Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina.

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

A single cell-based computational platform to identify chemical compounds targeting desired sets of transcription factors for cellular conversion.

Cellular conversion can be induced by perturbing a handful of key transcription factors (TFs). Replacement of direct manipulation of key TFs with chemical compounds offers a less laborious and safer strategy to drive cellular conversion for regenerative medicine. Nevertheless, identifying optimal chemical compounds currently requires large-scale screening of chemical libraries, which is resource intensive. Existing computational methods aim at predicting cell conversion TFs, but there are no methods for identifying chemical compounds targeting these TFs. Here, we develop a single cell-based platform (SiPer) to systematically prioritize chemical compounds targeting desired TFs to guide cellular conversions. SiPer integrates a large compendium of chemical perturbations on non-cancer cells with a network model and predicted known and novel chemical compounds in diverse cell conversion examples. Importantly, we applied SiPer to develop a highly efficient protocol for human hepatic maturation. Overall, SiPer provides a valuable resource to efficiently identify chemical compounds for cell conversion.

ChemPert: mapping between chemical perturbation and transcriptional response for non-cancer cells.

Prior knowledge of perturbation data can significantly assist in inferring the relationship between chemical perturbations and their specific transcriptional response. However, current databases mostly contain cancer cell lines, which are unsuitable for the aforementioned inference in non-cancer cells, such as cells related to non-cancer disease, immunology and aging. Here, we present ChemPert (https://chempert.uni.lu/), a database consisting of 82 270 transcriptional signatures in response to 2566 unique perturbagens (drugs, small molecules and protein ligands) across 167 non-cancer cell types, as well as the protein targets of 57 818 perturbagens. In addition, we develop a computational tool that leverages the non-cancer cell datasets, which enables more accurate predictions of perturbation responses and drugs in non-cancer cells compared to those based onto cancer databases. In particular, ChemPert correctly predicted drug effects for treating hepatitis and novel drugs for osteoarthritis. The ChemPert web interface is user-friendly and allows easy access of the entire datasets and the computational tool, providing valuable resources for both experimental researchers who wish to find datasets relevant to their research and computational researchers who need comprehensive non-cancer perturbation transcriptomics datasets for developing novel algorithms. Overall, ChemPert will facilitate future in silico compound screening for non-cancer cells.

Neural network learning defines glioblastoma features to be of neural crest perivascular or radial glia lineages.

Glioblastoma is believed to originate from nervous system cells; however, a putative origin from vessel-associated progenitor cells has not been considered. We deeply single-cell RNA-sequenced glioblastoma progenitor cells of 18 patients and integrated 710 bulk tumors and 73,495 glioma single cells of 100 patients to determine the relation of glioblastoma cells to normal brain cell types. A novel neural network-based projection of the developmental trajectory of normal brain cells uncovered two principal cell-lineage features of glioblastoma, neural crest perivascular and radial glia, carrying defining methylation patterns and survival differences. Consistently, introducing tumorigenic alterations in naïve human brain perivascular cells resulted in brain tumors. Thus, our results suggest that glioblastoma can arise from the brains' vasculature, and patients with such glioblastoma have a significantly poorer outcome.

HuR/ELAVL1 drives malignant peripheral nerve sheath tumor growth and metastasis.

Cancer cells can develop a strong addiction to discrete molecular regulators, which control the aberrant gene expression programs that drive and maintain the cancer phenotype. Here, we report the identification of the RNA-binding protein HuR/ELAVL1 as a central oncogenic driver for malignant peripheral nerve sheath tumors (MPNSTs), which are highly aggressive sarcomas that originate from cells of the Schwann cell lineage. HuR was found to be highly elevated and bound to a multitude of cancer-associated transcripts in human MPNST samples. Accordingly, genetic and pharmacological inhibition of HuR had potent cytostatic and cytotoxic effects on tumor growth, and strongly suppressed metastatic capacity in vivo. Importantly, we linked the profound tumorigenic function of HuR to its ability to simultaneously regulate multiple essential oncogenic pathways in MPNST cells, including the Wnt/ß-catenin, YAP/TAZ, RB/E2F, and BET pathways, which converge on key transcriptional networks. Given the exceptional dependency of MPNST cells on HuR for survival, proliferation, and dissemination, we propose that HuR represents a promising therapeutic target for MPNST treatment.

Computational Strategies for Niche-Dependent Cell Conversion to Assist Stem Cell Therapy.

The field of regenerative medicine has blossomed in recent decades. However, the ultimate goal of tissue regeneration - replacing damaged or aged cells with healthy functioning cells - still faces a number of challenges. In particular, better understanding of the role of the cellular niche in shaping stem cell phenotype and conversion would aid in improving current protocols for stem cell therapies. In this regard, the implementation of novel computational approaches that consider the niche effect on stem cells would be valuable. Here we discuss current problems in stem cell transplantation and rejuvenation, and we propose computational strategies to control niche-dependent cell conversion to overcome them.

A complex of the ubiquitin ligase TRIM32 and the deubiquitinase USP7 balances the level of c-Myc ubiquitination and thereby determines neural stem cell fate specification.

The balance between stem cell maintenance and differentiation has been proposed to depend on antagonizing ubiquitination and deubiquitination reactions of key stem cell transcription factors (SCTFs) mediated by pairs of E3 ubiquitin ligases and deubiquitinating enzymes. Accordingly, increased ubiquitination results in proteasomal degradation of the SCTF, thereby inducing cellular differentiation, whereas increased deubiquitination stabilizes the SCTF, leading to maintenance of the stem cell fate. In neural stem cells, one of the key SCTFs is c-Myc. Previously, it has been shown that c-Myc is ubiquitinated by the E3 ligase TRIM32, thereby targeting c-Myc for proteasomal degradation and inducing neuronal differentiation. Accordingly, TRIM32 becomes upregulated during adult neurogenesis. This upregulation is accompanied by subcellular translocation of TRIM32 from the cytoplasm of neuroblasts to the nucleus of neurons. However, we observed that a subpopulation of proliferative type C cells already contains nuclear TRIM32. As these cells do not undergo neuronal differentiation, despite containing TRIM32 in the nucleus, where it can ubiquitinate c-Myc, we hypothesize that antagonizing factors, specifically deubiquitinating enzymes, are present in these particular cells. Here we show that TRIM32 associates with the deubiquitination enzyme USP7, which previously has been implicated in neural stem cell maintenance. USP7 and TRIM32 were found to exhibit a dynamic and partially overlapping expression pattern during neuronal differentiation both in vitro and in vivo. Most importantly, we are able to demonstrate that USP7 deubiquitinates and thereby stabilizes c-Myc and that this function is required to maintain neural stem cell fate. Accordingly, we propose the balanced ubiquitination and deubiquitination of c-Myc by TRIM32 and USP7 as a novel mechanism for stem cell fate determination.

Proteome and Secretome Characterization of Glioblastoma-Derived Neural Stem Cells.

Glioblastoma multiforme (GBM) (grade IV astrocytoma) is the most common and aggressive primary brain tumor. GBM consists of heterogeneous cell types including a subset of stem cell-like cells thought to sustain tumor growth. These tumor-initiating glioblastoma multiforme-derived neural stem (GNS) cells as well as their genetically normal neural stem (NS) counterparts can be propagated in culture as relatively pure populations. Here, we perform quantitative proteomics to globally characterize and compare total proteome plus the secreted proteome (secretome) between GNS cells and NS cells. Proteins and pathways that distinguish malignant cancer (GNS) stem cells from their genetically normal counterparts (NS cells) might have value as new biomarkers or therapeutic targets. Our analysis identified and quantified ∼7,500 proteins in the proteome and ∼2,000 in the secretome, 447 and 138 of which were differentially expressed, respectively. Notable tumor-associated processes identified using gene set enrichment analysis included: extracellular matrix interactions, focal adhesion, cell motility, and cell signaling. We focused on differentially expressed surface proteins, and identified 26 that participate in ligand-receptor pairs that play a prominent role in tumorigenesis. Immunocytochemistry and immunoblotting confirmed that CD9, a recently identified marker of adult subventricular zone NS cells, was consistently enriched across a larger set of primary GNS cell lines. CD9 may, therefore, have value as a GNS-specific surface marker and a candidate therapeutic target. Altogether, these findings support the notion that increased cell-matrix and cell-cell adhesion molecules play a crucial role in promoting the tumor initiating and infiltrative properties of GNS cells. Stem Cells 2017;35:967-980.

Regulation of Fn14 Receptor and NF-κB Underlies Inflammation in Meniere's Disease.

Meniere's disease (MD) is a rare disorder characterized by episodic vertigo, sensorineural hearing loss, tinnitus, and aural fullness. It is associated with a fluid imbalance between the secretion of endolymph in the cochlear duct and its reabsorption into the subarachnoid space, leading to an accumulation of endolymph in the inner ear. Epidemiological evidence, including familial aggregation, indicates a genetic contribution and a consistent association with autoimmune diseases (AD). We conducted a case-control study in two phases using an immune genotyping array in a total of 420 patients with bilateral MD and 1,630 controls. We have identified the first locus, at 6p21.33, suggesting an association with bilateral MD [meta-analysis leading signal rs4947296, OR = 2.089 (1.661-2.627); p = 1.39 × 10-09]. Gene expression profiles of homozygous genotype-selected peripheral blood mononuclear cells (PBMCs) demonstrated that this region is a trans-expression quantitative trait locus (eQTL) in PBMCs. Signaling analysis predicted several tumor necrosis factor-related pathways, the TWEAK/Fn14 pathway being the top candidate (p = 2.42 × 10-11). This pathway is involved in the modulation of inflammation in several human AD, including multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis. In vitro studies with genotype-selected lymphoblastoid cells from patients with MD suggest that this trans-eQTL may regulate cellular proliferation in lymphoid cells through the TWEAK/Fn14 pathway by increasing the translation of NF-κB. Taken together; these findings suggest that the carriers of the risk genotype may develop an NF-κB-mediated inflammatory response in MD.

A systems biology approach to identify niche determinants of cellular phenotypes.

Recent reports indicate a dominant role for cellular microenvironment or niche for stably maintaining cellular phenotypic states. Identification of key niche mediated signaling that maintains stem cells in specific phenotypic states remains a challenge, mainly due to the complex and dynamic nature of stem cell-niche interactions. In order to overcome this, we consider that stem cells maintain their phenotypic state by experiencing a constant effect created by the niche by integrating its signals via signaling pathways. Such a constant niche effect should induce sustained activation/inhibition of specific stem cell signaling pathways that controls the gene regulatory program defining the cellular phenotypic state. Based on this view, we propose a computational approach to identify the most likely receptor mediated signaling responsible for transmitting niche signals to the transcriptional regulatory network that maintain cell-specific gene expression patterns, termed as niche determinants. We demonstrate the utility of our method in different stem cell systems by identifying several known and novel niche determinants. Given the key role of niche in several degenerative diseases, identification of niche determinants can aid in developing strategies for potential applications in regenerative medicine.

A Generalized Gene-Regulatory Network Model of Stem Cell Differentiation for Predicting Lineage Specifiers.

Identification of cell-fate determinants for directing stem cell differentiation remains a challenge. Moreover, little is known about how cell-fate determinants are regulated in functionally important subnetworks in large gene-regulatory networks (i.e., GRN motifs). Here we propose a model of stem cell differentiation in which cell-fate determinants work synergistically to determine different cellular identities, and reside in a class of GRN motifs known as feedback loops. Based on this model, we develop a computational method that can systematically predict cell-fate determinants and their GRN motifs. The method was able to recapitulate experimentally validated cell-fate determinants, and validation of two predicted cell-fate determinants confirmed that overexpression of ESR1 and RUNX2 in mouse neural stem cells induces neuronal and astrocyte differentiation, respectively. Thus, the presented GRN-based model of stem cell differentiation and computational method can guide differentiation experiments in stem cell research and regenerative medicine.

Parkinsonism Improved With Levodopa After Endoscopic Third Ventriculostomy in Shunted Hydrocephalus Due to Aqueductal Stenosis.

INTRODUCTION: Levodopa-responsive parkinsonism has been reported following ventriculoperitoneal (VP) shunt in patients with obstructive hydrocephalus due to aqueductal stenosis. It has been thought to arise from injury to the global rostral midbrain including the nigrostriatal pathway by a transtentorial pressure gradient. We present a similar patient, but his parkinsonism resisted levodopa administration during the initial therapy. CASE REPORT: A 51-year-old man suffered from hydrocephalus due to secondary aqueductal stenosis presumably attributed to massive bleeding during surgery for a fourth ventricle hemangioblastoma. After resolution of the hydrocephalus with VP shunt, he developed severe parkinsonism, Parinaud syndrome, and hyperreflexia, suggesting global rostral midbrain dysfunction, but high-dosage levodopa therapy was not effective. An inverted transtentorial pressure gradient suggested by his unilateral slit-like ventricle was assumed to be the cause of the levodopa resistance. Also based on an assumption that the absorption of cerebrospinal fluid was impaired due to the intraoperative bleeding, a lumbar peritoneal shunt was added to the preexisting VP shunt, but it failed to control the ventricular size. Instead, endoscopic third ventriculostomy stabilized it, characteristically inducing levodopa responsiveness in our patient. An increase of the levodopa dosage led to clinical improvement, which needed a maintenance dosage because of dependency. CONCLUSION: The details of this patient suggest that a transtentorial pressure gradient may have impaired more distal basal ganglia connections over a global rostral midbrain including the nigrostriatal pathway, and that aggressive levodopa therapy after endoscopic third ventriculostomy can be effective for refractory parkinsonism.

IgG4-related disease initially presented as an orbital mass lesion mimicking optic nerve sheath meningioma.

We report a case of an optic nerve mass lesion associated with IgG4-related disease. A 39-year-old man presented with right blurred vision and proptosis 8 years before admission. Magnetic resonance imaging showed a mass lesion in the center of the right orbit, which was diagnosed as optic nerve sheath meningioma by neuroradiologists and neurosurgeons. Irradiation was selected for treatment of the lesion on the basis of the radiological diagnosis; subsequently, the lesion gradually reduced in size. However, regrowth of an optic nerve mass lesion observed during the previous 2 years caused remarkable exophthalmos, and removal of the orbital mass lesion was performed via a transcranial orbital approach. Pathological examinations resulted in a diagnosis of IgG4-related disease, and hematological tests revealed an elevated level of serum IgG4. Additional radiological examinations showed mass lesions in the left maxillary nerve, bilateral inferior alveolar nerves, paravertebral tissue, and left kidney. Treatment with oral steroids has produced a reduction in the size of these lesions.

The RNA helicase DDX6 regulates cell-fate specification in neural stem cells via miRNAs.

In neural stem cells (NSCs), the balance between stem cell maintenance and neuronal differentiation depends on cell-fate determinants such as TRIM32. Previously, we have shown that TRIM32 associates with the RNA-induced silencing complex and increases the activity of microRNAs such as Let-7a. However, the exact mechanism of microRNA regulation by TRIM32 during neuronal differentiation has yet to be elucidated. Here, we used a mass spectrometry approach to identify novel protein-protein interaction partners of TRIM32 during neuronal differentiation. We found that TRIM32 associates with proteins involved in neurogenesis and RNA-related processes, such as the RNA helicase DDX6, which has been implicated in microRNA regulation. We demonstrate, that DDX6 colocalizes with TRIM32 in NSCs and neurons and that it increases the activity of Let-7a. Furthermore, we provide evidence that DDX6 is necessary and sufficient for neuronal differentiation and that it functions in cooperation with TRIM32.

A differential network analysis approach for lineage specifier prediction in stem cell subpopulations.

BACKGROUND: Stem cell differentiation is a complex biological process. Cellular heterogeneity, such as the co-existence of different cell subpopulations within a population, partly hampers our understanding of this process. The modern single-cell gene expression technologies, such as single-cell RT-PCR and RNA-seq, have enabled us to elucidate such heterogeneous cell subpopulations. However, the identification of a transcriptional regulatory network (TRN) for each cell subpopulation within a population and genes determining specific cell fates (lineage specifiers) remains a challenge due to the slower development of appropriate computational and experimental workflows. Here, we propose a computational differential network analysis approach for predicting lineage specifiers in binary-fate differentiation events. METHODS: The proposed method is based on a model that considers each stem cell subpopulation being in a stable state maintained by its specific TRN stability core, and cell differentiation involves changes in these stability cores between parental and daughter cell subpopulations. The method first reconstructs topologically different cell-subpopulation specific TRNs from single-cell gene expression data, literature knowledge and transcription factor (TF)-DNA binding-site prediction. Then, it systematically predicts lineage specifiers by identifying genes in the TRN stability cores in both parental and daughter cell subpopulations. RESULTS: Application of this method to different stem cell differentiation systems was able to predict known and putative novel lineage specifiers. These examples include the differentiation of inner cell mass into either primitive endoderm or epiblast, different progenitor cells in the hematopoietic system, and the lung alveolar bipotential progenitor into either alveolar type 1 or alveolar type 2. CONCLUSIONS: The method is generally applicable to any binary-fate differentiation system, for which single-cell gene expression data are available. Therefore, it should aid in understanding stem cell lineage specification, and in the development of experimental strategies for regenerative medicine.

Tolosa-hunt syndrome associated with cytomegalovirus infection.

We herein present the case of a 38-year-old woman with left-sided oculomotor paralysis with ocular pain that developed after a respiratory infection. Her serum was positive for IgM against GM2 and GalNAc-GD1a gangliosides and cytomegalovirus. Thin-slice magnetic resonance imaging revealed enhanced abnormal tissue located primarily in the superolateral part of the left-sided cavernous sinus, which corticosteroids subsequently obscured with immediate resolution of the patient's ocular symptoms. These clinical features were consistent with those of Tolosa-Hunt syndrome (THS). Our findings in the present patient suggest that cytomegalovirus may provoke granuloma formation in the cavernous sinus, as reported in other various organs, thereby leading to the development of THS.

Properties of isotope patterns and their utility for peptide identification in large-scale proteomic experiments.

RATIONALE: In proteomic experiments, isotope patterns are routinely generated for all detected peptides. While this pattern is determined by peptide composition, it has not been evaluated as a parameter that can help in the process of peptide identification. METHODS: First, we investigated how the relative isotope abundance (RIA) accuracy in proteomic data sets depends on the spectral intensity, resolution, and the number of mass spectrometry (MS) 1 scans, using an Orbitrap Velos mass spectrometer. Next, we explored the discriminatory power of isotope patterns in the context of proteome analyses of various complexities, either alone or in combination with a Mascot database search. Finally, we provide a theoretical framework for the required accuracies of both peptide mass and RIA for peptide identification. RESULTS: We demonstrate that the RIA error obtained in routine proteome analyses is 4-5%, and that this is only modestly influenced by spectral intensity, resolution, and the number of MS1 scans. While RIA alone has no discriminatory power, in a Mascot search isotope patterns can distinguish top scoring hits from runner-up hits in 70-95% of cases. Our theoretical approach shows that RIA accuracy needs to be ~0.2% in order to uniquely identify peptides in full proteomes. CONCLUSIONS: Our results demonstrate that isotope patterns can have discriminatory power when used in combination with a classical database search. Inclusion of this parameter in proteomic workflows may help to increase confidence in peptide identification, but in practical terms this will be limited to small proteomes.

Long-term follow up of thymus in patients with myasthenia gravis.

We examined transversely the thymus of 33 myasthenia gravis (MG) patients followed up for more than 5 years and found three thymomas. One was found 21 years after thymoma resection (Masaoka I, WHO Type B2 thymoma) and extended thymectomy. The other two were non-thymomatous at onset, and they were not treated with extended thymectomy. Therapeutic guidelines should mention the importance of follow-up in MG thymus.

Subcellular localization and possible functions of gamma-glutamyltransferase in the radish (Raphanus sativus L.) plant.

Previously we reported the purification of soluble gamma-glutamyltransferases (GGTs) from radish cotyledon. Subcellular fractionation of radish cells revealed that soluble GGT is a vacuolar enzyme. Acivicin, a GGT inhibitor, mediated the in vivo catabolism inhibition of the glutathione S-conjugate generated from endogenous glutathione and exogenously supplied monochlorobimane. Thus soluble GGT is possibly involved in the catabolism of glutathione S-conjugates.