Het tweede BaMa-symposium was weer een succes!

We waren dit jaar te gast op de Hogeschool Leiden. De voorzieningen waren uitstekend en de organisatie liep geolied.
Introductie en welkom werden verzorgd door John van der Willik, directeur Techniek van de Hogeschool Leiden.  Hij waardeerde het zeer dat zijn opleidingsinstituut kon bijdragen aan dit belangrijke aspect van het bachelor- en masteronderwijs.

 

Sprekers, aanwezige docenten en studenten waren zeer enthousiast over het programma, de kwaliteit van de voordrachten en de ervaring om buiten de onderwijsinstelling te presenteren en mensen te spreken. Voor zover we mensen gesproken hebben is er grote eenstemmigheid om het BAMA symposium jaarlijks te continueren. De presentatie van de iGEM winnaars over de chip om verrotting van vlees aan te tonen liet de studenten zien dat in een kort project veel te bereiken valt.

 

Programma

09.30 – 09.30u           Aankomst, registratie, koffie en thee

09.30 – 09.40u           Welkom en korte introductie

09.40 – 10.40u           Sessie 1: presentaties 1 t/m 3 (Chair : Annelies van Goor, HLO Leiden)

10.40 – 11.10u           Koffie en thee

11.30 – 12.30u           Sessie 2: presentaties 4 t/m 6 ((Chair : Annelies van Goor, HLO Leiden)

12.30 – 13.30u           Lunch in het restaurant van de HL

13.30 – 14.50u            Sessie 3: presentaties 7 t/m 10 (chair Loek van Alphen)

14.50 – 16.00u           Afsluiting met drankjes en award voor de beste presetatie

15.00 – 16.30u           Drankjes en uitreiking prijs voor de beste presentatie

Presentaties

Muriëlle van der Horst
Fontys Hogeschool Eindhoven Toegepaste  natuurwetenschappen, Applied Science

Validation of serological diagnosis of Hepatitis E virus infections.

 Aim:
 A clinical validation of the serological detection of Hepatitis E virus (HEV) was performed in two serum panels.

 Approach:
 The first panel (positive-, negative- and crossreactive samples) was tested by two commercial ELISA assays (IgG/IgM;     Wantai and Recomwell). The second panel (90 children <2 years) was included in order to improve our estimate of the  specificity.

 Results:
 Considering IgM, the specificity of Wantai was higher compared to Recomwell, 94% and 87,8% respectively.   Considering IgG, Recomwell appeared better than Wantai, 81% and 72%, respectively. The HEV IgG seroprevalence in  children was 2.2 % in both commercial assays. 

 Conclusion:
 Wantai-IgM and Recomwell-IgG ELISA showed the best analytical performance. A  high number of false positives was observed in both IgG assays. However this observation was made only in adult samples and not in young children who are less likely to be HEV exposed. This suggests that the HEV seroprevalence in Dutch adults may be around 20%.
 

Alba Torrents
Medical Microbiology, Universiteit van Amsterdam

Stabilizing HIV-1 protein vaccines by disulfide bonds.

Aim:
Soluble HIV-1 envelope glycoprotein complex (Env), consisting of gp120 and gp41, can be the best option for vaccine design. Env structure has been extensively studied but there is a lack of information on gp120-gp41 interface. The aim of this study is to map gp120-gp41 interface and to improve stabilization of soluble Env.

Approach:
Soluble Env can be stabilized by introducing a disulfide bond between gp120-gp41 (SOS-gp140), but this alters Env structure. A panel of new SOS-gp140 was generated, relocating the disulfide bond based on directed evolution data.

Results:
The panel of new SOS-gp140 indicates that Env mutants can trimerize and be cleaved with different efficiencies than the original SOS-gp140. HIV-1 specific antibodies bind these mutants with distinct antigenic profiles, showing that relocation of SOS induces structural changes. This information enables to determine closely interacting residues in gp120-gp41 interface.C

Conclusion:
These data will help to generate an accurate gp120-gp41 map and a stable soluble Env for the design of a protective vaccine.

Stefania Magnusdottir
Faculty of Earth and Life Sciences, VU University Amsterdam

Benefits of extra-cytosolic binding proteins in cell transport systems.

Aim:
Binding protein (BPs) dependent uptake systems are found in nearly all prokaryotes and are also functional in eukaryotes. These extra-cytosolic proteins bind to substrates and bring them to membrane-bound transporters which move the molecules to the cytosol. Synthesizing proteins requires usage of cell resources such as energy and amino acids. While the mechanical functions of BPs have been well studied, it is still uncertain how the cell benefits from this additional cost of binding protein synthesis.

Approach:
Compare simple mathematical models of BP and non-BP dependent transport networks.

Results:
At low substrate concentrations, the high affinity binding of BPs results in a high concentration of substrate-liganded BPs. This raises the effective substrate concentration for the transporters, resulting in substantially larger influx rate per transporter. Acquiring the same rate in a non-BP associated system would require the cell to produce more active transporters which are placed in the membrane.

Conclusion:
High-affinity BPs raise the effective concentration of substrate for transport and do not take up space in the membrane. When membrane space is limited, a BP-dependent transport can be highly beneficial to the cell.

Delia-Maria Goilo
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht

Genetic diversity of methicillin-resistant and susceptible Staphylococcus pseudintermedius in individual dogs.

 Aim:
Staphylococcus pseudintermedius is an opportunistic pathogen in dogs. Since 2006 a methicillin resistant variant (MRSP) with a multi-resistant phenotype is emerging. The aim of this study is to determine the phenotypic and genotypic diversity of S. pseudintermedius strains (methicillin resistant and susceptible) in a single host.

Approach:
For 20 dogs, 10 colonies of a primary culture, confirmed as  S. pseudintermedius by PCR-RFLP was characterized by PCR for the mecA gene and antimicrobial susceptibility. For each specific phenotype, one colony was characterized with PFGE, SCCmec typing, MLST and spa typing.

Results:
One dog carried three mecA positive genotypes and a mecA negative strain. PFGE analysis of these strains showed minor variation and revealed that they are highly related. In another dog two MRSP and two susceptible isolates were found that were identified as different strains with PFGE. Samples from 18 other dogs are currently being analyzed. 

Conclusion:
The first 2 dogs showed a co-colonization with susceptible and resistant S. pseudintermedius strains.

Yvette Gladdines
LUMC / Avans hogeschool

Protein export in hepatocytes in the liver stage of malaria in Plasmodium berghei.

Aim:
The aim is to explore the malaria parasite life cycle in the liver stage. Bio-informatic and literature searches have identified candidates, their expression and export will be analysed in hepatocytes. The ultimate goal is to develop intervention strategies against malaria, such as a vaccine or drugs.

Approach:
This will be done by designing and making DNA constructs with a fluorescent tag, mCherry. These constructs will be transfected into parasites that infect mice. Mosquitos will be infected to obtain the parasite stage that infects hepatocytes.The transfected parasites will be analysed in different ways, for example using PCR and fluorescent microscopy, to make sure the parasites have the altered DNA. Then hepatocytes can be infected to look at protein transport in the liver stage.

Results/future:
We have designed primers to amplify the candidate genes, done PCR cloning and made 4 complete constructs. We are now ready for the transfection step.

 

 

 

 Arjan Oldebesten and Elbrich Hendriks
University of Groningen

   Key lecture

   The Food Warden of iGEM Groningen 2012

    It's rotten and you know it!

The Food Warden is a system which detects meat spoilage. It uses Bacillus subtilis cells. Their natural genetic response to the gases of rotten meat has been identified and linked to a pigment production system. In this way, a consumer can easily see when the meat is spoiled.

Het team van elf master-studenten van de Rijksuniversiteit Groningen is in 2012 in Boston wereldkampioen geworden in de internationale iGEM-competitie!!

 

Maikel de Bresser
Fontys Hogeschool Toegepaste Natuurwetenschappen, Eindhoven

Tuf mRNA seems a promising marker to monitor Staphylococcus aureus bloodstream infection

Aim
Determine whether RNA can be used as a marker to monitor an active Staphylococcus aureus bloodstream infection.

Approach
S.aureus (100 CFU/ml) was spiked in TH broth and whole blood. Floxapen was used as antibiotic treatment. Samples were taken at day 0, 1, 3, and 6. To determine the viability of S.aureus, samples were plated on bloodagar and DNA and RNA presence of both Tuf and 16S were determined by real-time PCR.

Results
 At day 3 of floxapen treatment no growth was detectable on bloodagar plates, and Tuf mRNA was not detectable with real-time PCR. Tuf DNA and 16S rRNA /DNA were still detectable after 6 days of floxapen treatment, even when no bacteria were present on bloodagar.


Conclusion
Tuf mRNA seems to be a promising marker to measure S.aureus viability as compared to tuf DNA and 16S rRNA/DNA. This holds promise for bloodstream infection diagnostics.

Anouk Tengeler
Hogeschool Leiden & Universiteit Leiden

Contribution of increased efflux pump activity to fluoroquinolone resistance in Escherichia coli isolates

Aim
Efflux pumps are an essential component of all living cells because it removes toxic compounds from the cell. This study investigated whether increased activity of efflux pumps contributes to fluoroquinolone resistance in Escherichia coli (E. coli) isolates.

Approach
Efflux pump activity was determined in 50 E. coli isolates with reduced susceptibility against ciprofloxacin and cephalosporines. Efflux pump activity was studied with an optimised fluorometric method and with quantitative reverse transcription polymerase chain reaction (qRT-PCR) as well. Expression of efflux pump genes acrA, acrB, tolC and mdfA was investigated.

Results
A small, but significant, correlation was found between overexpression of the AcrAB-TolC pump and a multidrug-resistant phenotype.

Conclusion
Overactive efflux pump activity contributes to fluoroquinolone resistance in E. coli isolates. When the AcrAB-TolC pump is overexpressed, an isolate is more likely to be multidrug-resistant. MICs were not affected by increased efflux pump activity.

Mike Ververs
Applied Sciences, Fontys Hogeschool, Eindhoven

Validating RT-PCR's for whole blood detection of S.pneumoniae, P aeruginosa and C. albicans

Aim
Validating real-time-PCRs for S. pneumoniae (SP), P. aeruginosa (PA) and C. albicans (CA) for direct detection from whole blood.

Approach
RT-PCRs were validated using 44 bacterial/fungal strains to determine the specificity of the assays. Additionally, SP, CA and PA were spiked in Tris-EDTA buffer and whole blood to determine analytical and clinical sensitivity of the assays.

Results

The specificity of the SP and PA assay was found to be 100%. CA PCR showed cross hybridization with C. tropicalis (specificity 97,7%). Tenfold dilutions (1000-0 CFU/ml) were processed with the Polaris pathogen DNA isolation procedure. All samples until 10 CFU/ml were detected with a hitrate (% positive PCR) of 100%, The 1 CFU/ml samples showed a hitrate of 66% for both SP and PA, and a hitrate of 33% for CA.

Conclusion
Sensitive and specific real-time PCRs were developed to improve whole blood detection of these pathogens from patients suspected of having bacteremia.

 

Werner van Nijnatten
Avans Hogeschool, Breda

 

 

 

Vergelijkende studie van verschillende methoden ter identificatie van  toxinogene Clostridium difficile

(Presentatie van het projectplan)