, with total volumes ranging from 1 to 20 L. The onset of diarrhea occurs as soon as 20 minutes with clearing of the effluent as early as 90 minutes. Faster administration rates appear to shorten overall cleansing time. Two PEG-electrolyte lavage solutions (ELSs) have recently gained FDA approval. The recommended dosage rate is 1.2-1.8 L/hr orally or by nasogastric tube until rectal effluent is clear. In most patients, this requires a maximum of 4-6 L. Initial data indicate that PEG-ELSs are safe for elderly patients and for patients who have an increased risk of fluid overload, but these solutions have not been evaluated in children, pregnant women, or patients with inflammatory bowel disease. WBI is an effective alternative to other regimens for removing fecal material and reducing bowel lumen bacterial counts before colonoscopy and colorectal surgery. Retention of bacterial counts before colonoscopy and colorectal surgery. Retention of excess WBI solution may interfere with the quality of barium enema radiographs; this can be minimized by completing the irrigation the evening before the examination. Gastrointestinal side effects occur in about one third of the patients following WBI, but do not generally require discontinuing the irrigation. Solutions containing PEG with sodium sulfate as the primary electrolyte result in the least net water and electrolyte movement and are preferred over other solutions.

Effect of hemorrhagic shock on cefazolin and gentamicin pharmacokinetics in dogs.

The physiologic response to traumatic injury may alter the disposition of drugs and thereby affect their therapeutic or toxic potential. A study was conducted in 10 mongrel dogs to determine the effect of experimental hemorrhagic shock with resuscitation on the pharmacokinetics of gentamicin and cefazolin. Single simultaneous intravenous doses of gentamicin (3 mg/kg) and cefazolin (25 mg/kg) were administered to each animal on an initial study day, after which serial blood and urine collections were performed. After 1 week, a standard hemorrhagic shock model was applied to each animal. Shock was continued for 1 h, after which the animal was resuscitated with either whole blood or saline. After stabilization for 20 min, a second dose of gentamicin and cefazolin was administered, and blood and urine were again collected. Drug clearance was not significantly altered, except for that of cefazolin after saline resuscitation, for which there was a significant increase in drug clearance. After both methods of resuscitation an increase in the volume of distribution was noted for cefazolin and gentamicin. Drug half-life was noted to be increased after shock for cefazolin by both resuscitation methods and for gentamicin after shock by saline resuscitation. Although alterations of pharmacokinetic parameters were noted, mean concentrations of gentamicin and cefazolin in serum were similar for pre- and postshock phases.

The evaluation of postoperative function of the adrenal gland.

The cosyntropin (Cortrosyn) stimulation test has been adapted for use in the postoperative period. The normal adrenal gland response to 200 micrograms of cosyntropin given intravenously has been quantified at six, 12, 24, 48 and 72 hours after extensive general surgical procedures. The value of the test in quickly and accurately diagnosing postoperative acute insufficiency of the adrenal gland remains to be established.

The serum and urinary cortisol response to operative trauma.

Serum and urinary cortisol levels were monitored throughout the perioperative period in eight patients who underwent elective extensive general surgical procedures. Significant elevations of serum cortisol levels occurred four and eight hours after anesthetic induction and were reflected by elevated urinary cortisol production on the day of operation. Cortisol serum levels and urinary production rapidly returned toward normal as all subsequent measurements were insignificantly different from preoperative values. Adrenal gland stimulation resulting from general surgical procedures is intense but short-lived.