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Papers by Martien Broekhuijsen

Research paper thumbnail of Gunnar Skogan Dory van de Meent

Validation of the air collector SASS 2000PLUS in a bioaerosol test (BAT) chamber at TNO Forsvaret... more Validation of the air collector SASS 2000PLUS in a bioaerosol test (BAT) chamber at TNO Forsvarets forskningsinstitutt

Research paper thumbnail of Validation of the air collector SASS 2000PLUS in a bioaerosol test (BAT) chamber at TNO

Research paper thumbnail of Microarrays for Genomotyping of Pathogens

Detection of Highly Dangerous Pathogens

Research paper thumbnail of Novel Dutch Self-Assessment Biosecurity Toolkit to Identify Biorisk Gaps and to Enhance Biorisk Awareness

Frontiers in Public Health, 2014

Research paper thumbnail of Synthesis of fusion proteins containing antigenic determinants of foot-and-mouth disease virus

Vaccine, 1986

Part of the genome of foot-and-mouth disease virus (FMDV) type 01,BFS, including the sequence enc... more Part of the genome of foot-and-mouth disease virus (FMDV) type 01,BFS, including the sequence encoding the capsid polypeptide VP1, was cloned in Escherichia coli following a new cloning strategy. The clone containing the VP1 sequence was used for the construction of two expression plasmids encoding VP1 fusion proteins. Subsequently, substantial amounts of the two VP1-beta-galactosidase fusion proteins, containing either one (amino acid region 140-160) or two (amino acid regions 140-160 and 200-213) antigenic determinants of the virus, were synthesized by E. coli bacteria. The protein containing the amino acid region 140-160 of VP1 fused to beta-galactosidase efficiently induced antibodies in rabbits specifically reacting with FMDV type 01,BFS. The same protein was also capable of eliciting neutralizing antibodies. The fusion protein containing both antigenic determinants did not efficiently induce antibodies reacting with FMDV.

Research paper thumbnail of The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: A convenient and general selection system for filamentous fungal transformation

Molecular and General Genetics MGG, 1989

The development of an efficient and homologous transformation system for Aspergillus oryzae is de... more The development of an efficient and homologous transformation system for Aspergillus oryzae is described. This is based on nitrate reductase (niaD) of the nitrate assimilation pathway. The niaD system offers a number of inherent advantages over many other systems and may be of general use for nitrate-utilising filamentous fungi. Transformation frequencies of up to 800 transformants per microgram DNA are observed with A. oryzae. The preponderance of integration events take place at the resident niaD locus either by gene conversion (41%), single integration (23%) or multiple tandem integration (36%). Heterologous expression of the A. oryzae niaD gene in the filamentous fungi A. nidulans, A. niger and Penicillium chrysogenum is observed. That heterologous putative niaD hybridisation signals are seen with other fungal DNAs affords the opportunity to isolate the corresponding niaD from various fungi in order to develop homolgous transformation. Co-transformation with the introduction of the non-selected markers pyrG, tub-2, and uidA has been achieved.

Research paper thumbnail of Genome-Wide DNA Microarray Analysis of Francisella tularensis Strains Demonstrates Extensive Genetic Conservation within the Species but Identifies Regions That Are Unique to the Highly Virulent F. tularensis subsp. tularensis

Journal of Clinical Microbiology, 2003

Francisella tularensis is a potent pathogen and a possible bioterrorism agent. Little is known, h... more Francisella tularensis is a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies, F. tularensis subsp. tularensis , F. tularensis subsp. mediasiatica , F. tularensis subsp. holarctica , and F. tularensis subsp. novicida . We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain F. tularensis subsp. tularensis Schu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species F. tularensis , and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e., F. tularensis subsp. tularensis and F. tulare...

Research paper thumbnail of Secretion of heterologous proteins by Aspergillus niger: Production of active human interleukin-6 in a protease-deficient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein

Journal of Biotechnology, 1993

To develop improved methods for heterologous protein production in Aspergillus niger, we studied ... more To develop improved methods for heterologous protein production in Aspergillus niger, we studied the secretion of human interleukin-6 (hlL6). Since in vitro experiments with culture medium revealed that hlL6 was rapidly degraded, several protease-deficient strains of A. niger were isolated and tested for reduced degradation of hlL6 compared with the wild-type strain. The mutant strain giving the least degradative effect on hlL6 (designated ABI.13) was transformed with several hlL6-expression plasmids. Initially, hlL6 was expressed using various signal sequences fused to the sequence of mature hlL6. The resulting transformants did not produce detectable amounts of hlL6, despite high transcription levels in one transformant. We hypothesized that hlL6 was not efficiently processed during passage along the secretion pathway. Therefore, hlL6 was expressed as a fusion protein with glucoamylase, a protein which is efficiently secreted by A. niger

Research paper thumbnail of Synthesis of fusion proteins with multiple copies of an antigenic determinant of foot-and-mouth disease virus antibodies

Gene, 1986

A series of four expression plasmids coding for fusion proteins containing foot-and-mouth disease... more A series of four expression plasmids coding for fusion proteins containing foot-and-mouth disease virus (FMDV) sequences was constructed. The fusion proteins contain a large part of beta-galactosidase from Escherichia coli preceded (N-terminal) by 1, 2, 4 or 8 repeats of the antigenic determinant of FMDV consisting of amino acids 137-162 of the capsid polypeptide VP1. All four fusion proteins were efficiently produced in E. coli host bacteria. Immunization of rabbits resulted in FMDV-specific, neutralizing antibodies, the response being dependent on the number of repeats. With enzyme-linked immunosorbent-assay techniques it was shown that the FMDV antigenic determinants are exposed on the surface of the fusion proteins under non-denaturing conditions.

Research paper thumbnail of A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae

Current Genetics, 1989

A homologous transformation system for Aspergillus oryzae is described. The system is based on an... more A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. owzae strain deficient in orotidine-5'-phosphate decarboxylase (pyrG) and the vector pAO4-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. owzae pyrG mutant with circular pAO4-2 resulted in the appearance of Pyr + transformants at a frequency of up to 20 per lag of DNA, whereas with linear pAO4-2 up to 200 transformants per btg DNA were obtained. In 75 % of the Pyr + transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pAO4-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding 13-galactosidase, and uidA, encoding 13-glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.

Research paper thumbnail of Preliminary validation of real-time PCR assays for the identification of Yersinia pestis

Clinical Chemistry and Laboratory Medicine, 2008

Background: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents ... more Background: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high casefatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis. Methods: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values. Results: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results. Conclusions: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples.

Research paper thumbnail of Gunnar Skogan Dory van de Meent

Validation of the air collector SASS 2000PLUS in a bioaerosol test (BAT) chamber at TNO Forsvaret... more Validation of the air collector SASS 2000PLUS in a bioaerosol test (BAT) chamber at TNO Forsvarets forskningsinstitutt

Research paper thumbnail of Validation of the air collector SASS 2000PLUS in a bioaerosol test (BAT) chamber at TNO

Research paper thumbnail of Microarrays for Genomotyping of Pathogens

Detection of Highly Dangerous Pathogens

Research paper thumbnail of Novel Dutch Self-Assessment Biosecurity Toolkit to Identify Biorisk Gaps and to Enhance Biorisk Awareness

Frontiers in Public Health, 2014

Research paper thumbnail of Synthesis of fusion proteins containing antigenic determinants of foot-and-mouth disease virus

Vaccine, 1986

Part of the genome of foot-and-mouth disease virus (FMDV) type 01,BFS, including the sequence enc... more Part of the genome of foot-and-mouth disease virus (FMDV) type 01,BFS, including the sequence encoding the capsid polypeptide VP1, was cloned in Escherichia coli following a new cloning strategy. The clone containing the VP1 sequence was used for the construction of two expression plasmids encoding VP1 fusion proteins. Subsequently, substantial amounts of the two VP1-beta-galactosidase fusion proteins, containing either one (amino acid region 140-160) or two (amino acid regions 140-160 and 200-213) antigenic determinants of the virus, were synthesized by E. coli bacteria. The protein containing the amino acid region 140-160 of VP1 fused to beta-galactosidase efficiently induced antibodies in rabbits specifically reacting with FMDV type 01,BFS. The same protein was also capable of eliciting neutralizing antibodies. The fusion protein containing both antigenic determinants did not efficiently induce antibodies reacting with FMDV.

Research paper thumbnail of The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: A convenient and general selection system for filamentous fungal transformation

Molecular and General Genetics MGG, 1989

The development of an efficient and homologous transformation system for Aspergillus oryzae is de... more The development of an efficient and homologous transformation system for Aspergillus oryzae is described. This is based on nitrate reductase (niaD) of the nitrate assimilation pathway. The niaD system offers a number of inherent advantages over many other systems and may be of general use for nitrate-utilising filamentous fungi. Transformation frequencies of up to 800 transformants per microgram DNA are observed with A. oryzae. The preponderance of integration events take place at the resident niaD locus either by gene conversion (41%), single integration (23%) or multiple tandem integration (36%). Heterologous expression of the A. oryzae niaD gene in the filamentous fungi A. nidulans, A. niger and Penicillium chrysogenum is observed. That heterologous putative niaD hybridisation signals are seen with other fungal DNAs affords the opportunity to isolate the corresponding niaD from various fungi in order to develop homolgous transformation. Co-transformation with the introduction of the non-selected markers pyrG, tub-2, and uidA has been achieved.

Research paper thumbnail of Genome-Wide DNA Microarray Analysis of Francisella tularensis Strains Demonstrates Extensive Genetic Conservation within the Species but Identifies Regions That Are Unique to the Highly Virulent F. tularensis subsp. tularensis

Journal of Clinical Microbiology, 2003

Francisella tularensis is a potent pathogen and a possible bioterrorism agent. Little is known, h... more Francisella tularensis is a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies, F. tularensis subsp. tularensis , F. tularensis subsp. mediasiatica , F. tularensis subsp. holarctica , and F. tularensis subsp. novicida . We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain F. tularensis subsp. tularensis Schu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species F. tularensis , and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e., F. tularensis subsp. tularensis and F. tulare...

Research paper thumbnail of Secretion of heterologous proteins by Aspergillus niger: Production of active human interleukin-6 in a protease-deficient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein

Journal of Biotechnology, 1993

To develop improved methods for heterologous protein production in Aspergillus niger, we studied ... more To develop improved methods for heterologous protein production in Aspergillus niger, we studied the secretion of human interleukin-6 (hlL6). Since in vitro experiments with culture medium revealed that hlL6 was rapidly degraded, several protease-deficient strains of A. niger were isolated and tested for reduced degradation of hlL6 compared with the wild-type strain. The mutant strain giving the least degradative effect on hlL6 (designated ABI.13) was transformed with several hlL6-expression plasmids. Initially, hlL6 was expressed using various signal sequences fused to the sequence of mature hlL6. The resulting transformants did not produce detectable amounts of hlL6, despite high transcription levels in one transformant. We hypothesized that hlL6 was not efficiently processed during passage along the secretion pathway. Therefore, hlL6 was expressed as a fusion protein with glucoamylase, a protein which is efficiently secreted by A. niger

Research paper thumbnail of Synthesis of fusion proteins with multiple copies of an antigenic determinant of foot-and-mouth disease virus antibodies

Gene, 1986

A series of four expression plasmids coding for fusion proteins containing foot-and-mouth disease... more A series of four expression plasmids coding for fusion proteins containing foot-and-mouth disease virus (FMDV) sequences was constructed. The fusion proteins contain a large part of beta-galactosidase from Escherichia coli preceded (N-terminal) by 1, 2, 4 or 8 repeats of the antigenic determinant of FMDV consisting of amino acids 137-162 of the capsid polypeptide VP1. All four fusion proteins were efficiently produced in E. coli host bacteria. Immunization of rabbits resulted in FMDV-specific, neutralizing antibodies, the response being dependent on the number of repeats. With enzyme-linked immunosorbent-assay techniques it was shown that the FMDV antigenic determinants are exposed on the surface of the fusion proteins under non-denaturing conditions.

Research paper thumbnail of A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae

Current Genetics, 1989

A homologous transformation system for Aspergillus oryzae is described. The system is based on an... more A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. owzae strain deficient in orotidine-5'-phosphate decarboxylase (pyrG) and the vector pAO4-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. owzae pyrG mutant with circular pAO4-2 resulted in the appearance of Pyr + transformants at a frequency of up to 20 per lag of DNA, whereas with linear pAO4-2 up to 200 transformants per btg DNA were obtained. In 75 % of the Pyr + transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pAO4-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding 13-galactosidase, and uidA, encoding 13-glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.

Research paper thumbnail of Preliminary validation of real-time PCR assays for the identification of Yersinia pestis

Clinical Chemistry and Laboratory Medicine, 2008

Background: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents ... more Background: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high casefatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis. Methods: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values. Results: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results. Conclusions: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples.