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Cloning, expression, and immunoblotting of recombinant calpain family cysteine protease-like protein and hypothetical protein 2. (A and D) Polymerase chain reaction amplification of targeted gene. Lanes 1 and 2 with duplicate samples of amplified gene. (B and E) Expressed targeted proteins. Lanes 1 and 2 with duplicate samples of expressed protein. (C and F) Western blotting of recombinant proteins against sera of three groups: miltefosine resistant (R1, R2, R3, and R4), miltefosine sensitive (S1, S2, S3, and S4), and healthy control (HC).
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Miltefosine is the only orally administrable drug for the treatment of leishmaniasis. But in recent years, a decline in its efficacy points toward the emergence of resistance to this drug. Knowledge of biomarkers for miltefosine resistance may be beneficial for proper selection of treatment regimen. Splenic aspirates were collected and parasites cu...
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... expression, and purification of recombinant protein. Recombinant forms of calpain family cysteine protease-like protein and hypothetical proteins were success- fully expressed and purified (Figure ...
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Citations
... DNA microarrays are used to diagnose microbial pathogens in patients, food samples rapidly, and water samples [3][4][5][6]. Microarrays demonstrate the application in resolving antimicrobial resistance in clinically-relevant microbes [7][8] and biomarker elucidation [9][10]. Microbial-based microarrays are cost-effective compared to other microbe detection techniques and offer the added benefit of providing valid identification of test samples in less time. ...
... Following treatment with miltefosine, infected patients were evaluated. Comparing differential gene expression characteristics of parasites from relapsed and cured patients, Tiwary, Kumar, and Sundar [10] showed that a cysteine protease-like protein was highly upregulated from relapsed patients, suggesting that the cysteine proteaselike protein could serve as a biomarker to monitor patient relapse. ...
Human microbial infections are symbiotic processes between pathogens and humans that often lead to human disease and death. Microbial infections involve the attachment, growth, and survival of microorganisms on human skin, inside the body, or inside specific cells. Microbial infections can be localized to one body region or migrate to secondary body locations utilizing various transport mechanisms. Understanding host-pathogen interactions related to the expression of essential genes during and after infection can lead to valuable information for biologists and clinicians. Microarray technologies allow researchers to perform genomic characterization experiments rapidly and efficiently. Microarray experiments support the resolution of underlying molecular events that play a role in normal and aberrant physiologic activities in living systems. Microarray technology, coupled with bioinformatics analysis, generates comprehensive insights into relevant genes, proteins, and protein-protein interactions. This review article explores recent microarray research studies from select protozoan and bacterial pathogens to illustrate how researchers utilize microarray technology to examine microbial infection aspects. Microarray studies of pathogen and host genomes at various stages of the infection process will generate a more precise understanding of pathogenic life cycles and pathogen survival strategies. Detailed knowledge of the genes involved in the microbial infection process will lead to discovering disease biomarkers and potent therapeutic solutions.
... Moreover, in this study out of 669 upregulated genes, only a calpain family cystein protease like protein (GenBank: CBZ34784) was found to be significantly overexpressed in VL resistant parasites and showed the presence of anti-calpain antibody in the serum of miltefosine-relapsed patients. Thus this protein can be further used as a potential biomarker for miltefosine resistance in VL (Lari et al., 2019;Tiwary et al., 2018). These results advocated the promoted use of DNA microarray in identifying diagnostic, prognostic, predictive biomarkers, and new drug targets for treatment in VL. ...
... The investigators only identified a CALP-like protein with significant over expression in relapsed patients. It was concluded that Leishmania CALP can be used as a potential biomarker of miltefosine resistance [85] . ...
Emergence of drug resistance is major concern for combating against Cutaneous Leishmaniasis, a neglected tropical disease affecting 98 countries including India. Miltefosine is the only oral drug available for the disease and Miltefosine transporter proteins play pivotal role in the emergence of drug resistant Leishmania major. The cause of resistance is less accumulation of drug inside the parasite either by less uptake of drug due to decrease in the activity of P4ATPase-CDC50 complex or by increased efflux of the drug by P-glycoprotein (P-gp, an ABC transporter). In this paper, we are trying to allosterically modulate the behavior of resistant parasite (L. major) towards its sensitivity for the existing drug (Miltefosine, a phosphatidylcholine analog). We have used computational approaches to deal with the conservedness of the proteins and apparently its 3D structure prediction through ab initio modeling. Long scale membrane embedded molecular dynamics simulations were carried out to study the structural interaction and stability. Parasite specific motifs of these proteins were identified based on machine learning technique, against which a peptide library was designed. The protein-peptide docking shows good binding energy of peptides Pg5F, Pg8F and PC2 with specific binding to the motifs. These peptides were tested both in vitro and in vivo, where Pg5F in combination with PC2 showed 50-60% inhibition in resistant L. major’s promastigote and amastigote forms and 80-90% decrease in parasite load in mice. We posit a model system wherein the data provides sufficient impetus for being novel therapeutics in order to counteract the drug resistance phenotype in Leishmania parasites.
