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Proteomic and Genomic Analysis of Leishmania donovani Parasites for their Survival in Mammalian Host and Designing Therapeutic Strategies to Combat the Disease
No full textVisceral leishmaniasis (VL) is an immunosuppressive disease which is fatal if left untreated. Non-availability of vaccine, invasive methods of diagnosis and limitations of currently available therapies further complicate the problem. Designing effective therapies need detailed
understanding of both parasite and host factors responsible for infection. Leishmania secrete virulence factors which modulate host immunity for their survival. We observed loss of virulence in Leishmania donovani promastigotes as a result of continuous axenic cultivation, resulting in
differential modulation of macrophage microbicidal activity in vitro and higher immunostimulation in animals. Comparative proteomic screening of L. donovani AG83
promastigotes of early and late passages identified decrease in expression of some important virulence factors viz., gp63, HSP70, LACK, EF1-, dihydrolipoamide acetyltransferase, etc. providing the basis for loss of virulence. Post-transcriptional and post-translational modifications determine the ultimate protein repertoire in Leishmania. We generated RNA-Seq data to compare the transcript abundance in early (2nd), intermediate (11th), and late (25th) passages, and found altered metabolism, signaling, DNA repair and RNA editing processes which may account for the gross changes in protein expression when grown for longer periods in culture. We could
cite similarity of parasite multiplication and dissemination with cancer cell division and metastasis in terms of gene expression pattern. Thus we propose that common pathway or target based clinical interventions can be designed for VL and cancer. Based on our understanding of
the parasite and host factors of VL we did mechanistic study on previously developed cationic drug free and pentavalent antimonial (SbV) entrapped phosphatidylcholine (PC) - stearylamine (SA) liposomes and compared their efficacy with PC-dioctadecyldimethylammonium bromide
(DDAB) vesicles. We observed increased retention and slow release of liposomal drug responsible for resistance reversal and phosphatidylserine-mediated direct leishmanicidal activity in vitro and host-protective immomodulation for effective therapy in vivo, with PC-DDAB being a better antileishmanial requiring a 4-times less dose for equivalent activity. To further improve
this formulation we entrapped TGF-β signaling inhibitor SB-505124 (SB), earlier used in cancer therapy, in PC-SA and encapsulated globally used form of SbV, meglumine antimoniate (MA) into it (PC-SA-SB-MA). After successful in vitro screening we observed near sterile cure in a
mice model of infection with a dose of 10 mg/kg b.w. w.r.t. PC of PC-SA entrapping 50mg/kg b.w of SB and encapsulating 6 mg/kg b.w. of MA, whereas 20 mg/kg b.w of only PC-SA-MA
was inferior in clearing parasites from liver, spleen and bone marrow of infected mice. Clearance of parasites was accompanied with higher IFN-/IL-10 ratio for successful cure. Thus our liposomal formulations can act as dual modulators of parasite and host. To conclude, similarities
between VL and cancer can be exploited in identifying drug and vaccine targets and designing common clinical interventions for these diseases
Recent Advances in Nucleic Acid Binding Aspects of Berberine Analogs and Implications for Drug Design
No full textBerberine is one of the most widely known alkaloids belonging to the protoberberine group exhibiting myriad therapeutic properties. The anticancer potency of berberine appears to derive from its multiple actions including strong interaction with nucleic acids exhibiting adenine-thymine base pair specificity, inhibition of the enzymes topoisomerases and telomerases, and stabilizing the
quadruplex structures. It was realized that the development of berberine as a potential anticancer agent necessitates enhancing its nucleic acid binding efficacy through appropriate structural modifications.More recently a number of such approaches have been attempted in various laboratories with great success. Several derivatives have been synthesized mostly with substitutions at the 8, 9 and 13 positions of the isoquinoline chromophore, and studied for enhanced nucleic acid binding activity. In this article, we present an up to date review of the details of the interaction of berberine and several of its important synthetic 8, 9 and 13 substituted derivatives with various nucleic acid structures reported recently. These studies provide interesting knowledge on the mode, mechanism, sequence and structural specificity of the binding of berberine derivatives and correlate structural and
energetic aspects of the interaction providing better understanding of the structure- activity relations for designing and development of berberine based therapeutic agents with higher efficacy and therapeutic potential
Selective detection of Escherichia coli DNA using fluorescent carbon spindles
No full textWe investigate the interaction of hydrophilic blue emitting carbon spindles with various deoxyribonucleic acids (DNA) having different base pair compositions, such as Herring testes (HT), calf thymus (CT),Escherichia coli (EC) and Micrococcus lysodeikticus (ML) DNA, to understand the mode of interaction. Interestingly, the fluorescent carbon spindles selectively interacted with E. coli DNA resulting in enhanced fluorescence of the former. Interaction of the same carbon with other DNAs exhibited insignificant changes in fluorescence. In addition, in the presence of EC DNA, the D band in the Raman spectrum attributed to the defect state completely disappeared, resulting in enhanced
crystallinity. Microscopy images confirmed the wrapping of DNA on the carbon spindles leading to the assembly of spindles in the form of flowers. Dissociation of double-stranded DNA occurred upon interaction with carbon spindles, resulting in selective E. coli DNA interaction. The carbon spindles also exhibited a similar fluorescence enhancement upon treating with E. coli bacteria. These
results confirm the possibility of E. coli detection in water and other liquid foods using such fluorescent
carbo
A novel spirooxindole derivative inhibits the growth of Leishmania donovani parasite both in vitro and in vivo by targeting type IB topoisomerase
No full textVisceral Leishmaniasis is a fatal parasitic disease and there is an emergent need for development of effective drugs against this neglected tropical disease. We report here development of a novel spirooxindole derivative N-benzyl 2, 2’ α 3, 3’, 5’, 6’, 7’, 7α,α'-octahydro-2methoxycarbonyl-spiro [indole-3, 3’ -pyrrolizidine]-2 one (Compound 4c) which inhibits Leishmania donovani topoisomerase IB (LdTopIB) and kills the wild type as well as drug-resistant parasite strains. This compound inhibits catalytic activity of LdTopIB in competitive manner. Unlike Camptothecin, the compound does not stabilize the DNA-topoisomerase IB cleavage complex; rather, they hinder drug-DNA-enzyme covalent complex formation. Fluorescence studies show stoichiometry of this compound binding to LdTopIB is 2:1 (mole/mole) with a dissociation constant of 6.65 μM. Molecular docking with LdTopIB using the stereoisomers of Compound 4c produced two probable hits for binding site: one in small subunit and the other in the hinge region of the large subunit of LdTopIB. This spirooxindole is highly cytotoxic to promastogotes of L. donovani and also induces apoptosis-like cell death in parasite. Treatment with compound 4c causes depolarization of mitochondrial membrane potential, formation of reactive oxygen species inside parasites and ultimately fragmentation of nuclear DNA. Compound 4c also effectively clears amastigote forms of wild type and drug-resistant parasites from infected mouse peritoneal macrophages but has less effect on host macrophages. Moreover compound 4c showed strong antileishmanial efficacies in BALB/c mice model of leishmaniasis. Potentially this compound can be used as a lead for developing excellent anileishmanial agent against emerging drug resistant strains of the parasite
Spectral mapping of 3D multi-cellular tumor spheroids: time-resolved confocal microscopy
No full textA tumor-like multi-cellular spheroid (3D) differs from a 2D cell in a number of ways. This is
demonstrated using time resolved confocal microscopy. Two different tumor spheroids – HeLa (cervical
cancer) and A549 (lung cancer) – are studied using 3 different fluorescent dyes – C153 (non-covalent),
CPM (covalent) and doxorubicin (non-covalent, anti-cancer drug). The pattern of localization of these
three fluorescent probes in the 3D tumor cell exhibits significant differences from that in the
conventional 2D cells. For both the cells (HeLa and A549), the total uptake of doxorubicin in the 3D cell
is much lower than that in the 2D cell. The uptake of doxorubicin molecules in the A549 spheroid is
significantly different compared to the HeLa spheroid. The local polarity (i.e. emission maxima) and
solvation dynamics in the 3D tumor cell differ from those in 2D cells. The covalent probe CPM exhibits
intermittent fluorescence oscillations in the 1–2 s time scale. This is attributed to redox processes. These
results may provide new insights into 3D tumors
Characterization of Hydroxyl-hydrogen Bond and its Role in Protein Stability and Function
No full textHydrogen bonding involving hydroxyl (OH) group has been recognized as playing a fundamental role in determining the structural stability, function and dynamics of many chemical and biological systems. Intramolecular and intermolecular H-bonds provide ample structural stability to proteins and nucleic acids. It also plays an
important role in enzyme-catalysis and molecular recognition processes. It is one of the most important noncovalent interactions between substrate and enzyme active site residues. One of our objectives was to investigate the role of hydrogen bonding interaction in ribonuclease A-mediated hydrolysis of RNA. We have chosen ribose, an integral analog of nucleic acid (deoxyribose in DNA and ribose in RNA) as our model compound. We employed deuterium isotope effect to probe the intramolecular
and intermolecular H-bonding interactions and to elucidate enzymatic reaction mechanism of RNAse A. We have utilized NMR and Raman spectroscopic methods as a primary tool to determine the strength of H-bond and related thermodynamic
parameters in free ribose and in ribonucleotide, uridine monophosphate (UMP). We have also supported our study with extensive density functional theory (DFT)
calculations
Investigations on Immunomodulatory and ParasiticidaL Efficacies of Individualistic and Combinatorial Chemotherapeutic Agents against Visceral Leishmaniasis
No full textLeishmaniasis is a collection of broad spectrum of diseases caused by obligate intracellular parasites belonging to genus Leishmania. It has emerged as the third most
prevalent parasite-borne disease worldwide after malaria and filariasis (Pourahmad et al., 2009). Manifestations of this disorder can range from less severe cutaneous leishmaniasis (CL) characterized by self-resolving local cutaneous lesions and mucocutaneous leishmaniasis (MCL) affecting the mucus membranes of mouth, nose and throat to the more serious visceral leishmaniasis (VL), which is potentially fatal. An estimated 12 million people worldwide have some form of leishmaniasis, and another 350 million
people are at risk (WHO Technical Report, 2010). There are approximately 0.2-0.4 million new cases of VL each year and over 90% of these occur in India, Bangladesh, Nepal, Brazil, Sudan and South Sudan, although these numbers are likely to be an underestimation since majority of the cases are not even reported in these and many of the other 88 endemic countries (Alvar et al., 2010). Post-kala-azar dermal leishmaniasis (PKDL), characterized by dispigmented macule, erythematous rashes, indurated plaques, papules and nodules, develops in a small percentage of Indian VL patients generally after 1 to 7 years, and in >50% Sudanese VL patients during or within 6 months of successful
antimonial therapy (Zijlstra et al., 2003). This chronic skin condition is probably a reservoir of parasites especially during inter-epidemic periods of kala-aza
Rugose atypical Vibrio cholerae O1 El Tor responsible for 2009 cholera outbreak in India
No full textVibrio cholerae causes cholera outbreaks in endemic regions where the water quality and sanitation facilities remain poor. Apart from biotype and serotype changes, V. cholerae
undergoes phase variation, which results in the generation of two morphologically different variants termed smooth and rugose. In this study, 12 rugose (R-VC) and 6 smooth (S-VC) V. cholerae O1 Ogawa isolates were identified in a cholera outbreak that occurred in Hyderabad, India. Antimicrobial susceptibility results showed that all the isolates were resistant to ampicillin, furazolidone and nalidixic acid. In addition, R-VC isolates were resistant to ciprofloxacin (92 %), streptomycin (92 %), erythromycin (83 %), trimethoprim-sulfamethoxazole (75 %) and tetracycline
(75 %). Based on the ctxB gene analysis, all the isolates were identified as El Tor variant with mutation in two positions of ctxB, similar to the classical biotype. The R-VC isolates specifically showed excessive biofilm formation and were comparatively less motile. In addition, the majority of these isolates (~83 %) displayed random mutations in the hapR gene, which encodes haemagglutinin protease regulatory protein. In the PFGE analysis, R-VC and S-VC were placed in distinct clusters but remained clonally related. In the ribotyping analysis, all the R-VC isolates
exhibited R-III pattern, which is a prevailing type among the current El Tor isolates. A hapR deletion mutant generated using an S-VC isolate expressed rugose phenotype. To our knowledge, this is the first report on the association of rugose V. cholerae O1 in a large cholera
outbreak with extended antimicrobial resistance and random mutations in the haemagglutinin protease regulatory protein encoding gene (hapR)
Mechanism Of Macrophage Activation And Enhancement Of Leishmanicidal Effect By An Attenuated Leishmania Donovani And A Membrane Protein Of The Same Attenuated Leishmania Strain
No full textLeishmania is a trypanosomatid protozoan that is responsible for the disease leishmaniasis. Promastigote form of pathogenic Leishmania, an intracellular pathogen, delays phagosome maturation and represses normal function of macrophages (MΦs).
In the first chapter, attenuated Leishmania strain (ALS) UR6 exposed RAW 264.7 cells showed an enhanced respiratory burst and enhanced production of pro-inflammatory mediators. We observed low parasite burden in ALS primed MΦs when co-infected with PLD, compared to control. In our study, increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani (PLD) AG83 with Lysosome was found. Moreover, increased co-localization was observed between PLD late phagosomal markers viz. Rab7, LAMP1, Cathepsin D, and V-ATPase which indicate phagosome maturation. It was also observed that V-type and p38 MAPK is the key player in acidification and maturation of phagosome in ALS pre-exposed MΦs. We conclude pre-exposure of MΦs to ALS could potentiate short-term prophylactic response in the host.
In the second chapter, bioactivity of a Leishmanial integral membrane glycol-lipoprotein (LIMP) isolated from the attenuated Leishmania donovani UR6 was assessed. LIMP is well capable of inducing the generation of ROS in RAW 264.7 cells. LIMP also capable driving pro-inflammatory cytokine viz. IL-12, TNF-α, and IL-1β expression. The overall expression and nuclear accumulation of p65 subunit of NF-kB after treatment with LIMP were found. LIMP was also found to be activating TLR-2-CD14 axis.
In the third chapter, we observe Lipid Bodies (LB) or lipid droplets (LD) generation in L. donovani infected MΦs in a time-dependent manner. The size of LD was also increased along with time. We provide novel insight on the signaling molecules i.e. ERK½, which is responsible for LB accumulation. Alongside ERK1/2, the paracrine action of IL-10 may also important to induce LB generation. Interestingly LPG-deficient UR6 failed to induce LB generation. But inhibition of phagosome maturation drastically stimulates LB accumulation in UR6 infected MΦs. Aspirin treatment in AG83 infected MΦs does not only lowers LD load but also favors phagolysosome biogenesis and correct cytokine balance, hence aspirin could be used as a therapeutic tool to resist parasite growth in the early hour of infection
Cancer Cell Imaging Using in Situ Generated Gold Nanoclusters
No full textIn situ generated fluorescent gold nanoclusters (Au-NCs) are
used for bio-imaging of three human cancer cells, namely, lung (A549), breast (MCF7), and colon (HCT116), by confocal microscopy. The amount of Au-NCs in non-cancer cells (WI38 and MCF10A) is 20–40 times less than those in the corresponding cancer cells. The presence of a larger amount of glutathione (GSH) capped Au-NCs in the cancer cell is ascribed to a higher glutathione level in cancer cells. The Au-NCs exhibit fluorescence maxima at 490–530 nm inside the cancer cells. The fluorescence maxima and matrix-assisted laser desorption ionization (MALDI) mass spectrometry suggest that the fluorescent Au-NCs consist of GSH capped clusters with a core structure (Au8-13). Time-resolved confocal microscopy indicates a nanosecond (1–3 ns) lifetime of the Au-NCs inside the cells. This rules
out the formation of aggregated Au–thiolate complexes,
which typically exhibit microsecond (�1000 ns) lifetimes. Fluorescence correlation spectroscopy (FCS) in live cells indicates that the size of the Au-NCs is �1–2 nm. For in situ generation,we used a conjugate consisting of a room-temperature ionic liquid (RTIL, [pmim][Br]) and HAuCl4. Cytotoxicity studies indicate that the conjugate, [pmim][AuCl4], is non-toxic for both cancer and non-cancer cells