Shailza Singh
Savitribai Phule Pune University
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Biochimica et Biophysica Acta | 2014
Milsee Mol; Milind S. Patole; Shailza Singh
BACKGROUND Modulated immune signal (CD14-TLR and TNF) in leishmaniasis can be linked to EGFR pathway involved in wound healing, through crosstalk points. This signaling network can be further linked to a synthetic gene circuit acting as a positive feedback loop to elicit a synchronized intercellular communication among the immune cells which may contribute to a better understanding of signaling dynamics in leishmaniasis. METHODS Network reconstruction with positive feedback loop, simulation (ODE 15s solver) and sensitivity analysis of CD14-TLR, TNF and EGFR was done in SimBiology (MATLAB 7.11.1). Cytoscape and adjacency matrix were used to calculate network topology. PCA was extracted by using sensitivity coefficient in MATLAB. Model reduction was done using time, flux and sensitivity score. RESULTS Network has five crosstalk points: NIK, IκB-NFκB and MKK (4/7, 3/6, 1/2) which show high flux and sensitivity. PI3K in EGFR pathway shows high flux and sensitivity. PCA score was high for cytoplasmic ERK1/2, PI3K, Atk, STAT1/3 and nuclear JNK. Of the 125 parameters, 20% are crucial as deduced by model reduction. CONCLUSIONS EGFR can be linked to CD14-TLR and TNF through the MAPK crosstalk points. These pathways may be controlled through Ras and Raf that lie upstream of signaling components ERK ½ (c) and JNK (n) that have a high PCA score via a synthetic gene circuit for activating cell-cell communication to elicit an inflammatory response. Also a disease resolving effect may be achieved through PI3K in the EGFR pathway. GENERAL SIGNIFICANCE The reconstructed signaling network can be linked to a gene circuit with a positive feedback loop, for cell-cell communication resulting in synchronized response in the immune cell population, for disease resolving effect in leishmaniasis.
Journal of Nanomedicine & Nanotechnology | 2015
Sougata Ghosh; Piyush More; Rahul Nitnavare; Soham Jagtap; Rohan Chippalkatti; Abhishek Derle; Rohini Kitture; Adersh Asok; S. N. Kale; Shailza Singh; Mahemud L. Shaikh; Boppana Ramanamurthy; Jayesh R. Bellare; Balu A. Chopade
Background: Biological route for synthesis of copper nanoparticles (CuNPs) with therapeutic potential is a major challenge. In this study, CuNPs were synthesized by D. bulbifera tuber extract (DBTE) which were further evaluated for antidiabetic and free radical scavenging activity. Methods: CuNPs synthesized by DBTE were characterized by UV-visible spectroscopy, transmission electron microscopy, energy dispersive spectroscopy and dynamic light scattering. CuNPs were checked for α-amylase and α-glucosidase inhibition along with interaction studies employing fluroscence spectroscopy, circular dichroism spectroscopy and computational docking. DPPH, nitric oxide and superoxide radical scavenging activities of CuNPs were also checked. Results: Spherical monodispersed CuNPs were synthesized within 5 h that was indicated by a colour change from pale blue to brown. Majority of the nanoparticles synthesized were found to be between 12 to 16 nm as showed in DLS which grew till a final size of 86 to 126 nm as indicated in TEM. Bioreduced CuNPs showed 38.70 ± 1.45% and 34.72 ± 1.22% inhibition against porcine and murine pancreatic amylase, respectively with an uncompetitive mode that was further confirmed by docking studies. Fluorescence spectroscopy confirmed the interaction of CuNPs to the enzyme via Trp residues while CD spectra indicated the structural and conformational changes on binding of CuNPs to the enzyme. CuNPs exhibited 99.09 ± 0.15% inhibition against α-glucosidase while 90.67 ± 0.33% inhibition against murine intestinal glucosidase, respectively. CuNPs showed 40.81 ± 1.44%, 79.06 ± 1.02% and 48.39 ± 1.46% scavenging activity against DPPH, nitric oxide and superoxide radicals respectively. Conclusion: D.bulbifera tuber extract mediated bioreduction is most rapid route to synthesize novel CuNPs with promising antidiabetic and antioxidant properties. This is the first detailed report which provides intense scientific rationale for the use of CuNPs as nanomedicine for efficient control of T2DM and oxidative stress.
Human Immunology | 2013
Mohammed Mumtaz Naiyer; S. Saha; Vijayshree Hemke; Somenath Roy; Shailza Singh; Krishnasastry V. Musti; Bhaskar Saha
Interleukin-10 (IL-10) is an anti-inflammatory cytokine that works through IL-10 receptor alpha subunit- and suppresses immune responses in many infectious diseases such as leishmaniasis as well as in cancer. Therefore, in order to restore the host-protective immune responses in such diseases, an antagonist to this cytokine is a pressing need. Herein, using phage peptide library display, we have identified a dodecameric peptide that functions as an antagonist to human IL-10 receptor in an IL-10-induced STAT3 phosphorylation assay. The peptide antagonists ability to restore anti-leishmanial function in CD40-activated macrophages was also tested. We observed that the peptide reduced IL-10-induced STAT-3 phosphorylation and enhanced CD40-activated leishmanial functions in macrophages.
BMC Evolutionary Biology | 2014
Vineetha Mandlik; Sonali Shinde; Shailza Singh
BackgroundSelection pressure governs the relative mutability and the conservedness of a protein across the protein family. Biomolecules (DNA, RNA and proteins) continuously evolve under the effect of evolutionary pressure that arises as a consequence of the host parasite interaction. IPCS (Inositol phosphorylceramide synthase), SPL (Sphingosine-1-P lyase) and SPT (Serine palmitoyl transferase) represent three important enzymes involved in the sphingolipid metabolism of Leishmania. These enzymes are responsible for maintaining the viability and infectivity of the parasite and have been classified as druggable targets in the parasite metabolome.ResultsThe present work relates to the role of selection pressure deciding functional conservedness and divergence of the drug targets. IPCS and SPL protein families appear to diverge from the SPT family. The three protein families were largely under the influence of purifying selection and were moderately conserved baring two residues in the IPCS protein which were under the influence of positive selection. To further explore the selection pressure at the codon level, codon usage bias indices were calculated to analyze genes for their synonymous codon usage pattern. IPCS gene exhibited slightly lower codon bias as compared to SPL and SPT protein families.ConclusionEvolutionary tracing of the proposed drug targets has been done with a viewpoint that the amino-acids lining the drug binding pocket should have a lower evolvability. Sites under positive selection (HIS20 and CYS30 of IPCS) should be avoided during devising strategies for inhibitor design.
PLOS ONE | 2016
Vineetha Mandlik; Sohan Patil; Ramanamurthy Bopanna; Sudipta Basu; Shailza Singh
Cutaneous leishmaniasis affects nearly 0.7 to 1.3 million people annually. Treatment of this disease is difficult due to lack of appropriate medication and the growing problem of drug resistance. Natural compounds such as coumarins serve as complementary therapeutic agents in addition to the current treatment modalities. In this study, we have performed an in-silico screening of the coumarin derivatives and their anti-leishmanial properties has been explored both in-vitro and in-vivo. One of the compounds (compound 2) exhibited leishmanicidal activity and to further study its properties, nanoliposomal formulation of the compound was developed. Treatment of cutaneous lesions in BALB/c mice with compound 2 showed significantly reduced lesion size as compared to the untreated mice (p<0.05) suggesting that compound 2 may possess anti-leishmanial properties.
F1000Research | 2016
Vineetha Mandlik; Shailza Singh
Inositol phosphorylceramide synthase (IPCS) has emerged as an important, interesting and attractive target in the sphingolipid metabolism of Leishmania. IPCS catalyzes the conversion of ceramide to IPC which forms the most predominant sphingolipid in Leishmania. IPCS has no mammalian equivalent and also plays an important role in maintaining the infectivity and viability of the parasite. The present study explores the possibility of targeting IPCS; development of suitable inhibitors for the same would serve as a treatment strategy for the infectious disease leishmaniasis. Five coumarin derivatives were developed as inhibitors of IPCS protein. Molecular dynamics simulations of the complexes of IPCS with these inhibitors were performed which provided insights into the binding modes of the inhibitors. In vitro screening of the top three compounds has resulted in the identification of one of the compounds (compound 3) which shows little cytotoxic effects. This compound therefore represents a good starting point for further in vivo experimentation and could possibly serve as an important drug candidate for the treatment of leishmaniasis.
Journal of Immunology | 2015
Pallavi R. Gangalum; Waldionê de Castro; Leda Q. Vieira; Ranadhir Dey; Luis Rivas; Shailza Singh; Subrata Majumdar; Bhaskar Saha
Miltefosine [hexadecylphosphocholine (HPC)] is the only orally bioavailable drug for the disease visceral leishmaniasis, which is caused by the protozoan parasite Leishmania donovani. Although miltefosine has direct leishmanicidal effects, evidence is mounting for its immune system–dependent effects. The mechanism of such indirect antileishmanial effects of miltefosine remains to be discovered. As platelet-activating factor and HPC share structural semblances and both induce killing of intracellular Leishmania, we surmised that platelet-activating factor (PAF) receptor had a significant role in the antileishmanial function of miltefosine. The proposition was supported by molecular dynamic simulation of HPC docking into PAF receptor and by comparison of its leishmanicidal function on PAF receptor–deficient macrophages and mice under HPC treatment. We observed that compared with wild-type macrophages, the PAF receptor–deficient macrophages showed 1) reduced binding of a fluorescent analog of HPC, 2) decreased TNF-α production, and 3) lower miltefosine-induced killing of L. donovani. Miltefosine exhibited significantly compromised leishmanicidal function in PAF receptor–deficient mice. An anti-PAF receptor Ab led to a significant decrease in miltefosine-induced intracellular Leishmania killing and IFN-γ production in a macrophage–T cell coculture system. These results indicate significant roles for PAF receptor in the leishmanicidal activity of HPC. The findings open new avenues for a more rational understanding of the mechanism of action of this drug as well as for improved therapeutic strategies.
Journal of Immunology | 2015
Sushmita Chakraborty; Ankita Srivastava; Mukesh Kumar Jha; Arathi Nair; Surya Prakash Pandey; Neetu Srivastava; Sangeeta Kumari; Shailza Singh; M.V. Krishnasastry; Bhaskar Saha
Leishmania major is a parasite that resides and replicates in macrophages. We previously showed that the parasite enhanced CD40-induced Raf-MEK-ERK signaling but inhibited PI3K-MKK-p38MAPK signaling to proleishmanial effects. As Raf and PI3K have a Ras-binding domain but exert opposite effects on Leishmania infection, we examined whether Ras isoforms had differential roles in Leishmania infection. We observed that L. major enhanced N-Ras and H-Ras expression but inhibited K-Ras expression in macrophages. L. major infection enhanced N-Ras activity but inhibited H-Ras and K-Ras activity. TLR2 short hairpin RNA or anti-TLR2 or anti-lipophosphoglycan Abs reversed the L. major–altered N-Ras and K-Ras expressions. Pam3CSK4, a TLR2 ligand, enhanced N-Ras expression but reduced K-Ras expression, indicating TLR2-regulated Ras expression in L. major infection. Whereas N-Ras silencing reduced L. major infection, K-Ras and H-Ras silencing enhanced the infection both in macrophages in vitro and in C57BL/6 mice. BALB/c-derived macrophages transduced with lentivirally expressed N-Ras short hairpin RNA and pulsed with L. major–expressed MAPK10 enhanced MAPK10-specific Th1-type response. CD40-deficient mice primed with these macrophages had reduced L. major infection, accompanied by higher IFN-γ but less IL-4 production. As N-Ras is activated by Sos, a guanine nucleotide exchange factor, we modeled the N-Ras–Sos interaction and designed two peptides from their interface. Both the cell-permeable peptides reduced L. major infection in BALB/c mice but not in CD40-deficient mice. These data reveal the L. major–enhanced CD40-induced N-Ras activation as a novel immune evasion strategy and the potential for Ras isoform–targeted antileishmanial immunotherapy and immunoprophylaxis.
Journal of Chemical Biology | 2013
Vineetha Mandlik; Dixita Limbachiya; Sonali Shinde; Milsee Mol; Shailza Singh
Building circuits and studying their behavior in cells is a major goal of systems and synthetic biology. Synthetic biology enables the precise control of cellular states for systems studies, the discovery of novel parts, control strategies, and interactions for the design of robust synthetic systems. To the best of our knowledge, there are no literature reports for the synthetic circuit construction for protozoan parasites. This paper describes the construction of genetic circuit for the targeted enzyme inositol phosphorylceramide synthase belonging to the protozoan parasite Leishmania. To explore the dynamic nature of the circuit designed, simulation was done followed by circuit validation by qualitative and quantitative approaches. The genetic circuit designed for inositol phosphorylceramide synthase (Biomodels Database—MODEL1208030000) shows responsiveness, oscillatory and bistable behavior, together with intrinsic robustness.
Chemical Biology & Drug Design | 2011
Shailza Singh; Priyanka Joshi; Balu A. Chopade
Acinetobacter spp., source of numerous nosocomial infections, deserves a close attention as various multidrug resistance strains are being discovered worldwide. Acinetobacter baylyi is chosen because of its high competence for natural transformation, and its ability to undergo direct homology‐based recombination. An in silico comparative analysis of the metabolic pathways of the host Homo sapiens and the pathogen Acinetobacter baylyi was performed by using BLASTp search. This search is against the non‐redundant database restricted to the Homo sapiens subset. Sixteen unique pathways identified enlisted a total of 183 drug targets of which 31 belong to the metabolic pathways unique to pathogen having no human homolog. Of these potential drug targets enlisted, RmlA enzyme (d‐glucose‐1 phosphate thymidylyltransferase) is the first enzyme in the polyketide sugar unit synthesis metabolic pathway, which leads to the formation of l‐rhamnose. In gram‐negative bacteria, l‐rhamnose is one of the important residues of the O‐antigen of lipopolysaccharide, a key determinant factor for the virulence of these species. Moreover, these proteins are highly conserved among microorganisms, and therefore, conclusions drawn from the structure of a protein from one species will have strong implications for the corresponding enzyme structure of another origin. Homology modeling of RmlA was performed by MODELLER and the PMDB ID obtained is PM0076419. Further, molecular docking studies were performed for RmlA, which might aid drug design for nosocomial infections.