Lipid-peptide nanocomplexes for mRNA delivery in vitro and in vivo

Despite recent advances in the field of mRNA therapy, the lack of safe and efficacious delivery vehicles with pharmaceutically developable properties remains a major limitation. Here, we describe the systematic optimisation of lipid-peptide nanocomplexes for the delivery of mRNA in two murine cancer cell types, B16-F10 melanoma and CT26 colon carcinoma as well as NCI-H358 human lung bronchoalveolar cells. Different combinations of lipids and peptides were screened from an original lipid-peptide nanocomplex formulation for improved luciferase mRNA transfection in vitro by a multi-factorial screening approach. This led to the identification of key structural elements within the nanocomplex associated with substantial improvements in mRNA transfection efficiency included alkyl tail length of the cationic lipid, the fusogenic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. The peptide component (K16GACYGLPHKFCG) was further improved by the inclusion of a linker, RVRR, that is cleavable by the endosomal enzymes cathepsin B and furin, and a hydrophobic motif (X-S-X) between the mRNA packaging (K16) and receptor targeting domains (CYGLPHKFCG). Nanocomplex transfections of a murine B16-F10 melanoma tumour supported the inclusion of cholesterol for optimal transfection in vivo as well as in vitro. In vitro transfections were also performed with mRNA encoding interleukin-15 as a potential immunotherapy agent and again, the optimised formulation with the key structural elements demonstrated significantly higher expression than the original formulation. Physicochemical characterisation of the nanocomplexes over time indicated that the optimal formulation retained biophysical properties such as size, charge and mRNA complexation efficiency for 14 days upon storage at 4 °C without the need for additional stabilising agents. In summary, we have developed an efficacious lipid-peptide nanocomplex with promising pharmaceutical development properties for the delivery of therapeutic mRNA

Unilateral high division of the Sciatic Nerve with divided Piriformis

Abstract While doing the routine dissection for the undergraduate students in the department of Anatomy, Nil Ratan Sircar Medical College, Kolkata, few variations were found in the gluteal region of a 70 years old male cadaver, in the year 2013. On the right side the sciatic nerve (SN) pierced the piriformis muscle dividing it into superior and inferior slips and then, after a short distance, divided into tibial and common peroneal nerves in the gluteal region. On the left side, sciatic nerve divided into two terminal branches (common peroneal and tibial nerves) in the lower part of the back of the thigh near the apex of the popliteal fossa as usual. This high division of the sciatic nerve may result in nerve injury during deep intramuscular injections in gluteal region, piriformis syndrome due to compression of the nerve, failed SN block in anesthesia and surgical complications

Post-outburst evolution of bonafide FUor V2493 Cyg: A Spectro-photometric monitoring

We present here the results of eight years of our near-simultaneous optical/near-infrared spectro-photometric monitoring of bonafide FUor candidate `V2493 Cyg' starting from 2013 September to 2021 June. During our optical monitoring period (between October 16, 2015 and December 30, 2019), the V2493 Cyg is slowly dimming with an average dimming rate of $\sim$26.6 $\pm$ 5.6 mmag/yr in V band. Our optical photometric colors show a significant reddening of the source post the second outburst pointing towards a gradual expansion of the emitting region post the second outburst. The mid infra-red colors, on the contrary, exhibits a blueing trend which can be attributed to the brightening of the disc due to the outburst. Our spectroscopic monitoring shows a dramatic variation of the H$\alpha$ line as it transitioned from absorption feature to the emission feature and back. Such transition can possibly be explained by the variation in the wind structure in combination with accretion. Combining our time evolution spectra of the Ca II infra-red triplet lines with the previously published spectra of V2493 Cyg, we find that the accretion region has stabilised compared to the early days of the outburst. The evolution of the O I $\lambda$7773 \AA~ line also points towards the stabilization of the circumstellar disc post the second outburst.Comment: 34 pages, 12 figures, 6 tables, accepted for publication in Ap

Axonal response of mitochondria to demyelination and complex IV activity within demyelinated axons in experimental models of multiple sclerosis

AIMS: Axonal injury in multiple sclerosis (MS) and experimental models is most frequently detected in acutely demyelinating lesions. We recently reported a compensatory neuronal response, where mitochondria move to the acutely demyelinated axon and increase the mitochondrial content following lysolecithin-induced demyelination. We termed this homeostatic phenomenon, which is also evident in MS, the axonal response of mitochondria to demyelination (ARMD). The aim of this study is to determine whether ARMD is consistently evident in experimental demyelination and how its perturbation relates to axonal injury.METHODS: In the present study, we assessed axonal mitochondrial content as well as axonal mitochondrial respiratory chain complex IV activity (cytochrome c oxidase or COX) of axons and related these to axonal injury in nine different experimental disease models. We used immunofluorescent histochemistry as well as sequential COX histochemistry followed by immunofluorescent labelling of mitochondria and axons.RESULTS: We found ARMD a consistent and robust phenomenon in all experimental disease models. The increase in mitochondrial content within demyelinated axons, however, was not always accompanied by a proportionate increase in complex IV activity, particularly in highly inflammatory models such as experimental autoimmune encephalomyelitis (EAE). Axonal complex IV activity inversely correlated with the extent of axonal injury in experimental disease models.CONCLUSIONS: Our findings indicate that ARMD is a consistent and prominent feature and emphasise the importance of complex IV activity in the context of ARMD, especially in autoimmune inflammatory demyelination, paving the way for the development of novel neuroprotective therapies.</p

C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we show using patient stem cell-derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, our study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.</p