Browsing by Author "Das, Ashis Kumar"

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Conserved Plasmodium Protein (PF3D7_0406000) of Unknown Function: In-silico Analysis and Cellular Localization
(Elsevier, 2021-08) Das, Ashis Kumar
In spite of a decrease in malaria cases, the threat of malaria due to Plasmodium falciparum still prevails. The sequencing of Plasmodium falciparum reveals that approximately 60% of the Plasmodium genes code for hypothetical/putative proteins. Here we report an in silico characterization and localization of one such protein. This was encoded by one of the hub genes, in a weighted gene co-expression based systems network, from in-vivo samples of patients suffering from uncomplicated malaria or complicated malaria disease like jaundice and jaundice with renal failure. Interestingly, the protein PF3D7_0406000 (PFD0300w) is classified as a conserved protein of unknown function and shows no identity with any protein from the human host. The transcriptomic data shows up-regulation of transcripts in cases of malaria induced disease complications. PFD0300w peptide antibody based immunolocalization studies using a, gametocyte producing P. falciparum strain RKL-9, shows presence of the protein in the cytoplasm of both asexual and sexual stage parasites.
Conserved Plasmodium Protein (PF3D7_0406000) of Unknown Function: In-silico Analysis and Cellular Localization
(Elsiever, 2021-08) Das, Ashis Kumar
In spite of a decrease in malaria cases, the threat of malaria due to Plasmodium falciparum still prevails. The sequencing of Plasmodium falciparum reveals that approximately 60% of the Plasmodium genes code for hypothetical/putative proteins. Here we report an in silico characterization and localization of one such protein. This was encoded by one of the hub genes, in a weighted gene co-expression based systems network, from in-vivo samples of patients suffering from uncomplicated malaria or complicated malaria disease like jaundice and jaundice with renal failure. Interestingly, the protein PF3D7_0406000 (PFD0300w) is classified as a conserved protein of unknown function and shows no identity with any protein from the human host. The transcriptomic data shows up-regulation of transcripts in cases of malaria induced disease complications. PFD0300w peptide antibody based immunolocalization studies using a, gametocyte producing P. falciparum strain RKL-9, shows presence of the protein in the cytoplasm of both asexual and sexual stage parasites.
Design, construction and validation of a Plasmodium vivax microarray for the transcriptome profiling of clinical isolates
(Elsiever, 2016-12) Das, Ashis Kumar
High density oligonucleotide microarrays have been used on Plasmodium vivax field isolates to estimate whole genome expression. However, no microarray platform has been experimentally optimized for studying the transcriptome of field isolates. In the present study, we adopted both bioinformatics and experimental testing approaches to select best optimized probes suitable for detecting parasite transcripts from field samples and included them in designing a custom 15K P. vivax microarray. This microarray has long oligonucleotide probes (60 mer) that were in-situ synthesized onto glass slides using Agilent SurePrint technology and has been developed into an 8X15K format (8 identical arrays on a single slide). Probes in this array were experimentally validated and represents 4180 P. vivax genes in sense orientation, of which 1219 genes have also probes in antisense orientation. Validation of the 15K array by using field samples (n = 14) has shown 99% of parasite transcript detection from any of the samples. Correlation analysis between duplicate probes (n = 85) present in the arrays showed perfect correlation (r2 = 0.98) indicating the reproducibility. Multiple probes representing the same gene exhibited similar kind of expression pattern across the samples (positive correlation, r ≥ 0.6). Comparison of hybridization data with the previous studies and quantitative real-time PCR experiments were performed to highlight the microarray validation procedure. This array is unique in its design, and results indicate that the array is sensitive and reproducible. Hence, this microarray could be a valuable functional genomics tool to generate reliable expression data from P. vivax field isolates.
Disease specific modules and hub genes for intervention strategies: A co-expression network based approach for Plasmodium falciparum clinical isolates
(Elsiever, 2015-10) Das, Ashis Kumar
Systems biology approaches that are based on gene expression and bioinformatics analysis have been successful in predicting the functions of many genes in Plasmodium falciparum, a protozoan parasite responsible for most of the deaths due to malaria. However, approaches that can provide information about the biological processes that are active in this parasite in vivo during complicated malaria conditions have been scarcely deployed. Here we report the analysis of a weighted gene co-expression based network for P. falciparum, from non-cerebral clinical complications. Gene expression profiles of 20 P. falciparum clinical isolates were utilized to construct the same. A total of 20 highly interacting modules were identified post network creation. In 12 of these modules, at least 10% of the member genes, were found to be differentially regulated in parasites from patient isolates showing complications, when compared with those from patients with uncomplicated disease. Enrichment analysis helped identify biological processes like oxidation–reduction, electron transport chain, protein synthesis, ubiquitin dependent catabolic processes, RNA binding and purine nucleotide metabolic processes as associated with these modules. Additionally, for each module, highly connected hub genes were identified. Detailed functional analysis of many of these, which have known annotated functions underline their importance in parasite development and survival. This suggests, that other hub genes with unknown functions may also be playing crucial roles in parasite biology, and, are potential candidates for intervention strategies.
A glimpse into the clinical proteome of human malaria parasites Plasmodium falciparum and Plasmodium vivax
(Wiley, 2009-11-09) Das, Ashis Kumar; Garg, Shilpi
Malaria causes a worldwide annual mortality of about a million people. Rapidly evolving drug-resistant species of the parasite have created a pressing need for the identification of new drug targets and vaccine candidates. By developing fractionation protocols to enrich parasites from low-parasitemia patient samples, we have carried out the first ever proteomics analysis of clinical isolates of early stages of Plasmodium falciparum (Pf) and P. vivax. Patient-derived malarial parasites were directly processed and analyzed using shotgun proteomics approach using high-sensitivity MS for protein identification. Our study revealed about 100 parasite-coded gene products that included many known drug targets such as Pf hypoxanthine guanine phosphoribosyl transferase, Pf L-lactate dehydrogenase, and Plasmepsins. In addition, our study reports the expression of several parasite proteins in clinical ring stages that have never been reported in the ring stages of the laboratory-cultivated parasite strain. This proof-of-principle study represents a noteworthy step forward in our understanding of pathways elaborated by the parasite within the malaria patient and will pave the way towards identification of new drug and vaccine targets that can aid malaria therapy.
Microfluidic paper device with on-site heating to produce reactive peroxide species for enhanced smartphone enabled chemiluminescence signal
(Elsevier, 2022-01) Das, Ashis Kumar
Chemiluminescence signal amplification (CLSA) is of huge interest because of its sensitive detection in various applications such as food analysis, biomedical diagnosis and environmental monitoring. Due to this, there is a manifold attention to develop rapidly prototyped and miniaturized devices for CLSA. In this context, herein, a novel CLSA approach is demonstrated on a 3D printed microfluidic paper-based analytical device (μPADs), fabricated using Fused deposition modeling (FDM) printing technology. Influence of working temperature, ranging 30 °C–110 °C, on CL signal generation from well-established Luminol/Co+2 – H2O2 reaction was analyzed using a screen-printed flexible heater onto the 3D printed reaction platform. A smartphone-based capturing/detection system provided the amenability for a point-of-care testing system. For the first time, strong and stable CLSA was found with about 255% ± 5% increase in its signal intensity without using any additional external enhancers. The on-site working temperature was directly in proportional to the intensity of CL signal generated from Luminol/Co+2 – H2O2 reaction under optimum conditions, wherein the device had a wide linear range from 50 nM to 1 μM with a detection limit of 35 nM for H2O2 detection. The reliability of the developed amplification method was tested for practicability to detect the concentration of H2O2 in milk as real sample analysis. Overall, such CLSA mechanism in miniaturized μPADs will have strong potential for multiple CL based detection and monitoring application.
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.
Plasmodium falciparum complicated malaria: Modulation and connectivity between exportome and variant surface antigen gene families
(Elsiever, 2015-05) Das, Ashis Kumar
In temperate and sub-tropical regions of Asia and Latin America, complicated malaria manifested as hepatic dysfunction or renal dysfunction is seen in all age groups. There has been a concerted focus on understanding the patho-physiological and molecular basis of complicated malaria in children, much less is known about it in adults. We report here, the analysis of data from a custom, cross strain microarray (Agilent Platform) using material from adult patient samples, showing hepatic dysfunction or renal failure. These are the most common manifestations seen in adults along with cerebral malaria. The data has been analyzed with reference to variant surface antigens, encoded by the var, rifin and stevor gene families. The differential regulation profiles of key genes (comparison between Plasmodium falciparum complicated and uncomplicated isolates) have been observed. The exportome has been analyzed using similar parameters. Gene ontology term based functional enrichment of differentially regulated genes identified, up-regulated genes statistically enriched (P < 0.05) to critical biological processes like generation of precursor metabolite and energy, chromosome organization and electron transport chain. Systems network based functional enrichment of overall differentially regulated genes yielded a similar result. We are reporting here, up-regulation of var group B and C genes whose proteins are predicted to interact with CD36 receptor in the host, the up-regulation of domain cassette 13 (DC13) containing var group A, as also the up-regulation of group A rifins and many of the stevors. This is contrary to most other reports from pediatric patients, with cerebral malaria where the up-regulation of mostly var A group genes have been seen. A protein–protein interaction based network has been created and analysis performed. This co-expression and text mining based network has shown overall connectivity between the variant surface antigens (VSA) and the exportome. The up-regulation of var group B and C genes encoding PfEMP1 with different domain architecture would be important for deciding strategies for disease prevention.
Plasmodium vivax Malaria
(Emerging Infectious Diseases, 2005-01) Das, Ashis Kumar
We report 11 cases of severe Plasmodium vivax malaria in Bikaner (western India). Patients exhibited cerebral malaria, renal failure, circulatory collapse, severe anemia, hemoglobinurea, abnormal bleeding, acute respiratory distress syndrome, and jaundice. Peripheral blood microscopy, parasite antigen–based assays, and parasite 18s rRNA gene–based polymerase chain reaction showed the presence of P. vivax and absence of P. falciparum.
Reduction of Sphingosine Kinase 1 Phosphorylation and Activity in Plasmodium-Infected Erythrocytes
(Frontiers, 2020-03-03) Das, Ashis Kumar
Sphingosine-1-phosphate (S1P), a bioactive lipid mediator is involved in an array of biological processes and linked to pathological manifestations. Erythrocyte is known as the major reservoir for S1P as they lack S1P-degrading enzymes (S1P lyase and S1P phosphohydrolase) and harbor sphingosine kinase-1 (SphK-1) essential for sphingosine conversion to S1P. Reduced S1P concentration in serum was correlated with disease severity in patients with Plasmodium falciparum and Plasmodium vivax infections. Herein, we aimed to identify the underlying mechanism and contribution of host erythrocytes toward depleted S1P levels in Plasmodium-infected patients vs. healthy individuals. The level and activity of SphK-1 were measured in vitro in both uninfected and cultured P. falciparum-infected erythrocytes. Infected erythrocytes demonstrated a significant decrease in SphK-1 level in a time-dependent manner. We found that 10–42 h post invasion (hpi), SphK1 level was predominantly reduced to ∼50% in rings, trophozoites, and schizonts compared to uninfected erythrocytes. We next analyzed the phosphorylation status of SphK-1, a modification responsible for its activity and S1P production, in both uninfected control and Plasmodium-infected erythrocytes. Almost ∼50% decrease in phosphorylation of SphK-1 was observed that could be corroborated with significant reduction in the production and release of S1P in infected erythrocytes. Serum S1P levels were studied in parallel in P. falciparum (N = 15), P. vivax (N = 36)-infected patients, and healthy controls (N = 6). The findings revealed that S1P concentration was significantly depleted in uncomplicated malaria cases and was found to be lowest in complicated malaria and thrombocytopenia in both P. falciparum and P. vivax-infected groups (∗∗p < 0.01). The lower serum S1P level could be correlated with the reduced platelet count defining the role of S1P level in platelet formation. In conclusion, erythrocyte SphK-1 and S1P levels were studied in Plasmodium-infected individuals and erythrocytes that helped in characterizing the complications associated with malaria and thrombocytopenia, providing insights into the contribution of host erythrocyte biology in malaria pathogenesis. Finally, this study proposes the use of S1P and its analog as a novel adjunct therapy for malaria complications.
Revealing natural antisense transcripts from Plasmodium vivax isolates: Evidence of genome regulation in complicated malaria
(Elsiever, 2013-12) Das, Ashis Kumar; Garg, Shilpi; Saxena, Vishal
Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130–435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is less studied and poorly understood, in spite of these facts. Emerging evidence of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating the parasite directly from infected patients is the best way to study its biology and pathogenic mechanisms. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. The mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of Natural Antisense Transcripts (NATs) in Plasmodium falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed microarray with strand
A Tale of Two Parasites: A Glimpse into the RNA Methylome of Patient-derived P. falciparum and P. vivax isolates
(2024) Das, Ashis Kumar
Understanding the molecular mechanisms of the malarial parasites in hosts is crucial for developing effective treatments. Epitranscriptomic research on pathogens has unveiled the significance of RNA methylation in gene regulation and pathogenesis. We present the first report investigating methylation patterns determined using Nanopore Direct RNA Sequencing, to single-base resolution across RNA of P. falciparum and P. vivax from Malaria patients with hepatic dysfunction. Distinct RNA methylation profiles of N6-methyladenosine and 5-methylcytosine in the parasites suggest species-specific regulatory mechanisms. These modifications are unevenly present in the annotated regions of the mRNA, potentially influencing mRNA export and translation. Methylated antisense transcripts, splicing events and modified transcripts originating from apicoplast and mitochondrial genomes have been detected. We observe a striking overlap of differential methylation of isoforms with either of the modifications in both species. The correlation of these regulatory layers will decipher the post-transcriptional environment of malaria parasites in vivo and elucidate their inherent proteome plasticity.
An unexpected cause of fever and seizures
(Elsiever, 2008-09) Das, Ashis Kumar