Impact of queuosine modification of endogenous tRNAs on sense codon reassignment.

The extent to which alteration of endogenous tRNA modifications may be exploited to improve genetic code expansion efforts has not been broadly investigated. Modifications of tRNAs are strongly conserved evolutionarily, but the vast majority of tRNA modifications are not essential. We identified queuosine (Q), a non-essential, hypermodified guanosine nucleoside found in position 34 of the anticodons of four  tRNAs as a modification that could potentially be utilized to improve sense codon reassignment.

Directed Evolution Pipeline for the Improvement of Orthogonal Translation Machinery for Genetic Code Expansion at Sense Codons.

The expansion of the genetic code beyond a single type of noncanonical amino acid (ncAA) is hindered by inefficient machinery for reassigning the meaning of sense codons. A major obstacle to using directed evolution to improve the efficiency of sense codon reassignment is that fractional sense codon reassignments lead to heterogeneous mixtures of full-length proteins with either a ncAA or a natural amino acid incorporated in response to the targeted codon. In stop codon suppression systems, missed incorporations lead to truncated proteins; improvements in activity may be inferred from increased protein yields or the production of downstream reporters.

The Influence of Competing tRNA Abundance on Translation: Quantifying the Efficiency of Sense Codon Reassignment at Rarely Used Codons.

A quantitative understanding of how system composition and molecular properties conspire to determine the fidelity of translation is lacking. Our strategy directs an orthogonal tRNA to directly compete against endogenous tRNAs to decode individual targeted codons in a GFP reporter. Sets of directed sense codon reassignment measurements allow the isolation of particular factors contributing to translational fidelity.

Mapping the Plasticity of the Escherichia coli Genetic Code with Orthogonal Pair-Directed Sense Codon Reassignment.

The relative quantitative importance of the factors that determine the fidelity of translation is largely unknown, which makes predicting the extent to which the degeneracy of the genetic code can be broken challenging. Our strategy of using orthogonal tRNA/aminoacyl tRNA synthetase pairs to precisely direct the incorporation of a single amino acid in response to individual sense and nonsense codons provides a suite of related data with which to examine the plasticity of the code. Each directed sense codon reassignment measurement is an in vivo competition experiment between the introduced orthogonal translation machinery and the natural machinery in Escherichia coli.

Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment.

Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M.

Evaluating Sense Codon Reassignment with a Simple Fluorescence Screen.

Understanding the interactions that drive the fidelity of the genetic code and the limits to which modifications can be made without breaking the translational system has practical implications for understanding the molecular mechanisms of evolution as well as expanding the set of encodable amino acids, particularly those with chemistries not provided by Nature. Because 61 sense codons encode 20 amino acids, reassigning the meaning of sense codons provides an avenue for biosynthetic modification of proteins, furthering both fundamental and applied biochemical research. We developed a simple screen that exploits the absolute requirement for fluorescence of an active site tyrosine in green fluorescent protein (GFP) to probe the pliability of the degeneracy of the genetic code.

Malignant Course of Anomalous Left Coronary Artery Causing Sudden Cardiac Arrest: A Case Report and Review of the Literature.

Sudden cardiac arrest has been reported to occur in patients with congenital anomalous coronary artery disease. About 80% of the anomalies are benign and incidental findings at the time of catheterization. We present a case of sudden cardiac arrest caused by anomalous left anterior descending artery.

Nurse retention in a correctional facility: a study of the relationship between the nurses' perceived barriers and benefits.

Retention of nursing staff is more complex in a correctional facility. After a period of 3 years, only 20% of the staff remained employed at this study facility. Without retention of qualified correctional nurses, there are decreases in access to care, gaps in continuity of care, and less time for mentorship.

Gastroesophageal reflux disease: current treatment approaches.

Gastroesophageal reflux disease is a common, usually lifelong, disorder resulting from chronic abnormal exposure of the lower esophagus to gastric contents. Motor dysfunction of the lower esophageal sphincter is the primary cause of this disease. At this writing, no medical therapies can completely resolve abnormal lower esophageal sphincter function; therefore, the treatment of gastroesophageal reflux disease centers on suppression of intragastric acid secretion.

Acetylcysteine in the prevention of contrast-induced nephropathy after coronary angiography.

Background: Contrast-induced nephropathy (CIN) after coronary angiography is associated with increased morbidity and mortality rates. Preliminary studies with N-acetylcysteine (NAC) have found conflicting results in the prevention of CIN in patients undergoing coronary angiography. This study was designed to evaluate the efficacy and safety of NAC in the prevention of CIN in patients undergoing coronary angiography.

Isolated noncompaction cardiomyopathy presenting with paroxysmal supraventricular tachycardia--case report and literature review.

Isolated noncompaction cardiomyopathy is an exceedingly rare congenital cardiomyopathy. A case of isolated noncompaction cardiomyopathy is reported and the literature on the subject collected through a comprehensive literature search is reviewed. Fewer than 100 cases of this condition have been reported.

Medical student and faculty perceptions of desirable primary care teaching site characteristics.

Background: As the need to expand and improve primary care teaching experiences has mushroomed, the need to identify desirable preceptor and site characteristics has also grown.

Purpose: The current study was designed to assess the relative importance students and preceptors place on site versus preceptor characteristics.

Methods: After a required year-long primary care experience, third-year medical students (n=39) and primary care preceptors (n=20) completed a Q-sort exercise.

Growth of NS0 cells in protein-free, chemically defined medium.

Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein-free media. Compared with serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum-supplemented medium or a serum-free medium supplemented with an animal-derived lipoprotein.

Animal serum-free culture of purified human CD34+ cells: amplification of progenitors from G-CSF and GM-CSF-mobilized peripheral blood.

The isolation and culture of human CD34+ cells could have broad clinical application for hematologic support following high-dose chemotherapy or bone marrow transplantation. The need for reproducible, animal product-free conditions for the culture of progenitors is crucial to the widespread clinical implementation of ex vivo cell therapies. In these studies, we explored the use of animal serum-free (ASF) medium for the culture of isolated human bone marrow and peripheral blood CD34+ cells.

Medical students' perceptions of desirable characteristics of primary care teaching sites.

Purpose: With the increased need to establish primary care sites, we must identify the key characteristics of effective primary care training experiences.

Methods: After completing a required year-long primary care experience, third-year medical students participated in focus groups (n = 17) and completed a Q-sort exercise with three sets of items: preceptor characteristics, site characteristics, and a combination of preceptor and site characteristics (n = 59).

Results: Thirty-eight concepts were identified by content analysis, which were grouped into six major categories: patients, preceptors' teaching behaviors, characteristics of office and staff, preceptors' personal characteristics, programmatic issues, and educational opportunities.

Rapid isolation and serum-free expansion of human CD34+ cells.

Human CD34+ cells were isolated from bone marrow from normal volunteers and expanded under serum-free culture conditions. CD34+ cells were cultured with interleukin-3 (IL-3), IL-1, and stem cell factor and expanded in granulocyte-macrophage colony-forming units, erythroid blast-forming units, and CD34+ cell number during the first 7-14 days of incubation. By contrast, cultures maintained in fetal calf serum under identical conditions showed much reduced expansion, as measured by all of the above parameters.

Terminal ileal mucosal mast cells in irritable bowel syndrome.

Terminal ileal biopsies were prospectively obtained and stained specifically for mast cells in 20 patients with irritable bowel syndrome (IBS) and 15 controls. The number of terminal ileal mast cells per high powered field (MC/HPF) (mean +/- SEM) was 23.3 +/- 3.

Evaluation of anorectal physiology in patients with increased mast cells.

Diarrhea, urgency, and fecal incontinence are common complaints in systemic mastocytosis and in patients with increased gastrointestinal mucosal mast cells. We performed anorectal manometry on six patients with clinical symptoms of mastocytosis and histologic evidence of increased mast cells and compared the results to anorectal manometry of six age- and sex-matched controls, with no bowel symptoms. Standard techniques with balloon volumes were used to measure maximal basal pressure, maximal squeeze pressure, smallest volume sensed, degree of relaxation of the internal sphincter, and the volume causing: (1) a strong urge to defecate and (2) pain.

Ten-year experience with mitral valve replacement in the elderly.

Limited data are available on mitral valve replacement in the elderly patient. Therefore we report our 10-year experience including predictors of perioperative mortality and subsequent long-term cardiac mortality in elderly patients with mitral valve replacement compared to younger patients. Of the 126 consecutive patients with mitral valve replacement, 26 were older (77 +/- 4, group 1) and 100 were younger (62 +/- 9, group 2) than 70 years.

Colonic mucosal mast cell distribution at line of demarcation of active ulcerative colitis.

We examined the distribution of colonic mucosal mast cells in 25 patients with active ulcerative colitis, with a clear line of demarcation separating active inflammation from normal mucosa. Biopsies, at least one adjacent to the line of demarcation, one in inflamed mucosa, and one above were obtained during colonoscopy. Eight patients had elevated mast cells throughout the colon, and 12 had increased numbers at the line of demarcation of disease.