Department of Biological Sciences

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Author: search.filters.author.Garg, ShilpiSubject: search.filters.subject.Plasmodium falciparum

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    Plasmodium iron-sulfur [fe-s] Cluster assembly protein dre2 as a plausible target of artemisinin: mechanistic insights derived in a prokaryotic heterologous system
    (Elsevier, 2023-06) Saxena, Vishal; Garg, Shilpi
    Iron-sulfur (Fe-S) cluster containing proteins have been assigned roles in various essential cellular processes, such as regulation of gene expression, electron transfer, sensing of oxygen and balancing free radical chemistry. However, their role as the drug target remains sparse. Recently the screening of protein alkylation targets for artemisinin in Plasmodium falciparum led to identification of Dre2, a protein involved in redox mechanism for the cytoplasmic Fe-S cluster assembly in different organisms. In the present study, to further explore the interaction between artemisinin and Dre2, we have expressed the Dre2 protein of both P. falciparum and P. vivax in E. coli. The opaque brown colour of the IPTG induced recombinant Plasmodium Dre2 bacterial pellet, suggested iron accumulation as confirmed by the ICP-OES analysis. In addition, overexpression of rPvDre2 in E. coli reduced its viability, growth and increased the ROS levels of bacterial cells, which in turn led to an increase in expression of stress response genes of E. coli such as recA, soxS, mazF. Moreover, the overexpression of rDre2 induced cell death could be rescued by treatment with Artemisinin derivatives suggesting their interaction. The interaction between DHA and PfDre2 was later demonstrated by CETSA and microscale thermophoresis. Overall, this study suggests that Dre2 is the probable target of Artemisinin and the antimalarial activity of DHA/Artemether could also be due to yet unidentified molecular mechanism altering the Dre2 activity in addition to inducing DNA and protein damage.
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    A prospective study on adult patients of severe malaria caused by Plasmodium falciparum, Plasmodium vivax and mixed infection from Bikaner, northwest India
    (JVBD, 2014-10) Garg, Shilpi; Das, Ashis
    Description of severe vivax malaria and mixed species infection requires good clinical study. The present study was undertaken to evalute the characteristics of severe malaria patients in Bikaner, northwest India.
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    Novel point mutations in sulfadoxine resistance genes of Plasmodium falciparum from India
    (Elsiever, 2009-04) Garg, Shilpi; Saxena, Vishal; Das, Ashis
    Point mutations in the dhfr and dhps genes of Plasmodium falciparum are associated with pyrimethamine and sulfadoxine resistance respectively. In this study we have analyzed these genes from Bikaner (situated in North-West region of India), where both uncomplicated and severe manifestations of P. falciparum malaria are seen. A majority of isolates showed double mutant allele for DHFR. In contrast, the only reported mutation present in DHPS was A437G in few isolates. In addition, three novel non-synonymous mutations were observed in the PfDHPS from this region viz., S587F, N666K and C668W. The mutations at the 666 and 668 codon seem to form a bend in the big loop region of the DHPS enzyme and may affect the binding of the drug to the enzyme. Molecular docking of sulfadoxine to this mutated structure indicates reduction in its binding affinity to this enzyme
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    Thrombocytopenia in childhood malaria with special reference to P. vivax monoinfection: A study from Bikaner (Northwestern India)
    (Taylor & Francis, 2011-08-24) Garg, Shilpi; Das, Ashis
    Thrombocytopenia is commonly seen in Plasmodium vivax malaria, but its prognostic value has not been addressed in children. This prospective study included 676 admitted children of malaria [Plasmodium falciparum (Pf) monoinfection 262, Plasmodium vivax (Pv) monoinfection 380, and mixed (Pf + Pv) infection 34], in which thrombocytopenia (platelet count <150 × 103/mm3 on admission) was found in 442 (65.38%) children [Pf monoinfection 55.3% (145/262), Pv monoinfection 73.16% (278/380), and mixed infection 55.88% (19/34)]. The association of thrombocytopenia was statistically significant with Pv monoinfection [73.16% (278/380)] in comparison to either Pf monoinfection [55.34% (145/262); odds ratio (OR) = 2.199 (95% confidence interval (CI) 1.577–3.068), p < 0.0001] or mixed infection [55.88% (19/34); OR = 2.152 (95%CI 1.054–4.394), p = 0.032]. In Pv monoinfection, thrombocytopenia was highest in 0–5 years age group and subsequently decreased with advancing age, whereas in Pf monoinfection it was reverse. Severe thrombocytopenia (platelet count <20 × 103/mm3) was present in 16.52% (73/442) children [Pv monoinfection 21.58% (60/278) and Pf monoinfection 8.97% (13/145)]. The risk of developing severe thrombocytopenia was also highest in Pv monoinfection [15.79% (60/380)] in comparison to Pf monoinfection [10.59% (13/262); OR = 3.591 (95%CI 1.928–6.690), p < 0.0001]. Bleeding manifestations were associated in 21.27% (94/442) children [Pf monoinfection 9.92% (26/262), Pv monoinfection 16.58% (63/380), and mixed malaria 14.71% (5/34)]. All the children having bleeding manifestations had thrombocytopenia but low platelet counts were not always associated with abnormal bleeding. The association of severe malaria was significantly more among children having Pv monoinfection with platelet counts <20 × 103/mm3 [OR = 2.569 (95%CI 1.196–5.517), p < 0.014] with specificity of 88.3% and positive predictive value of 85%. Till today, thrombocytopenia is not included in severe malaria criterion described by WHO, but when platelet counts <20 × 103/mm3, we advocate it to include as one of the severe malaria criteria.
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    An in vivo transcriptome data set of natural antisense transcripts from Plasmodium falciparum clinical isolates
    (Elsiever, 2014-12) Garg, Shilpi; Saxena, Vishal; Das, Ashis
    Antisense transcription is pervasive among biological systems and one of the products of antisense transcription is natural antisense transcripts (NATs). Emerging evidences suggest that they are key regulators of gene expression. With the discovery of NATs in Plasmodium falciparum, it has been suggested that these might also be playing regulatory roles in this parasite. However, all the reports describing the diversity of NATs have come from parasites in culture condition except for a recent study published by us. In order to explore the in vivo diversity of NATs in P. falciparum clinical isolates, we performed a whole genome expression profiling using a strand-specific 244 K microarray that contains probes for both sense and antisense transcripts. In this report, we describe the experimental procedure and analysis thereof of the microarray data published recently in Gene Expression Omnibus (GEO) under accession number GSE44921. This published data provide a wealth of information about the prevalence of NATs in P. falciparum clinical isolates from patients with diverse malaria related disease conditions. Supplementary information about the description and interpretation of the data can be found in a recent publication by Subudhi et al. in Experimental Parasitology (2014).
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    Natural antisense transcripts in Plasmodium falciparum isolates from patients with complicated malaria
    (Elsiever, 2014-06) Das, Ashis Kumar; Saxena, Vishal; Garg, Shilpi
    Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how the parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which are best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. Using a custom designed strand specific whole genome microarray, a total of 797 NATs targeted against annotated loci have been detected. Out of these, 545 NATs are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. However, 96 genes showed a change in sense/antisense ratio on comparison between uncomplicated and complicated disease conditions. The antisense transcripts map to a broad range of biochemical/metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action of NATs, which would further help in understanding the in vivo pathological adaptations of these parasites.