[Learning to see things from a different perspective: interns and residents collaborate with artists to become better doctors]. / Anders leren kijken en de ander leren zien.

In 2016 and 2017, we started an innovative learning track in the Radboudumc that combines arts and medical education, and appraised the learning processes involved. The voluntary track was followed by 32 and 30 participants respectively, mostly interns and a few residents. The initiative built upon the ideas of several American educational developments which incorporated museum visits. We extended the format by having participants join artists in their studios, to allow students to have an immersive experience of a different discipline, rather than only observing its end products. The track did not have specific learning objectives. However, participants were encouraged to set personal goals and to reflect on what they learned in terms of observation skills, creative thinking, personalized health care, and frame reflection. Here we report the rationale of the track, and illustrate preliminary conclusions with participants' quotes.

[Leading cause of death in young Dutch people: the cigarette]. / Doodsoorzaak nr. 1 bij jonge Nederlanders: de sigaret.

OBJECTIVE: To investigate the impact of smoking on premature death in the Netherlands and the difference between causes of death for smokers and non-smokers. DESIGN: Observational cohort study. METHOD: Data on smoking behaviour were obtained from 40,000 people who participated in the CBS (Statistics Netherlands) health survey between 2001-2006. These data were linked to data on death and cause of death for the 10 years following this questionnaire. Hazard ratios were calculated for premature deaths among smokers, classified into smoking intensity, and ex-smokers as compared with those who had never smoked. These data were used to estimate cumulative death of smokers versus non-smokers. RESULTS: The hazard ratio for premature death was 3.8 (95% CI: 3.2-4.5) for heavy smokers, 2.6 (95% CI: 2.2-3.0) for moderate smokers and 1.7 (95% CI: 1.3-2.3) for light smokers. Lifelong heavy smokers had a chance of 23% of dying before the age of 65. For moderate and light smokers and for non-smokers, the chance was respectively 16, 11 and 7%. For half of all people who died relatively young, cancer was the underlying cause of death. This was mainly lung cancer for smokers. Heavy smokers are estimated to have lost 13 years of life, moderate smokers 9 and light smokers 5. Smoking cessation at any age still benefited health. Ex-smokers who had quit before an approximate age of 35 had the same life expectancy as lifelong non-smokers. CONCLUSION: An estimated four in ten premature deaths can be attributed to smoking in the Netherlands. Cancer is the predominant cause of death amongst smokers. Smoking cessation increases life expectancy. Therefore, the earlier a smoker stops, the better.

A cluster-randomised trial of screening for language disorders in toddlers.

OBJECTIVE: To assess the screening performance of a specific language-screening instrument at 18 and 24 months of age and to assess its effect on the early detection and prognosis of language delay. DESIGN: Child health care physicians were randomised to the intervention group, in which specific language screening was conducted twice (at age 18 months and 24 months), or to the control group (usual care). The specific screening instrument consisted of a uniform set of questions for the parents and test elements for the child, with scaled scores to assess responses. SETTING: Child health care in the Netherlands and referral of screen-positive children. SUBJECTS: 5734 children in the intervention group and 4621 in the control group. MAIN OUTCOME MEASURES: Test characteristics and disorders at 24 months, and confirmed diagnoses of a language disorder before 36 months in both groups. Gold standard based on reports of parents, specialists and expert panel. Prognosis estimated from two diagnostic language development performance scores at 36 months (in questionnaire). RESULTS: In the intervention group, 3147 of the 5734 children (55%) were screened with the specific screening instrument and 73 of the screened children (2.3%) were screen-positive. Of the screen-positive children, 41 (55%) had confirmed language delay (diagnostic assessment and/or reported treatment). The estimated sensitivity of the test ranged between 24-52% depending on the severity of language disorders. The prevalence of language disorders in three-year olds was estimated to be 2.4-5.3%. In the intervention group, 1.25-2 times more children with language delay had been diagnosed before 36 months. The assessment of language development at 36 months showed no statistically significant differences between the intervention and the control groups. CONCLUSIONS: The inclusion of a specific language-screening instrument in child health centre activities resulted in the earlier detection of children with language delay. Short-term health benefits could not be demonstrated. Large-scale introduction cannot be recommended on the basis of this information alone.

Breast cancer screening in Navarra: interpretation of a high detection rate at the first screening round and a low rate at the second round.

Our objective was to evaluate the on-going European pilot project for breast cancer screening in Navarra, Spain, and to predict the effects and costs of the programme in the long run. Observed results in Navarra, consisting of more than 100,000 screens, were compared with expected results. A microsimulation screening analysis model was used that included demographical, epidemiological and screening characteristics of Navarra. Alternative assumptions on epidemiological and screening characteristics were also addressed. The observed detection rate (5.9 per 1,000 screened women) in the first round was 18% higher than expected; the observed rate in the subsequent round (2.9) was 17% lower than expected. Longer pre-clinical durations, lower sensitivity or the existence of a high-risk group in Navarra could not satisfactorily explain the first and second round results together. Nevertheless, the programme will have an important health benefit for the women involved, due to an important trend in incidence in recent years and the relatively unfavourable clinical stage distribution in Navarra. The proportion T2+ cancers that will be prevented after 10 years of screening amounts to 36%. The annual mortality reduction in steady state is expected to range between 17% (if the observed rates in the second round indicate real screening performance) to 23% (if the first round indicates real performance). Our results demonstrate that a high detection rate in the first round is insufficient to evaluate the quality of a programme. Interval cancer rates, results of the subsequent round and size distributions are also crucial indicators of the quality of the screening programme and should be analysed in their specific context.

[Malaria: under-notification and risk assessment for travelers to the tropics]. / Malaria: onderrapportage en risicoschatting voor tropenreizigers.

OBJECTIVE: To define the risk of contracting malaria for travellers to malaria-endemic areas and to calculate the under-notification under the current notification system. DESIGN: Retrospective epidemiological analysis. SETTING: Agricultural University Wageningen, The Netherlands. METHODS: Risks of contracting malaria were estimated by calculating the incidence per 100,000 travellers. This incidence was found using an estimated under-notification calculated on the basis of data from the "SIG Zorginformatie' (hospital cases) and the Medical Health Inspectorate (notified cases). RESULTS: The average under-notification in the Netherlands was at least 59% in the period January 1988 to June 1993 inclusive and increased by 10% each year. Judging by this estimation at least 3170 travellers returned to the Netherlands with malaria in this period. The risk in Africa appeared to be decreasing. In Asia the malaria incidence increased slightly in recent years while no trend was found for America. The imported malaria was mainly caused by Plasmodium falciparum. CONCLUSION: In order to improve the malaria notification system in the Netherlands a notification obligation for all microbiological laboratories should be introduced as almost all malaria is diagnosed there.

The use of genetic engineering to obtain efficient production of porcine pancreatic phospholipase A2 by Saccharomyces cerevisiae.

We have developed an efficient production system for porcine pancreatic phospholipase A2 in Saccharomyces cerevisiae (baker's yeast). The cDNA encoding the prophospholipase A2 was expressed under the control of the galactose inducible GAL7 promotor, and secretion was directed by the secretion signals of yeast invertase. This construct yielded up to 6 mg prophospholipase A2 activity per 1 fermentation broth, secreted as a glycosylated invertase prophospholipase A2 hybrid protein. Upon genetically deleting the glycosylation site, the level of secretion decreased to 3.6 mg prophospholipase A2 per 1. Changing the invertase secretion signals for an invertase/alpha-mating factor prepro sequence-fusion increased the secretion level up to 8 mg per 1. The secreted non-glycosylated prophospholipase A2 species was correctly processed. Our results demonstrate the promises and limitations for rational design to obtain high level expression and secretion of heterologous proteins by S. cerevisiae.

Conversion of pig pancreas phospholipase A2 by protein engineering into enzyme active against Escherichia coli treated with the bactericidal/permeability-increasing protein.

Phospholipases A2 (PLA-2) are conserved enzymes that can vary widely in their activity toward certain biological targets. Activity of PLA-2 toward Escherichia coli treated with the bactericidal/permeability-increasing protein (BPI) of granulocytes has been detected only in "Group II" PLA-2 (lacking Cys11-Cys77) and correlates with overall basicity and the presence of a cluster of basic amino acids within a variable surface region near the NH2 terminus (including residues 6, 7, 10, 11, and 15). We now show that of five pancreatic PLA-2 ("Group I" enzymes) tested from different species of mammals, the human enzyme that is most basic both globally (pI 8.7) and locally (Arg-6, Lys-7, and Lys-10) is active toward BPI-treated E. coli (approximately 1-2% activity of the most active Group II PLA-2) whereas the other four PLA-2 are essentially inactive (less than 0.1%). The cDNA of the pig pancreatic PLA-2 (pI 6.4; Arg-6, Ser-7, Lys-10) has been modified by site-specific mutagenesis and the wild-type and mutant PLA-2 have been expressed in and purified from either E. coli or Saccharomyces cerevisiae to determine more precisely the structural determinants of PLA-2 activity toward BPI-treated E. coli. The single substitution of lysine (or arginine) for Ser-7 transformed the pig pancreatic PLA-2 into an active enzyme toward BPI-treated E. coli possessing 25-50% the activity of the human PLA-2. Additional modifications to increase global basicity (increase in net charge up to +4) caused a further (up to 2-fold) increase in activity. All mutant PLA-2 still containing Ser-7 possessed little or no activity toward BPI-treated E. coli. Changes in activity toward BPI-treated E. coli were accompanied by parallel changes in enzyme binding to this target. In contrast, substitution of lysine (or arginine) for Ser-7 caused little or no alteration of enzyme activity toward either autoclaved E. coli or egg yolk lipoproteins indicating no major effects on the catalytic properties of the PLA-2. This study demonstrates directly the role of NH2-terminal basic residues in the action of PLA-2 on BPI-treated E. coli and suggests that these properties mainly facilitate PLA-2 binding to this biological target.

Regulation and role of phospholipases in host-bacteria interaction.

Deacylating phospholipases play essential roles in numerous biological events, requiring tight control of hydrolytic activity. Most cells, unless stimulated or perturbed, exhibit little phospholipid turnover. Activation of phospholipases A (PLA) is usually triggered by membrane perturbing conditions or agents. Some activators indiscriminately activate any PLA, others are highly specific. Our studies concern an activator that is a potent bactericidal protein with membrane-perturbing properties, isolated from polymorphonuclear leukocytes (PMN), that is only cytotoxic for gram-negative bacteria and primarily responsible for the fate of several gram-negative bacterial species, ingested and killed by the PMN. It is this protein that activates the hydrolysis of the phospholipids of the killed bacteria (E. coli) by three PLA: 1) an E. coli PLA, the pldA gene product; 2) a PLA2 of PMN; 3) a soluble PLA2 in the extracellular fluid of an inflammatory exudate. However, this activator protein does not trigger the action of many other PLA2, all members of a highly conserved class of PLA. Our structural studies (including genetic engineering) of both responsive and non-responsive PLA2 have revealed that the amino acid composition and sequence of the NH2-terminal alpha-helix of the PLA2 molecule are major determinants of the ability of the PMN protein to activate a given PLA2. Our results provide another demonstration that these important enzymes have diverged during evolution to perform different biological functions.

Glutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A2.

In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium-binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387-5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site-directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66----Asn, Glu71----Asn and Glu92----Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low-affinity calcium binding.

The role of Asp-49 and other conserved amino acids in phospholipases A2 and their importance for enzymatic activity.

The role of aspartic acid-49 (Asp-49) in the active site of porcine pancreatic phospholipase A2 was studied by recombinant DNA techniques: two mutant proteins were constructed containing either glutamic acid (Glu) or lysine (Lys) at position 49. Enzymatic characterization indicated that the presence of Asp-49 is essential for effective hydrolysis of phospholipids. Conversion of Asp-49 to either Glu or Lys strongly reduces the binding of Ca2+ ions, in particular for the lysine mutant, but the affinity for substrate analogues is hardly affected. Extensive purification of naturally occurring Lys-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein that was nearly inactive. Inhibition studies showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself has no enzymatic activity. Our results indicate that Asp-49 is essential for the catalytic action of phospholipase A2. The importance of Asp-49 was further evaluated by comparison of the primary sequences of 53 phospholipases A2 and phospholipase homologues showing that substitutions at position 49 are accompanied by structural variations of otherwise conserved residues. The occurrence of several nonconserved substitutions appeared to be a general characteristic of nonactive phospholipase A2 homologues.

The role of aspartic acid-49 in the active site of phospholipase A2. A site-specific mutagenesis study of porcine pancreatic phospholipase A2 and the rationale of the enzymatic activity of [lysine49]phospholipase A2 from Agkistrodon piscivorus piscivorus' venom.

In order to probe the role of Asp-49 in the active site of porcine pancreatic phospholipase A2 two mutant proteins were constructed containing either Glu or Lys at position 49. Their enzymatic activities and their affinities for substrate and for Ca2+ ions were examined in comparison with the native enzyme. Enzymatic characterization indicated that the presence of Asp-49 is essential for effective hydrolysis of phospholipids. Conversion of Asp-49 to either Glu or Lys strongly reduces the binding of Ca2+ ions in particular for the lysine mutant but the affinity for substrate analogues is hardly affected. Extensive purification of [Lys49]phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein which was 4000 times less active than the basic [Asp49]phospholipase A2 from this venom. Inhibition studies with p-bromophenacyl bromide showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself is inactive. The results obtained both with the porcine pancreatic phospholipase A2 mutants and with the native venom enzymes show that Asp-49 is essential for the catalytic action of phospholipase A2.

Secretion of biologically active porcine prophospholipase A2 by Saccharomyces cerevisiae. Use of the prepro sequence of the alpha-mating factor.

The cDNA coding for porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in Saccharomyces cerevisiae. Expression and secretion of proPLA could only be obtained after fusing the proPLA to the prepro sequence of the yeast alpha-mating factor. Upon secretion, the fusion protein was cleaved by the KEX2 protease yielding a 140-amino-acid zymogen-like form of the phospholipase A2. This protein was purified in high yield by ion-exchange chromatography. Limited proteolysis with trypsin cleaved the 'zymogen' to yield active phospholipase A2, which was indistinguishable from the authentic porcine pancreatic enzyme. These results show that a protein with a disulphide bridge content as high as 7 per 124 amino acid residues can be correctly processed by the yeast secretory apparatus.

Expression of porcine pancreatic phospholipase A2. Generation of active enzyme by sequence-specific cleavage of a hybrid protein from Escherichia coli.

The cDNA coding for the porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in E. coli. Expression of proPLA could only be obtained in the form of intracellular aggregates after fusing the 15 kDa proPLA to a large (greater than or equal to 45 kDa) bacterial peptide. The fusion protein was readily purified from cell lysates, and specifically cleaved. Cleavage of the fusion protein was achieved with either hydroxylamine (at Asn/Gly sequences in the denatured protein), or trypsin (between the pro- and the mature PLA in the renatured fusion protein). The former method releases a proPLA-like enzyme, while the latter directly yields PLA. Renaturation of the fusion protein was made possible by the use of a recently reported new S-sulphonation method. The released (pro)PLA was purified (yields of 2-3 mg/ltr of culture medium), and showed identical properties compared to native (pro)PLA.

Synthesis of phosphorothioate-containing DNA fragments by a modified hydroxybenzotriazole phosphotriester approach.

The phosphorothioylating agent which was obtained by treating 2,5-dichlorophenyl phosphorodichloridothioate with 1-hydroxy-6-nitrobenzotriazole proved to be very effective for the synthesis in solution and on a solid support of phosphorothioate-containing DNA fragments.