Biochemistry and Molecular Biology Education, 2012
In the current laboratory assignment, technical aspects of the polymerase chain reaction (PCR) ar... more In the current laboratory assignment, technical aspects of the polymerase chain reaction (PCR) are integrated in the context of six different bacterial outbreak scenarios. The "Enterobacterial Repetitive Intergenic Consensus Sequence" (ERIC) PCR was used to analyze different outbreak scenarios. First, groups of 2-4 students determined optimal ERIC-PCR conditions to validate the protocol and subsequently applied ERIC-PCR to identify genetic relatedness among bacterial strains. Based on these genetic fingerprints, students selected the outbreak cases from the patient samples and assessed the risk factors for the outbreak scenario. Finally, students presented their findings during a classroom presentation. The results indicated that the assignment successfully facilitated student learning on the technical aspects of (ERIC) PCR and clearly demonstrated the practical application of PCR in a clinical diagnostic setting. Additionally, the assignment was highly appreciated by the students.
Burkholderia cepacia complex (Bcc) and Pseudomonas aeruginosa strains, colonize the respiratory t... more Burkholderia cepacia complex (Bcc) and Pseudomonas aeruginosa strains, colonize the respiratory tract of cyctic fibrosis patients. These strains are phenotypically difficult to discriminate, but differ greatly in their pathogenic potential and species identification is relevant. Here, three methods were compared for their diagnostic capacity. A Bcc collection was analyzed with Raman spectroscopy, AFLP and rep-PCR analysis. Raman spectroscopy of 40 strains revealed high similarity. Rep-PCR and AFLP of respectively 96 and 112 strains revealed that Bcc strains could be distinguished from Pseudomonas strains. Both molecular methods allowed the identification of most Bcc species according to previous phenotypic and molecular characterization. Both AFLP and rep-PCR method data correspond with the previously reported species identification. However, Raman spectroscopy does not discriminate among P. aeruginosa and Bcc species and is therefore not useful as a diagnostic tool.
Recent studies in rodents have shown that there are significant differences in gene expression pr... more Recent studies in rodents have shown that there are significant differences in gene expression profiles between the hippocampal subregions CA1, CA3, and DG. These differences in molecular make-up within the hippocampus most likely underlie the differences in morphology, physiology, and vulnerability to insults that exist between the subregions of the hippocampus and are as such part of the basic molecular architecture of the hippocampus. The aim of this study was to investigate at large scale whether these subregional differences in gene expression are conserved in the hippocampus of a nonhuman primate, the common marmoset. This study is very timely, given the recent development of the first marmoset-specific DNA microarray, exclusively containing sequences targeting transcripts derived from the marmoset hippocampus. Hippocampal subregions CA1, CA3, and DG were isolated by laser microdissection and RNA was isolated, amplifed, and hybridized to the marmoset-specific microarray (EUMAMA) containing more than 1,500 transcripts expressed in the adult marmoset hippocampus. Large differences in expression were observed in particular between the DG region and both pyramidal subregions. Moreover, the subregion-specific patterns of gene expression showed a remarkable conservation with the rodent brain both in terms of individual genes and degree of differential expression. To our knowledge, this is the first study investigating large scale hippocampal gene expression in a nonhuman primate. The obtained expression profiles not only provide novel data on the expression of more than 1,500 transcripts per hippocampal subregion but also are of potential interest to neuroscientists interested in the role of the different subregions in learning and memory in the nonhuman primate brain. V V C 2009 Wiley-Liss, Inc. Abbreviations used: Adcy1, adenylate cyclase 1; Aff2, AF4/FMR2 family, member 2; Ap1m1, adaptor-related protein complex AP-1, mu subunit 1; Ap4b1, adaptor-related protein complex AP-4, beta 1; Arc, activity regulated cytoskeletal-associated protein; Atp2a2, ATPase, Ca11 transporting, cardiac muscle, slow twitch 2; Bcl2l2, Bcl2-like; Bdnf, brain derived neurotrophic factor; CA1, cornu ammonis 1; CA3, cornu ammonis 3; Celsr2, cadherin EGF LAG seven-pass G-type receptor 2; Chgb, chromogranin B; Cnn3, calponin 3, acidic; Comt, catechol-O-methyltransferase; DG, dentate gyrus; Dgcr6, DiGeorge syndrome critical region gene 6; Dusp6, dual specificity phosphatase 6; Eif2ak1, eukaryotic translation initiation factor 2 alpha kinase 1; Etv5, ets variant gene 5; EUMAMA, European Marmoset MicroArray; FDR, false discovery rate; Fyn, Fyn proto-oncogene; Gabarapl1, gamma-aminobutyric acid (GABA(A)) receptor-associated protein-like 1; Gabrd, gamma-aminobutyric acid (GABA-A) receptor, subunit delta; Gabrg2, gammaaminobutyric acid (GABA-A) receptor, subunit gamma 2; Glo1, glyoxalase 1; Gpr125, G protein-coupled receptor 125; Gpx3, glutathione peroxidase 3; Gucy1a3, Guanylate cyclase 1, soluble, alpha 3; Hapln4, hyaluronan and proteoglycan link protein 4; Hnmt, histamine N-methyltransferase; Hspa9b, heat shock 70kDa protein 9B; Ihpk2, inositol hexaphosphate kinase 2; ISH, in situ hybridization; Jam3, junction adhesion molecule 3; Klc2, kinesin light chain 2; Ldb2, LIM domain binding 2; Lphn2, latrophilin 2; LTP, long term potentiation; Map3k5, mitogen activated protein kinase kinase kinase 5; Mapk8ip1, mitogen activated protein kinase 8 interacting protein 1; Mica, MHC class I polypeptide-related sequence A; Ncald, neurocalcin delta; Ncam, neural cell adhesion molecules; Npy1r, neuropeptide Y receptor Y1; Nrcam, neuronal cell adhesion molecule; Ntrk2, neurotrophic tyrosine kinase, receptor, type 2; Opcml, opioid binding protein/cell adhesion molecule-like; Pcdha4, protocadherin alpha 4; Pkp1, plakophilin 1; Prkca, protein kinase C, alpha; Psen2, presenilin 2; qPCR, quantitative PCR; Rabep1, rabaptin, RAB GTPase binding effector protein 1; Rap1gds1, RAP1, GTP-GDP dissociation stimulator 1; Rassf5, Ras association (RalGDS/AF-6) domain family 5; Rims4, regulating synaptic membrane exocytosis 4; Rnd1, Rho family GTPase 1; Scotin, scotin gene; Serpini1, serine (or cysteine) peptidase inhibitor, clade I, member 1; Sh3gl2, SH3-domain GRB2-like 2; Snap25, synaptosomal-associated protein 25; Snph, syntaphilin; Sphk2, sphingosine kinase 2; Stambp, Stam binding protein; Stx3, syntaxin 3; Syn2, synapsin II; Synj2, synaptojanin 2; Vamp1, vesicleassociated membrane protein 1; Vamp3, vesicle-associated membrane protein 3; Vav3, vav 3 oncogene; Vdac1, voltage-dependent anion channel 1.
The aim of the current study was (i) to examine the overlap in the pattern of glucocorticoid rece... more The aim of the current study was (i) to examine the overlap in the pattern of glucocorticoid receptor (GR)-mediated transcriptional responses between different neuronal substrates and (ii) to assess the nature of these responses by differentiating between primary and downstream GR-responsive genes. For this purpose, nerve growth factor-differentiated catecholaminergic PC12 cells were used in which endogenous GRs were activated briefly with a high dose of corticosterone followed by gene expression profiling 1 and 3 h afterwards using Affymetrix GeneChips. The results revealed a strikingly similar temporal pattern to that which was reported previously in hippocampus, with only down-regulated genes 1 h after GR activation and the majority of genes up-regulated 3 h after GR activation. Real-time quantatitive PCR of tran-scripts in cycloheximide-treated cells showed that all five GRresponsive genes selected from the 1-h time point were primary responsive, whereas all four GR-responsive genes selected from the 3-h time point were downstream responsive. At the level of individual genes, the overlap with the previously generated hippocampal data sets was small, illustrating the cell-type specifity of GR-mediated genomic responses. Finally, we identified a number of interesting genes, such as SWI/SNF, synaptosomal-associated protein 25 and certain Rab proteins which may play a role in the effects of glucocorticoids on catecholaminergic neuronal functioning.
A semi-quantitative Real-Time PCR strategy was developed to identify indicator organisms for an i... more A semi-quantitative Real-Time PCR strategy was developed to identify indicator organisms for an indication on anastomotic leakage in peritoneal drainage fluid, Escherichia coli and Enterococcus faecalis. Materials and Methods: The analytical performance of the amplification method was validated with 10 culture-positive and 7 culture negative peritoneal drain fluid samples, obtained from 9 different patients with a colorectal anastomosis. Results: Real-Time PCR results were fully concordant with the microbiological culture results. However, among the culture negative samples, four false-positive RT-PCR results were found. All false-positives originated from a single patient with a surgical site infection. This may indicate an elevated sensitivity of the RT-PCR method.
Expression profiling in the hippocampus is hampered by its cellular heterogeneity. The aim of thi... more Expression profiling in the hippocampus is hampered by its cellular heterogeneity. The aim of this study was to evaluate the feasibility of using laser-microdissected hippocampal subregions for expression profiling to improve detection of transcripts with a subregion-specific expression. Cornu ammonis (CA)3 and dentate gyrus (DG) subregions were isolated from rat brain slices using laser microdissection, subjected to two rounds of linear amplification and hybridized to rat GeneChips containing approximately 8000 transcripts (RG_U34A; Affymetrix). Analysis of the data using significance analysis of microarrays revealed 724 genes with a significant difference in expression between CA3 and DG with a false discovery rate of 2.1%, of which 264 had higher expression in DG and 460 in CA3. Several transcripts with known differential expression between the subregions were included in the dataset, as well as numerous novel mRNAs and expressed sequence tags. Sorting of the differentially expressed genes according to gene ontology classification revealed that genes involved in glycolysis and general metabolism, neurogenesis and cell adhesion were consistently expressed at higher levels in CA3. Conversely, a large cluster of genes involved in protein biosynthesis were expressed at higher levels in DG. In situ hybridization was used to validate differential expression of a selection of genes. The results of this study demonstrate that the combination of laser microdissection and GeneChip technology is both technically feasible and very promising. Besides providing an extensive inventory of genes showing differential expression between CA3 and DG, this study supports the idea that profiling in hippocampal subregions should improve detection of genes with a subregion-specific expression or regulation.
The common marmoset monkey (Callithrix jacchus), a small non-endangered New World primate native ... more The common marmoset monkey (Callithrix jacchus), a small non-endangered New World primate native to eastern Brazil, is becoming increasingly used as a non-human primate model in biomedical research, drug development and safety assessment. In contrast to the growing interest for the marmoset as an animal model, the molecular tools for genetic analysis are extremely limited.
Biochemistry and Molecular Biology Education, 2012
In the current laboratory assignment, technical aspects of the polymerase chain reaction (PCR) ar... more In the current laboratory assignment, technical aspects of the polymerase chain reaction (PCR) are integrated in the context of six different bacterial outbreak scenarios. The "Enterobacterial Repetitive Intergenic Consensus Sequence" (ERIC) PCR was used to analyze different outbreak scenarios. First, groups of 2-4 students determined optimal ERIC-PCR conditions to validate the protocol and subsequently applied ERIC-PCR to identify genetic relatedness among bacterial strains. Based on these genetic fingerprints, students selected the outbreak cases from the patient samples and assessed the risk factors for the outbreak scenario. Finally, students presented their findings during a classroom presentation. The results indicated that the assignment successfully facilitated student learning on the technical aspects of (ERIC) PCR and clearly demonstrated the practical application of PCR in a clinical diagnostic setting. Additionally, the assignment was highly appreciated by the students.
Burkholderia cepacia complex (Bcc) and Pseudomonas aeruginosa strains, colonize the respiratory t... more Burkholderia cepacia complex (Bcc) and Pseudomonas aeruginosa strains, colonize the respiratory tract of cyctic fibrosis patients. These strains are phenotypically difficult to discriminate, but differ greatly in their pathogenic potential and species identification is relevant. Here, three methods were compared for their diagnostic capacity. A Bcc collection was analyzed with Raman spectroscopy, AFLP and rep-PCR analysis. Raman spectroscopy of 40 strains revealed high similarity. Rep-PCR and AFLP of respectively 96 and 112 strains revealed that Bcc strains could be distinguished from Pseudomonas strains. Both molecular methods allowed the identification of most Bcc species according to previous phenotypic and molecular characterization. Both AFLP and rep-PCR method data correspond with the previously reported species identification. However, Raman spectroscopy does not discriminate among P. aeruginosa and Bcc species and is therefore not useful as a diagnostic tool.
Recent studies in rodents have shown that there are significant differences in gene expression pr... more Recent studies in rodents have shown that there are significant differences in gene expression profiles between the hippocampal subregions CA1, CA3, and DG. These differences in molecular make-up within the hippocampus most likely underlie the differences in morphology, physiology, and vulnerability to insults that exist between the subregions of the hippocampus and are as such part of the basic molecular architecture of the hippocampus. The aim of this study was to investigate at large scale whether these subregional differences in gene expression are conserved in the hippocampus of a nonhuman primate, the common marmoset. This study is very timely, given the recent development of the first marmoset-specific DNA microarray, exclusively containing sequences targeting transcripts derived from the marmoset hippocampus. Hippocampal subregions CA1, CA3, and DG were isolated by laser microdissection and RNA was isolated, amplifed, and hybridized to the marmoset-specific microarray (EUMAMA) containing more than 1,500 transcripts expressed in the adult marmoset hippocampus. Large differences in expression were observed in particular between the DG region and both pyramidal subregions. Moreover, the subregion-specific patterns of gene expression showed a remarkable conservation with the rodent brain both in terms of individual genes and degree of differential expression. To our knowledge, this is the first study investigating large scale hippocampal gene expression in a nonhuman primate. The obtained expression profiles not only provide novel data on the expression of more than 1,500 transcripts per hippocampal subregion but also are of potential interest to neuroscientists interested in the role of the different subregions in learning and memory in the nonhuman primate brain. V V C 2009 Wiley-Liss, Inc. Abbreviations used: Adcy1, adenylate cyclase 1; Aff2, AF4/FMR2 family, member 2; Ap1m1, adaptor-related protein complex AP-1, mu subunit 1; Ap4b1, adaptor-related protein complex AP-4, beta 1; Arc, activity regulated cytoskeletal-associated protein; Atp2a2, ATPase, Ca11 transporting, cardiac muscle, slow twitch 2; Bcl2l2, Bcl2-like; Bdnf, brain derived neurotrophic factor; CA1, cornu ammonis 1; CA3, cornu ammonis 3; Celsr2, cadherin EGF LAG seven-pass G-type receptor 2; Chgb, chromogranin B; Cnn3, calponin 3, acidic; Comt, catechol-O-methyltransferase; DG, dentate gyrus; Dgcr6, DiGeorge syndrome critical region gene 6; Dusp6, dual specificity phosphatase 6; Eif2ak1, eukaryotic translation initiation factor 2 alpha kinase 1; Etv5, ets variant gene 5; EUMAMA, European Marmoset MicroArray; FDR, false discovery rate; Fyn, Fyn proto-oncogene; Gabarapl1, gamma-aminobutyric acid (GABA(A)) receptor-associated protein-like 1; Gabrd, gamma-aminobutyric acid (GABA-A) receptor, subunit delta; Gabrg2, gammaaminobutyric acid (GABA-A) receptor, subunit gamma 2; Glo1, glyoxalase 1; Gpr125, G protein-coupled receptor 125; Gpx3, glutathione peroxidase 3; Gucy1a3, Guanylate cyclase 1, soluble, alpha 3; Hapln4, hyaluronan and proteoglycan link protein 4; Hnmt, histamine N-methyltransferase; Hspa9b, heat shock 70kDa protein 9B; Ihpk2, inositol hexaphosphate kinase 2; ISH, in situ hybridization; Jam3, junction adhesion molecule 3; Klc2, kinesin light chain 2; Ldb2, LIM domain binding 2; Lphn2, latrophilin 2; LTP, long term potentiation; Map3k5, mitogen activated protein kinase kinase kinase 5; Mapk8ip1, mitogen activated protein kinase 8 interacting protein 1; Mica, MHC class I polypeptide-related sequence A; Ncald, neurocalcin delta; Ncam, neural cell adhesion molecules; Npy1r, neuropeptide Y receptor Y1; Nrcam, neuronal cell adhesion molecule; Ntrk2, neurotrophic tyrosine kinase, receptor, type 2; Opcml, opioid binding protein/cell adhesion molecule-like; Pcdha4, protocadherin alpha 4; Pkp1, plakophilin 1; Prkca, protein kinase C, alpha; Psen2, presenilin 2; qPCR, quantitative PCR; Rabep1, rabaptin, RAB GTPase binding effector protein 1; Rap1gds1, RAP1, GTP-GDP dissociation stimulator 1; Rassf5, Ras association (RalGDS/AF-6) domain family 5; Rims4, regulating synaptic membrane exocytosis 4; Rnd1, Rho family GTPase 1; Scotin, scotin gene; Serpini1, serine (or cysteine) peptidase inhibitor, clade I, member 1; Sh3gl2, SH3-domain GRB2-like 2; Snap25, synaptosomal-associated protein 25; Snph, syntaphilin; Sphk2, sphingosine kinase 2; Stambp, Stam binding protein; Stx3, syntaxin 3; Syn2, synapsin II; Synj2, synaptojanin 2; Vamp1, vesicleassociated membrane protein 1; Vamp3, vesicle-associated membrane protein 3; Vav3, vav 3 oncogene; Vdac1, voltage-dependent anion channel 1.
The aim of the current study was (i) to examine the overlap in the pattern of glucocorticoid rece... more The aim of the current study was (i) to examine the overlap in the pattern of glucocorticoid receptor (GR)-mediated transcriptional responses between different neuronal substrates and (ii) to assess the nature of these responses by differentiating between primary and downstream GR-responsive genes. For this purpose, nerve growth factor-differentiated catecholaminergic PC12 cells were used in which endogenous GRs were activated briefly with a high dose of corticosterone followed by gene expression profiling 1 and 3 h afterwards using Affymetrix GeneChips. The results revealed a strikingly similar temporal pattern to that which was reported previously in hippocampus, with only down-regulated genes 1 h after GR activation and the majority of genes up-regulated 3 h after GR activation. Real-time quantatitive PCR of tran-scripts in cycloheximide-treated cells showed that all five GRresponsive genes selected from the 1-h time point were primary responsive, whereas all four GR-responsive genes selected from the 3-h time point were downstream responsive. At the level of individual genes, the overlap with the previously generated hippocampal data sets was small, illustrating the cell-type specifity of GR-mediated genomic responses. Finally, we identified a number of interesting genes, such as SWI/SNF, synaptosomal-associated protein 25 and certain Rab proteins which may play a role in the effects of glucocorticoids on catecholaminergic neuronal functioning.
A semi-quantitative Real-Time PCR strategy was developed to identify indicator organisms for an i... more A semi-quantitative Real-Time PCR strategy was developed to identify indicator organisms for an indication on anastomotic leakage in peritoneal drainage fluid, Escherichia coli and Enterococcus faecalis. Materials and Methods: The analytical performance of the amplification method was validated with 10 culture-positive and 7 culture negative peritoneal drain fluid samples, obtained from 9 different patients with a colorectal anastomosis. Results: Real-Time PCR results were fully concordant with the microbiological culture results. However, among the culture negative samples, four false-positive RT-PCR results were found. All false-positives originated from a single patient with a surgical site infection. This may indicate an elevated sensitivity of the RT-PCR method.
Expression profiling in the hippocampus is hampered by its cellular heterogeneity. The aim of thi... more Expression profiling in the hippocampus is hampered by its cellular heterogeneity. The aim of this study was to evaluate the feasibility of using laser-microdissected hippocampal subregions for expression profiling to improve detection of transcripts with a subregion-specific expression. Cornu ammonis (CA)3 and dentate gyrus (DG) subregions were isolated from rat brain slices using laser microdissection, subjected to two rounds of linear amplification and hybridized to rat GeneChips containing approximately 8000 transcripts (RG_U34A; Affymetrix). Analysis of the data using significance analysis of microarrays revealed 724 genes with a significant difference in expression between CA3 and DG with a false discovery rate of 2.1%, of which 264 had higher expression in DG and 460 in CA3. Several transcripts with known differential expression between the subregions were included in the dataset, as well as numerous novel mRNAs and expressed sequence tags. Sorting of the differentially expressed genes according to gene ontology classification revealed that genes involved in glycolysis and general metabolism, neurogenesis and cell adhesion were consistently expressed at higher levels in CA3. Conversely, a large cluster of genes involved in protein biosynthesis were expressed at higher levels in DG. In situ hybridization was used to validate differential expression of a selection of genes. The results of this study demonstrate that the combination of laser microdissection and GeneChip technology is both technically feasible and very promising. Besides providing an extensive inventory of genes showing differential expression between CA3 and DG, this study supports the idea that profiling in hippocampal subregions should improve detection of genes with a subregion-specific expression or regulation.
The common marmoset monkey (Callithrix jacchus), a small non-endangered New World primate native ... more The common marmoset monkey (Callithrix jacchus), a small non-endangered New World primate native to eastern Brazil, is becoming increasingly used as a non-human primate model in biomedical research, drug development and safety assessment. In contrast to the growing interest for the marmoset as an animal model, the molecular tools for genetic analysis are extremely limited.
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