Biology subjects

Ghoshal, B.

Publications and source records attributed to Ghoshal, B..

8 recordsLinked to original sources

bioRxiv2025

Targeting dCas9-SunTag to a susceptibility gene promoter is sufficient for CRISPR interference

Cassava production in sub-Saharan Africa is severely impacted by diseases. Most pathogens require interaction with host susceptibility factors to complete their life cycles and cause disease. Targeted DNA methylation is an epigenetic strategy to alter gene expression in plants and we previously reported that a zinc-finger fused to DMS3 could establish methylation at the promoter of MeSWEET10a, a bacterial susceptibility gene, and this resulted in decreased disease. Here, we attempt a similar strategy for cassava brown streak disease. This disease is caused by the ipomoviruses CBSV and UCBSV. These viruses belong to the family Potyviridae, which has been shown extensively to require host eIF4E-family proteins to infect plants and cause disease. We previously found that cassava plants with simultaneous knockout mutations in two eIF4E genes, nCBP-1 and nCBP-2, resulted in decreased susceptibility to CBSD. Here, we report successful simultaneous targeting of both promoters with methylation using a dCas9-DMRcd-SunTag system. However, in contrast to our previous work with MeSWEET10a, controls indicate that CRISPR interference is occurring in these lines and is sufficient for reduction of gene expression. Future research will use genetic crosses to segregate away the DNA methylation reagents and, if DNA methylation proves heritable, assess whether methylation alone is sufficient increase resistance to CBSD.

plant biology↗

bioRxiv2024

Ex Vivo Delivery of mRNA to Immune Cells Via a Non-Endosomal Route Obviates the Need for Nucleoside Modification

Base modification and the use of lipid nanoparticles (LNPs) are thought to be essential for efficient in vivo delivery and expression of mRNA. However, for ex vivo immune cell engineering, the need for either of the two is unclear. Previous reports have suggested that nucleic acids may be efficiently delivered to immune cells ex vivo, through a non-endosomal delivery route, but the need for base modification has not been determined. Herein, we demonstrate that when a non-endosomal delivery method is used, unmodified mRNA performs equally well to the commonly used base-modified mRNA, including the N1 methyl pseudo-uridine modification, in terms of protein expression and inflammatory response in cells. However, if an endosomal delivery route is used, then N1 methyl pseudo-uridine modification is necessary for high expression and low inflammatory response, as demonstrated by others as well. Overall, we show that non-endosomal mRNA delivery renders nucleoside modifications non-essential, and that unmodified mRNA combined with non-endosomal delivery route may be used for efficient ex vivo mRNA-based engineering of immune cells.

bioengineering↗

bioRxiv2024

Crosstalk between plasma membrane and Staphylococcus α-hemolysin during oligomerization

The infectious microbe Staphylococcus aureus releases an array of cytotoxic pore-forming toxins (PFTs) that severely damage the cell membrane during bacterial infection. However, the interaction interfaces between the host cell and toxin were merely explored. Herein, we monitored the active oligomeric states facilitated membrane disruption processes such as lysis, and protrusion in the plasma membrane and lipid membrane. Furthermore, necrosis was triggered in the neutrophil-like cells upon synergistic binding and oligomerization of the monomeric -HL. Additionally, we solved RBC membrane stabilized structure of different conformational states of this {beta}-PFT using a single-particle cryo-EM. We further confirmed that internal membrane fluidity was the deterministic factor associated with the formation of intermediate pre-pores, heptameric pore-like, and complete pore species. Together, this is the first study to unveil the structure-function analysis of pre-pore to pore transition of any small {beta}-PFT during its crosstalk with the cell. HighlightsO_LI-HL promotes necrosis in HL60 cells and lysis of shorter lipid bilayer region. C_LIO_LICryo-EM of small PFT in the cellular environment. C_LIO_LIStructural characterization of heptameric pore, pore-like, and pre-pore complex in the presence of RBCs. C_LIO_LIBilayer phase behavior (Ld/Lo) governs different conformational and geometrical variants of -HL. C_LI

biophysics↗

bioRxiv2022

Improving disease resistance in plants by editing the epigenome

Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas phaseoli pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. In this work, we direct methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display decreased CBB symptoms while maintaining normal growth and development. This work therefore presents an epigenome editing approach useful for crop improvement.

plant biology↗

bioRxiv2021

Leishmania Hijacks microRNA Import-Export Machinery of Infected Macrophage and Survives by Cross-Communicating Host miR-146a to Subjugate HuR and miR-122 in Neighbouring cells

Leishmania donovani, the causative agent of visceral leishmaniasis, infects and resides within tissue macrophage cells of the mammalian host. It is not clear how the parasite infected cells cross-talk with the non-infected cells in the infection niche to regulate the infection process. Interestingly, miRNAs, the regulatory small RNAs of the host, could get trafficked into and out of infected cells as part of extracellular vesicles to ensure exchange of the epigenetic signals and can regulate the expression of their target genes in both donor and recipient cells. Leishmania, for its survival in host macrophage, adopts a dual strategy to regulate the intercellular transport of host miRNAs. The parasite, by preventing mitochondrial function of the host cells, restricts the entry of liver cell derived miR-122 containing extracellular vesicles in infected macrophage to curtail the inflammatory response by miR-122. The parasite reciprocally upregulates the extracellular export of anti-inflammatory miR-146a from the infected cells. The exported miR-146a restricts miR-122 production in liver cells and polarizes neighbouring naive macrophages to the M2 state. miR-146a upregulates IL-10 in neighbouring macrophages where miR-146a dominates the RNA binding and miRNA suppressor protein HuR to inhibit the expression of proinflammatory cytokine mRNAs having HuR-interacting AU-rich elements and polarized the recipient cells to M2 stage. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/459146v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@32e32dorg.highwire.dtl.DTLVardef@e0d3c6org.highwire.dtl.DTLVardef@6f77e6org.highwire.dtl.DTLVardef@289e49_HPS_FORMAT_FIGEXP M_FIG C_FIG Key FindingsO_LILeishmania stops vesicular entry of inflammatory miR-122 in infected cells by causing mitochondrial depolarization C_LIO_LILeishmania secrets miR-146a from infected cells to stop miR-122 production in neighbouring hepatocytes C_LIO_LImiR-146a containing vesicles secreted by infected cells stops inflammatory response in recipient naive macrophage C_LIO_LImiR-146 targets RNA binding protein HuR to stop inflammatory cytokine production C_LI

immunology↗

bioRxiv2020

GW182 Proteins Restrict Extracellular Vesicle Mediated Selective Export of Used miRNAs In Mammalian Cancer Cells

MicroRNAs are small regulatory RNAs of relatively long half-life in non-proliferative human cells. However, in cancer cells the half-lives of miRNAs are comparatively short. To understand the mechanism of rapid miRNA turn over in cancer cells, we explored the effect of "usage" of specific miRNAs for translation repression of their targets on the abundance of that miRNA. We have noted an accelerated extracellular vesicle mediated export of "used" miRNAs in mammalian cells and the miRNA-export process get retarded by Ago2 interacting protein GW182B. The GW182 group of proteins are localized to GW182 Bodies or RNA Processing Bodies in mammalian cells and GW182B dependent retardation of miRNA export depends on GW-body integrity and is independent of HuR mediated auxiliary pathway of miRNA export. Our data thus support existence of a HuR independent pathway of miRNA export in human cells that can be targeted to increase cellular miRNA levels in cancer cells to induce senescence. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/294488v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1d6e30dorg.highwire.dtl.DTLVardef@d4ab06org.highwire.dtl.DTLVardef@282ad4org.highwire.dtl.DTLVardef@113ef76_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIUsage of miRNA is proportional with its export and turnover C_LIO_LIGW182 proteins cause phase separation of Ago2 to P-bodies and restrict miRNA export via extracellular vesicles (EVs) C_LIO_LIHuR driven auxiliary path of miRNA export is independent of GW182 controlled main path of miRNA exclusion C_LI

molecular biology↗

bioRxiv2020

Non-Canonical Ago Loading of EV-Derived Exogenous miRNA Generates Foreign miRNPs on Endosomes to Arbitrate Gene Expression in Recipient Cells

MicroRNAs, the tiny regulators of gene expression, can be transferred between neighbouring cells via Extracellular Vesicles (EV) to control the expression of genes in both donor and recipient cells. How the Extracellular Vesicles-derived miRNAs are getting internalized and become functional in target cells is an unresolved question. We found that EV-associated miRNAs are primarily single stranded and, to become functional, get loaded with the Ago proteins present in the recipient cells without requiring host Dicer1. Following endocytosis of miRNA-containing extracellular vesicles, loading of host Ago occurs on the endosomal membrane where pH-dependent membrane fusion triggers the release of internalized miRNAs to form exogenous miRNP pool. In addition, loading of exogenous miRNA to host Ago also depends on the mitochondrial activity of recipient cells. Internalization of hepatocyte derived miR-122 containing EVs in macrophage activates the recipient cell by targeting cytokine expression. Leishmania donovani, a protozoan parasite known to affect endocytosis and to cause mitochondrial depolarization in infected macrophages, restricts the EV-internalization process and thereby preventing inflammatory cytokine expression and ensuring internalized pathogen survival in macrophage.

cell biology↗

bioRxiv2020

Robust Production of Uniform Human Cerebral Organoids from Pluripotent Stem Cells

Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human specific cortical development and pathophysiology in an experimentally tractable system. While diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent (ie uniform) hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single cell profiling, that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.

neuroscience↗