Multiplexed transcriptome discovery of RNA-binding protein binding sites by antibody-barcode eCLIP.

Ultraviolet crosslinking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process several RBPs in parallel. We present a new method termed antibody-barcode eCLIP that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate several RBPs simultaneously. We observe performance comparable with that of eCLIP with the advantage of dramatically increased scaling while maintaining the same material requirement of a single eCLIP experiment.

Multicenter Cohort Study of Infliximab Pharmacokinetics and Therapy Response in Pediatric Acute Severe Ulcerative Colitis.

BACKGROUND & AIMS: We aimed to model infliximab (IFX) pharmacokinetics (PK) in pediatric acute severe ulcerative colitis (ASUC) and assess the association between PK parameters, including drug exposure, and clinical response. METHODS: We studied a multicenter prospective cohort of hospitalized children initiating IFX for ASUC or IBD-unclassified. Serial IFX serum concentrations over 26 weeks were used to develop a PK model. We tested the association of PK parameter estimates with day 7 clinical response, week 8 clinical remission, week 26 corticosteroid-free clinical remission (CSF-CR) (using the Pediatric Ulcerative Colitis Activity Index), and colectomy-free survival. RESULTS: Thirty-eight participants received IFX (median initial dose, 9.9 mg/kg). Day 7 clinical response, week 8 clinical remission, and week 26 CSF-CR occurred in 71%, 55%, and 43%, respectively. Albumin, C-reactive protein, white blood cell count, platelets, weight, and antibodies to IFX were significant covariates incorporated into a PK model. Week 26 non-remitters exhibited faster IFX clearance than remitters (P = .013). However, cumulative IFX exposure did not differ between clinical response groups. One (2.7%) and 4 (10.8%) participants underwent colectomy by week 26 and 2 years, respectively. Day 3 IFX clearance >0.02 L/h was associated with colectomy (hazard ratio, 58.2; 95% confidence interval, 6.0-568.6; P < .001). CONCLUSIONS: At median higher-than-label IFX dosing for pediatric ASUC, baseline faster IFX CL was associated with colectomy and at week 26 with lack of CSF-CR. IFX exposure was not predictive of clinical outcomes. Higher IFX dosing may sufficiently optimize early outcomes in pediatric ASUC. Larger studies are warranted to determine whether sustained intensification can overcome rapid clearance and improve later outcomes. CLINICALTRIALS: gov identifier: NCT02799615.

Short-term safety of mTOR inhibitors in infants and very young children with tuberous sclerosis complex (TSC): Multicentre clinical experience.

OBJECTIVE: To evaluate the safety of mTOR inhibitors (sirolimus or everolimus) in infants and very young children with tuberous sclerosis complex (TSC) under two years of age. METHODS: Study design was retrospective to capture medical record data from 52 international TSC Centres who initiated treatment with sirolimus or everolimus in TSC children before the age of two years. Data collection included demographic and clinical information including reason(s) for initiating treatment with mTOR inhibitors, treatment duration, dosing, and corresponding serum trough levels, response to treatment, and adverse events (AE). RESULTS: 19 of 52 (37%) TSC Centres reported treatment of at least one child with TSC under the age of two years with everolimus or sirolimus. Treatment-related data were provided for 45 patients meeting inclusion criteria. Everolimus was utilised 87% of the time, compared to 24% for sirolimus (5 subjects, 11%, were treated separately with both). Refractory epilepsy (45%) was the most common primary reason for initiating treatment and treatment was initiated on average at 11.6 ± 7.6 months of age. At least one AE, suspected or definitely treatment-related, occurred in 35 of 45 (78%) treated subjects. Most AEs were mild (Grade 1) or moderate (Grade 2) in severity and most commonly related to infections. Severe AE (Grade 3) was reported in 7 subjects (20%) and no life-threatening AE (Grade 4) or death/disability (Grade 5) was reported. Treatment was discontinued due to an AE in 9 of 45 (20%). CONCLUSIONS: Everolimus, and to a lesser extent sirolimus, are increasingly being used to treat TSC infants and very young children for multiple TSC-associated clinical indications. While AEs were common, most were not severe and did not prevent continued treatment in the majority of this younger population.

Lipopeptides rather than lipopolysaccharide favor the development of dendritic cell dysfunction similar to polymicrobial sepsis in mice.

OBJECTIVE: We investigated whether the dysfunction of dendritic cells (DC) that develops during polymicrobial sepsis is mimicked by systemic administration of the Toll-like receptor (TLR) 4 agonist lipopolysaccharide (LPS) or of the TLR2 agonist Pam3-Cys-Ser-Lys4 (P3CSK4). MATERIALS AND METHODS: BALB/c mice underwent cecal ligation and puncture (CLP) or sham operation or received a single i.p. injection of LPS (30 mg/kg body weight), P3CSK4 (10 mg/kg body weight), or saline as control. Purified splenic DC and in-vitro-generated DC from bone marrow were analyzed in terms of surface marker expression, cytokine secretion, and antigen-specific T-cell activation in vivo. RESULTS: Splenic DC were suppressed in IL-12 secretion 12 h after LPS and P3CSK4 administration but released increased levels of IL-12 4 days after TLR agonist application, unlike DC from CLP mice. Polymicrobial sepsis and TLR agonists caused a loss of DC in the spleen but led to the expansion of diverse DC subsets. DC that differentiated from bone marrow after P3CSK4 but not after LPS application resembled DC from CLP mice regarding cytokine secretion and impaired Th1-cell polarization. CONCLUSIONS: The development of DC dysfunction during sepsis is at least partly mimicked by TLR2 agonists rather than TLR4 agonists.

Polysialic acid immobilized on silanized glass surfaces: a test case for its use as a biomaterial for nerve regeneration.

The immobilization of polysialic acid (polySia) on glass substrates has been investigated with regard to the applicability of this polysaccharide as a novel, biocompatible and bioresorbable material for tissue engineering, especially with regard to its use in nerve regeneration. PolySia, a homopolymer of alpha-2,8-linked sialic acid, is involved in post-translational modification of the neural cell adhesion molecule (NCAM). The degradation of polySia can be controlled which makes it an interesting material for coating and for scaffold construction in tissue engineering. Here, we describe the immobilization of polySia on glass surfaces via an epoxysilane linker. Whereas glass surfaces will not actually be used in nerve regeneration scaffolds, they provide a simple and efficient means for testing various methods for the investigation of immobilized polySia. The modified surfaces were investigated with contact angle measurements and the quantity of immobilized polySia was examined by the thiobarbituric acid assay and a specific polySia-ELISA. The interactions between the polySia-modified surface and immortalized Schwann cells were evaluated via cell adhesion and cell viability assays. The results show that polySia can be immobilized on glass surfaces via the epoxysilane linker and that surface-bound polySia has no toxic effects on Schwann cells. Therefore, as a key substance in the development of vertebrates and as a favourable substrate for the cultivation of Schwann cells, it offers interesting features for the use in nerve guidance tubes for treatment of peripheral nerve injuries.

A study on polysialic acid as a biomaterial for cell culture applications.

Polysialic acid (PSA) was investigated for its applicability as coating material for mammalian cell cultivation. PSA is involved in post-translational modification of the vertebrate neural cell adhesion molecule (NCAM). It is biocompatible and degradation-controlled. Thus, it becomes interesting for use as a coating and scaffold material for tissue engineering applications, especially for peripheral nerve regeneration. As a preliminary study of the use of PSA as scaffold material it was tested in its soluble form as coating material. The cytotoxicity was investigated and compared to another polysaccharide beta-glucan, to widely used coating substances (collagen I, poly-L-lysine, hyaluronic acid) and uncoated tissue culture plastic material. The interactions between the modified cell culture surface and the cells were investigated using a model liver cell line Hep-G2 and a neurobiological cell line PC-12. The PSA coating itself was analyzed by immunoanalysis. Viability of the cells was investigated by the MTT assay. The number and distribution of adhered cells were studied by cell nuclei staining. Furthermore, the differentiation status of the PC-12 cells was monitored, as well as glucose and lactate levels in the cell culture medium from the Hep-G2 cells. Comparable viability and similar numbers of attached cells were observed. Growth in cell clusters was observed for PSA, beta-glucan, and hyaluronic acid coated materials. In general, the results indicate that PSA is comparable to other well-established coating materials (e.g. collagen I, hyaluronic acid, and poly-L-lysine). Moreover, as a key substance in vertebrate development it offers interesting features for nerve regeneration, especially as an insoluble, modified scaffold material.

Collagen biomaterial doped with colominic acid for cell culture applications with regard to peripheral nerve repair.

Colominic acid (CA) is a homopolymer of sialic acid residues and is solely composed of polymerised units of alpha-2,8-linked N-acetylneuraminic acid. CA is a specific derivative of polysialic acid (PSA), produced as the capsular polysaccharide of Escherichia coli K1 derived molecule of PSA. PSA in vivo plays a significant role in synaptic plasticity and neural development. The use of collagen materials doped with defined CA is presented for the cultivation of various cell lines relevant for possible applications in Tissue Engineering. First, the release behaviour under culture conditions of the collagen-based (C-CA) materials was investigated by thiobarbituric acid assay. Additionally, the established cell lines, PC-12 and immortalised Schwann cells (ISC), used for neurobiological and neurochemical studies and the model liver cell line Hep-G2 as indicator for biocompatibility testing, were cultured on the C-CA matrix. Cell proliferation (MTT-test) and cell adhesion (DAPI-staining) of the cell lines on the matrices were observed. Likewise, gene expression of the marker genes thyrosine hydroxylase for the PC-12 cells, and albumin, transferrin and CYP3A4 for the Hep-G2 cells was evaluated via RT-PCR. The results indicate that CA integration in established biomaterial constructs enhances cell proliferation and offers promising features as conduits additive in regarding peripheral nerve regeneration.

Fast and efficient screening system for new biomaterials in tissue engineering: a model for peripheral nerve regeneration.

The use of three-dimensional biodegradable matrices is one major issue in tissue engineering. Numerous materials, fabrication techniques, and modifications have been used and tested in different areas of tissue engineering recently. But nevertheless, technology is far from being optimized and optimal constructs with bioidentical and mechanical properties have not been described in the literature so far. Hence, there is great demand of new suitable biomaterials for tissue engineering applications. In this study, a fast and efficient screening system for initial testing of biomaterials for cell culture application was developed. The set up for the screening system and the decision criteria applied for the determination of suitability of new materials are presented. Hep-G2 and PC-12 cells were seeded onto different matrices and cultured over a period of 2 weeks. The viability of the cells was monitored via the MTT assay. Cell spreading was investigated by DAPI-staining of cell nuclei. Furthermore, the adhesion of the cells on the different matrices was examined by counting the number of attached cells. With these general assays a classification of materials is possible with regard to their suitability. Optimal cell models must be chosen for the defined applications and at least two cell lines are necessary for a differentiating interpretation.