Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning.

Loss of tuberin, the product of TSC2 gene, increases mammalian target of rapamycin (mTOR) signaling, promoting cell growth and tumor development. However, in cells expressing tuberin, it is not known how repression of mTOR signaling is relieved to activate this pathway in response to growth factors and how hamartin participates in this process. We show that hamartin colocalizes with hypophosphorylated tuberin at the membrane, where tuberin exerts its GTPase-activating protein (GAP) activity to repress Rheb signaling. In response to growth signals, tuberin is phosphorylated by AKT and translocates to the cytosol, relieving Rheb repression. Phosphorylation of tuberin at serines 939 and 981 does not alter its intrinsic GAP activity toward Rheb but partitions tuberin to the cytosol, where it is bound by 14-3-3 proteins. Thus, tuberin bound by 14-3-3 in response to AKT phosphorylation is sequestered away from its membrane-bound activation partner (hamartin) and its target GTPase (Rheb) to relieve the growth inhibitory effects of this tumor suppressor.

Comparison of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR assay to culture by use of BBL CHROMagar MRSA for detection of MRSA in nasal surveillance cultures from intensive care unit patients.

This study compared the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) real-time PCR assay to culture by the use of BBL CHROMagar MRSA for the detection of MRSA in 627 nasal surveillance specimens collected from intensive care unit (ICU) patients. The PCR assay had a sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 96.7%, 70.3%, and 100%, respectively. Nine of 19 false-positive PCR specimens grew methicillin-susceptible S. aureus (MSSA) from broth enrichment culture, of which two demonstrated evidence of mecA gene dropout. Compared to culture by the use of BBL CHROMagar MRSA, the BD GeneOhm MRSA PCR assay demonstrated sensitivity and specificity above 95% for the detection of MRSA nasal colonization and provided shorter turnaround time in generating positive and negative final results.

A step-by-step procedure to analyze the efficacy of siRNA using real-time PCR.

Knockdown of cellular RNA using short interfering RNA has enabled researchers to perform loss-of-function (LOF) experiments in a wide variety of cell types and model systems. RNA interference techniques and reagents have made possible experiments that test everything from the analysis of function of single genes to screening for genes that are involved in critical biological pathways on a genome-wide scale. Although siRNA experiments are generally common practice in research laboratories, it is still important to keep in mind that many factors can influence efficacy of knockdown. A properly designed siRNA, optimized protocols of siRNA delivery, and an appropriate and well-optimized readout are all critical parameters for ensuring the success of your experiment. In this chapter, we provide step-by-step procedures for performing an siRNA knockdown experiment from cell culture to analysis of knockdown using quantitative real-time PCR.

Prospective, randomized evaluation of a personal digital assistant-based research tool in the emergency department.

BACKGROUND: Personal digital assistants (PDA) offer putative advantages over paper for collecting research data. However, there are no data prospectively comparing PDA and paper in the emergency department. The aim of this study was to prospectively compare the performance of PDA and paper enrollment instruments with respect to time required and errors generated. METHODS: We randomized consecutive patients enrolled in an ongoing prospective study to having their data recorded either on a PDA or a paper data collection instrument. For each method, we recorded the total time required for enrollment, and the time required for manual transcription (paper) onto a computer database. We compared data error rates by examining missing data, nonsensical data, and errors made during the transcription of paper forms. Statistical comparisons were performed by Kruskal-Wallis and Poisson regression analyses for time and errors, respectively. RESULTS: We enrolled 68 patients (37 PDA, 31 paper). Two of 31 paper forms were not available for analysis. Total data gathering times, inclusive of transcription, were significantly less for PDA (6:13 min per patient) compared to paper (9:12 min per patient; p < 0.001). There were a total of 0.9 missing and nonsense errors per paper form compared to 0.2 errors per PDA form (p < 0.001). An additional 0.7 errors per paper form were generated during transcription. In total, there were 1.6 errors per paper form and 0.2 errors per PDA form (p < 0.001). CONCLUSION: Using a PDA-based data collection instrument for clinical research reduces the time required for data gathering and significantly improves data integrity.

Pretest probability assessment derived from attribute matching.

BACKGROUND: Pretest probability (PTP) assessment plays a central role in diagnosis. This report compares a novel attribute-matching method to generate a PTP for acute coronary syndrome (ACS). We compare the new method with a validated logistic regression equation (LRE). METHODS: Eight clinical variables (attributes) were chosen by classification and regression tree analysis of a prospectively collected reference database of 14,796 emergency department (ED) patients evaluated for possible ACS. For attribute matching, a computer program identifies patients within the database who have the exact profile defined by clinician input of the eight attributes. The novel method was compared with the LRE for ability to produce PTP estimation <2% in a validation set of 8,120 patients evaluated for possible ACS and did not have ST segment elevation on ECG. 1,061 patients were excluded prior to validation analysis because of ST-segment elevation (713), missing data (77) or being lost to follow-up (271). RESULTS: In the validation set, attribute matching produced 267 unique PTP estimates [median PTP value 6%, 1st-3rd quartile 1-10%] compared with the LRE, which produced 96 unique PTP estimates [median 24%, 1st-3rd quartile 10-30%]. The areas under the receiver operating characteristic curves were 0.74 (95% CI 0.65 to 0.82) for the attribute matching curve and 0.68 (95% CI 0.62 to 0.77) for LRE. The attribute matching system categorized 1,670 (24%, 95% CI = 23-25%) patients as having a PTP < 2.0%; 28 developed ACS (1.7% 95% CI = 1.1-2.4%). The LRE categorized 244 (4%, 95% CI = 3-4%) with PTP < 2.0%; four developed ACS (1.6%, 95% CI = 0.4-4.1%). CONCLUSION: Attribute matching estimated a very low PTP for ACS in a significantly larger proportion of ED patients compared with a validated LRE.

Prospective study of clinician-entered research data in the Emergency Department using an Internet-based system after the HIPAA Privacy Rule.

BACKGROUND: Design and test the reliability of a web-based system for multicenter, real-time collection of data in the emergency department (ED), under waiver of authorization, in compliance with HIPAA. METHODS: This was a phase I, two-hospital study of patients undergoing evaluation for possible pulmonary embolism. Data were collected by on-duty clinicians on an HTML data collection form (prospective e-form), populated using either a personal digital assistant (PDA) or personal computer (PC). Data forms were uploaded to a central, offsite server using secure socket protocol transfer. Each form was assigned a unique identifier, and all PHI data were encrypted, but were password-accessible by authorized research personnel to complete a follow-up e-form. RESULTS: From April 15, 2003-April 15 2004, 1022 prospective e-forms and 605 follow-up e-forms were uploaded. Complexities of PDA use compelled clinicians to use PCs in the ED for data entry for most forms. No data were lost and server log query revealed no unauthorized entry. Prospectively obtained PHI data, encrypted upon server upload, were successfully decrypted using password-protected access to allow follow-up without difficulty in 605 cases. Non-PHI data from prospective and follow-up forms were available to the study investigators via standard file transfer protocol. CONCLUSIONS: Data can be accurately collected from on-duty clinicians in the ED using real-time, PC-Internet data entry in compliance with the Privacy Rule. Deidentification-reidentification of PHI was successfully accomplished by a password-protected encryption-deencryption mechanism to permit follow-up by approved research personnel.

Accuracy of very low pretest probability estimates for pulmonary embolism using the method of attribute matching compared with the Wells score.

OBJECTIVES: Attribute matching matches an explicit clinical profile of a patient to a reference database to estimate the numeric value for the pretest probability of an acute disease. The authors tested the accuracy of this method for forecasting a very low probability of venous thromboembolism (VTE) in symptomatic emergency department (ED) patients. METHODS: The authors performed a secondary analysis of five data sets from 15 hospitals in three countries. All patients had data collected at the time of clinical evaluation for suspected pulmonary embolism (PE). The criterion standard to exclude VTE required no evidence of PE or deep venous thrombosis (DVT) within 45 days of enrollment. To estimate pretest probabilities, a computer program selected, from a large reference database of patients previously evaluated for PE, patients who matched 10 predictor variables recorded for each current test patient. The authors compared the outcome frequency of having VTE [VTE(+)] in patients with a pretest probability estimate of <2.5% by attribute matching, compared with a value of 0 from the Wells score. RESULTS: The five data sets included 10,734 patients, and 747 (7.0%, 95% confidence interval [CI] = 6.5% to 7.5%) were VTE(+) within 45 days. The pretest probability estimate for PE was <2.5% in 2,975 of 10,734 (27.7%) patients, and within this subset, the observed frequency of VTE(+) was 48 of 2,975 (1.6%, 95% CI = 1.2% to 2.1%). The lowest possible Wells score (0) was observed in 3,412 (31.7%) patients, and within this subset, the observed frequency of VTE(+) was 79 of 3,412 (2.3%, 95% CI = 1.8% to 2.9%) patients. CONCLUSIONS: Attribute matching categorizes over one-quarter of patients tested for PE as having a pretest probability of <2.5%, and the observed rate of VTE within 45 days in this subset was <2.5%.

Hydrogen transfer from supercritical methanol over a solid base catalyst: a model for lignin depolymerization.

A (super)critical transfer: The consecutive hydrogenolysis and hydrogenation of the lignin model compound dihydrobenzofuran was studied in supercritical methanolic solutions using porous metal oxide catalysts. These catalysts promote H(2) production from methanol followed by hydrogenolysis of the ether linkages and reduction of the aromatic rings, leading principally to a mixture of cyclohexanols.

Frequency of thromboprophylaxis and incidence of in-hospital venous thromboembolism in a cohort of emergency department patients.

OBJECTIVE: Prior work suggests that in-hospital pulmonary and venous thromboembolism (VTE) could be decreased if the rate of prophylaxis for VTE in high-risk patients were increased at the time of admission. Our objective was to quantify the rate of thromboprophylaxis and incidence of in-hospital VTE, based upon risk of VTE, in a cohort of patients admitted through the emergency department (ED). METHODS: We performed a prospective cohort study at an urban ED with >100,000 visits. All medical patients >17 years admitted from the ED were prospectively identified on a random sample of days for one year. Using a structured data form we collected each patient's risk factors for VTE, and prophylaxis measures. We computed a validated risk score of each patient, with a score >or=4 high-risk (HR) and a score <4 low risk (LR). The main outcome was VTE during the hospitalization, diagnosed after admission from ED. RESULTS: Of 4732 patients, VTE was diagnosed during hospitalization in 44 (0.9%). 437 (9%) patients were HR for VTE and HR patients had significantly higher frequency of VTE vs. LR patients, 1.8 vs. 0.8% (95% CI for difference of 1% = 0.1-3%). Only 36% of HR patients received thromboprophylaxis. There were no significant differences in the frequency of observed inpatient VTE events between patients who were prescribed prophylaxis compared with those who were not prescribed prophylaxis in either risk group. CONCLUSION: These data suggest only a modest opportunity for ED-based policy for thromboprophylaxis in admitted medical patients.

AMP-activated protein kinase signaling results in cytoplasmic sequestration of p27.

Tuberin, the Tsc2 gene product, integrates the phosphatidylinositol 3-kinase/mitogen-activated protein kinase (mitogenic) and LKB1/AMP-activated protein kinase (AMPK; energy) signaling pathways, and previous independent studies have shown that loss of tuberin is associated with elevated AMPK signaling and altered p27 function. In Tsc2-null tumors and tumor-derived cells from Eker rats, we observed elevated AMPK signaling and concordant cytoplasmic mislocalization of p27. Cytoplasmic localization of p27 in Tsc2-null cells was reversible pharmacologically using inhibitors of the LKB1/AMPK pathway, and localization of p27 to the cytoplasm could be induced directly by activating AMPK physiologically (glucose deprivation) or genetically (constitutively active AMPK) in Tsc2-proficient cells. Furthermore, AMPK phosphorylated p27 in vitro on at least three sites including T170 near the nuclear localization signal, and T170 was shown to determine p27 localization in response to AMPK signaling. p27 functions in the nucleus to suppress cyclin-dependent kinase-2 (Cdk2) activity and has been reported to mediate an antiapoptotic function when localized to the cytoplasm. We found that cells with elevated AMPK signaling and cytoplasmic p27 localization exhibited elevated Cdk2 activity, which could be suppressed by inhibiting AMPK signaling. In addition, cells with elevated AMPK signaling and cytoplasmic p27 localization were resistant to apoptosis, which could be overcome by inhibition of AMPK signaling and relocalization of p27 to the nucleus. These data show that AMPK signaling determines the subcellular localization of p27, and identifies loss of integration of pathways controlling energy balance, the cell cycle, and apoptosis due to aberrant AMPK and p27 function as a feature of cells that have lost the Tsc2 tumor suppressor gene.