Protein interaction mapping with ribosome-displayed using PLATO ORF libraries

Identifying physical interactions between proteins and other molecules is a critical aspect of biological analysis. Here we describe PLATO, an in vitro method for mapping such interactions by affinity enrichment of a library of full-length open reading frames displayed on ribosomes, followed by massively parallel analysis using DNA sequencing. We demonstrate the broad utility of the method by identifying known and new interacting partners of LYN kinase, patient autoantibodies and the small molecules gefitinib and dasatinib

Osteopontin is linked with AKT, FoxO1, and myostatin in skeletal muscle cells

Introduction: Osteopontin (OPN) polymorphisms are associated with muscle size and modify disease progression in Duchenne muscular dystrophy (DMD). We hypothesized that OPN may share a molecular network with myostatin (MSTN). Methods: Studies were conducted in the golden retriever (GRMD) and mdx mouse models of DMD. Follow-up in-vitro studies were employed in myogenic cells and the mdx mouse treated with recombinant mouse (rm) or human (Hu) OPN protein. Results: OPN was increased and MSTN was decreased and levels correlated inversely in GRMD hypertrophied muscle. RM-OPN treatment led to induced AKT1 and FoxO1 phosphorylation, microRNA-486 modulation, and decreased MSTN. An AKT1 inhibitor blocked these effects, whereas an RGD-mutant OPN protein and an RGDS blocking peptide showed similar effects to the AKT inhibitor. RMOPN induced myotube hypertrophy and minimal Feret diameter in mdx muscle. Discussion: OPN may interact with AKT1/MSTN/FoxO1 to modify normal and dystrophic muscle

Clinical feasibility of umbilical cord tissue-derived mesenchymal stem cells in the treatment of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a progressively debilitating neurological condition in which the immune system abnormally erodes the myelin sheath insulating the nerves. Mesenchymal stem cells (MSC) have been used in the last decade to safely treat certain immune and inflammatory conditions. METHODS: A safety and feasibility study was completed on the use of umbilical cord MSC (UCMSC) as a treatment for MS. In this 1-year study, consenting subjects received seven intravenous infusions of 20 × 10 RESULTS: Twenty subjects were enrolled in this study. No serious adverse events were reported. Of the mild AEs denoted as possibly related to treatment, most were headache or fatigue. Symptom improvements were most notable 1 month after treatment. Improvements were seen in EDSS scores (p \u3c 0.03), as well as in bladder, bowel, and sexual dysfunction (p \u3c 0.01), in non-dominant hand average scores (p \u3c 0.01), in walk times (p \u3c 0.02) and general perspective of a positive health change and improved quality of life. MRI scans of the brain and the cervical spinal cord showed inactive lesions in 15/18 (83.3%) subjects after 1 year. CONCLUSIONS: Treatment with UCMSC intravenous infusions for subjects with MS is safe, and potential therapeutic benefits should be further investigated. Trial registration ClinicalTrials.gov NCT02034188. Registered Jan 13, 2014. https://clinicaltrials.gov/ct2/show/NCT02034188

Application of a synthetic human proteome to autoantigen discovery through PhIP-Seq

In this study, we improve on current autoantigen discovery approaches by creating a synthetic representation of the complete human proteome, the T7 “peptidome” phage display library (T7-Pep), and use it to profile the autoantibody repertoires of individual patients. We provide methods for 1) designing and cloning large libraries of DNA microarray-derived oligonucleotides encoding peptides for display on bacteriophage, and 2) analysis of the peptide libraries using high throughput DNA sequencing. We applied phage immunoprecipitation sequencing (PhIP-Seq) to identify both known and novel autoantibodies contained in the spinal fluid of three patients with paraneoplastic neurological syndromes. We also show how our approach can be used more generally to identify peptide-protein interactions and point toward ways in which this technology will be further developed in the future. We envision that PhIP-Seq can become an important new tool in autoantibody analysis, as well as proteomic research in general

IRE1α and IGF signaling predict resistance to an endoplasmic reticulum stress-inducing drug in glioblastoma cells.

To date current therapies of glioblastoma multiforme (GBM) are largely ineffective. The induction of apoptosis by an unresolvable unfolded protein response (UPR) represents a potential new therapeutic strategy. Here we tested 12ADT, a sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, on a panel of unselected patient-derived neurosphere-forming cells and found that GBM cells can be distinguished into "responder" and "non-responder". By RNASeq analysis we found that the non-responder phenotype is significantly linked with the expression of UPR genes, and in particular ERN1 (IRE1) and ATF4. We also identified two additional genes selectively overexpressed among non-responders, IGFBP3 and IGFBP5. CRISPR-mediated deletion of the ERN1, IGFBP3, IGFBP5 signature genes in the U251 human GBM cell line increased responsiveness to 12ADT. Remarkably, >65% of GBM cases in The Cancer Genome Atlas express the non-responder (ERN1, IGFBP3, IGFBP5) gene signature. Thus, elevated levels of IRE1α and IGFBPs predict a poor response to drugs inducing unresolvable UPR and possibly other forms of chemotherapy helping in a better stratification GBM patients

Olig2-Regulated Lineage-Restricted Pathway Controls Replication Competence in Neural Stem Cells and Malignant Glioma

Recent studies have identified stem cells in brain cancer. However, their relationship to normal CNS progenitors, including dependence on common lineage-restricted pathways, is unclear. We observe expression of the CNS-restricted transcription factor, OLIG2, in human glioma stem and progenitor cells reminiscent of type C transit-amplifying cells in germinal zones of the adult brain. Olig2 function is required for proliferation of neural progenitors and for glioma formation in a genetically relevant murine model. Moreover, we show p21^(WAF1/CIP1), a tumor suppressor and inhibitor of stem cell proliferation, is directly repressed by OLIG2 in neural progenitors and gliomas. Our findings identify an Olig2-regulated lineage-restricted pathway critical for proliferation of normal and tumorigenic CNS stem cells

Breast cancer surface receptors predict risk for developing brain metastasis and subsequent prognosis

Determining the status of breast cancer surface receptors (estrogen receptor, progesterone receptor, HER2/neu) has become routine in the care of patients with this disease and has proven to be helpful in guiding treatment. For this reason, breast cancer has become a model for molecularly guided therapy in solid tumors. Emerging data support that these receptors are associated with risk for developing brain metastases. Additionally, once brain metastases have occurred these receptors may also correlate with prognosis

miR-21 in the Extracellular Vesicles (EVs) of Cerebrospinal Fluid (CSF): A Platform for Glioblastoma Biomarker Development

Glioblastoma cells secrete extra-cellular vesicles (EVs) containing microRNAs (miRNAs). Analysis of these EV miRNAs in the bio-fluids of afflicted patients represents a potential platform for biomarker development. However, the analytic algorithm for quantitative assessment of EV miRNA remains under-developed. Here, we demonstrate that the reference transcripts commonly used for quantitative PCR (including GAPDH, 18S rRNA, and hsa-miR-103) were unreliable for assessing EV miRNA. In this context, we quantitated EV miRNA in absolute terms and normalized this value to the input EV number. Using this method, we examined the abundance of miR-21, a highly over-expressed miRNA in glioblastomas, in EVs. In a panel of glioblastoma cell lines, the cellular levels of miR-21 correlated with EV miR-21 levels (p<0.05), suggesting that glioblastoma cells actively secrete EVs containing miR-21. Consistent with this hypothesis, the CSF EV miR-21 levels of glioblastoma patients (n=13) were, on average, ten-fold higher than levels in EVs isolated from the CSF of non-oncologic patients (n=13, p<0.001). Notably, none of the glioblastoma CSF harbored EV miR-21 level below 0.25 copies per EV in this cohort. Using this cut-off value, we were able to prospectively distinguish CSF derived from glioblastoma and non-oncologic patients in an independent cohort of twenty-nine patients (Sensitivity=87%; Specificity=93%; AUC=0.91, p<0.01). Our results suggest that CSF EV miRNA analysis of miR-21 may serve as a platform for glioblastoma biomarker development

Evidence of Coronavirus (CoV) Pathogenesis and Emerging Pathogen SARS-CoV-2 in the Nervous System: A Review on Neurological Impairments and Manifestations.

The coronavirus disease 2019 (COVID-19) pandemic is an issue of global significance that has taken the lives of many across the world. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for its pathogenesis. The pulmonary manifestations of COVID-19 have been well described in the literature. Initially, it was thought to be limited to the respiratory system; however, we now recognize that COVID-19 also affects several other organs, including the nervous system. Two similar human coronaviruses (CoV) that cause severe acute respiratory syndrome (SARS-CoV-1) and Middle East respiratory syndrome (MERS-CoV) are also known to cause disease in the nervous system. The neurological manifestations of SARS-CoV-2 infection are growing rapidly, as evidenced by several reports. There are several mechanisms responsible for such manifestations in the nervous system. For instance, post-infectious immune-mediated processes, direct virus infection of the central nervous system (CNS), and virus-induced hyperinflammatory and hypercoagulable states are commonly involved. Guillain-Barré syndrome (GBS) and its variants, dysfunction of taste and smell, and muscle injury are numerous examples of COVID-19 PNS (peripheral nervous system) disease. Likewise, hemorrhagic and ischemic stroke, encephalitis, meningitis, encephalopathy acute disseminated encephalomyelitis, endothelialitis, and venous sinus thrombosis are some instances of COVID-19 CNS disease. Due to multifactorial and complicated pathogenic mechanisms, COVID-19 poses a large-scale threat to the whole nervous system. A complete understanding of SARS-CoV-2 neurological impairments is still lacking, but our knowledge base is rapidly expanding. Therefore, we anticipate that this comprehensive review will provide valuable insights and facilitate the work of neuroscientists in unfolding different neurological dimensions of COVID-19 and other CoV associated abnormalities