Mado Vandewoestyne

Tracking the progression of the human inner cell mass during embryonic stem cell derivation

The different pluripotent states of mouse embryonic stem cells (ESCs) in vitro have been shown to correspond to stages of mouse embryonic development. For human cells, little is known about the events that precede the generation of ESCs or whether they correlate with in vivo developmental stages. Here we investigate the cellular and molecular changes that occur during the transition from the human inner cell mass (ICM) to ESCs in vitro. We demonstrate that human ESCs originate from a post-ICM intermediate (PICMI), a transient epiblast-like structure that has undergone X-inactivation in female cells and is both necessary and sufficient for ESC derivation. The PICMI is the result of progressive and defined ICM organization in vitro and has a distinct state of cell signaling. The PICMI can be cryopreserved without compromising ESC derivation capacity. As a closer progenitor of ESCs than the ICM, the PICMI provides insight into the pluripotent state of human stem cells.

Progranulin expression correlates with dense-core amyloid plaque burden in Alzheimer disease mouse models.

Amyloid‐β (Aβ) plaques are pathological hallmarks of Alzheimer disease (AD). In addition, innate inflammatory responses, such as those mediated by microglia, are integral to the pathogenesis of AD. Interestingly, only dense‐core plaques and not diffuse plaques are associated with neuritic and inflammatory pathology in AD patients as well as in mouse AD models. However, the precise neuropathological changes that occur in the brain in response to amyloid deposition are largely unknown. To study the molecular mechanism(s) responsible for Aβ‐mediated neuropathology, we performed a gene expression analysis on laser‐microdissected brain tissue of Tg2576 and APPPS1 mice that are characterized by different types of amyloid plaques and genetic backgrounds. Data were validated by image and biochemical analyses on different ages of Tg2576, APPPS1, and Aβ42‐depositing BRI‐Aβ42 mice. Consistent with an important role of inflammatory responses in AD, we identified progranulin (mouse Grn; human GRN) as one of the top ten up‐regulated molecules in Tg2576 (≈8‐fold increased) and APPPS1 (≈2‐fold increased) mice compared to littermate controls, and among the eight significantly up‐regulated molecules common to both mouse models. In addition, Grn levels correlated significantly with amyloid load, especially the dense‐core plaque pathology (p < 0.001). We further showed that Grn is up‐regulated in microglia and neurons and neurites around dense‐core plaques, but not in astrocytes or oligodendrocytes, as has been shown in AD patients. Our data therefore support the ongoing use of these mouse models in drug trials, especially those with anti‐inflammatory compounds. Moreover, the correlation of Grn with increasing disease severity in AD mouse models prompts human studies exploring the viability of GRN as a disease biomarker. Because loss of GRN has recently been shown to cause frontotemporal dementia and serves as a risk factor for AD, the strong GRN reactivity around dense‐core plaques is consistent with an important role of this factor in AD pathogenesis. Copyright

My-Forensic-Loci-queries (MyFLq) framework for analysis of forensic STR data generated by massive parallel sequencing

Forensic scientists are currently investigating how to transition from capillary electrophoresis (CE) to massive parallel sequencing (MPS) for analysis of forensic DNA profiles. MPS offers several advantages over CE such as virtually unlimited multiplexy of loci, combining both short tandem repeat (STR) and single nucleotide polymorphism (SNP) loci, small amplicons without constraints of size separation, more discrimination power, deep mixture resolution and sample multiplexing. We present our bioinformatic framework My-Forensic-Loci-queries (MyFLq) for analysis of MPS forensic data. For allele calling, the framework uses a MySQL reference allele database with automatically determined regions of interest (ROIs) by a generic maximal flanking algorithm which makes it possible to use any STR or SNP forensic locus. Python scripts were designed to automatically make allele calls starting from raw MPS data. We also present a method to assess the usefulness and overall performance of a forensic locus with respect to MPS, as well as methods to estimate whether an unknown allele, which sequence is not present in the MySQL database, is in fact a new allele or a sequencing error. The MyFLq framework was applied to an Illumina MiSeq dataset of a forensic Illumina amplicon library, generated from multilocus STR polymerase chain reaction (PCR) on both single contributor samples and multiple person DNA mixtures. Although the multilocus PCR was not yet optimized for MPS in terms of amplicon length or locus selection, the results show excellent results for most loci. The results show a high signal-to-noise ratio, correct allele calls, and a low limit of detection for minor DNA contributors in mixed DNA samples. Technically, forensic MPS affords great promise for routine implementation in forensic genomics. The method is also applicable to adjacent disciplines such as molecular autopsy in legal medicine and in mitochondrial DNA research.

Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua.

BackgroundThe medicinal plant Artemisia annua is covered with filamentous trichomes and glandular, artemisinin producing trichomes. A high artemisinin supply is needed at a reduced cost for treating malaria. Artemisinin production in bioreactors can be facilitated if a better insight is obtained in the biosynthesis of artemisinin and other metabolites. Therefore, metabolic activities of glandular and filamentous trichomes were investigated at the transcriptome level.ResultsBy laser pressure catapulting, glandular and filamentous trichomes as well as apical and sub-apical cells from glandular trichomes were collected and their transcriptome was sequenced using Illumina RNA-Seq. A de novo transcriptome was assembled (Trinity) and studied with a differential expression analysis (edgeR).A comparison of the transcriptome from glandular and filamentous trichomes shows that MEP, MVA, most terpene and lipid biosynthesis pathways are significantly upregulated in glandular trichomes. Conversely, some transcripts coding for specific sesquiterpenoid and triterpenoid enzymes such as 8-epi-cedrol synthase and an uncharacterized oxidosqualene cyclase were significantly upregulated in filamentous trichomes. All known artemisinin biosynthesis genes are upregulated in glandular trichomes and were detected in both the apical and sub-apical cells of the glandular trichomes. No significant differential expression could be observed between the apical and sub-apical cells.ConclusionsOur results underscore the vast metabolic capacities of A. annua glandular trichomes but nonetheless point to the existence of specific terpene metabolic pathways in the filamentous trichomes. Candidate genes that might be involved in artemisinin biosynthesis are proposed based on their putative function and their differential expression level.

Laser capture microdissection in forensic research: a review.

In forensic sciences, short tandem repeat (STR) analysis has become the prime tool for DNA-based identification of the donor(s) of biological stains and/or traces. Many traces, however, contain cells and, hence, DNA, from more than a single individual, giving rise to mixed genotypes and the subsequent difficulties in interpreting the results. An even more challenging situation occurs when cells of a victim are much more abundant than the cells of the perpetrator. Therefore, the forensic community seeks to improve cell-separation methods in order to generate single-donor cell populations from a mixed trace in order to facilitate DNA typing and identification. Laser capture microdissection (LCM) offers a valuable tool for precise separation of specific cells. This review summarises all possible forensic applications of LCM, gives an overview of the staining and detection options, including automated detection and retrieval of cells of interest, and reviews the DNA extraction protocols compatible with LCM of cells from forensic samples.

Automatic detection of spermatozoa for laser capture microdissection

In sexual assault crimes, differential extraction of spermatozoa from vaginal swab smears is often ineffective, especially when only a few spermatozoa are present in an overwhelming amount of epithelial cells. Laser capture microdissection (LCM) enables the precise separation of spermatozoa and epithelial cells. However, standard sperm-staining techniques are non-specific and rely on sperm morphology for identification. Moreover, manual screening of the microscope slides is time-consuming and labor-intensive. Here, we describe an automated screening method to detect spermatozoa stained with Sperm HY-LITER™. Different ratios of spermatozoa and epithelial cells were used to assess the automatic detection method. In addition, real postcoital samples were also screened. Detected spermatozoa were isolated using LCM and DNA analysis was performed. Robust DNA profiles without allelic dropout could be obtained from as little as 30 spermatozoa recovered from postcoital samples, showing that the staining had no significant influence on DNA recovery.

Unbreakable Codes in Electrospun Fibers: Digitally Encoded Polymers to Stop Medicine Counterfeiting

Fluorescent polymer solutions can easily be electrospun into micrometer-sized fibers and subsequently encoded with long lasting digital codes by a photobleaching process. Such encoded fibers may find various applications; as illustrated in this report, placing encoded fibers in drug tablets may become a strategy to protect them from counterfeiting.

Presence and potential of cell free DNA in different types of forensic samples

Extracellular or cell free DNA has been found to exist in many biological media such as blood and saliva. To check whether cell free DNA is present in the supernatant which is normally discarded during several DNA extraction processes, such as Chelex(®) extraction, DNA profiles of cell pellet and concentrated supernatant from 30 artificial case like samples and from 100 real forensic samples were compared. Presence of cell free DNA was shown in all investigated sample types. Moreover, in some samples additional alleles, not detected during analysis of the cell pellet, were detected, offering valuable information which would normally have been discarded together with the supernatant. The results presented here indicate that cell free DNA deserves further consideration since it has the potential to increase the DNA yield in forensic casework samples in general and in contact traces in particular.

Suspension fluorescence in situ hybridization (S-FISH) combined with automatic detection and laser microdissection for STR profiling of male cells in male/female mixtures

Laser microdissection is a valuable tool for isolating specific cells from mixtures, such as male cells in a mixture with female cells, e.g., in cases of sexual assault. These cells can be stained with Y-chromosome-specific probes. We developed an automatic screening method to detect male cells after fluorescence in situ hybridization in suspension (S-FISH). To simulate forensic casework, the method was tested on female saliva after cataglottis (a kiss involving tongue-to-tongue contact) and on licking traces (swabs of dried male saliva on female skin) even after drying. After isolation of the detected cells, short tandem repeat profiling was performed. Full DNA profiles could consistently be obtained from as little as ten buccal cells. Isolation of five cells resulted in a mean of 98% (SD of 3.4%) of the alleles detected, showing that the developed S-FISH staining had no significant negative influence on DNA recovery and can be used in forensic casework.

Laser capture microdissection: should an ultraviolet or infrared laser be used?

Laser capture microdissection (LCM) is a well-established cell separation technique. It combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that need to be separated from others. Consequently, this biological material can be used for genome or transcriptome analyses. Appropriate methods of sample preparation, however, are crucial for the success of downstream molecular analysis. The aim of this study was to objectively compare the two main LCM systems, one based on an ultraviolet (UV) laser and the other based on an infrared (IR) laser, on different criteria ranging from user-friendliness to sample quality. The comparison was performed on two types of samples: peripheral blood mononuclear cells and blastocysts. The UV laser LCM system had several advantages over the IR laser LCM system. Not only does the UV system allow faster and more precise sample collection, but also the obtained samples-even single cell samples-can be used for DNA extraction and downstream polymerase chain reaction (PCR) applications. RNA-based applications are more challenging for both LCM systems. Although sufficient RNA can be extracted from as few as 10 cells for reverse transcription quantitative PCR (RT-qPCR) analysis, the low RNA quality should be taken into account when designing the RT-qPCR assays.