LysoTracker is a marker of differentiated alveolar type II cells

Background

LysoTracker Green DND-26 is a fluorescent dye that stains acidic compartments in live cells and has been shown to selectively accumulate in lamellar bodies in alveolar type II (AT2) cells in the lung. The aim of this study was to determine whether the accumulation of LysoTracker in lamellar bodies can be used to isolate viable AT2 cells by flow cytometry and track their differentiation in live-cell culture by microscopy.

Methods

Mouse lung cells were sorted on the basis of CD45^negCD31^negEpCAM^posLysoTracker^pos expression and characterized by immunostaining for SP-C and cultured in a three-dimensional epithelial colony-forming unit (CFU-Epi) assay. To track AT2 cell differentiation, lung epithelial stem and progenitor cells were cultured in a CFU-Epi assay with LysoTracker-supplemented media.

Results

The purity of sorted AT2 cells as determined by SP-C staining was 97.4% and viability was 85.3%. LysoTracker^pos AT2 cells generated SP-C^pos alveolar epithelial cell colonies in culture, and when added to the CFU-Epi culture medium, LysoTracker marked the differentiation of stem/progenitor-derived AT2 cells.

Conclusions

This study describes a novel method for isolating AT2 cells from mouse lungs. The high purity and viability of cells attained by this method, makes them suitable for functional analysis in vitro. The application of LysoTracker to live cell cultures will allow better assessment of the cellular and molecular mechanisms that regulate AT2 cell differentiation.     

TBP2 is a substitute for TBP in Xenopus oocyte transcription

Background  

TATA-box-binding protein 2 (TBP2/TRF3) is a vertebrate-specific paralog of TBP that shares with TBP a highly conserved carboxy-terminal domain and the ability to bind the TATA box. TBP2 is highly expressed in oocytes whereas TBP is more abundant in embryos.     

Isolabelling is a radiation-induced phenomenon

Human lymphocytes were incubated during two mitotic cycles in the presence of 5-bromodeoxyuridine and differentiation between chromatids was obtained with combined Hoechst 33258 and azur-eosine staining. Analysis of non-irradiated cells revealed numerous sister chromatid exchanges (SCE) and no abnormalities of harlequine appearance of chromosomes. When, however, the cells were irradiated, an identical staining (IS, isostaining) of some chromosomes or chromosome segments were observed. Production of IS was accompanied by decrease of the frequency of SCE, the total frequency of SCE+IS remained, however, the same as in control. An antagonism between SCE and IS was established: the frequency of SCE decreased in the cells with multiple IS, and chromosomes with both SCE and IS were only rarely observed. Thus, IS is neither an artifact nor a physiologic event but a phenomenon induced by radiation. The reliable existence of IS is considered as an evidence for binemic structure of chromatid. It is suggested that some mechanism of lateral spread of genetic information is involved in the production of SCE. If delayed by radiation, the spread could be restricted only to a fraction of chromosome cross-section resulting in IS.     

Gadd45a is an RNA binding protein and is localized in nuclear speckles

Background

The Gadd45 proteins play important roles in growth control, maintenance of genomic stability, DNA repair, and apoptosis. Recently, Gadd45 proteins have also been implicated in epigenetic gene regulation by promoting active DNA demethylation. Gadd45 proteins have sequence homology with the L7Ae/L30e/S12e RNA binding superfamily of ribosomal proteins, which raises the question if they may interact directly with nucleic acids.

Principal Findings

Here we show that Gadd45a binds RNA but not single- or double stranded DNA or methylated DNA in vitro. Sucrose density gradient centrifugation experiments demonstrate that Gadd45a is present in high molecular weight particles, which are RNase sensitive. Gadd45a displays RNase-sensitive colocalization in nuclear speckles with the RNA helicase p68 and the RNA binding protein SC35. A K45A point mutation defective in RNA binding was still active in DNA demethylation. This suggests that RNA binding is not absolutely essential for demethylation of an artificial substrate. A point mutation at G39 impared RNA binding, nuclear speckle localization and DNA demethylation, emphasizing its relevance for Gadd45a function.

Significance

The results implicate RNA in Gadd45a function and suggest that Gadd45a is associated with a ribonucleoprotein particle.     

Mir-34a is upregulated during liver regeneration in rats and is associated with the suppression of hepatocyte proliferation

Background

MicroRNAs are a class of small regulatory RNAs that modulate a variety of biological processes, including cellular differentiation, apoptosis, metabolism and proliferation. This study aims to explore the effect of miR-34a in hepatocyte proliferation and its potential role in liver regeneration termination.

Methodology/Principal Finding

MiR-34a was highly induced after partial hepatectomy. Overexpression of miR-34a in BRL-3A cells could significantly inhibit cell proliferation and down-regulate the expression of inhibin βB (INHBB) and Met. In BRL-3A cells, INHBB was identified as a direct target of miR-34a by luciferase reporter assay. More importantly, INHBB siRNA significantly repressed cell proliferation. A decrease of INHBB and Met was detected in regenerating liver.

Conclusion/Significance

MiR-34a expression was upregulated during the late phase of liver regeneration. MiR-34a-mediated regulation of INHBB and Met may collectively contribute to the suppression of hepatocyte proliferation.     

The shine-through masking paradigm is a potential endophenotype of schizophrenia

Background

To understand the genetics of schizophrenia, a hunt for so-called intermediate phenotypes or endophenotypes is ongoing. Visual masking has been proposed to be such an endophenotype. However, no systematic study has been conducted yet to prove this claim. Here, we present the first study showing that masking meets the most important criteria for an endophenotype.

Methodology/Principal Findings

We tested 62 schizophrenic patients, 39 non-affected first-degree relatives, and 38 healthy controls in the shine-through masking paradigm and, in addition, in the Continuous Performance Test (CPT) and the Wisconsin Card Sorting Test (WCST). Most importantly, masking performance of relatives was significantly in between the one of patients and controls in the shine-through paradigm. Moreover, deficits were stable throughout one year. Using receiver operating characteristics (ROC) methods, we show that the shine-through paradigm distinguishes with high sensitivity and specificity between schizophrenic patients, first-order relatives and healthy controls.

Conclusions/Significance

The shine-through paradigm is a potential endophenotype.     

Lipocalin-7 is a matricellular regulator of angiogenesis

Background

Matricellular proteins are extracellular regulators of cellular adhesion, signaling and performing a variety of physiological behaviors such as proliferation, migration and differentiation. Within vascular microenvironments, matricellular proteins exert both positive and negative regulatory cues to vascular endothelium. The relative balance of these matricellular cues is believed to be critical for vascular homeostasis, angiogenesis activation or angiogenesis resolution. However, our knowledge of matricellular proteins within vascular microenvironments and the mechanisms by which these proteins impact vascular function remain largely undefined. The matricellular protein lipocalin-7 (LCN7) is found throughout vascular microenvironments, and circumstantial evidence suggests that LCN7 may be an important regulator of angiogenesis. Therefore, we hypothesized that LCN7 may be an important regulator of vascular function.

Methodology and Principal Findings

To test this hypothesis, we examined the effect of LCN7 overexpression, recombinant protein and gene knockdown in a series of in vitro and in vivo models of angiogenesis. We found that overexpression of LCN7 in MB114 and SVEC murine endothelial cell lines or administration of highly purified recombinant LCN7 protein increased endothelial cell invasion. Similarly, LCN7 increased angiogenic sprouting from quiescent endothelial cell monolayers and ex vivo aortic rings. Moreover, LCN7 increased endothelial cell sensitivity to TGF-β but did not affect sensitivity to other pro-angiogenic growth factors including bFGF and VEGF. Finally, morpholino based knockdown of LCN7 in zebrafish embryos specifically inhibited angiogenic sprouting but did not affect vasculogenesis within injected embryos.

Conclusions and Significance

No functional analysis has previously been performed to elucidate the function of LCN7 in vascular or other cellular processes. Collectively, our results show for the first time that LCN7 is an important pro-angiogenic matricellular protein of vascular microenvironments.     

Jak2 is a negative regulator of ubiquitin-dependent endocytosis of the growth hormone receptor

Background

Length and intensity of signal transduction via cytokine receptors is precisely regulated. Degradation of certain cytokine receptors is mediated by the ubiquitin ligase SCF(βTrCP). In several instances, Janus kinase (Jak) family members can stabilise their cognate cytokine receptors at the cell surface.

Principal Findings

In this study we show in Hek293 cells that Jak2 binding to the growth hormone receptor prevents endocytosis in a non-catalytic manner. Following receptor activation, the detachment of phosphorylated Jak2 induces down-regulation of the growth hormone receptor by SCF(βTrCP). Using γ2A human fibroblast cells we show that both growth hormone-induced and constitutive growth hormone receptor endocytosis depend on the same factors, strongly suggesting that the modes of endocytosis are identical. Different Jak2 RNA levels in HepG2, IM9 and Hek293 cells indicate the importance of cellular concentration on growth hormone receptor function. Both Jak2 and βTrCP bind to neighbouring linear motifs in the growth hormone receptor tail without the requirement of modifications, indicating that growth hormone sensitivity is regulated by the cellular level of non-committed Jak2.

Conclusions/Significance

As signal transduction of many cytokine receptors depends on Jak2, the study suggests an integrative role of Jak2 in cytokine responses based on its enzyme activity as well as its stabilising properties towards the receptors.     

Indian hedgehog gene transfer is a chondrogenic inducer of human mesenchymal stem cells

Introduction

Anti-endothelial cell antibodies (AECAs) are thought to be critical for vasculitides in collagen diseases, but most were directed against molecules localized within the cell and not expressed on the cell surface. To clarify the pathogenic roles of AECAs, we constructed a retroviral vector system for identification of autoantigens expressed on the endothelial cell surface.

Methods

AECA activity in sera from patients with collagen diseases was measured with flow cytometry by using human umbilical vein endothelial cells (HUVECs). A cDNA library of HUVECs was retrovirally transfected into a rat myeloma cell line, from which AECA-positive clones were sorted with flow cytometry. cDNA of the cells was analyzed to identify an autoantigen, and then the clinical characteristics and the functional significance of the autoantibody were evaluated.

Results

Two distinct AECA-positive clones were isolated by using serum immunoglobulin G (IgG) from a patient with systemic lupus erythematosus (SLE). Both clones were identical to cDNA of fibronectin leucine-rich transmembrane protein 2 (FLRT2). HUVECs expressed FLRT2 and the prototype AECA IgG bound specifically to FLRT2-transfected cells. Anti-FLRT2 antibody activity accounted for 21.4% of AECAs in SLE. Furthermore, anti-FLRT2 antibody induced complement-dependent cytotoxicity against FLRT2-expressing cells.

Conclusions

We identified the membrane protein FLRT2 as a novel autoantigen of AECAs in SLE patients by using the retroviral vector system. Anti-FLRT2 antibody has the potential to induce direct endothelial cell cytotoxicity in about 10% of SLE patients and could be a novel molecular target for intervention. Identification of such a cell-surface target for AECAs may reveal a comprehensive mechanism of vascular injury in collagen diseases.     

Esophagitis in a high H. pylori prevalence area: severe disease is rare but concomitant peptic ulcer is frequent

Background

Few data are available on the prevalence of erosive and severe esophagitis in Western countries.

Objective

To retrospectively determine the prevalence and the factors predicting erosive esophagitis and severe esophagitis in a large series of endoscopies in Spain.

Design

Retrospective observational study. A multivariate analysis was performed to determine variables predicting severe esophagitis.

Setting

Databases of 29 Spanish endoscopy units.

Patients

Patients submitted to a diagnostic endoscopy during the year 2005.

Interventions

Retrospective review of the databases.

Main Outcome Measurements

Esophagitis severity (graded according to the Los Angeles classification) and associated endoscopic findings.

Results

Esophagitis was observed in 8.7% of the 93,699 endoscopies reviewed. Severe esophagitis (LA grade C or D) accounted for 22.5% of cases of the disease and was found in 1.9% of all endoscopies. Incidences of esophagitis and those of severe esophagitis were 86.2 and 18.7 cases per 100,000 inhabitants per year respectively. Male sex (OR 1.89) and advanced age (OR 4.2 for patients in the fourth age quartile) were the only variables associated with severe esophagitis. Associated peptic ulcer was present in 8.8% of cases.

Limitations

Retrospective study, no data on individual proton pump inhibitors use.

Conclusions

Severe esophagitis is an infrequent finding in Spain. It occurs predominantly in males and in older individuals. Peptic ulcer disease is frequently associated with erosive esophagitis.     

Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential

Background

Cone degeneration is the hallmark of the inherited retinal disease retinitis pigmentosa. We have previously identified a trophic factor "Rod-derived Cone Viability Factor (RdCVF) that is secreted by rods and promote cone viability in a mouse model of the disease.

Results

Here we report the bioinformatic identification and the experimental analysis of RdCVF2, a second trophic factor belonging to the Rod-derived Cone Viability Factor family. The mouse RdCVF gene is known to be bifunctional, encoding both a long thioredoxin-like isoform (RdCVF-L) and a short isoform with trophic cone photoreceptor viability activity (RdCVF-S). RdCVF2 shares many similarities with RdCVF in terms of gene structure, expression in a rod-dependent manner and protein 3D structure. Furthermore, like RdCVF, the RdCVF2 short isoform exhibits cone rescue activity that is independent of its putative thiol-oxydoreductase activity.

Conclusion

Taken together, these findings define a new family of bifunctional genes which are: expressed in vertebrate retina, encode trophic cone viability factors, and have major therapeutic potential for human retinal neurodegenerative diseases such as retinitis pigmentosa.     

Vasospasm is a significant factor in cyclosporine-induced neurotoxicity: Case report

Background

The aetiology of central nervous system lesions observed in cerebral cyclosporine neurotoxicity remains controversial.

Case presentation

We report a 48-year-old woman with a non-severe aplastic anaemia who presented with stroke-like episodes while on cyclosporine treatment.Transcranial Doppler ultrasound revealed severely elevated flow velocities in several cerebral vessels, consistent with vasospasm. Immediately after reducing the cyclosporine dose, the stroke-like episodes disappeared. Only after cyclosporine withdrawal the transcranial Doppler ultrasound abnormalities fully resolved.

Conclusions

This case demonstrates a significant role of vasospasm in the pathway of cyclosporine-induced neurotoxicity. Transcranial Doppler ultrasound is an effective tool for the diagnosis and follow-up of cyclosporine-induced vasospasm.

     

Temporal variability is a personalized feature of the human microbiome

Background

It is now apparent that the complex microbial communities found on and in the human body vary across individuals. What has largely been missing from previous studies is an understanding of how these communities vary over time within individuals. To the extent to which it has been considered, it is often assumed that temporal variability is negligible for healthy adults. Here we address this gap in understanding by profiling the forehead, gut (fecal), palm, and tongue microbial communities in 85 adults, weekly over 3 months.

Results

We found that skin (forehead and palm) varied most in the number of taxa present, whereas gut and tongue communities varied more in the relative abundances of taxa. Within each body habitat, there was a wide range of temporal variability across the study population, with some individuals harboring more variable communities than others. The best predictor of these differences in variability across individuals was microbial diversity; individuals with more diverse gut or tongue communities were more stable in composition than individuals with less diverse communities.

Conclusions

Longitudinal sampling of a relatively large number of individuals allowed us to observe high levels of temporal variability in both diversity and community structure in all body habitats studied. These findings suggest that temporal dynamics may need to be considered when attempting to link changes in microbiome structure to changes in health status. Furthermore, our findings show that, not only is the composition of an individual’s microbiome highly personalized, but their degree of temporal variability is also a personalized feature.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0531-y) contains supplementary material, which is available to authorized users.     

Succinate dehydrogenase is a direct target of sirtuin 3 deacetylase activity

Background

Sirtuins (SIRT1-7) are a family of NAD-dependent deacetylases and/or ADP-ribosyltransferases that are involved in metabolism, stress responses and longevity. SIRT3 is localized to mitochondria, where it deacetylates and activates a number of enzymes involved in fuel oxidation and energy production.

Methodology/Principal Findings

In this study, we performed a proteomic screen to identify SIRT3 interacting proteins and identified several subunits of complex II and V of the electron transport chain. Two subunits of complex II (also known as succinate dehydrogenase, or SDH), SDHA and SDHB, interacted specifically with SIRT3. Using mass spectrometry, we identified 13 acetylation sites on SDHA, including six novel acetylated residues. SDHA is hyperacetylated in SIRT3 KO mice and SIRT3 directly deacetylates SDHA in a NAD-dependent manner. Finally, we found that SIRT3 regulates SDH activity both in cells and in murine brown adipose tissue.

Conclusions/Significance

Our study identifies SDHA as a binding partner and substrate for SIRT3 deacetylase activity. SIRT3 loss results in decreased SDH enzyme activity, suggesting that SIRT3 may be an important physiological regulator of SDH activity.     

Ophthalmic acid is a marker of oxidative stress in plants as in animals

Background

Ophthalmic acid (OPH), γ-glutamyl-L-2-aminobutyryl-glycine, a tripeptide analogue of glutathione (GSH), has recently captured considerable attention as a biomarker of oxidative stress in animals. The OPH and GSH biosynthesis, as well as some biochemical behaviors, are very similar. Here, we sought to investigate the presence of OPH in plants and its possible relationship with GSH, known to possess multiple functions in the plant development, growth and response to environmental changes.

IL-13 induces a bronchial epithelial phenotype that is profibrotic

Background

Inflammatory cytokines (e.g. IL-13) and mechanical perturbations (e.g. scrape injury) to the epithelium release profibrotic factors such as TGF-β₂, which may, in turn, stimulate subepithelial fibrosis in asthma. We hypothesized that prolonged IL-13 exposure creates a plastic epithelial phenotype that is profibrotic through continuous secretion of soluble mediators at levels that stimulate subepithelial fibrosis.

Methods

Normal human bronchial epithelial cells (NHBE) were treated with IL-13 (0, 0.1, 1, or 10 ng/ml) for 14 days (day 7 to day 21 following seeding) at an air-liquid interface during differentiation, and then withdrawn for 1 or 7 days. Pre-treated and untreated NHBE were co-cultured for 3 days with normal human lung fibroblasts (NHLF) embedded in rat-tail collagen gels during days 22–25 or days 28–31.

Results

IL-13 induced increasing levels of MUC5AC protein, and TGF-β₂, while decreasing β-Tubulin IV at day 22 and 28 in the NHBE. TGF-β₂, soluble collagen in the media, salt soluble collagen in the matrix, and second harmonic generation (SHG) signal from fibrillar collagen in the matrix were elevated in the IL-13 pre-treated NHBE co-cultures at day 25, but not at day 31. A TGF-β₂ neutralizing antibody reversed the increase in collagen content and SHG signal.

Conclusion

Prolonged IL-13 exposure followed by withdrawal creates an epithelial phenotype, which continuously secretes TGF-β₂ at levels that increase collagen secretion and alters the bulk optical properties of an underlying fibroblast-embedded collagen matrix. Extended withdrawal of IL-13 from the epithelium followed by co-culture does not stimulate fibrosis, indicating plasticity of the cultured airway epithelium and an ability to return to a baseline. Hence, IL-13 may contribute to subepithelial fibrosis in asthma by stimulating biologically significant TGF-β₂ secretion from the airway epithelium.     

Allelic mapping bias in RNA-sequencing is not a major confounder in eQTL studies

Background

RNA sequencing (RNA-seq) is the current gold-standard method to quantify gene expression for expression quantitative trait locus (eQTL) studies. However, a potential caveat in these studies is that RNA-seq reads carrying the non-reference allele of variant loci can have lower probability to map correctly to the reference genome, which could bias gene quantifications and cause false positive eQTL associations. In this study, we analyze the effect of this allelic mapping bias in eQTL discovery.

Results

We simulate RNA-seq read mapping over 9.5 M common SNPs and indels, with 15.6% of variants showing biased mapping rate for reference versus non-reference reads. However, removing potentially biased RNA-seq reads from an eQTL dataset of 185 individuals has a very small effect on gene and exon quantifications and eQTL discovery. We detect only a handful of likely false positive eQTLs, and overall eQTL SNPs show no significant enrichment for high mapping bias.

Conclusion

Our results suggest that RNA-seq quantifications are generally robust against allelic mapping bias, and that this does not have a severe effect on eQTL discovery. Nevertheless, we provide our catalog of putatively biased loci to allow better controlling for mapping bias to obtain more accurate results in future RNA-seq studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0467-2) contains supplementary material, which is available to authorized users.