The delivery of personalised, precision medicines via synthetic proteins

Introduction: The design of advanced drug delivery systems based on synthetic and su-pramolecular chemistry has been very successful. Liposomal doxorubicin (Caelyx®), and liposomal daunorubicin (DaunoXome®), estradiol topical emulsion (EstrasorbTM) as well as soluble or erodible polymer systems such as pegaspargase (Oncaspar®) or goserelin acetate (Zoladex®) represent considerable achievements. The Problem: As deliverables have evolved from low molecular weight drugs to biologics (currently representing approximately 30% of the market), so too have the demands made of advanced drug delivery technology. In parallel, the field of membrane trafficking (and endocytosis) has also matured. The trafficking of specific receptors i.e. material to be recycled or destroyed, as well as the trafficking of protein toxins has been well characterized. This, in conjunction with an ability to engineer synthetic, recombinant proteins provides several possibilities. The Solution: The first is using recombinant proteins as drugs i.e. denileukin diftitox (Ontak®) or agalsidase beta (Fabrazyme®). The second is the opportunity to use protein toxin architecture to reach targets that are not normally accessible. This may be achieved by grafting regulatory domains from multiple species to form synthetic proteins, engineered to do multiple jobs. Examples include access to the nucleocytosolic compartment. Herein the use of synthetic proteins for drug delivery has been reviewed

Current methods to analyze lysosome morphology, positioning, motility and function

Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.Medical Biochemistr

Highly-parallelized simulation of a pixelated LArTPC on a GPU

The rapid development of general-purpose computing on graphics processing units (GPGPU) is allowing the implementation of highly-parallelized Monte Carlo simulation chains for particle physics experiments. This technique is particularly suitable for the simulation of a pixelated charge readout for time projection chambers, given the large number of channels that this technology employs. Here we present the first implementation of a full microphysical simulator of a liquid argon time projection chamber (LArTPC) equipped with light readout and pixelated charge readout, developed for the DUNE Near Detector. The software is implemented with an end-to-end set of GPU-optimized algorithms. The algorithms have been written in Python and translated into CUDA kernels using Numba, a just-in-time compiler for a subset of Python and NumPy instructions. The GPU implementation achieves a speed up of four orders of magnitude compared with the equivalent CPU version. The simulation of the current induced on 10^3 pixels takes around 1 ms on the GPU, compared with approximately 10 s on the CPU. The results of the simulation are compared against data from a pixel-readout LArTPC prototype

One antigen, one gold? A quantitative analysis of immunogold labeling of plasma membrane 5'-nucleotidase in frozen thin sections

We present an evaluation of the efficiency of immunogold labeling for a low abundance plasma membrane protein. Several independent methods were used to determine the density of 5'-nucleotidase on the plasma membrane of the Fao cell. These methods include morphometry in combination with either enzymology or cell surface radiometric assay. Immunocytochemistry of frozen thin sections with either single or double layers of antibody and visualized with protein A complexed with 5 nm colloidal gold was used to estimate the same density. The application of a balance sheet to immunogold labeling demonstrates that the labeling is never quantitative. For example, labeling of the cell surface is always greater than labeling on the section. We show that departures from the "one antigen, one gold" ideal are systematic, so that an efficiency can be calculated and quantitative results can be obtained. The ability to obtain reliable quantitative results from immunogold labeling extends the utility of this already powerful technique

A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export. Lysosomes play an important role in cellular LDL-cholesterol uptake. Here, the authors perform a genome-wide genetic screen for cholesterol regulators and identify C18orf8 as a conserved subunit of a trimeric Rab7 GEF that controls LDL trafficking and NPC1-dependent lysosomal cholesterol export