Batakrishna Jana

Indolicidin Targets Duplex DNA: Structural and Mechanistic Insight through a Combination of Spectroscopy and Microscopy

Indolicidin (IR13), a 13‐residue antimicrobial peptide from the cathelicidin family, is known to exhibit a broad spectrum of antimicrobial activity against various microorganisms. This peptide inhibits bacterial DNA synthesis resulting in cell filamentation. However, the precise mechanism remains unclear and requires further investigation. The central PWWP motif of IR13 provides a unique structural element that can wrap around, and thus stabilize, duplex B‐type DNA structures. Replacements of the central Trp‐Trp pair with Ala‐Ala, His‐His, or Phe‐Phe residues in the PxxP motif significantly affects the ability of the peptide to stabilize duplex DNA. Results of microscopy studies in conjunction with spectroscopic data confirm that the DNA duplex is stabilized by IR13, thereby inhibiting DNA replication and transcription. In this study we provide high‐resolution structural information on the interaction between indolicidin and DNA, which will be beneficial for the design of novel therapeutic antibiotics based on peptide scaffolds.

Selective Killing of Breast Cancer Cells by Doxorubicin‐Loaded Fluorescent Gold Nanoclusters: Confocal Microscopy and FRET

Fluorescent gold nanoclusters (AuNCs) capped with lysozymes are used to deliver the anticancer drug doxorubicin to cancer and noncancer cells. Doxorubicin-loaded AuNCs cause the highly selective and efficient killing (90 %) of breast cancer cells (MCF7) (IC50 =155 nm). In contrast, the killing of the noncancer breast cells (MCF10A) by doxorubicin-loaded AuNCs is only 40 % (IC50 =4500 nm). By using a confocal microscope, the fluorescence spectrum and decay of the AuNCs were recorded inside the cell. The fluorescence maxima (at ≈490-515 nm) and lifetime (≈2 ns), of the AuNCs inside the cells correspond to Au10-13 . The intracellular release of doxorubicin from AuNCs is monitored by Förster resonance energy transfer (FRET) imaging.

Excited state proton transfer in the lysosome of live lung cells: normal and cancer cells.

Dynamics of excited state proton transfer (ESPT) in the lysosome region of live lung cells (normal and cancer) is studied by picosecond time-resolved confocal microscopy. For this, we used a fluorescent probe, pyranine (8-hydroxy-pyrene-1,3,6-trisulfonate, HPTS). From the colocalization of HPTS with a lysotracker dye (lysotracker yellow), we confirmed that HPTS resides in the lysosome for both of the cells. The diffusion coefficient (Dt) in the lysosome region was obtained from fluorescence correlation spectroscopy (FCS). From Dt, the viscosity of lysosome is estimated to be ∼40 and ∼30 cP in the cancer and normal cells, respectively. The rate constants of the elementary steps of ESPT in a normal lung cell (WI38) are compared with those in a lung cancer cell (A549). It is observed that the time constant of the initial proton transfer process in a normal cell (τ(PT) = 40 ps) is similar to that in a cancer cell. The recombination of the geminate ion pair is slightly faster (τ(rec) = 25 ps) in the normal cell than that (τ(rec) = 30 ps) in a cancer cell. The time constant of the dissociation (τ(diss)) of the geminate ion pair for the cancer cell (τ(diss) = 80 ps) is 1.5 times faster compared to that (τ(diss) = 120 ps) in a normal cell.

Confocal microscopy of cytoplasmic lipid droplets in a live cancer cell: number, polarity, diffusion and solvation dynamics

Time resolved confocal microscopy indicates that the cytoplasmic lipid droplets (CLDs) in live cells (normal and cancer lung cells) are less polar, and exhibit slower diffusion (motility) and solvation dynamics than the cytoplasm. The number of CLDs in a human lung cancer cell (A549) is ∼10 times higher than in a non-cancer lung fibroblast cell (WI38). This may result in accumulation of non-polar cell signaling agents in the CLDs of the cancer cell.

Dual Functionalized Graphene Oxide Serves as a Carrier for Delivering Oligohistidine- and Biotin-Tagged Biomolecules into Cells†

A versatile method of dual chemical functionalization of graphene oxide (GO) with Tris-[nitrilotris(acetic acid)] (Tris-NTA) and biotin for cellular delivery of oligohistidine- and biotin-tagged biomolecules is reported. Orthogonally functionalized GO surfaces with Tris-NTA and biotin to obtain a dual-functionalized GO (DFGO) are prepared and characterized by various spectroscopic and microscopic techniques. Fluorescence microscopic images reveal that DFGO surfaces are capable of binding oligohistidine-tagged biomolecules/proteins and avidin/biotin-tagged biomolecules/proteins orthogonally. The DFGO nanoparticles are non-cytotoxic in nature and can deliver oligohistidine- and biotin-tagged biomolecules simultaneously into the cell.

Targeted delivery of a novel peptide–docetaxel conjugate to MCF-7 cells through neuropilin-1 receptor: reduced toxicity and enhanced efficacy of docetaxel

We have designed a novel peptide–docetaxel conjugate, which delivers docetaxel specifically to cancer cell targeting neuropilin-1 (NRP-1) receptor, thus enhancing its efficacy and acts more aggressively in breast cancer cells.

α-Cyclodextrin Interacts Close to Vinblastine Site of Tubulin and Delivers Curcumin Preferentially to the Tubulin Surface of Cancer Cell

Tubulin is the key cytoskeleton component, which plays a crucial role in eukaryotic cell division. Many anticancer drugs have been developed targeting the tubulin surface. Recently, it has been shown that few polyhydroxy carbohydrates perturb tubulin polymerization. Cyclodextrin (CD), a polyhydroxy carbohydrate, has been extensively used as the delivery vehicle for delivery of hydrophobic drugs to the cancer cell. However, interaction of CD with intracellular components has not been addressed before. In this Article, we have shown for the first time that α-CD interacts with tubulin close to the vinblastine site using molecular docking and Förster resonance energy transfer (FRET) experiment. In addition, we have shown that α-CD binds with intracellular tubulin/microtubule. It delivers a high amount of curcumin onto the cancer cell, which causes severe disruption of intracellular microtubules. Finally, we have shown that the inclusion complex of α-CD and curcumin (CCC) preferentially enters into the human lung cancer cell (A549) as compared to the normal lung fibroblast cell (WI38), causes apoptotic death, activates tumor suppressor protein (p53) and cyclin-dependent kinase inhibitor 1 (p21), and inhibits 3D spheroid growth of cancer cell.

A biotin micropatterned surface generated by photodestruction serves as a novel platform for microtubule organisation and DNA hybridisation.

MASK AND CAPTURE: Photodestruction of parts of a biotin-functionalised surface by shining UV light through a photomask produces a biotin micropattern. These micropatterns can selectively capture functional biotin-tagged biomolecules/proteins such as microtubules and molecular beacons.

Cancer Cell Specific Delivery of Photosystem I Through Integrin Targeted Liposome Shows Significant Anticancer Activity

Many anticancer drugs are developed for the treatment of cancer from natural sources. Photosystem I (PSI), a protein complex present in the chloroplast, is involved in photosynthesis and generates reactive oxygen species (ROS) in plant. Here, we used the ROS generation property of PSI for cancer therapy. We show that PSI can enter into different kinds of cancer cell like human lung carcinoma (A549) and mouse melanoma (B16F10) cell lines and generate ROS inside the cells. It inhibits the proliferation of cancer cell and causes apoptotic death of cancer cells. We also show that PSI induces apoptosis through mitochondria-dependent internal pathway, induces caspase3, causes DNA fragmentation, and arrests cell cycle at SubG0 phase. We also prepared, using C16-LDV lipopeptide [C16 long chain attached on the N-terminal of the tripeptide containing amino acids leucine (L), aspartic acid (D), and valine (V) abbreviated as NH2-LDV-COOH], α4β1 integrin targeted liposomal formulation of PSI, which specifically kills the cancer cell without affecting normal cells, and it is found to be more potent compared to clinically used drug doxorubicin. Finally, we found that LDV liposomal formulation of PSI inhibits the growth of tumor in C57BL/6J mice model.

Novel hexapeptide interacts with tubulin and microtubules, inhibits Aβ fibrillation, and shows significant neuroprotection.

Herein, we report a novel hexapeptide, derived from activity dependent neuroprotective protein (ADNP), that spontaneously self-assembles to form antiparallel β-sheet structure and produces nanovesicles under physiological conditions. This peptide not only strongly binds with β-tubulin in the taxol binding site but also binds with the microtubule lattice in vitro as well as in intracellular microtubule networks. Interestingly, it shows inhibition of amyloid fibril formation upon co-incubation with Aβ peptide following an interesting mechanistic pathway and excellent neuroprotection in PC12 cells treated with anti-nerve growth factor (NGF). The potential of this hexapeptide opens up a new paradigm in design and development of novel therapeutics for AD.