Biology subjects

Manna, D.

Publications and source records attributed to Manna, D..

4 recordsLinked to original sources

bioRxiv2024

IVISc-L: A quick and simple in vivo assay to study the regulation of gene expression

Several methods are available to study the regulation of gene expression at cellular and molecular levels. Adaptation of these methods in vivo is cumbersome and often requires animal sacrifice. Here, we report an assay (IVISc-L, In Vivo Imaging of Subcutaneous Luminescence) to study gene regulation in vivo. This assay involves subcutaneous injection of a plasmid DNA encoding firefly luciferase, whose expression is under the regulatory mechanism to be investigated. We could infer its regulated expression by detecting the subcutaneous luminescence using an in vivo imaging system. Using this assay, we have demonstrated the regulation of gene expression mediated by a promoter, micro-RNAs, stop codon readthrough, and rare codons. This minimally invasive assay does not require animal sacrifice or any tissue extraction. The entire assay can be completed within 24 hours. Therefore, this assay will be useful in investigating the mechanisms of gene expression regulation, and screening molecules that can alter gene expression in vivo.

molecular biology↗

bioRxiv2024

Scrutinized lipid utilization disrupts Amphotericin-B responsiveness in clinical isolates of Leishmania donovani

The management of Leishmania donovani (LD), responsible for fatal visceral leishmaniasis (VL), faces increasing challenges due to rising drug-unresponsiveness, leading to increasing treatment failures. While hypolipidemia characterizes VL, LD, a cholesterol auxotroph, relies on host lipid scavenging for its intracellular survival. The aggressive pathology, in terms of increased organ parasite load, observed in hosts infected with antimony-unresponsive-LD (LD-R) as compared to their sensitive counterparts (LD-S), highlights LD-Rs heightened reliance on host lipids. Here we report that LD-R-infection promotes fluid-phase endocytosis in the host, selectively accumulating neutral lipids while excluding oxidized-LDL. LD-R enhances the fusion of endocytosed LDL-vesicles with its phagolysosomal membrane and inhibits cholesterol mobilization from these vesicles by suppressing NPC-1. This provides LD-R amastigotes with excess lipids, supporting their rapid proliferation and membrane synthesis. This excess LDL-influx leads to an eventual accumulation of neutral lipid droplets around LD-R amastigotes, thereby increasing their unresponsiveness towards Amphotericin-B, a second-line amphiphilic antileishmanial. Notably, VL patients showing relapse with Amphotericin-B treatment exhibited significantly lower serum LDL and cholesterol than cured cases. Treatment with Aspirin, a lipid droplet blocker, reduced lipid droplets around LD-R amastigotes, restoring Amphotericin-B responsiveness.

microbiology↗

bioRxiv2022

Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses

Pancreatic -cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human -cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality -cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live -cells from dissociated human islet cells with > 97% purity. The -cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form -pseudoislets. The -pseudoislets could be maintained in culture without significant loss of viability, and responded to glucose challenge by secreting appropriate levels of glucagon. RNA-sequencing analyses (RNA-seq) revealed that expression levels of key -cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in -pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary -cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.

cell biology↗

bioRxiv2022

Factor VII activating protease (FSAP) inhibits the outcome of ischemic stroke in mouse models.

Factor VII activating protease (FSAP) is a circulating serine protease, and individuals with the Marburg I (MI) single nucleotide polymorphism (SNP), which results in an inactive enzyme, have an increased risk of stroke. The outcome of ischemic stroke is more marked in FSAP-deficient mice compared to their wild-type (WT) counterparts. Plasma FSAP levels are raised in patients as well as mice after stroke. In vitro, FSAP promotes fibrinolysis by cleavage of fibrinogen, activates protease-activated receptors and decreases the cellular cytotoxicity of histones. Since these are desirable properties in stroke treatment, we tested the effect of recombinant serine protease domain of FSAP (FSAP-SPD) on ischemic stroke in mice. A combination of tissue plasminogen activator (tPA) and FSAP-SPD enhanced clot lysis, improved microvascular perfusion and neurological outcome and reduced infarct volumes in a mouse model of thromboembolic stroke. In the tail bleeding model FSAP-SPD treatment provoked a faster clotting time indicating that it has a pro-coagulant effect that is described before. FSAP-SPD improved stroke outcome and diminished the negative effects of co-treatment with tPA in the transient middle cerebral artery occlusion model. The inactive MI-isoform of FSAP did not have any effects in either model. In mice with FSAP deficiency there were minor differences in the outcomes of stroke but the treatment with FSAP-SPD was equally effective. Thus, FSAP represents a promising novel therapeutic strategy in the treatment of ischemic stroke that requires further evaluation.

biochemistry↗