Virupakshi Soppina

A dicationic styrylpyridinium fluorophore for selective staining of mitochondria and investigation of the associated physiological processes is described

The endoplasmic reticulum (ER) and lipid droplets (LDs) intricately interact in cellular processes, with the ER serving as a hub for lipid synthesis and LDs acting as storage organelles for lipids.

Studying the viscosity of lipid droplets (LDs) provides insights into various diseases associated with LD viscosity. Ferroptosis is one such process in which LD viscosity increases due to the abnormal accumulation of lipid ROS (reactive oxygen species) caused by peroxidation. For investigating the LD imaging and ferroptosis, we developed two molecules (NNS and DNS) that show significant Stokes shifts (182–232 nm) and utilized them for sub-cellular imaging. Excellent localization is noted with the lipid droplets. Subsequently, DNS was used to monitor the variations in the LD viscosity during erastin-induced ferroptosis followed by ferroptosis inhibition. Additionally, we explored variations in the LD quantity, size, and accumulation when subjected to oleic acid stimulation. Extensive DFT and TDDFT investigations have been employed to understand the effect of NO₂ substitution on the linear and branched molecular derivatives. Our results with the improved lipophilic fluorophore, exhibiting excellent colocalization with LDs, offer valuable insights into sensing erastin-induced ferroptosis and have the potential for real-time diagnostic applications.

Lipid droplets (LDs) have emerged as major regulators of cellular metabolism, encompassing lipid storage, membrane synthesis, viral replication, and protein degradation. Exclusive studies have suggested a direct link between LDs and cancer, as a notable abundance of LDs is found in cancerous cells. Therefore, monitoring the location, distribution, and movements of LDs is of paramount importance for understanding their involvement in biological processes. To target LDs, we designed and synthesized fluorophores with a styryl scaffold bearing electron-donating amino groups and pyridine-N-oxide, a zwitterionic acceptor moiety. We explored their photophysical properties in various solvents and conducted systematic DFT calculations on the synthesized fluorescent molecules, comparing them with neutral pyridine and cationic pyridinium styryl dyes. The results demonstrate that diphenylaminostyryl pyridine-N-oxide (TNO) shows excellent imaging of LDs, in contrast to the behavior of cationic styrylpyridinium (TNC), which primarily localizes within the mitochondria. Notably, pyridine N-oxide offers several benefits: an increased dipole moment facilitating charge separation between donors and acceptors, substantial HOMO and LUMO stabilization, improved water solubility, favorable redox properties, and bathochromic-shifted absorption/emission spectra, showing promise as a fluorescent tool for probing the cellular-biological realm.

Sensing proteins and imaging sub-cellular organelles with N -sulfonyl pyridinium fluorophores.

Hypochlorous acid (HOCl) is critical for maintaining immune system balance, but it can harm mitochondria by hindering enzyme activity, leading to decreased ATP and increased cell death. In this study, we have designed a fluorophore with a pyridinium scaffold for selective staining of the mitochondria and to detect hypochlorite. The fluorophore exhibits strong solvatochromic emission due to intramolecular charge transfer and excellent sub‐cellular localization in the mitochondria. Additionally, it shows a rapid response to HOCl with high selectivity among different reactive oxygen/nitrogen compounds with a detection limit of 2.31 μM. Moreover, it is also utilized for the exogenous and endogenous detection of HOCl in live cells, which may help study the role of hypochlorite in organelles at the cellular level. DFT and TDDFT calculations have been carried out to understand the relationship between the structure and properties of the cationic probes with respect to the α‐cyano substitution and extension of π‐conjugation. The selective detection of HOCl by C4 over other cationic probes has also been well‐demonstrated, showing how the binding of HOCl affects the electronic properties of C4 through the analysis of non‐bonding orbitals (NBO) population, electrostatic potential surface (ESP), and density of states (DOS) projected DOS investigations.

KIF1A is involved in fast axonal transport of synaptic vesicle precursor, neurofilament, and dense-core vesicle, essential for neuronal development and maintenance. Several point mutations in the KIF1A motor domain have been identified in patients with various motor neuron diseases. Recent studies have shown that these mutations affected the motor and cargo localization in cultured hippocampal and C.elegans neurons. However, a detailed analysis of these mutations on KIF1A motility, force generation, and cargo transport is largely unexplored. Here, we have analyzed the effect of 16 point mutations and showed that these mutations significantly decreased the motor velocity and landing rates compared to wild-type motors. Except for A255V, mutations V144F, V220I, and E233D mildly affected motor mechanical outputs. S58L, A202P, R216P, R216H, L249Q, T312M, and R316W mutants exhibited drastic impairments in the motility properties, force generation, and cargo transport. Notably, T46M, T99M, G102D, S215R, and E253K mutants showed strong microtubule binding, resulting in complete disruption of cargo transport. Our study provides the first comprehensive demonstration of KIF1A disease mutations at the molecular level. The observed changes in motility properties and cargo transport align with the severity of disease phenotype observed in KIF1A-associated neurological disorders)

Trifluoromethyl-substituted fluorophores with naphthalene and julolidine groups were utilized imaging and quantifying lipid droplets in COS-7 cells.

Two rhodamine–phenothiazine conjugates, RP1 and RP2, were synthesized, and their photophysical properties, subcellular localization, and photocytotoxicity were investigated. We observed robust localization of RP1 in mitochondria and dual localization in mitochondria and lysosomes with RP2 in live cells. Live cell imaging with these probes allowed us to track the dynamics of mitochondria and lysosomes during ROS-induced mitochondrial damage and the subsequent lysosomal digestion of the damaged mitochondria. The fluorophores also demonstrated preferential accumulation in cancer cells compared to normal cells and had strong photo-cytotoxicity. However, no cytotoxicity was observed in the dark. The mitochondrial staining and light-induced ROS production were not limited to mammalian cell lines, but were also observed in the animal model C. elegans. The study demonstrated the potential applications of these probes in visualizing the mitochondria–lysosome cross-talk after ROS production and for photodynamic therapy.

Alcoholic liver disease (ALD) alters gut microbiota and tight junctions, causing bacterial components to enter the portal vein and induce oxidative stress-induced inflammation in the liver. Only corticosteroids and liver transplants are treatment options for severe alcoholic hepatitis. ALD’s pathophysiology is unknown. However, acetaldehyde’s toxic effects cause oxidative stress and intestinal permeability. This study investigates the influence of a synbiotic (a combination of aged garlic extract (AGE) and Lactobacillus rhamnosus MTCC1423) on colonic oxidative stress and inflammation in ALD male Wistar rats and Caco2 cells. MDA measurement by HPLC in CaCo2 cells, blood serum, and colon tissue demonstrated that synbiotic treatment in the ALD model reduces oxidative stress. Further, fecal high-throughput 16S rRNA gene sequencing revealed the microbiome’s shift towards Firmicutes in the synbiotic group compared to ethanol. In addition, DCFDA labeling and H/E staining demonstrate that the synbiotic is beneficial in inhibiting the development of ALD. In the colon, the synbiotic reduces the activation of CYP2E1 and the inflammatory markers TNF-a and IL-6 while elevating the mRNA expression of ZO-1, occludin, and IL-10. Synbiotics colonize Lactobacillus to restore barrier function and microbiota and reduce colon oxidative stress. Thus, a synbiotic combination can be used in ALD treatment.