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1.  Next-generation sequencing-based genome diagnostics across clinical genetics centers: implementation choices and their effects 
European Journal of Human Genetics  2015;23(9):1142-1150.
Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found ‘no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of ‘greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care.
PMCID: PMC4538197  PMID: 25626705
3.  New fluorescent cholesterol analogs as membrane probes 
Biochimica et biophysica acta  1999;1420(0):189-202.
New fluorescent cholesterol analogs, (22E,20R)-3β-hydroxy-23-(9-anthryl)-24-norchola-5,22-diene (R-AV-Ch), and the 20S-isomer (S-AV-Ch) were synthesized, their spectral and membrane properties were characterized. The probes bear a 9-anthrylvinyl (AV) group instead of C22–C27 segment of the cholesterol alkyl chain. Computer simulations show that both of the probes have bulkier tail regions than cholesterol and predict some perturbation in the packing of membranes, particularly for R-AV-Ch. In monolayer experiments, the force–area behavior of the probes was compared with that of cholesterol, pure and in mixtures with palmitoyloleoyl phosphatidylcholine (POPC) and N-stearoyl sphingomyelin (SSM). The results show that pure R-AV-Ch occupies 35–40% more cross-sectional area than cholesterol at surface pressures below film collapse (0–22 mN/m); whereas S-AV-Ch occupies nearly the same molecular area as cholesterol. Isotherms of POPC or SSM mixed with 0.1 mol fraction of either probe are similar to isotherms of the corresponding mixtures of POPC or SSM with cholesterol. The probes show typical AV absorption (λ 386, 368, 350 and 256 nm) and fluorescence (λ 412–435 nm) spectra. Steady-state anisotropies of R-AV-Ch and S-AV-Ch in isotropic medium or liquid–crystalline bilayers are higher than the values obtained for other AV probes reflecting hindered intramolecular mobility of the fluorophore and decreased overall rotational rate of the rigid cholesterol derivatives. This suggestion is confirmed by time-resolved fluorescence experiments which show also, in accordance with monolayer data, that S-AV-Ch is better accommodated in POPC–cholesterol bilayers than R-AV-Ch. Model and natural membranes can be labeled by either injecting the probes via a water-soluble organic solvent or by co-lyophilizing probe and phospholipid prior to vesicle production. Detergent-solubilization studies involving `raft' lipids showed that S-AV-Ch almost identically mimicked the behavior of cholesterol and that of R-AV-Ch was only slightly inferior. Overall, the data suggest that the AV-labeled cholesterol analogs mimic cholesterol behavior in membrane systems and will be useful in related studies.
PMCID: PMC4004019  PMID: 10446302
Sterol analog; Fluorescence parameter; Anthrylvinyl; Monolayer; Model bilayer; Detergent-insolubility
4.  Lack of adherence to hypertension treatment guidelines among GPs in southern Sweden-A case report-based survey 
BMC Family Practice  2012;13:34.
General practitioners (GPs) often fail to correctly adhere to guidelines for the treatment of hypertension. The reasons for this are unclear, but could be related to lack of knowledge in assessing individual patients' cardiovascular disease risk. Our aim was to investigate how GPs in southern Sweden adhere to clinical guidelines for the treatment of hypertension when major cardiovascular risk factors are taken into consideration.
A questionnaire with five genuine cases of hypertension with different cardiovascular risk profiles was sent to a random sample of GPs in southern Sweden (n = 109) in order to investigate the attitude towards blood pressure (BP) treatment when major cardiovascular risk factors were present.
In general, GPs who responded tended to focus on the absolute target BP rather than assessing the entire cardiovascular risk factor profile. Thus, cases with the highest risk of cardiovascular disease were not treated accordingly. However, there was also a tendency to overtreat the lowest risk individuals. Furthermore, the BP levels for initiating pharmacological treatment varied widely (systolic BP 140-210 mmHg). ACE inhibitors (70%) were the most common first choice of pharmacological treatment.
In this study, GPs in Southern Sweden were suggesting, for different cases, either under- or overtreatment in relation to current guidelines for treatment of hypertension. On reason may be that they failed to correctly assess individual cardiovascular risk factor profiles.
PMCID: PMC3391982  PMID: 22536853
Hypertension; Adherence; Guidelines; Treatment; Primary care
5.  Förster Resonance Energy Transfer (FRET) between Heterogeneously Distributed Probes: Application to Lipid Nanodomains and Pores 
The formation of membrane heterogeneities, e.g., lipid domains and pores, leads to a redistribution of donor (D) and acceptor (A) molecules according to their affinity to the structures formed and the remaining bilayer. If such changes sufficiently influence the Förster resonance energy transfer (FRET) efficiency, these changes can be further analyzed in terms of nanodomain/pore size. This paper is a continuation of previous work on this theme. In particular, it is demonstrated how FRET experiments should be planned and how data should be analyzed in order to achieve the best possible resolution. The limiting resolution of domains and pores are discussed simultaneously, in order to enable direct comparison. It appears that choice of suitable donor/acceptor pairs is the most crucial step in the design of experiments. For instance, it is recommended to use DA pairs, which exhibit an increased affinity to pores (i.e., partition coefficients KD,A > 10) for the determination of pore sizes with radii comparable to the Förster radius R0. On the other hand, donors and acceptors exhibiting a high affinity to different phases are better suited for the determination of domain sizes. The experimental setup where donors and acceptors are excluded from the domains/pores should be avoided.
PMCID: PMC3546683  PMID: 23203189
FRET; lipid domains; pores; lipid bilayer; rafts; fluorescent probes
6.  Barriers to adherence to hypertension guidelines among GPs in southern Sweden: A survey 
To evaluate barriers to adherence to hypertension guidelines among publicly employed general practitioners (GPs).
Questionnaire-based survey distributed to GPs in 24 randomly selected primary care centres in the Region of Skåne in southern Sweden.
A total of 109 GPs received a self-administered questionnaire and 90 of them responded.
Main outcome measures
Use of risk assessment programmes. Reasons to postpone or abstain from pharmacological treatment for the management of hypertension.
Reported managing of high blood pressure (BP) varied. In all, 53% (95% CI 42–64%) of the GPs used risk assessment programmes and nine out of 10 acknowledged blood pressure target levels. Only one in 10 did not inform the patients about these levels. The range for immediate initiating pharmacological treatment was a systolic BP 140–220 (median 170) mmHg and diastolic BP 90–110 (median 100) mmHg. One-third (32%; 95% CI 22–42%) of the GPs postponed or abstained from pharmacological treatment of hypertension due to a patient's advanced age. No statistically significant associations were observed between GPs’ gender, professional experience (i.e. in terms of specialist family medicine and by number of years in practice), and specific reasons to postpone or abstain from pharmacological treatment of hypertension.
These data suggest that GPs accept higher blood pressure levels than recommended in clinical guidelines. Old age of the patient seems to be an important barrier among GPs when considering pharmacological treatment for the management of hypertension.
PMCID: PMC3409603  PMID: 18609250
Barriers; family practice; guidelines; hypertension; primary care; survey
7.  Design and Synthesis of Fluorescent Pilicides and Curlicides: Bioactive Tools to Study Bacterial Virulence Mechanisms 
Pilicides and curlicides are compounds that block the formation of the virulence factors pili and curli, respectively. To facilitate studies of the interaction between these compounds and the pili and curli assembly systems, fluorescent pilicides and curlicides have been synthesized. This was achieved by using a strategy based on structure–activity knowledge, in which key pilicide and curlicide substituents on the ring-fused dihydrothiazolo 2-pyridone central fragment were replaced by fluorophores. Several of the resulting fluorescent compounds had improved activities as measured in pili- and curli-dependent biofilm assays. We created fluorescent pilicides and curlicides by introducing coumarin and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores at two positions on the peptidomimetic pilicide and curlicide central fragment. Fluorescence images of the uropathogenic Escherichia coli (UPEC) strain UTI89 grown in the presence of these compounds shows that the compounds are strongly associated with the bacteria with a heterogeneous distribution.
PMCID: PMC3569613  PMID: 22431310
antivirulence; biological activity; coumarin; fluorescence; structure–activity relationships

Results 1-7 (7)