Search

Aims: Risk assessment tools are needed for timely identification of patients with heart failure (HF) with reduced ejection fraction (HFrEF) who are at high risk of adverse events. In this study, we aim to derive a small set out of 4210 repeatedly measured proteins, which, along with clinical characteristics and established biomarkers, carry optimal prognostic capacity for adverse events, in patients with HFrEF., Methods and Results: In 382 patients, we performed repeated blood sampling (median follow-up: 2.1 years) and applied an aptamer-based multiplex proteomic approach. We used machine learning to select the optimal set of predictors for the primary endpoint (PEP: composite of cardiovascular death, heart transplantation, left ventricular assist device implantation, and HF hospitalization). The association between repeated measures of selected proteins and PEP was investigated by multivariable joint models. Internal validation (cross-validated c -index) and external validation (Henry Ford HF PharmacoGenomic Registry cohort) were performed. Nine proteins were selected in addition to the MAGGIC risk score, N-terminal pro-hormone B-type natriuretic peptide, and troponin T: suppression of tumourigenicity 2, tryptophanyl-tRNA synthetase cytoplasmic, histone H2A Type 3, angiotensinogen, deltex-1, thrombospondin-4, ADAMTS-like protein 2, anthrax toxin receptor 1, and cathepsin D. N-terminal pro-hormone B-type natriuretic peptide and angiotensinogen showed the strongest associations [hazard ratio (95% confidence interval): 1.96 (1.17-3.40) and 0.66 (0.49-0.88), respectively]. The multivariable model yielded a c -index of 0.85 upon internal validation and c -indices up to 0.80 upon external validation. The c -index was higher than that of a model containing established risk factors ( P = 0.021)., Conclusion: Nine serially measured proteins captured the most essential prognostic information for the occurrence of adverse events in patients with HFrEF, and provided incremental value for HF prognostication beyond established risk factors. These proteins could be used for dynamic, individual risk assessment in a prospective setting. These findings also illustrate the potential value of relatively 'novel' biomarkers for prognostication., Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT01851538?term=nCT01851538&draw=2&rank=1 24., Competing Interests: Conflict of interest: R.O. is employed by Somalogic Inc. D.L. reports non-financial support from Somalogic Inc., during the conduct of the study; grants and personal fees from Janssen, personal fees from Ortho Diagnostics, personal fees from DCRI (Novartis), grants from Bayer, grants from Astra Zeneca, grants from Critical Diagnostics, non-financial support from Somalogic, grants from Lilly, personal fees from ACI (Abbott Laboratories), personal fees from Martin Pharmaceuticals, personal fees from Illumina, personal fees from Vicardia, other from Hridaya, grants and personal fees from Amgen, personal fees from Cytokinetics, outside the submitted work. In addition, D.L. has a patent genomic predictors of BB response issued. The other authors have no disclosures to report., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Fusion genes are typically identified by RNA sequencing (RNA-seq) without elucidating the causal genomic breakpoints. However, non-poly(A)-enriched RNA-seq contains large proportions of intronic reads that also span genomic breakpoints., Results: We have developed an algorithm, Dr. Disco, that searches for fusion transcripts by taking an entire reference genome into account as search space. This includes exons but also introns, intergenic regions, and sequences that do not meet splice junction motifs. Using 1,275 RNA-seq samples, we investigated to what extent genomic breakpoints can be extracted from RNA-seq data and their implications regarding poly(A)-enriched and ribosomal RNA-minus RNA-seq data. Comparison with whole-genome sequencing data revealed that most genomic breakpoints are not, or minimally, transcribed while, in contrast, the genomic breakpoints of all 32 TMPRSS2-ERG-positive tumours were present at RNA level. We also revealed tumours in which the ERG breakpoint was located before ERG, which co-existed with additional deletions and messenger RNA that incorporated intergenic cryptic exons. In breast cancer we identified rearrangement hot spots near CCND1 and in glioma near CDK4 and MDM2 and could directly associate this with increased expression. Furthermore, in all datasets we find fusions to intergenic regions, often spanning multiple cryptic exons that potentially encode neo-antigens. Thus, fusion transcripts other than classical gene-to-gene fusions are prominently present and can be identified using RNA-seq., Conclusion: By using the full potential of non-poly(A)-enriched RNA-seq data, sophisticated analysis can reliably identify expressed genomic breakpoints and their transcriptional effects., (© The Author(s) 2021. Published by Oxford University Press GigaScience.)

Background: In order to identify suitable therapeutic targets for glioma anti-angiogenic therapy, the process of neovascularization mediated by circulating angiogenic cells (CACs) needs to be scrutinized., Methods: In the present study, we compared the expression of neovascularization-related genes by 3 circulating CAC subsets (hematopoietic progenitor cells [HPCs], CD34 + , and KDR + cells; internal controls: peripheral blood mononuclear cells and circulating endothelial cells) of treatment-naïve patients with glioblastoma (GBM) to those of patients undergoing reactive neovascularization (myocardial infarction (MI). CACs from umbilical cord (representing developmental neovascularization) and healthy subjects served as controls. Fluorescent-activated cell sorting was used to isolate CACs, RT-PCR to determine the expression levels of a panel of 48 neovascularization-related genes, and Luminex assays to measure plasma levels of 21 CAC-related circulating molecules., Results: We found essential differences in gene expression between GBM and MI CACs. GBM CACs had a higher expression of proangiogenic factors (especially, KITL , CXCL12 , and JAG1 ), growth factor and chemotactic receptors ( IGF1R , TGFBR2 , CXCR4 , and CCR2 ), adhesion receptor monomers ( ITGA5 and ITGA6 ), and matricellular factor POSTN . In addition, we found major differences in the levels of neovascularization-related plasma factors. A strong positive correlation between plasma MMP9 levels and expression of CXCR4 in the CAC subset of HPCs was found in GBM patients., Conclusions: Our findings indicate that CAC-mediated neovascularization in GBM is characterized by more efficient CAC homing to target tissue and a more potent proangiogenic response than in physiologic tissue repair in MI. Our findings can aid in selecting targets for therapeutic strategies acting against GBM-specific CACs., (© The Author(s) 2020. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Context: Thyroid hormone is important for normal brain development. The type 2 deiodinase (D2) controls thyroid hormone action in the brain by activating T4 to T3. The enzymatic activity of D2 depends on the incorporation of selenocysteine for which the selenocysteine-insertion sequence (SECIS) element located in the 3' untranslated region is indispensable. We hypothesized that mutations in the SECIS element could affect D2 function, resulting in a neurocognitive phenotype., Objective: To identify mutations in the SECIS element of DIO2 in patients with intellectual disability and to test their functional consequences., Design, Setting, and Patients: The SECIS element of DIO2 was sequenced in 387 patients with unexplained intellectual disability using a predefined pattern of thyroid function tests. SECIS element read-through in wild-type or mutant D2 was quantified by a luciferase reporter system in transfected cells. Functional consequences were assessed by quantifying D2 activity in cell lysate or intact cell metabolism studies., Results: Sequence analysis revealed 2 heterozygous mutations: c.5703C>T and c.5730A>T, which were also present in the unaffected family members. The functional evaluation showed that both mutations did not affect D2 enzyme activity in cell lysates or intact cells, although the 5730A>T mutation decreased SECIS element read-through by 75%. In the patient harboring the c.5730A>T variant, whole genome sequencing revealed a pathogenic deletion of the STXBP1 gene., Conclusions: We report on two families with mutations in the SECIS element of D2. Although functional analysis showed that nucleotide 5730 is important for normal SECIS element read-through, the two variants did not segregate with a distinct phenotype., (Copyright © 2019 Endocrine Society.)

Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Background: The determination of microbial communities using the mothur tool suite (https://www.mothur.org) is well established. However, mothur requires bioinformatics-based proficiency in order to perform calculations via the command-line. Galaxy is a project dedicated to providing a user-friendly web interface for such command-line tools (https://galaxyproject.org/)., Results: We have integrated the full set of 125+ mothur tools into Galaxy as the Galaxy mothur Toolset (GmT) and provided a set of workflows to perform end-to-end 16S rRNA gene analyses and integrate with third-party visualization and reporting tools. We demonstrate the utility of GmT by analyzing the mothur MiSeq standard operating procedure (SOP) dataset (https://www.mothur.org/wiki/MiSeq_SOP)., Conclusions: GmT is available from the Galaxy Tool Shed, and a workflow definition file and full Galaxy training manual for the mothur SOP have been created. A Docker image with a fully configured GmT Galaxy is also available., (© The Author(s) 2018. Published by Oxford University Press.)

Context: Despite the well-recognized clinical features resulting from insufficient or excessive thyroid hormone (TH) levels in humans, it is largely unknown which genes are regulated by TH in human tissues., Objective: To study the effect of TH on human gene expression profiles in whole blood, mainly consisting of T3 receptor (TR) α-expressing cells., Methods: We performed next-generation RNA sequencing on whole blood samples from eight athyroid patients (four females) on and after 4 weeks off levothyroxine replacement. Gene expression changes were analyzed through paired differential expression analysis and confirmed in a validation cohort. Weighted gene coexpression network analysis (WGCNA) was applied to identify thyroid state-related networks., Results: We detected 486 differentially expressed genes (fold-change >1.5; multiple testing corrected P value < 0.05), of which 76% were positively and 24% were negatively regulated. Gene ontology (GO) enrichment analysis revealed that three biological processes were significantly overrepresented, of which the process translational elongation showed the highest fold enrichment (7.3-fold, P = 1.8 × 10-6). WGCNA analysis independently identified various gene clusters that correlated with thyroid state. Further GO analysis suggested that thyroid state affects platelet function., Conclusions: Changes in thyroid state regulate numerous genes in human whole blood, predominantly TRα-expressing leukocytes. In addition, TH may regulate gene transcripts in platelets., (Copyright © 2017 Endocrine Society)

FINDbase (http://www.findbase.org) is a comprehensive data repository that records the prevalence of clinically relevant genomic variants in various populations worldwide, such as pathogenic variants leading mostly to monogenic disorders and pharmacogenomics biomarkers. The database also records the incidence of rare genetic diseases in various populations, all in well-distinct data modules. Here, we report extensive data content updates in all data modules, with direct implications to clinical pharmacogenomics. Also, we report significant new developments in FINDbase, namely (i) the release of a new version of the ETHNOS software that catalyzes development curation of national/ethnic genetic databases, (ii) the migration of all FINDbase data content into 90 distinct national/ethnic mutation databases, all built around Microsoft's PivotViewer (http://www.getpivot.com) software (iii) new data visualization tools and (iv) the interrelation of FINDbase with DruGeVar database with direct implications in clinical pharmacogenomics. The abovementioned updates further enhance the impact of FINDbase, as a key resource for Genomic Medicine applications., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Unlabelled: A new generation of tools that identify fusion genes in RNA-seq data is limited in either sensitivity and or specificity. To allow further downstream analysis and to estimate performance, predicted fusion genes from different tools have to be compared. However, the transcriptomic context complicates genomic location-based matching. FusionMatcher (FuMa) is a program that reports identical fusion genes based on gene-name annotations. FuMa automatically compares and summarizes all combinations of two or more datasets in a single run, without additional programming necessary. FuMa uses one gene annotation, avoiding mismatches caused by tool-specific gene annotations. FuMa matches 10% more fusion genes compared with exact gene matching due to overlapping genes and accepts intermediate output files that allow a stepwise analysis of corresponding tools., Availability and Implementation: The code is available at: https://github.com/ErasmusMC-Bioinformatics/fuma and available for Galaxy in the tool sheds and directly accessible at https://bioinf-galaxian.erasmusmc.nl/galaxy/, Contact: y.hoogstrate@erasmusmc.nl or a.stubbs@erasmusmc.nl, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Study Question: Are first trimester trophoblast volume (TV) and placental bed vascular volume (PBVV) different in IVF or IVF/ICSI pregnancies in comparison with spontaneously conceived pregnancies?, Summary Answer: Any possible abnormal placentation in IVF or IVF/ICSI pregnancies in comparison with spontaneously conceived pregnancies is not detected by a difference in PBVV or TV at an early gestational age (GA)., What Is Known Already: Assisted reproductive technology pregnancies have been associated with an increased risk of placenta-related adverse pregnancy outcomes. It is unclear whether these effects originate from infertility or from the technique itself., Study Design, Size, Duration: We performed a retrospective cohort study in which 154 pregnant patients qualified for participation., Participants/materials, Setting, Methods: Out of 154 pregnant patients, 84 conceived spontaneously and 70 conceived after IVF or IVF/ICSI. We determined the TV at 10 weeks GA by Virtual Organ Computer-aided AnaLysis measuring application and the PBVV at 12 weeks GA by the virtual reality operating system of BARCO I-Space in both subgroups. The investigators were blinded to the mode of conception during the measurements. Analysis was limited to singleton pregnancies with only one sac ever detectable., Main Results and the Role of Chance: There were no differences in TV (mean 42.7, SD 15.9 versus mean 41.2, SD 13.9, P = 0.70) and PBVV (mean 27.6, SD 16.9 versus mean 24.8, SD 19.9, P = 0.20) between IVF or IVF/ICSI pregnancies and spontaneously conceived pregnancies. There was a significant correlation between TV and PBVV (rs = 0.283, P = 0.004)., Limitations, Reasons for Caution: The limitations of the present study concern the small size of the study groups., Wider Implications of the Findings: IVF or IVF/ICSI does not seem to be associated with abnormal placentation., Study Funding/competing Interests: This study was financially supported by the Erasmus Trustfonds, the Meindert de Hoop foundation and the Fonds NutsOhra. No competing interests are declared., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Summary: The discovery of functionally related groups in a set of significantly abundant proteins from a mass spectrometry experiment is an important step in a proteomics analysis pipeline. Here we describe NetWeAvers (Network Weighted Averages) for analyzing groups of regulated proteins in a network context, e.g. as defined by clusters of protein-protein interactions. NetWeAvers is an R package that provides a novel method for analyzing proteomics data integrated with biological networks. The method includes an algorithm for finding dense clusters of proteins and a permutation algorithm to calculate cluster P-values. Optional steps include summarizing quantified peptide values to single protein values and testing for differential expression, such that the data input can simply be a list of identified and quantified peaks., Availability and Implementation:  The NetWeAvers package is written in R, is open source and is freely available on CRAN and from netweavers.erasmusmc.nl under the GPL-v2 license., Contact: e.mcclellan@erasmusmc.nl

Background: Ustekinumab is a fully human anti-p40 monoclonal antibody which neutralizes interleukin (IL)-12 and IL-23, thereby interfering with T-helper (Th)1/Th17 pathways and keratinocyte activation, and is highly effective in the treatment of psoriasis. During ustekinumab treatment, some of our patients noticed reduced koebnerization of noninvolved skin and less new plaque formation., Objectives: To determine whether ustekinumab improves psoriasis-related gene expression and tape-strip responses in noninvolved skin., Methods: Before and 4 weeks after ustekinumab treatment, noninvolved skin was tape-stripped. After 5 h, biopsies were taken from untouched and tape-stripped skin. The mRNA expression of psoriasis-related markers such as NGF, GATA3 and IL-22RA1, and several antimicrobial peptides (AMP) was quantified. Leucocyte counts and a broad range of inflammatory serum proteins were analysed to gain insight into the systemic alterations., Results: Four weeks following a single ustekinumab injection, NGF showed a significant decrease, whereas GATA3 and IL-22RA1 expression increased, indicative of reduced responsiveness to epidermal triggering. This was accompanied by an increase of the inflammation-related serum proteins GPNMB, MST1 and TRADD. The baseline and tape-strip-induced mRNA expression of the AMP human β-defensin-2 (hBD-2), S100A7 and LL-37 remained unaltered. Clinically, after 4 weeks, eight out of 11 patients showed a 50% psoriasis area and severity index (PASI) improvement, which was accompanied by a significant reduction in serum hBD-2 levels. No changes were noted in total leucocytes, C-reactive protein and erythrocyte sedimentation rate., Conclusions: These findings indicate that ustekinumab reduces psoriasis-related gene expression in noninvolved psoriatic skin, making it more resistant to exogenous triggering, without disturbing its antimicrobial response. In parallel, ustekinumab modulates important circulating inflammation-related proteins., (© 2013 The Authors. BJD © 2013 British Association of Dermatologists.)

Study Question: Is it possible to evaluate first trimester brain ventricle development in human pregnancies using an innovative virtual reality (VR) application and to analyze the relation of the embryonic volume (EV) and brain ventricle fluid volume (BVFV) with gestational age (GA), crown-rump length (CRL) and the Carnegie stage?, Summary Answer: Volumetry and staging of the human embryo using a VR application make it possible to obtain unique information about in-vivo embryonic normal and abnormal development and about the sizes of the ventricles and body., What Is Known Already: Human brain development is complex and has a rapidly changing anatomy during the first trimester of pregnancy. New insights will enable early detection of cerebral pathology., Study Design, Size, Duration: In a prospective cohort study, we weekly performed three-dimensional (3D) ultrasound examinations in 112 uncomplicated pregnancies between 6 + 0 and 12 + 6 weeks GA., Materials, Setting, Methods: The examinations resulted in 696 3D ultrasound scans that were transferred to the I-Space VR system and analyzed using V-Scope volume rendering software. V-Scope is used to create a 'hologram' of the ultrasound image and allows depth perception and interaction with the rendered objects. The CRL measurements were performed with a tracing tool, and the volume measurements were automatically performed with a segmentation algorithm. The embryos were staged according to the internal and external characteristics of the Carnegie staging system. All longitudinal outcomes were analyzed using repeated measures ANOVA., Main Results and the Role of Chance: CRL could be measured in 91% of the datasets and ranged from 2.5 to 79.0 mm. EV could be measured in 66% of the datasets and ranged from 2.4 to 23 812.0 mm&sup3;, whereas the BVFV could be measured in 38% of the datasets and ranged from 10.4 to 226.3 mm&sup3;. Finally, in 74% of the datasets, the embryos were staged according to the Carnegie criteria, starting as early as stage 12. Reference charts of volumes versus GA, CRL and stage were constructed. There was no significant relationship between the CRL or EV and the birthweight., Limitations, Reasons for Cautions: The low success rate is a limitation of this study that can be explained mainly by non-targeted scanning of the embryonic head., Wider Implications of the Findings: The I-Space VR system and the V-Scope software enable automatic EV and BVFV measurements and 3D observations of embryonic development in the first trimester. This allows in-vivo staging of human embryos based on both internal and external morphological characteristics., Study Funding, Competing Interests: None.

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.

Aims: There is an increasing need for translational studies identifying molecular targets contributing to atherosclerotic plaque destabilization. Local molecular plaque markers that are related to plaque vulnerability may hold predictive value to identify patients who are at increased risk to suffer from cardiovascular events. Animal studies revealed that adipocyte fatty acid binding protein (FABP4) is associated with the progression of atherosclerosis; however, FABP4 expression studies in human atherosclerotic plaques are lacking. We investigated FABP4 expression in carotid atherosclerotic lesions in relation to plaque composition and future cardiovascular events., Methods and Results: Atherosclerotic plaques were obtained from 561 patients undergoing carotid endarterectomy (CEA). Plaques were analysed for the presence of macrophages, lipid core, smooth-muscle cells, collagen, calcification, and intraplaque haemorrhage. Patients were followed for 3 years after CEA. The primary outcome was defined as the composite of vascular death, vascular event, and surgical or percutaneous vascular intervention. Fatty acid binding protein levels correlated with unstable plaque characteristics and symptomatic lesions. Patients with increased FABP4 plaque levels showed a two-fold increased risk [HR = 1.99, 95% confidence interval (95% CI) (1.30-3.04)] (P = 0.005) to reach the primary outcome during follow-up. Increased FABP4 levels related to primary outcome, independent from general cardiovascular risk factors [HR = 1.33, 95% CI (1.08-1.65)] (P = 0.008)., Conclusion: FABP4 levels in atherosclerotic lesions are associated with an unstable plaque phenotype and an increased risk for cardiovascular events during follow-up. Besides risk stratification for adverse future cardiovascular events, the outcome of the present study supports the relevance of exploring FABP4 antagonists as a potential pharmaceutical intervention to treat atherosclerotic disease progression.

Background: The recent introduction of virtual reality (VR) enables us to use all three dimensions in a three-dimensional (3D) image. The aim of this prospective study was to evaluate an innovative VR technique for automated 3D volume measurements of the human embryo and yolk sac in first trimester pregnancies., Methods: We analysed 180 3D first trimester ultrasound scans of 42 pregnancies. Scans were transferred to an I-Space VR system and visualized as 3D 'holograms' with the V-Scope volume-rendering software. A semi-automatic segmentation algorithm was used to calculate the volumes. The logarithmically transformed outcomes were analysed using repeated measurements ANOVA. Interobserver and intraobserver agreement was established by calculating intraclass correlation coefficients (ICCs)., Results: Eighty-eight embryonic volumes (EVs) and 118 yolk sac volumes (YSVs) were selected and measured between 5(+5) and 12(+6) weeks of gestational age (GA). EV ranged from 14 to 29 877 mm(3) and YSV ranged from 33 to 424 mm(3). ANOVA calculations showed that when the crown-rump length (CRL) doubles, the mean EV increases 6.5-fold and when the GA doubles, the mean EV increases 500-fold (P < 0.001). Furthermore, it was found that a doubling in GA results in a 3.8-fold increase of the YSV and when the CRL doubles, the YSV increases 1.5-fold (P < 0.001). Interobserver and intraobserver agreement were both excellent with ICCs of 0.99., Conclusion: We measured the human EV and YSV in early pregnancy using a VR system. This innovative technique allows us to obtain unique information about the size of the embryo using all dimensions, which may be used to differentiate between normal and abnormal human development.

Background: Innovative imaging techniques, using up-to-date ultrasonic equipment, necessitate specific biometry. The aim of our study was to test the possibility of detailed human embryonic biometry using a virtual reality (VR) technique., Methods: In a longitudinal study, three-dimensional (3D) measurements were performed from 6 to 14 weeks gestational age in 32 pregnancies (n = 16 spontaneous conception, n = 16 IVF/ICSI). A total of 125 3D volumes were analysed in the I-Space VR system, which allows binocular depth perception, providing a realistic 3D illusion. Crown-rump length (CRL), biparietal diameter (BPD), occipito-frontal diameter (OFD), head circumference (HC) and abdominal circumference (AC) were measured as well as arm length, shoulder width, elbow width, hip width and knee width., Results: CRL, BPD, OFD and HC could be measured in more than 96% of patients, and AC in 78%. Shoulder width, elbow width, hip width and knee width could be measured in more than 95% of cases, and arm length in 82% of cases. Growth curves were constructed for all variables. Ear and foot measurements were only possible beyond 9 weeks gestation., Conclusions: This study provides a detailed, longitudinal description of normal human embryonic growth, facilitated by a VR system. Growth curves were created for embryonic biometry of the CRL, BPD, HC and AC early in pregnancy and also of several 'new' biometric measurements. Applying virtual embryoscopy will enable us to diagnose growth and/or developmental delay earlier and more accurately. This is especially important for pregnancies at risk of severe complications, such as recurrent late miscarriage and early growth restriction.

Context: Skeletal muscle is an important target tissue for thyroid hormone (TH). It is currently unknown which genes are regulated by physiological TH levels., Objective: We examined the effects of l-thyroxine on human skeletal muscle transcriptome., Design: Microarray analysis of transcript levels was performed using skeletal muscle biopsies from patients under euthyroid and hypothyroid conditions., Setting: The study was conducted in a university hospital laboratory., Patients: We studied skeletal muscle obtained from 10 thyroidectomized patients with differentiated thyroid carcinoma on and after 4 wk off L-thyroxine replacement., Mean Outcome Measures: Gene expression changes were measured using microarrays. Results were analyzed using dedicated statistical methods., Results: We detected 607 differentially expressed genes on L-thyroxine treatment, of which approximately 60% were positively and approximately 40% were negatively regulated. Representative genes were validated by quantitative PCR. Genes involved in energy and fuel metabolism were overrepresented among the up-regulated genes, of which a large number were newly associated with thyroid state. L-thyroxine therapy induced a large down-regulation of the primary transcripts of the noncoding microRNA pair miR-206/miR-133b., Conclusion: We demonstrated that physiological levels of TH regulate a myriad of genes in human skeletal muscle. The identification of novel putatively TH-responsive genes may provide the molecular basis of clinical effects in subjects with different TH status. The observation that TH regulates microRNAs reveals a new layer of complexity by which TH influences cellular processes.

BACKGROUND This study investigates whether dietary patterns, substantiated by biomarkers, are associated with semen quality. METHODS In 161 men of subfertile couples undergoing in vitro fertilization treatment in a tertiary referral clinic in Rotterdam, the Netherlands, we assessed nutrient intakes and performed principal component factor analysis to identify dietary patterns. Total homocysteine (tHcy), folate, vitamin B12 and B6 were measured in blood and seminal plasma. Semen quality was assessed by sperm volume, concentration, motility, morphology and DNA fragmentation index (DFI). Linear regression models analyzed associations between dietary patterns, biomarkers and sperm parameters, adjusted for age, body mass index (BMI), smoking, vitamins and varicocele. RESULTS The 'Health Conscious' dietary pattern shows high intakes of fruits, vegetables, fish and whole grains. The 'Traditional Dutch' dietary pattern is characterized by high intakes of meat, potatoes and whole grains and low intakes of beverages and sweets. The 'Health Conscious' diet was inversely correlated with tHcy in blood (beta = -0.07, P = 0.02) and seminal plasma (beta = -1.34, P = 0.02) and positively with vitamin B6 in blood (beta = 0.217, P = 0.01). An inverse association was demonstrated between the 'Health Conscious' diet and DFI (beta = -2.81, P = 0.05). The 'Traditional Dutch' diet was positively correlated with red blood cell folate (beta = 0.06, P = 0.04) and sperm concentration (beta = 13.25, P = 0.01). CONCLUSIONS The 'Health Conscious' and 'Traditional Dutch' dietary pattern seem to be associated with semen quality in men of subfertile couples.

Background: The aim of this study was to demonstrate that Carnegie Stages could be assigned to embryos visualized with a 3D virtual reality system., Methods: We analysed 48 3D ultrasound scans of 19 IVF/ICSI pregnancies at 7-10 weeks' gestation. These datasets were visualized as 3D 'holograms' in the BARCO I-Space virtual reality system. Embryos were staged according to external morphological features (i.e. mainly limb development). After staging, the crown rump length (CRL) was measured. Stage and CRL were compared with gestational age based on the date of oocyte retrieval and with the classical data on embryology from the Carnegie Collection., Results: Embryonic staging was relatively easy because the I-Space allows depth perception, which helps in the estimation of size and position. The presumed stages corresponded well with the measured CRL. However, in 28 out of 48 cases, the stages seemed to have been reached earlier than previously described for the Carnegie Collection., Conclusions: The I-Space, tentatively named Virtual Embryoscopy, is a promising non-invasive tool for early pregnancy evaluation. Combining embryonic growth with embryonic development opens a new area to study the relationship between embryonic growth, development and morphology, as well as second and third trimester pregnancy complications.

The human DNA repair protein ERCC1 resides in a complex together with the ERCC4, ERCC11 and XP-F correcting activities, thought to perform the 5' strand incision during nucleotide excision repair (NER). Its yeast counterpart, RAD1-RAD10, has an additional engagement in a mitotic recombination pathway, probably required for repair of DNA cross-links. Mutational analysis revealed that the poorly conserved N-terminal 91 amino acids of ERCC1 are dispensable for both repair functions, in contrast to a deletion of only four residues from the C-terminus. A database search revealed a strongly conserved motif in this C-terminus sharing sequence homology with many DNA break processing proteins, indicating that this part is primarily required for the presumed structure-specific endonuclease activity of ERCC1. Most missense mutations in the central region give rise to an unstable protein (complex). Accordingly, we found that free ERCC1 is very rapidly degraded, suggesting that protein-protein interactions provide stability. Survival experiments show that the removal of cross-links requires less ERCC1 than UV repair. This suggests that the ERCC1-dependent step in cross-link repair occurs outside the context of NER and provides an explanation for the phenotype of the human repair syndrome xeroderma pigmentosum group F.

The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER). The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al. (1994) EMBO J. 8, 1831-1843). Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC. In contrast, we cannot detect any bound HHR23A. Thus the HHR23 proteins may have an additional function independent of XPC. The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction. Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation. Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex. However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex. Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus.