P.J. van der Spek

A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A

DNA repair-deficient trichothiodystrophy (TTD) results from mutations in the XPD and XPB subunits of the DNA repair and transcription factor TFIIH. In a third form of DNA repair–deficient TTD, called group A, none of the nine subunits encoding TFIIH carried mutations; instead, the steady-state level of the entire complex was severely reduced. A new, tenth TFIIH subunit (TFB5) was recently identified in yeast. Here, we describe the identification of the human TFB5 ortholog and its association with human TFIIH. Microinjection of cDNA encoding TFB5 (GTF2H5, also called TTDA) corrected the DNA-repair defect of TTD-A cells, and we identified three functional inactivating mutations in this gene in three unrelated families with TTD-A. The GTF2H5 gene product has a role in regulating the level of TFIIH. The identification of a new evolutionarily conserved subunit of TFIIH implicated in TTD-A provides insight into TFIIH function in transcription, DNA repair and human disease.

High‐throughput microRNAome analysis in human germ cell tumours

Testicular germ cell tumours (GCTs) of adolescents and adults can be subdivided into seminomas (referred to as dysgerminomas of the ovary) and non‐seminomas, all referred to as type II GCTs. They originate from carcinoma in situ (CIS), being the malignant counterparts of primordial germ cells (PGCs)/gonocytes. The invasive components mimic embryogenesis, including the stem cell component embryonal carcinoma (EC), the somatic lineage teratoma (TE), and the extra‐embryonic tissues yolk sac tumour (YST) and choriocarcinoma (CH). The other type is the so‐called spermatocytic seminomas (SS, type III GCT), composed of neoplastic primary spermatocytes. We reported previously that the miRNAs hsa‐miR 371–373 cluster is involved in overruling cellular senescence induced by oncogenic stress, allowing cells to become malignant. Here we report the first high‐throughput screen of 156 microRNAs in a series of type II and III GCTs (n = 69, in duplicate) using a quantitative PCR‐based approach. After normalization to allow inter‐sample analysis, the technical replicates clustered together, and the previous hsa‐miRNA 371–373 cluster finding was confirmed. Unsupervised cluster analysis demonstrated that the cell lines are different from the in vivo samples. The in vivo samples, both normal and malignant, clustered predominantly based on their maturation status. This parallels normal embryogenesis, rather than chromosomal anomalies in the tumours. miRNAs within a single cluster showed a similar expression pattern, implying common regulatory mechanisms. Normal testicular tissue expressed most discriminating miRNAs at a higher level than SE and SS. Moreover, differentiated non‐seminomas showed overexpression of discriminating miRNAs. These results support the model that miRNAs are involved in regulating differentiation of stem cells, retained in GCTs. Copyright

Identification and characterization of XPC-binding domain of hHR23B.

hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.

Reliability of three‐dimensional sonographic measurements in early pregnancy using virtual reality

To establish the reliability of three‐dimensional (3D) ultrasound measurements in early pregnancy using a virtual reality system (the Barco I‐Space).

Collagenase matrix metalloproteinase-8 expressed in atherosclerotic carotid plaques is associated with systemic cardiovascular outcome

AIMS Atherosclerotic plaque rupture and subsequent thrombus formation are the major cause of acute cardiovascular events. Local plaque markers may facilitate detection of the vulnerable plaque and help identify the patient at risk for cardiovascular events. Matrix metalloproteinases (MMPs) are prevalent in the arterial wall throughout the arterial system and are associated with local plaque destabilization. We hypothesized that local MMP plaque levels are predictive for atherosclerotic cardiovascular events in other vascular territories. METHODS AND RESULTS Atherosclerotic plaques were obtained from 543 patients undergoing carotid endarterectomy (CEA). Plaques were analysed for the presence of macrophages, lipid-core, smooth muscle cells, collagen, calcification, and presence of plaque haemorrhage. MMP-2, MMP-8, and MMP-9 levels were assessed within the plaque. Following CEA, all patients underwent follow-up during 3 years. The primary outcome was defined as the composite of vascular death, non-fatal vascular event, and surgical or percutaneous vascular intervention. In contrast with MMP-2 plaque levels, MMP-8 and MMP-9 levels in the plaque were associated with an unstable carotid plaque composition and clinical presentation at baseline. Increased plaque MMP-8 level (>4.58) was associated with an increased risk for the occurrence of secondary manifestations of atherosclerotic disease during follow-up [hazard ratio = 1.76, 95% CI (1.25-2.48)] (P= 0.001), whereas plaque MMP-2 and MMP-9 levels were not predictive for systemic cardiovascular events. CONCLUSION In contrast with MMP-2, increased carotid MMP-8 and MMP-9 plaque levels are associated with an unstable plaque phenotype. High collagenase MMP-8 levels in the carotid plaque are associated with the occurrence of systemic cardiovascular outcome during follow-up.

Distinctive Phenotypic Abnormalities Associated with Submicroscopic 21q22 Deletion Including DYRK1A

Partial monosomy 21 has been reported, but the phenotypes described are variable with location and size of the deletion. We present 2 patients with a partially overlapping microdeletion of 21q22 and a striking phenotypic resemblance. They both presented with severe psychomotor delay, behavioral problems, no speech, microcephaly, feeding problems with frequent regurgitation, idiopathic thrombocytopenia, obesity, deep set eyes, down turned corners of the mouth, dysplastic ears, and small chin. Brain MRI showed cerebral atrophy mostly evident in frontal and temporal lobes, widened ventricles and thin corpus callosum in both cases, and in one patient evidence of a migration disorder. The first patient also presented with epilepsy and a ventricular septum defect. The second patient had a unilateral Peters anomaly. Microarray analysis showed a partially overlapping microdeletion spanning about 2.5 Mb in the 21q22.1–q22.2 region including the DYRK1A gene and excluding RUNX1. These patients present with a recognizable phenotype specific for this 21q22.1–q22.2 locus. We searched the literature for patients with overlapping deletions including the DYRK1A gene, in order to define other genes responsible for this presentation.

Using virtual reality for evaluation of fetal ambiguous genitalia

The utility of a virtual reality system was examined in the visualization of three‐dimensional (3D) ultrasound images of fetal ambiguous genitalia.

A virtual reality rendition of a fetal meningomyelocele at 32 weeks of gestation

Using a virtual reality system to render images obtained with three-dimensional (3D) ultrasound a fetal lumbosacral meningomyelocele (L3–S2) is shown here at 32 weeks’ gestation (Figure 1). The defect was originally observed in a 24-year-old primigravida during a routine sonogram at 22 weeks (Figure 2). After extensive counseling the patient decided not to have an amniocentesis and to continue with the pregnancy. In this image the meningomyelocele is seen from a coronal view with the sac partially ‘cut open’. The slitlike dark structure in the center is the actual midline defect of the osseous structure of the spine. The spinal nerve roots can be observed running from the vertebral column towards the inside wall of the meningomyelocele where they adhere to the neural placode (Figure 3). The neural placode is the flat plate of dysplastic neural tissue, which is elevated to the dome of the meningomyelocele by the pressure of cerebrospinal fluid. The image, originally obtained during a 3D ultrasound examination, is seen hovering in space in front of the investigator, giving the impression of a 3D structure with a diameter of approximately 80 × 80 × 60 cm. The image can be moved, resized and turned around in space with the help of a hand-held joystick. The joystick also allows a part of the volume to be ‘cut away’.

Vasculogenesis and Angiogenesis in the First Trimester Human Placenta: An Innovative 3D Study Using an Immersive Virtual Reality System

First trimester human villous vascularization is mainly studied by conventional two-dimensional (2D) microscopy. With this (2D) technique it is not possible to observe the spatial arrangement of the haemangioblastic cords and vessels, transition of cords into vessels and the transition of vasculogenesis to angiogenesis. The Confocal Laser Scanning Microscopy (CLSM) allows for a three-dimensional (3D) reconstruction of images of early pregnancy villous vascularization. These 3D reconstructions, however, are normally analyzed on a 2D medium, lacking depth perception. We performed a descriptive morphologic study, using an immersive Virtual Reality system to utilize the full third dimension completely. This innovative 3D technique visualizes 3D datasets as enlarged 3D holograms and provided detailed insight in the spatial arrangement of first trimester villous vascularization, the beginning of lumen formation within various junctions of haemangioblastic cords between 5 and 7 weeks gestational age and in the gradual transition of vasculogenesis to angiogenesis. This innovative immersive Virtual Reality system enables new perspectives for vascular research and will be implemented for future investigation.

Visual analytics in the pharmaceutical industry

With the flood of data across all aspects of the pharmaceutical industry, information visualization is emerging as a critical component of discovery, development, and business. But its a new class of visualizations that is having the greatest impact. Higher-level summaries that can provide a framework for understanding the immense volumes of data and that reveal unexpected relationships have come to the forefront. And, the ability to use these visualizations to cross-domains and data types provides the ability to integrate analyses and support fast, effective decisions.