Biology subjects

Sen, M.

Publications and source records attributed to Sen, M..

24 records · Page 2Linked to original sources

bioRxiv2021

Self-assembled nanoparticles enable delivery of VCP inhibitor, resulting in enhanced photoreceptor protection in retinal explants

1.Mutations in rhodopsin lead to its misfolding resulting in autosomal dominant retinitis pigmentosa (adRP). Pharmacological inhibition of the ATP-driven chaperone valosin- containing protein (VCP), a molecular checkpoint for protein quality control, slows down retinal degeneration in animal models. However, poor water-solubility of VCP inhibitors poses a challenge to their clinical translation as intravitreal injections for retinal treatment. In order to enable the delivery of VCP inhibitors, we have developed and investigated two formulations for the VCP inhibitor ML240. Nanoformulations of ML240 were obtained by using amphiphilic polymers methoxy-poly (ethylene glycol)5kDa-cholane (mPEG5kDa-cholane) and methoxy-poly (ethylene glycol)5kDa- cholesterol (mPEG5kDa-cholesterol). Both formulations increased the water-solubility of ML240 by two orders of magnitude and prolonged the drug released over ten days. Encapsulation of ML240 in mPEG5kDa-cholane showed superior photoreceptor protection at lower drug concentrations, normalized rhodopsin localization, and alleviated inflammatory microglial responses in an ex vivo rat model of retinal degeneration. The study demonstrates the potential of VCP inhibitor nanoformulations to treat adRP, a pharmacologically orphan disease.

pharmacology and toxicology↗

bioRxiv2020

Inhibition of VCP preserves retinal structure and function in autosomal dominant retinal degeneration

Due to continuously high production rates of rhodopsin (RHO) and high metabolic activity, photoreceptor neurons are especially vulnerable to defects in proteostasis. A proline to histidine substitution at position 23 (P23H) leads to production of structurally misfolded RHO, causing the most common form of autosomal dominant Retinitis Pigmentosa (adRP) in North America. The AAA-ATPase valosin-containing protein (VCP) extracts misfolded proteins from the ER membrane for cytosolic degradation. Here, we provide the first evidence that inhibition of VCP activity rescues degenerating P23H rod cells and improves their functional properties in P23H transgenic rat and P23H knock-in mouse retinae, both in vitro and in vivo. This improvement correlates with the restoration of the physiological RHO localization to rod outer segments (OS) and properly-assembled OS disks. As a single intravitreal injection suffices to deliver a long-lasting benefit in vivo, we suggest VCP inhibition as a potential therapeutic strategy for adRP patients carrying mutations in the RHO gene.

neuroscience↗

bioRxiv2020

Host Response to Bacterial Pathogens and Non-Pathogens is Determined by Wnt5A Mediated Actin Organization

Wnt5A signaling facilitates the killing of numerous bacterial pathogens but not non-pathogens. The basis of such distinction in killing remains unclear. Accordingly, we analyzed the influence of Wnt5A signaling on pathogenic E.coli K1 in relation to non-pathogenic E.coli K12-MG1655 and E.coli DH5. We found that bacterial killing by macrophages is dictated by the effect of Wnt5A aided actin assembly on the incumbent bacteria. Actin assembly mediated by Wnt5A signaling antagonized the disruptive influence of internalized E.coli K1 on cytoskeletal actin facilitating its eradication. However, internalized E.coli K12-MG1655 and E.coli DH5, which stabilize the actin cytoskleton remained unaffected by Wnt5A. Interestingly, actin assembly inhibitors altered bacterial phagosome compositions, supporting survival of K1, yet promoting killing of both K12-MG1655 and DH5, in Wnt5A activated macrophages. Taken together, our study reveals the importance of Wnt5A signaling dependent assembly of cytoskeletal actin in determining the outcome of host response to bacterial pathogens and non-pathogens.

immunology↗

bioRxiv2020

Complement receptor 3 forms a compact high affinity complex with iC3b

Complement receptor 3 (CR3, also known as Mac-1, integrin M{beta}2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment iC3b and to a lesser extent C3dg. While the interaction between the iC3b thioester domain and the ligand binding CR3 M I-domain is structurally and functionally well characterized, the nature of additional CR3-iC3b interactions required for phagocytosis of complement opsonized objects remain obscure. Here we analyzed the interaction between iC3b and the 150 kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of CR3 for iC3b compared to 515 nM for the iC3b thioester domain. Small angle x-ray scattering analysis revealed that iC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, iC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the iC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the iC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by iC3b than by C3dg and pave the way for development of specific therapeutics for treatment of inflammatory and neurodegenerative diseases that do not interfere with recognition of non-complement CR3 ligands.

biochemistry↗

bioRxiv2019

Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

DNA methylation (5mC) is central to cellular identity and the global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of terminally differentiated gametes to the pluripotent cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to genome-wide 5mC erasure remains unclear. Here, we report a new high-throughput single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, thereby allowing us to systematically probe the dynamics of global demethylation. First, when applied to hybrid mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, using scMspJI-seq in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, we found that human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse preimplantation embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells, thereby enabling quantitative investigation of methylation dynamics in developmental systems.

genomics↗

bioRxiv2018

Highly specific enrichment of rare nucleic acids using Thermus thermophilus Argonaute

Characterization of disease-associated, cell-free nucleic acids (liquid biopsy) provides a powerful, minimally-invasive means for early detection, genotyping, and personalized therapy; but is challenged by alleles of interest differing by single nucleotide from and residing among large abundance of wild-type alleles. We describe a new multiplexed enrichment assay, dubbed NAVIGATER, that utilizes short nucleic acid-guided endonucleases Argonaute (Ago), derived from the bacterium Thermus thermophilus (TtAgo), to specifically cleave complementary DNA and RNA while sparing alleles having single nucleotide mismatches with the guides. NAVIGATER greatly increases the fractions of rare alleles of interest in samples and enhances sensitivity of downstream procedures such ddPCR, sequencing, and clamped enzymatic amplification. We demonstrate 60-fold enrichment of KRAS G12D in blood samples from pancreatic cancer patients and detection of KRAS, EGFR, and BRAF mutants with XNA-PCR at 0.01% fraction.

molecular biology↗