Ron Kusters

Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides.

In Escherichia coli, components of a signal recognition particle (SRP) and its receptor have been identified which appear to be essential for efficient translocation of several proteins. In this study we use cross‐linking to demonstrate that E. coli SRP interacts with a variety of nascent presecretory proteins and integral inner membrane proteins. Evidence is presented that the interaction is correlated with the hydrophobicity of the core region of the signal sequence and thereby with its ability to promote transport in vivo. A second E. coli component, which is identified as trigger factor, can be efficiently cross‐linked to all tested nascent chains derived from both secreted and cytosolic proteins. We propose that SRP and trigger factor act as secretion‐specific and general molecular chaperone respectively, early in protein synthesis.

An alternative protein targeting pathway in Escherichia coli: studies on the role of FtsY

In Escherichia coli, a signal recognition particle (SRP) has been identified which binds specifically to the signal sequence of presecretory proteins and which appears to be essential for efficient translocation of a subset of proteins. In this study we have investigated the function of E. coli FtsY which shares sequence similarity with the alpha‐subunit of the eukaryotic SRP receptor (‘docking protein’) in the membrane of the endoplasmic reticulum. A strain was constructed which allows the conditional expression of FtsY. Depletion of FtsY is shown to cause the accumulation of the precursor form of beta‐lactamase, OmpF and ribose binding protein in vivo, whereas the processing of various other presecretory proteins is unaffected. Furthermore, FtsY‐depleted inverted cytoplasmic membrane vesicles are shown to be defective in the translocation of pre‐beta‐lactamase using an in vitro import assay. Subcellular localization studies revealed that FtsY is located in part at the cytoplasmic membrane with which it seems peripherally associated. These observations suggest that FtsY is the functional E. coli homolog of the mammalian SRP receptor.

Biomarkers and Molecular Analysis to Improve Bloodstream Infection Diagnostics in an Emergency Care Unit

Molecular pathogen detection from blood is still expensive and the exact clinical value remains to be determined. The use of biomarkers may assist in preselecting patients for immediate molecular testing besides blood culture. In this study, 140 patients with ≥ 2 SIRS criteria and clinical signs of infection presenting at the emergency department of our hospital were included. C-reactive protein (CRP), neutrophil-lymphocyte count ratio (NLCR), procalcitonin (PCT) and soluble urokinase plasminogen activator receptor (suPAR) levels were determined. One ml EDTA blood was obtained and selective pathogen DNA isolation was performed with MolYsis (Molzym). DNA samples were analysed for the presence of pathogens, using both the MagicPlex Sepsis Test (Seegene) and SepsiTest (Molzym), and results were compared to blood cultures. Fifteen patients had to be excluded from the study, leaving 125 patients for further analysis. Of the 125 patient samples analysed, 27 presented with positive blood cultures of which 7 were considered to be contaminants. suPAR, PCT, and NLCR values were significantly higher in patients with positive blood cultures compared to patients without (p < 0.001). Receiver operating characteristic curves of the 4 biomarkers for differentiating bacteremia from non-bacteremia showed the highest area under the curve (AUC) for PCT (0.806 (95% confidence interval 0.699–0.913)). NLCR, suPAR and CRP resulted in an AUC of 0.770, 0.793, and 0.485, respectively. When compared to blood cultures, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for SepsiTest and MagicPlex Sepsis Test were 11%, 96%, 43%, 80%, and 37%, 77%, 30%, 82%, respectively. In conclusion, both molecular assays perform poorly when one ml whole blood is used from emergency care unit patients. NLCR is a cheap, fast, easy to determine, and rapidly available biomarker, and therefore seems most promising in differentiating BSI from non-BSI patients for subsequent pathogen identification using molecular diagnostics.

The neutrophil-lymphocyte count ratio in patients with community-acquired pneumonia.

Study Objective The neutrophil-lymphocyte count ratio (NLCR) has been identified as a predictor of bacteremia in medical emergencies. The aim of this study was to investigate the value of the NLCR in patients with community-acquired pneumonia (CAP). Methods and Results Consecutive adult patients were prospectively studied. Pneumonia severity (CURB-65 score), clinical characteristics, complications and outcomes were related to the NLCR and compared with C-reactive protein (CRP), neutrophil count, white blood cell (WBC) count. The study cohort consisted of 395 patients diagnosed with CAP. The mean age of the patients was 63.4±16.0 years. 87.6% (346/395) of the patients required hospital admission, 7.8% (31/395) patients were admitted to the Intensive Care Unit (ICU) and 5.8% (23/395) patients of the study cohort died. The NLCR was increased in all patients, predicted adverse medical outcome and consistently increased as the CURB-65 score advanced. NLCR levels (mean ± SD) were significantly higher in non-survivors (23.3±16.8) than in survivors (13.0±11.4). The receiver-operating characteristic (ROC) curve for NLCR predicting mortality showed an area under the curve (AUC) of 0.701. This was better than the AUC for the neutrophil count, WBC count, lymphocyte count and CRP level (0.681, 0.672, 0.630 and 0.565, respectively). Conclusion Admission NLCR at the emergency department predicts severity and outcome of CAP with a higher prognostic accuracy as compared with traditional infection markers.

The functioning of the SRP receptor FtsY in protein‐targeting in E. coli is correlated with its ability to bind and hydrolyse GTP

In this study, we have established that FtsY, the E. coli homolog of the mammalian signal recognition particle (SRP) receptor, is a GTP‐binding protein which displays intrinsic GTPase activity. GTP was found to influence the protease sensitivity of FtsY indicative of a conformational change. FtsY mutated in the 4th GTP‐binding consensus element displayed reduced GTP‐binding and ‐hydrolysis which correlated with a reduced ability to interact with SRP. Overexpression of the mutant proteins had a stronger inhibitory effect on protein translocation than overexpression of wild‐type FtsY. These observations suggest that in E. coli GTP is important for proper functioning of FtsY in protein‐targeting.

Effect of Omega-3 Fatty Acids on Kidney Function after Myocardial Infarction: The Alpha Omega Trial

BACKGROUND AND OBJECTIVES Kidney function gradually decreases with age, and myocardial infarction accelerates this deterioration. Omega-3 (n-3) fatty acids may slow down the decline of kidney function. The effect of marine and plant-derived n-3 fatty acids on kidney function in patients after myocardial infarction was examined. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In the Alpha Omega Trial, 2344 patients with history of myocardial infarction ages 60-80 years old (81% men) were randomized to one of four trial margarines. The patients received an additional targeted amount of 400 mg/d eicosapentaenoic acid and docosahexaenoic acid, 2 g/d α-linolenic acid, eicosapentaenoic acid-docosahexaenoic acid plus α-linolenic acid, or placebo for 40 months. Serum cystatin C and serum creatinine were assessed at baseline and after 40 months. Creatinine-cystatin C-based GFR was estimated with the Chronic Kidney Disease Epidemiology Collaboration equation. RESULTS Patients consumed 19.9 g margarine/d, providing an additional 239 mg/d eicosapentaenoic acid with 159 mg/d docosahexaenoic acid, 1.99 g/d α-linolenic acid, or both in the active treatment groups. After 40 months, compared with baseline, mean (±SD) creatinine-cystatin C-based GFR was -6.9 (±12.6), -4.8 (±13.4), -6.2 (±12.8), and -6.0 (±13.0) ml/min per 1.73 m(2) in the placebo, eicosapentaenoic acid-docosahexaenoic acid, α-linolenic acid, and eicosapentaenoic acid-docosahexaenoic acid plus α-linolenic acid groups, respectively. After 40 months, in patients receiving eicosapentaenoic acid-docosahexaenoic acid compared with placebo, the decline in creatinine-cystatin C-based GFR was 2.1 less (95% confidence interval, 0.6 to 3.6; P<0.01) ml/min per 1.73 m(2); other comparisons were not statistical significant. Odds ratios (95% confidence intervals) of incident CKD (<60 ml/min per 1.73 m(2)) and rapid decline of kidney function (≥3 ml/min per year) for eicosapentaenoic acid-docosahexaenoic acid compared with placebo were 0.83 (0.58 to 1.18) and 0.85 (0.67 to 1.08), respectively. CONCLUSIONS Long-term supplementation with 400 mg/d eicosapentaenoic acid-docosahexaenoic acid provides a small beneficial effect on kidney function in patients with a history of myocardial infarction.

A PCT algorithm for discontinuation of antibiotic therapy is a cost-effective way to reduce antibiotic exposure in adult intensive care patients with sepsis

Abstract Objective: Procalcitonin (PCT) is a specific marker for differentiating bacterial from non-infective causes of inflammation. It can be used to guide initiation and duration of antibiotic therapy in intensive care unit (ICU) patients with suspected sepsis, and might reduce the duration of hospital stay. Limiting antibiotic treatment duration is highly important because antibiotic over-use may cause patient harm, prolonged hospital stay, and resistance development. Several systematic reviews show that a PCT algorithm for antibiotic discontinuation is safe, but upfront investment required for PCT remains an important barrier against implementation. The current study investigates to what extent this PCT algorithm is a cost-effective use of scarce healthcare resources in ICU patients with sepsis compared to current practice. Methods: A decision tree was developed to estimate the health economic consequences of the PCT algorithm for antibiotic discontinuation from a Dutch hospital perspective. Input data were obtained from a systematic literature review. When necessary, additional information was gathered from open interviews with clinical chemists and intensivists. The primary effectiveness measure is defined as the number of antibiotic days, and cost-effectiveness is expressed as incremental costs per antibiotic day avoided. Results: The PCT algorithm for antibiotic discontinuation is expected to reduce hospital spending by circa €3503 per patient, indicating savings of 9.2%. Savings are mainly due to reductions in length of hospital stay, number of blood cultures performed, and, importantly, days on antibiotic therapy. Probabilistic and one-way sensitivity analyses showed the model outcome to be robust against changes in model inputs. Conclusion: Proven safe, a PCT algorithm for antibiotic discontinuation is a cost-effective means of reducing antibiotic exposure in adult ICU patients with sepsis, compared to current practice. Additional resources required for PCT are more than offset by downstream cost savings. This finding is highly important given the aim of preventing widespread antibiotic resistance.

Thyrotropin versus age relation as an indicator of historical iodine intake.

BACKGROUND In populations with mild iodine deficiency, the serum level of thyrotropin (TSH) is negatively and the serum free thyroxine (FT4) is positively associated with age. An ongoing decrease of TSH and increase of FT4 can be found after iodine supplementation. The aim of this study was to investigate whether there are current differences in the relation between thyroid function and age in relation to differences in iodine intake in the past. METHODS Eight medical laboratories in several regions of The Netherlands, which are all iodine sufficient at present but with a difference in iodine status in the past, provided the results of all TSH and FT4 measurements performed from 2006 until 2011, resulting in 330,802 TSH and 103,940 FT4 measurements. RESULTS The negative association between TSH and age in the elderly is only present in areas with a historical iodine deficiency (regression coefficients [RC] -0.008, 95% confidence interval [CI] -0.009; -0.007). In the historically iodine-sufficient population, TSH shows no obvious increase or decrease with age. In both the historically iodine-sufficient and iodine-deficient populations, FT4 levels were positively associated with age in the elderly (RC 0.009, 95% CI 0.008; 0.010 and RC 0.008, 95% CI 0.007; 0.010, respectively). CONCLUSIONS There are differences in relation between thyroid function and age between populations with differences in iodine intake in the past, despite an adequate iodine status at present. This raises the question whether the present but also historical iodine status of a population should be taken into account when establishing the reference limits of TSH and FT4.

Rapidly rule out acute myocardial infarction by combining copeptin and heart-type fatty acid-binding protein with cardiac troponin

Background The rapid exclusion of acute myocardial infarction in patients with chest pain can reduce the length of hospital admission, prevent unnecessary diagnostic work-up and reduce the burden on our health-care systems. The combined use of biomarkers that are associated with different pathophysiological aspects of acute myocardial infarction could improve the early diagnostic assessment of patients presenting with chest pain. Methods We measured cardiac troponin I, copeptin and heart-type fatty acid-binding protein concentrations in 584 patients who presented to the emergency department with acute chest pain. The diagnostic performances for the diagnosis of acute myocardial infarction and NSTEMI were calculated for the individual markers and their combinations. Separate calculations were made for patients presenting to the emergency department <3 h, 3–6 h and 6–12 h after chest pain onset. Results For ruling out acute myocardial infarction, the net predictive values (95% CI) of cardiac troponin I, copeptin and heart-type fatty acid-binding protein were 90.4% (87.3–92.9), 84% (79.8–87.6) and 87% (83.5–90), respectively. Combining the three biomarkers resulted in a net predictive value of 95.8% (92.8–97.8). The improvement was most pronounced in the early presenters (<3 h) where the combined net predictive value was 92.9% (87.3–96.5) compared to 84.6% (79.4–88.9) for cardiac troponin I alone. The area under the receiver operating characteristic for the triple biomarker combination increased significantly (P < 0.05) compared to that of cardiac troponin I alone (0.880 [0.833–0.928] vs. 0.840 [0.781–0.898], respectively). Conclusions Combining copeptin, heart-type fatty acid-binding protein and cardiac troponin I measurements improves the diagnostic performance in patients presenting with chest pain. Importantly, in patients who present early (<3 h) after chest pain onset, the combination improves the diagnostic performance compared to the standard cardiac troponin I measurement alone.

No effect of n-3 fatty acids supplementation on NT-proBNP after myocardial infarction: The Alpha Omega Trial

Background heart failure is a major risk factor for cardiovascular mortality, for which n-3 fatty acids may have beneficial effects. We examined the effect of marine eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and plant-derived alpha-linolenic acid (ALA) on N-Terminal-pro Brain Natriuretic Peptide (NT-proBNP), a biomarker of heart failure. Methods we randomly assigned 4837 post-myocardial infarction patients, aged 60–80 years (82% men), to margarines supplemented with a targeted additional intake of 400 mg/day EPA and DHA, 2 g/day ALA, EPA-DHA plus ALA, or placebo for 40 months. In a random selection of 639 patients, NT-proBNP was determined both at baseline and at the end of follow-up. NT-proBNP was loge-transformed and analysed by type of treatment using analysis of covariance adjusting for baseline NT-proNBP. Results patients consumed on average 19.8 g margarine/day, providing an additional amount of 238 mg/day EPA with 158 mg/day DHA, 1.98 g/day ALA, or both, in the active-treatment groups. In the placebo group, the geometric mean level NT-proBNP increased from 245 ng/l (95%-confidence interval [CI]: 207–290) to 294 ng/l (95%-CI: 244–352) after 40 months (p = 0.001). NT-proBNP levels were not affected by ALA (+8% versus placebo; 95%-CI: −8% to +25%; p = 0.34), EPA-DHA (+2% versus placebo; 95%-CI: −14% to +18%; p = 0.78), nor EPA-DHA plus ALA (+9% versus placebo; 95%-CI: −8% to +25%; p = 0.31) treatment. Conclusions supplementation with modest amounts of EPA-DHA, with or without ALA, did not have a significant effect on NT-proBNP levels in patients with a history of myocardial infarction.