Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium

Identifying associations between diabetes and acute respiratory distress syndrome in patients with acute hypoxemic respiratory failure: an analysis of the LUNG SAFE database

Background: Diabetes mellitus is a common co-existing disease in the critically ill. Diabetes mellitus may reduce the risk of acute respiratory distress syndrome (ARDS), but data from previous studies are conflicting. The objective of this study was to evaluate associations between pre-existing diabetes mellitus and ARDS in critically ill patients with acute hypoxemic respiratory failure (AHRF). Methods: An ancillary analysis of a global, multi-centre prospective observational study (LUNG SAFE) was undertaken. LUNG SAFE evaluated all patients admitted to an intensive care unit (ICU) over a 4-week period, that required mechanical ventilation and met AHRF criteria. Patients who had their AHRF fully explained by cardiac failure were excluded. Important clinical characteristics were included in a stepwise selection approach (forward and backward selection combined with a significance level of 0.05) to identify a set of independent variables associated with having ARDS at any time, developing ARDS (defined as ARDS occurring after day 2 from meeting AHRF criteria) and with hospital mortality. Furthermore, propensity score analysis was undertaken to account for the differences in baseline characteristics between patients with and without diabetes mellitus, and the association between diabetes mellitus and outcomes of interest was assessed on matched samples. Results: Of the 4107 patients with AHRF included in this study, 3022 (73.6%) patients fulfilled ARDS criteria at admission or developed ARDS during their ICU stay. Diabetes mellitus was a pre-existing co-morbidity in 913 patients (22.2% of patients with AHRF). In multivariable analysis, there was no association between diabetes mellitus and having ARDS (OR 0.93 (0.78-1.11); p = 0.39), developing ARDS late (OR 0.79 (0.54-1.15); p = 0.22), or hospital mortality in patients with ARDS (1.15 (0.93-1.42); p = 0.19). In a matched sample of patients, there was no association between diabetes mellitus and outcomes of interest. Conclusions: In a large, global observational study of patients with AHRF, no association was found between diabetes mellitus and having ARDS, developing ARDS, or outcomes from ARDS. Trial registration: NCT02010073. Registered on 12 December 2013

Spontaneous Breathing in Early Acute Respiratory Distress Syndrome: Insights From the Large Observational Study to UNderstand the Global Impact of Severe Acute Respiratory FailurE Study

OBJECTIVES: To describe the characteristics and outcomes of patients with acute respiratory distress syndrome with or without spontaneous breathing and to investigate whether the effects of spontaneous breathing on outcome depend on acute respiratory distress syndrome severity. DESIGN: Planned secondary analysis of a prospective, observational, multicentre cohort study. SETTING: International sample of 459 ICUs from 50 countries. PATIENTS: Patients with acute respiratory distress syndrome and at least 2 days of invasive mechanical ventilation and available data for the mode of mechanical ventilation and respiratory rate for the 2 first days. INTERVENTIONS: Analysis of patients with and without spontaneous breathing, defined by the mode of mechanical ventilation and by actual respiratory rate compared with set respiratory rate during the first 48 hours of mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: Spontaneous breathing was present in 67% of patients with mild acute respiratory distress syndrome, 58% of patients with moderate acute respiratory distress syndrome, and 46% of patients with severe acute respiratory distress syndrome. Patients with spontaneous breathing were older and had lower acute respiratory distress syndrome severity, Sequential Organ Failure Assessment scores, ICU and hospital mortality, and were less likely to be diagnosed with acute respiratory distress syndrome by clinicians. In adjusted analysis, spontaneous breathing during the first 2 days was not associated with an effect on ICU or hospital mortality (33% vs 37%; odds ratio, 1.18 [0.92-1.51]; p = 0.19 and 37% vs 41%; odds ratio, 1.18 [0.93-1.50]; p = 0.196, respectively ). Spontaneous breathing was associated with increased ventilator-free days (13 [0-22] vs 8 [0-20]; p = 0.014) and shorter duration of ICU stay (11 [6-20] vs 12 [7-22]; p = 0.04). CONCLUSIONS: Spontaneous breathing is common in patients with acute respiratory distress syndrome during the first 48 hours of mechanical ventilation. Spontaneous breathing is not associated with worse outcomes and may hasten liberation from the ventilator and from ICU. Although these results support the use of spontaneous breathing in patients with acute respiratory distress syndrome independent of acute respiratory distress syndrome severity, the use of controlled ventilation indicates a bias toward use in patients with higher disease severity. In addition, because the lack of reliable data on inspiratory effort in our study, prospective studies incorporating the magnitude of inspiratory effort and adjusting for all potential severity confounders are required

Epidemiology and patterns of tracheostomy practice in patients with acute respiratory distress syndrome in ICUs across 50 countries

Background: To better understand the epidemiology and patterns of tracheostomy practice for patients with acute respiratory distress syndrome (ARDS), we investigated the current usage of tracheostomy in patients with ARDS recruited into the Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG-SAFE) study. Methods: This is a secondary analysis of LUNG-SAFE, an international, multicenter, prospective cohort study of patients receiving invasive or noninvasive ventilation in 50 countries spanning 5 continents. The study was carried out over 4 weeks consecutively in the winter of 2014, and 459 ICUs participated. We evaluated the clinical characteristics, management and outcomes of patients that received tracheostomy, in the cohort of patients that developed ARDS on day 1-2 of acute hypoxemic respiratory failure, and in a subsequent propensity-matched cohort. Results: Of the 2377 patients with ARDS that fulfilled the inclusion criteria, 309 (13.0%) underwent tracheostomy during their ICU stay. Patients from high-income European countries (n = 198/1263) more frequently underwent tracheostomy compared to patients from non-European high-income countries (n = 63/649) or patients from middle-income countries (n = 48/465). Only 86/309 (27.8%) underwent tracheostomy on or before day 7, while the median timing of tracheostomy was 14 (Q1-Q3, 7-21) days after onset of ARDS. In the subsample matched by propensity score, ICU and hospital stay were longer in patients with tracheostomy. While patients with tracheostomy had the highest survival probability, there was no difference in 60-day or 90-day mortality in either the patient subgroup that survived for at least 5 days in ICU, or in the propensity-matched subsample. Conclusions: Most patients that receive tracheostomy do so after the first week of critical illness. Tracheostomy may prolong patient survival but does not reduce 60-day or 90-day mortality. Trial registration: ClinicalTrials.gov, NCT02010073. Registered on 12 December 2013

Efficacy of treatment for hyperglycemic crisis in elderly diabetic patients in a day hospital

D Benaiges,1–3 JJ Chillarón,1–3 MJ Carrera,1,3 F Cots,3,4 J Puig de Dou,1 E Corominas,1 J Pedro-Botet,1–3 JA Flores-Le Roux,1–3 C Claret,1 A Goday,1–3 JF Cano1–3 1Department of Endocrinology and Nutrition, Hospital del Mar, 2Department of Medicine, Universitat Autònoma de Barcelona, 3Institut Hospital del Mar d’Investigacions Mèdiques, 4Epidemiology and Evaluation Department, Parc de Salut Mar, Barcelona, Spain Background: The purpose of this prospective cohort study was to compare the costs of day hospital (DH) care for hyperglycemic crisis in elderly diabetic patients with those of conventional hospitalization (CH). Secondary objectives were to compare these two clinical scenarios in terms of glycemic control, number of emergency and outpatient visits, readmissions, hypoglycemic episodes, and nosocomial morbidity. Methods: The study population comprised diabetic patients aged >74 years consecutively admitted to a tertiary teaching hospital in Spain for hyperglycemic crisis (sustained hyperglycemia [>300 mg/dL] for at least 3 days with or without ketosis). The patients were assigned to DH or CH care according to time of admission and were followed for 6 months after discharge. Exclusion criteria were ketoacidosis, hyperosmolar crisis, hemodynamic instability, severe intercurrent illness, social deprivation, or Katz index >D.Results: Sixty-four diabetic patients on DH care and 36 on CH care were included, with no differences in baseline characteristics. The average cost per patient was 1,345.1±793.6 € in the DH group and 2,212.4±982.5 € in the CH group (P<0.001). There were no differences in number of subjects with mild hypoglycemia during follow-up (45.3% DH versus 33.3% CH, P=0.24), nor in the percentage of patients achieving a glycated hemoglobin (HbA1c) <8% (67.2% DH versus 58.3% CH, P=0.375). Readmissions for hyperglycemic crisis and pressure ulcer rates were significantly higher in the CH group.Conclusion: DH care for hyperglycemic crises is more cost-effective than CH care, with a net saving of 1,418.4 € per case, lower number of readmissions and pressure ulcer rates, and similar short-term glycemic control and hypoglycemia rates. Keywords: day hospital, conventional hospitalization, hyperglycemic crisi

Efficacy of treatment for hyperglycemic crisis in elderly diabetic patients in a day hospital

The purpose of this prospective cohort study was to compare the costs of day hospital (DH) care for hyperglycemic crisis in elderly diabetic patients with those of conventional hospitalization (CH). Secondary objectives were to compare these two clinical scenarios in terms of glycemic control, number of emergency and outpatient visits, readmissions, hypoglycemic episodes, and nosocomial morbidity. The study population comprised diabetic patients aged >74 years consecutively admitted to a tertiary teaching hospital in Spain for hyperglycemic crisis (sustained hyperglycemia [>300 mg/dL] for at least 3 days with or without ketosis). The patients were assigned to DH or CH care according to time of admission and were followed for 6 months after discharge. Exclusion criteria were ketoacidosis, hyperosmolar crisis, hemodynamic instability, severe intercurrent illness, social deprivation, or Katz index >D. Sixty-four diabetic patients on DH care and 36 on CH care were included, with no differences in baseline characteristics. The average cost per patient was 1,345.1±793.6 € in the DH group and 2,212.4±982.5 € in the CH group (P <0.001). There were no differences in number of subjects with mild hypoglycemia during follow-up (45.3% DH versus 33.3% CH, P =0.24), nor in the percentage of patients achieving a glycated hemoglobin (HbA) <8% (67.2% DH versus 58.3% CH, P =0.375). Readmissions for hyperglycemic crisis and pressure ulcer rates were significantly higher in the CH group. DH care for hyperglycemic crises is more cost-effective than CH care, with a net saving of 1,418.4 € per case, lower number of readmissions and pressure ulcer rates, and similar short-term glycemic control and hypoglycemia rates

Pharmacokinetics in morbid obesity: influence of two bariatric surgery techniques on paracetamol and caffeine metabolism

PURPOSE: The purpose of the study was to study the impact of the two most common bariatric surgery techniques on paracetamol pharmacokinetics (a marker of gastric emptying) and caffeine metabolism (a marker of liver function). MATERIALS AND METHODS: In the present prospective study, we studied 24 morbid obese patients before, at 4 weeks, and 6 months after having undergone sleeve gastrectomy (n = 10) or Roux-en-Y gastric bypass (n = 14). For comparative purposes, 28 healthy controls (14 normal weights and 14 overweights) were also included in the study. RESULTS: Paracetamol pharmacokinetics was altered in the obese participants leading to lower bioavailability. Bariatric surgery resulted in faster absorption and normalized pharmacokinetic parameters, prompting an increase in paracetamol bioavailability. No differences were found between surgical procedures. In the case of caffeine, the ratio paraxanthine/caffeine did not differ between morbid obese and healthy individuals. This ratio remained unmodified after surgery, indicating that the liver function (assessed by cytochrome P450 1A2 activity) was unaffected by obesity or bariatric surgery. CONCLUSIONS: Paracetamol pharmacokinetics and caffeine plasma levels are altered in severely obese patients. The two studied bariatric surgical techniques normalize paracetamol oral bioavailability without impairing the liver function (measured by cytochrome P450 1A2 activity)