Tracking cell turnover in human brain using 15N-thymidine imaging mass spectrometry

Microcephaly is often caused by an impairment of the generation of neurons in the brain, a process referred to as neurogenesis. While most neurogenesis in mammals occurs during brain development, it thought to continue to take place through adulthood in selected regions of the mammalian brain, notably the hippocampus. However, the generality of neurogenesis in the adult brain has been controversial. While studies in mice and rats have provided compelling evidence for neurogenesis occurring in the adult rodent hippocampus, the lack of applicability in humans of key methods to demonstrate neurogenesis has led to an intense debate about the existence and, in particular, the magnitude of neurogenesis in the adult human brain. Here, we demonstrate the applicability of a powerful method to address this debate, that is, the in vivo labeling of adult human patients with 15N-thymidine, a non-hazardous form of thymidine, an approach without any clinical harm or ethical concerns. 15N-thymidine incorporation into newly synthesized DNA of specific cells was quantified at the single-cell level with subcellular resolution by Multiple-isotype imaging mass spectrometry (MIMS) of brain tissue resected for medical reasons. Two adult human patients, a glioblastoma patient and a patient with drug-refractory right temporal lobe epilepsy, were infused for 24 h with 15N-thymidine. Detection of 15N-positive leukocyte nuclei in blood samples from these patients confirmed previous findings by others and demonstrated the appropriateness of this approach to search for the generation of new cells in the adult human brain. 15N-positive neural cells were easily identified in the glioblastoma tissue sample, and the range of the 15N signal suggested that cells that underwent S-phase fully or partially during the 24 h in vivo labeling period, as well as cells generated therefrom, were detected. In contrast, within the hippocampus tissue resected from the epilepsy patient, none of the 2,000 dentate gyrus neurons analyzed was positive for 15N-thymidine uptake, consistent with the notion that the rate of neurogenesis in the adult human hippocampus is rather low. Of note, the likelihood of detecting neurogenesis was reduced because of (i) the low number of cells analyzed, (ii) the fact that hippocampal tissue was explored that may have had reduced neurogenesis due to epilepsy, and (iii) the labeling period of 24 h which may have been too short to capture quiescent neural stem cells. Yet, overall, our approach to enrich NeuN-labeled neuronal nuclei by FACS prior to MIMS analysis provides a promising strategy to quantify even low rates of neurogenesis in the adult human hippocampus after in vivo15N-thymidine infusion. From a general point of view and regarding future perspectives, the in vivo labeling of humans with 15N-thymidine followed by MIMS analysis of brain tissue constitutes a novel approach to study mitotically active cells and their progeny in the brain, and thus allows a broad spectrum of studies of brain physiology and pathology, including microcephaly

Vertebral body stenting: a new method for vertebral augmentation versus kyphoplasty

Vertebroplasty and kyphoplasty are well-established minimally invasive treatment options for compression fractures of osteoporotic vertebral bodies. Possible procedural disadvantages, however, include incomplete fracture reduction or a significant loss of reduction after balloon tamp deflation, prior to cement injection. A new procedure called “vertebral body stenting” (VBS) was tested in vitro and compared to kyphoplasty. VBS uses a specially designed catheter-mounted stent which can be implanted and expanded inside the vertebral body. As much as 24 fresh frozen human cadaveric vertebral bodies (T11-L5) were utilized. After creating typical compression fractures, the vertebral bodies were reduced by kyphoplasty (n = 12) or by VBS (n = 12) and then stabilized with PMMA bone cement. Each step of the procedure was performed under fluoroscopic control and analysed quantitatively. Finally, static and dynamic biomechanical tests were performed. A complete initial reduction of the fractured vertebral body height was achieved by both systems. There was a significant loss of reduction after balloon deflation in kyphoplasty compared to VBS, and a significant total height gain by VBS (mean ± SD in %, p < 0.05, demonstrated by: anterior height loss after deflation in relation to preoperative height [kyphoplasty: 11.7 ± 6.2; VBS: 3.7 ± 3.8], and total anterior height gain [kyphoplasty: 8.0 ± 9.4; VBS: 13.3 ± 7.6]). Biomechanical tests showed no significant stiffness and failure load differences between systems. VBS is an innovative technique which allows for the possibly complete reduction of vertebral compression fractures and helps maintain the restored height by means of a stent. The height loss after balloon deflation is significantly decreased by using VBS compared to kyphoplasty, thus offering a new promising option for vertebral augmentation

U-Compare bio-event meta-service: compatible BioNLP event extraction services

AbstractBackgroundBio-molecular event extraction from literature is recognized as an important task of bio text mining and, as such, many relevant systems have been developed and made available during the last decade. While such systems provide useful services individually, there is a need for a meta-service to enable comparison and ensemble of such services, offering optimal solutions for various purposes.ResultsWe have integrated nine event extraction systems in the U-Compare framework, making them inter-compatible and interoperable with other U-Compare components. The U-Compare event meta-service provides various meta-level features for comparison and ensemble of multiple event extraction systems. Experimental results show that the performance improvements achieved by the ensemble are significant. ConclusionsWhile individual event extraction systems themselves provide useful features for bio text mining, the U-Compare meta-service is expected to improve the accessibility to the individual systems, and to enable meta-level uses over multiple event extraction systems such as comparison and ensemble.This research was partially supported by KAKENHI 18002007 [YK, MM, JDK, SP, TO, JT]; JST PRESTO and KAKENHI 21500130 [YK]; the Academy of Finland and computational resources were provided by CSC -- IT Center for Science Ltd [JB, FG]; the Research Foundation Flanders (FWO) [SVL]; UK Biotechnology and Biological Sciences, Research Council (BBSRC project BB/G013160/1 Automated Biological Event Extraction from the Literature for Drug Discovery) and JISC, National Centre for Text Mining [SA]; the Spanish grant BIO2010-17527 [MN, APM]; NIH Grant U54 DA021519 [AO, DRR]Peer Reviewe

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Comparison of different normalization strategies for the analysis of glomerular microRNAs in IgA nephropathy

Small nucleolar RNAs (snoRNAs) have been used for normalization in glomerular microRNA (miRNA) quantification without confirmation of validity. Our aim was to identify glomerular reference miRNAs in IgA nephropathy. We compared miRNAs in human paraffin-embedded renal biopsies from patients with cellular-crescentic IgA-GN (n = 5; crescentic IgA-GN) and non-crescentic IgA-GN (n = 5; IgA-GN) to mild interstitial nephritis without glomerular abnormalities (controls, n = 5). Laser-microdissected glomeruli were used for expression profiling of 762 miRNAs by low-density TaqMan arrays (cards A and B). The comparison of different normalization methods (GeNormPlus, NormFinder, global mean and snoRNAs) in crescentic IgA-GN, IgA-GN and controls yielded similar results. However, levels of significance and the range of relative expression differed. In median, two normalization methods demonstrated similar results. GeNormPlus and NormFinder gave different top ranked reference miRNAs. Stability ranking for snoRNAs varied between cards A and B. In conclusion, we suggest the geometric mean of the most stable reference miRNAs found in GeNormPlus (miR-26b-5p), NormFinder (miR-28-5p) and snoRNAs (RNU44) as reference. It should be considered that significant differences could be missed using one particular normalization method. As a starting point for glomerular miRNA studies in IgA nephropathy we provide a library of miRNAs

Siponimod (BAF-312) Attenuates Perihemorrhagic Edema And Improves Survival in Experimental Intracerebral Hemorrhage

Background and Purpose- Perihemorrhagic edema (PHE) is associated with poor outcome after intracerebral hemorrhage (ICH). Infiltration of immune cells is considered a major contributor of PHE. Recent studies suggest that immunomodulation via S1PR (sphingosine-1-phosphate receptor) modulators improve outcome in ICH. Siponimod, a selective modulator of sphingosine 1-phosphate receptors type 1 and type 5, demonstrated an excellent safety profile in a large study of patients with multiple sclerosis. Here, we investigated the impact of siponimod treatment on perihemorrhagic edema, neurological deficits, and survival in a mouse model of ICH. Methods- ICH was induced by intracranial injection of 0.075 U of bacterial collagenase in 123 mice. Mice were randomly assigned to different treatment groups: vehicle, siponimod given as a single dosage 30 minutes after the operation or given 3x for 3 consecutive days starting 30 minutes after operation. The primary outcome of our study was evolution of PHE measured by magnetic resonance-imaging on T2-maps 72 hours after ICH, secondary outcomes included evolution of PHE 24 hours after ICH, survival and neurological deficits, as well as effects on circulating blood cells and body weight. Results- Siponimod significantly reduced PHE measured by magnetic resonance imaging (P=0.021) as well as wet-dry method (P=0.04) 72 hours after ICH. Evaluation of PHE 24 hours after ICH showed a tendency toward attenuated brain edema in the low-dosage group (P=0.08). Multiple treatments with siponimod significantly improved neurological deficits measured by Garcia Score (P=0.03). Survival at day 10 was improved in mice treated with multiple dosages of siponimod (P=0.037). Mice treated with siponimod showed a reduced weight loss after ICH (P=0.036). Conclusions- Siponimod (BAF-312) attenuated PHE after ICH, increased survival, and reduced ICH-induced sensorimotor deficits in our experimental ICH-model. Findings encourage further investigation of inflammatory modulators as well as the translation of BAF-312 to a human study of ICH patients