Quantifying the LAMP1 positive puncta
By onThis protocol describes quantifying the LAMP1 positive puncta.
Endosomal escape of RNA therapeutics: How do we solve this rate-limiting problem?
By onWith over 15 FDA approved drugs on the market and numerous ongoing clinical trials, RNA therapeutics, such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), have shown great potential to treat human disease. Their mechanism of action is based entirely on the sequence of validated disease-causing genes without the prerequisite knowledge of protein structure, activity or cellular location. In contrast to small molecule therapeutics that passively diffuse across the cell membrane's lipid bilayer, RNA therapeutics are too large, too charged, and/or too hydrophilic to passively diffuse across the cellular membrane and instead are taken up into cells by endocytosis. However, endosomes are also composed of a lipid bilayer barrier that results in endosomal capture and retention of 99% of RNA therapeutics with 1% or less entering the cytoplasm. Although this very low level of endosomal escape has proven sufficient for liver and some CNS disorders, it is insufficient for the vast majority of extra-hepatic diseases. Unfortunately, there are currently no acceptable solutions to the endosomal escape problem. Consequently, before RNA therapeutics can be used to treat widespread human disease, the rate-limiting delivery problem of endosomal escape must be solved in a nontoxic manner.
A step forward for LRRK2 inhibitors in Parkinson’s disease
By onIn common with the majority of neurodegenerative diseases, there is an urgent and pressing need for novel disease modifying therapies for Parkinson’s disease (PD). Reporting the results of the first human trial for kinase inhibitors of Leucine Rich Repeat Kinase 2 (LRRK2), Jennings and co-workers presented an important advance along the drug development pathway for a target that has long been a priority for the Parkinson’s research community. The focus discusses several topics including: functional characterisation of LRRK2 and the impact of mutations and a key role for altered kinase function in disease; the human genetics of the LRRK2 locus; the outcome of a first-in-human clinical trial for LRRK2 kinase inhibitors by Denali therapeutics, LRRK2 kinase inhibition as a therapeutic strategy in humans; the strategy of using antisense oligonucleotide knockdown approach and the challenges faced by clinical trials – measuring outcomes in chronic, slowly progressing disorders with variable rates of progression.
pCAG- WIPI2dC93E-cs- TEV-STREP
By onPlasmid: Mammalian expression of human WIPI2d C93E with C-terminal Strep.
Determination of NM Concentration
By onThis is the protocol for determining neuromelanin concentration and data.
cDNA clones for expression of LysoTag (TMEM192) in mammalian cells
By onPlasmids for expression of TMEM192 (LysoTag) in human and mouse cells.
Immunohistochemistry for Carbon Fiber Thread Electrodes
By onMethods for immunofluorescent staining of brain tissue with indwelling electrodes.
pCAG-OSF-ATG13 (2-197)-K15D
By onPlasmid for expression of human ATG13 HORMA K15D mutant in mammalian cells.
Nuclear Isolation of Post-Mortem Brain Tissue for snRNAseq
By onThis protocol details how to isolate nuclei from frozen brain tissue for single nuclear RNA sequencing using 10x Genomics GEM isolation using the Chromium accessory and Single Cell 3ʹ Reagent Kits.
3D-correlative FIB-milling and Cryo-ET of Autophagic structures in Yeast Cells
By onThis protocol describes how to plunge-freeze yeast on EM grids and how to target autophagic structures by combining cryo confocal fluorescence data to FIB-milling and tomogram acquisition.
Expansion Microscopy
By onExpansion microscopy is a technique to visualize biological structures with higher spatial resolution than traditional microscopy methods.
Immunofluorescent staining for neuronal marker MAP2
By onThis is the protocol for immunofluorescent staining for neuronal marker MAP2.
circRNA dataset
By onNew data set on circRNA in laser-captured neuron samples from 190 human brains of healthy controls, prodromal PD, and clinical PD (Dong et al., Nature Communications, in press). The RNA-seq raw FASTQ data and normalized expression matrix of all circRNAs in this study have been deposited in GEO under accession number GSE218203.
circRNA Custom Code
By onCustom code associated with (Dong et al., Nature Communications, in press) is publicly available at https://github.com/sterding/circRNA.
Unconventional Initiation of PINK1/Parkin Mitophagy by Optineurin
By onCargo sequestration is a fundamental step of selective autophagy in which cells generate a double membrane structure termed an autophagosome on the surface of cargoes. The authors uncover an unconventional path of PINK1/Parkin mitophagy initiation.
