Researchers reveal a new ALS hallmark, shedding light on a likely trigger of neurodegeneration kingscollegelon NatureComms
. Embryos were mounted anterior to the left, and longitudinal axon tracts just anterior to the otic vesicle were imaged. 10 μm z-projections were prepared and axon-localised intron puncta were quantified manually. Puncta score was calculated by normalising the number of puncta counted in an axon to the total axon length.
The RNeasy Micro Kit protocol was carried out, following minor modifications, on all samples. To ensure the removal of all traces of Buffer RW1, tubes were slowly rotated following the addition of 500 ml Buffer RPE to each column. After centrifugation, this step was repeated, with 500 ml Buffer RPE added to the tube, rotation, and centrifugation. RNA samples were stored at −70 °C. Concentrations were measured by Qubit RNA HS Kit using 2 ml of each sample.
For intron retention validation, intron-exon amplicons were made relative to either their respective compartment exon fragment expression for the gene or the expression of the intron-retaining fragment of the other compartment of the same genotype . Control fragments, of introns not retained, were also made relative to the expression of the retained intron-exon fragment.
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Frontiers | Modulating the microenvironment during FVIII uptake influences the nature of FVIII-peptides presented by antigen-presenting cellsBackground and Aims Hemophilia A is a severe bleeding disorder caused by the deficiency of functionally active coagulation factor VIII (FVIII). The induction of neutralizing anti-drug antibodies is a major complication in the treatment of hemophilia A patients with FVIII replacement therapies. Why some patients develop neutralizing antibodies (FVIII inhibitors) while others do not is not well understood. Previous studies indicated that the induction of FVIII inhibitors requires cognate interactions between FVIII-specific B cells and FVIII-specific CD4+ T cells in germinal center reactions. In this study, we investigated the FVIII peptide repertoire presented by antigen-presenting cells (APCs) under different microenvironment conditions that are expected to alter the uptake of FVIII by APCs. The aim of this study was to better understand the association between different microenvironment conditions during FVIII uptake and the FVIII peptide patterns presented by APCs. Methods We used a FVIII-specific CD4+ T cell hybridoma library derived from humanized HLA-DRB1*1501 (human MHC class II) hemophilic mice that were treated with human FVIII. APCs obtained from the same mouse strain were preincubated with FVIII under different conditions which are expected to alter the uptake of FVIII by APCs. Subsequently, these preincubated APCs were used to stimulate the FVIII-specific CD4+ T cell hybridoma library. Stimulation of peptide-specific CD4+ T-cell hybridoma clones was assessed by analyzing the IL-2 release into cell culture supernatants. Results The results of this study indicate that the specific microenvironment conditions during FVIII uptake by APCs determine the peptide specificities of subsequently activated FVIII-specific CD4+ T cell hybridoma clones. Incubation of APCs with FVIII complexed with von Willebrand Factor, FVIII activated by thrombin or FVIII combined with a blockade of receptors on APCs previously associated with FVIII uptake and clearance, resulted in dis
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A developmental atlas of somatosensory diversification and maturation in the dorsal root ganglia by single-cell mass cytometry - Nature NeuroscienceSomatosensory neurons detect pain, temperature and touch. Keeler et al. constructed a single-cell, protein-level atlas of nearly 3 million cells from the mouse dorsal root ganglia, covering 13 days of embryonic and postnatal development.
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