Annamarie D. Horan

Critical Care Nurse Practitioners Improve Compliance With Clinical Practice Guidelines in “semiclosed” Surgical Intensive Care Unit

This prospective study examined whether the integration of acute care nurse practitioners (ACNP) in a “semiclosed” surgical intensive care unit (SICU) model increased compliance with clinical practice guidelines (CPG). Patients were admitted to critical care services with a (a) “semiclosed”/ACNP team or (b) “mandatory consultation”/non-ACNP team. CPG compliance was significantly higher (P < .05) on the “semiclosed”/ACNP team for all 3 CPGs examined in the study.

The Km for Radiosensitization of Human Tumor Cells by Oxygen is Much Greater than 3 mmHg and is Further Increased by Elevated Levels of Cysteine

Abstract Horan, A. D. and Koch, C. J. The Km for Radiosensitization of Human Tumor Cells by Oxygen is Much Greater than 3 mmHg and is Further Increased by Elevated Levels of Cysteine. Radiat. Res. 156, 388–398 (2001). We studied the role of cysteine as an intracellular radiation protector under conditions in which both oxygen and thiols were monitored at 37°C. In HCT-116 human colon cancer cells, the intracellular cysteine content affects the radiation survival dramatically at intermediate oxygen levels, but not at zero or high oxygen levels. Using a spin-through-oil method with a dual radioactive label detection system, we measured intracellular cysteine and glutathione (GSH) levels for cells in suspension culture. A comparison of the cysteine levels of monolayer cells lysed in situ and of trypsinized monolayer cells in suspension (Horan et al., Cytometry 29, 76–82, 1997) revealed that, upon trypsinization from monolayer culture and transfer to a spinner apparatus at 37°C, HCT-116 cells lose most of their intracellular cysteine. Over the 60-min time course of control experiments, these cells do not recover intracellular cysteine despite the availability of cystine (the disulfide of cysteine) in the medium. When cells in spinner culture are provided with exogenous cysteine, they initially concentrate it to 10-fold the extracellular concentration, with the concentration factor decreasing to about 5-fold over the course of an hour. The intracellular GSH concentration changes little throughout this period, regardless of the changes in cysteine levels. The same apparatus was used to assess the survival of HCT-116 cells irradiated at 37°C under conditions of constant pO2 monitoring. For cells without added cysteine, the oxygen concentration for half-maximal radiation sensitivity was about 7.5 mmHg (intermediate hypoxia), more than twice the commonly accepted value (3 mmHg). At 7.5 mmHg, cells with added cysteine (intracellular concentration 3.5 mM) were almost as radioresistant as severely hypoxic cells (≈0.005% oxygen). Cells in parallel experiments in which the cells were grown in monolayers on glass Petri dishes had intermediate cysteine values and also intermediate radiosensitivity. We conclude that the radiation response of cells at intermediate oxygen levels is controlled predominantly by intracellular cysteine levels and that the cysteine levels commonly found in tumors may increase the Km for radiosensitivity to values much higher than suggested previously.

Radiation-sensitive tyrosine phosphorylation of cellular proteins: sensitive to changes in GSH content induced by pretreatment with n’acetyl-L-cysteine or L-buthionine-S,R-sulfoximine

PURPOSE At relatively high concentrations, ie., > 20 mM, N-acetyl-L-cysteine (NAC) scavenges reactive oxygen species produced by ionizing radiation in aqueous solution. Therefore, the ability of NAC to block signal transduction reactions in vivo, has lead to the suggestion that ROS are necessary for the normal propagation of these signals. In this paper we investigate the mechanism by which NAC alters signal transduction in whole cells. RESULTS Exposing CHO-K1 cells to ionizing radiation results in elevated pp59fyn kinase activity. Moreover, we observe changes in the phosphotyrosine content of multiple cellular proteins, including one prominent phosphotyrosyl protein with a Mr of 85 kDa. Both the radiation-induced changes in pp59fyn kinase activity and the changes in phosphotyrosine content of pp85 were not affected by exposing K1 cells to NAC during the time of irradiation, suggesting that ROS generated extracellularly are not involved in the radiation-induced changes observed in phosphotyrosyl proteins. We also demonstrate that the cell membrane is an effective barrier against negatively charged NAC. Therefore, it seems unlikely that NACs ability to block signal transduction reactions is related to scavenging of ROS intracellularly. Chronic exposure, ie., 1 h, to 20 mM NAC lead to a twofold elevation in GSH levels and resulted in a 17% decrease in the phosphotyrosine content of pp85 after exposure to 10 Gy. Moreover, pretreatment with L-buthionine-S,R-sulfoximine (BSO) decreased GSH levels and resulted in elevated phosphotyrosine levels in pp85 isolated from irradiated CHO-K1 cells. CONCLUSIONS Since many signaling molecules contain redox sensitive cysteine residues that regulate enzyme activity, we suggest that the effects of NAC on radiation-induced signal transduction are due to its ability to alter the intracellular reducing environment, and not related to direct scavenging of ROS.

Culture-independent pilot study of microbiota colonizing open fractures and association with severity, mechanism, location, and complication from presentation to early outpatient follow-up

Precise identification of bacteria associated with post‐injury infection, co‐morbidities, and outcomes could have a tremendous impact in the management and treatment of open fractures. We characterized microbiota colonizing open fractures using culture‐independent, high‐throughput DNA sequencing of bacterial 16S ribosomal RNA genes, and analyzed those communities with respect to injury mechanism, severity, anatomical site, and infectious complications. Thirty subjects presenting to the Hospital of the University of Pennsylvania for acute care of open fractures were enrolled in a prospective cohort study. Microbiota was collected from wound center and adjacent skin upon presentation to the emergency department, intraoperatively, and at two outpatient follow‐up visits at approximately 25 and 50 days following initial presentation. Bacterial community composition and diversity colonizing open fracture wounds became increasingly similar to adjacent skin microbiota with healing. Mechanism of injury, severity, complication, and location were all associated with various aspects of microbiota diversity and composition. The results of this pilot study demonstrate the diversity and dynamism of the open fracture microbiota, and their relationship to clinical variables. Validation of these preliminary findings in larger cohorts may lead to the identification of microbiome‐based biomarkers of complication risk and/or to aid in management and treatment of open fractures.

Effect of Oxygen on Radiation-Induced DNA Damage in Isolated Nuclei

In intact mammalian cells, ionizing radiation causes substantially less damage to DNA in the absence of oxygen than in the presence of oxygen. In contrast, when DNA is isolated (usually from viruses) and irradiated in solution, the absence of oxygen does not lead to a decrease in damage unless low-molecular-weight thiols are also present. We investigated an intermediate condition: that of DNA irradiated in isolated nuclei. Using an HPLC-based assay of thiols with electrochemical detection, we have determined that the nuclear isolation procedure leads to the elimination of virtually all low-molecular-weight thiols (predominantly glutathione and cysteine). Thus it was our expectation that the thiol-depleted state would concurrently eliminate the OER, and thereby mimic the isolated DNA system, while retaining structural characteristics of chromosomal DNA. We evaluated radiation-induced DNA damage in isolated nuclei by measuring single-strand breaks using alkaline elution and by measuring double-strand breaks using neutral elution and pulsed-field gel electrophoresis. Despite the removal of low-molecular-weight thiol compounds, the oxygen dependence of radiation-induced damage more closely paralleled that of whole cells than that of DNA in solution. Thus damage of DNA irradiated in isolated nuclei is dependent on oxygen.

Analysis of Tumor Thiol Concentrations: Comparison of Flow Cytometric With Chemical and Biochemical Techniques

The importance of glutathione (GSH) in contributing to cancer therapy resistance is well established. Various advantages may accrue from the ability to determine the distribution of GSH content in individual tumor cells disaggregated from solid tumors using flow cytometric techniques compared with biochemical or chemical measurements of the average GSH level in bulk tissue samples. The flow cytometric technique requires a thiol-reactive fluorescent adduct which is stable and which can differentiate cellular GSH from protein thiols. Thiol-reactive compounds specific for GSH require facilitated conjugation by endogenous cellular enzymes, but such compounds have not been found to accurately monitor GSH in human cells. Compounds which react generally with all thiols require a GSH-depleted calibration control to assess GSH vs. non-GSH components of fluorescent-adduct formation. Our report addresses this question, and compares three different thiol assays in several cell lines. We have found a simple way to control for non-GSH adduct formation. This involves a selective permeabilization process to release low molecular weight adducts (dominated by GSH and cysteine). In application to the desired goal of assessing the distribution of GSH in cells disaggregated from tumors, we have identified a problem with cell-line-specific thiol loss during the tumor cell disaggregation process.

The microbiota of traumatic, open fracture wounds is associated with mechanism of injury: Traumatic wound microbiome

Open fractures are characterized by disruption of the skin and soft tissue, which allows for microbial contamination and colonization. Preventing infection‐related complications of open fractures and other acute wounds remains an evolving challenge due to an incomplete understanding of how microbial colonization and contamination influence healing and outcomes. Culture‐independent molecular methods are now widely used to study human‐associated microbial communities without introducing culture biases. Using such approaches, the objectives of this study were to (1) define the long‐term temporal microbial community dynamics of open fracture wounds and (2) examine microbial community dynamics with respect to clinical and demographic factors. Fifty‐two subjects with traumatic open fracture wounds (32 blunt and 20 penetrating injuries) were enrolled prospectively and sampled longitudinally from presentation to the emergency department (ED) and at each subsequent inpatient or outpatient encounter. Specimens were collected from both the wound center and adjacent skin. Culture‐independent sequencing of the 16S ribosomal RNA gene was employed to identify and characterize microbiota. Upon presentation to the ED and time points immediately following, sample collection site (wound or adjacent skin) was the most defining feature discriminating microbial profiles. Microbial composition of adjacent skin and wound center converged over time. Mechanism of injury most strongly defined the microbiota after initial convergence. Further analysis controlling for race, gender, and age revealed that mechanism of injury remained a significant discriminating feature throughout the continuum of care. We conclude that the microbial communities associated with open fracture wounds are dynamic in nature until eventual convergence with the adjacent skin community during healing, with mechanism of injury as an important feature affecting both diversity and composition of the microbiota. A more complete understanding of the factors influencing microbial contamination and/or colonization in open fractures is a critical foundation for identifying markers indicative of outcome and deciphering their respective contributions to healing and/or complication.

Combined RT-qPCR of mRNA and microRNA Targets within One Fluidigm Integrated Fluidic Circuit

The ability to profile expression levels of a large number of mRNAs and microRNAs (miRNAs) within the same sample, using a single assay method, would facilitate investigations of miRNA effects on mRNA abundance and streamline biomarker screening across multiple RNA classes. A protocol is described for reverse transcription of long RNA and miRNA targets, followed by preassay amplification of the pooled cDNAs and quantitative PCR (qPCR) detection for a mixed panel of candidate RNA biomarkers. The method provides flexibility for designing custom target panels, is robust over a range of input RNA amounts, and demonstrated a high assay success rate.

Pilot Randomized Controlled Trial of Spinal Versus General Anesthesia for Hip Fracture Surgery

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Republication: Incidental findings on intensivist bedside ultrasonographic examinations: Why should we care?

Introduction: The primary goal of intensivist bedside ultrasonography (INBU) is the assessment of patient hemodynamic and volume status. Inevitably, INBU examinations provide views of various thoracic and abdominal structures. Despite the rapid recent increase in utilization of INBU, there are no published descriptions of incidental findings and/or their significance in this setting. Methods: Echocardiographic and vena cava examinations were performed by noncardiologist intensivists in 124 Surgical Intensive Care Unit (SICU) patients using hand-carried ultrasound. In addition, any findings that were deemed “incidental” were recorded. Information analyzed included patient demographics, time to complete INBU examination, and the nature of each incidental finding. Incidental findings were grouped into cardiac, pulmonary, and abdominal. To determine whether incidental INBU findings may have influenced subsequent diagnostic testing patterns, radiographic, and echocardiographic examinations directly relevant to the INBU findings and performed within 48 h of the INBU examination were reviewed. Results: Fifty-eight out of 124 (46.8%) patients in the study group had at least one incidental finding. There were 86 incidental findings, with 23 patients having more than one incidental finding. Forty-eight of 86 incidental findings (55.8%) were cardiac-related, 30 (34.9%) were pulmonary-related, and 8 (9.30%) were abdominal. There were significantly more diagnostic tests performed within 48 h of INBU in the incidental finding group (1.56/patient) than in the nonincidental group (1.18/patient, P< 0.04). The most common post-INBU diagnostic tests were chest radiogram (62%), formal trans-thoracic echocardiography (21%), and abdominal roentgenogram (14%). Computed tomography, formal abdominal ultrasonography, and trans-esophageal echocardiograms were performed less often. Four of 58 patients (6.9%) had a significant change in clinical management associated with the incidental INBU findings. One patient underwent percutaneous drainage of a pleural effusion; three underwent formal echocardiography, with subsequent change in medical management. Conclusions: Nearly half of all SICU patients who underwent INBU were found to have at least one incidental finding. The presence of an incidental finding may have influenced the subsequent pattern of clinical diagnostic testing. In addition, incidental findings on INBU were associated with a significant change in clinical management in nearly 7% of patients. The following core competencies are addressed in this article: Medical knowledge, Patient care, Practice based learning and improvement, Systems based practice. Republished with permission from: Stawicki SP, Shiroff AM, Hayden GE, Panebianco NL, Kirkpatrick JN, Horan AD, Gracias VH, Dean AJ. OPUS 12 Scientist 2008;2(3):11-14.