understanding-development-compensatory-pathways-mutant-malaria

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases

One of the mechanisms for subverting the effect of drugs by&#160;the malaria parasite is through rewiring of its transcriptome. The effect is more ...

Jawaharlal Nehru University

Despite recent advances, the role of ROS in mediating hypertrophic and apoptotic responses in cardiac myocytes elicited by norepinephrine (NE) is rather poorly understood. We demonstrate through our experiments that H9c2 cardiac myoblasts treated with 2 µM NE (hypertrophic dose) generate DCFH-DA positive ROS only for 2h; while those treated with 100 µM NE (apoptotic dose) sustains generation for 48 h, followed by apoptosis. Though the levels of DCFH fluorescence were comparable at early time points in the two treatment sets, its quenching by DPI, catalase and MnTmPyP suggested the existence of a different repertoire of ROS. Both doses of NE also induced moderate levels of H2O2 but with different kinetics. Sustained but intermittent generation of highly reactive species detectable by HPF was seen in both treatment sets but no peroxynitrite was generated in either conditions. Sustained generation of hydroxyl radicals with no appreciable differences were noticed in both treatment sets. Nevertheless, despite similar profile of ROS generation between the two conditions, extensive DNA damage as evident from the increase in 8-OH-dG content, formation of γ-H2AX and PARP cleavage was seen only in cells treated with the higher dose of NE. We therefore conclude that hypertrophic and apoptotic doses of NE generate distinct but comparable repertoire of ROS/RNS leading to two very distinct downstream responses.

Norepinephrine-induced apoptotic and hypertrophic responses in H9c2 cardiac myoblasts are characterized by different repertoire of reactive oxygen species generation.

Radiotherapy is the biggest force acting behind cancer treatment, yet the vast majority of patients get only modest benefit. The successive failure of targeted therapies in radiotherapy lies in the non-discriminative killing of both normal and cancer cells. However, there is still a reason for optimism due to recent advancement made in cancer biology which unrevealed many new deregulated pathways in cancer and their response towards drug and radiation. In this review, we comprehensively discussed novel and promising druggable target which can be exploited for tumor radiosensitization in addition to normal tissue radioprotection in radiotherapy, for better tumor controllability and patient quality of life. In the last part, we also discussed the radiation countermeasure agents in brief.

Emerging targets for radioprotection and radiosensitization in radiotherapy.

Aqueous bark extract of Terminalia arjuna (TA) has been in use as an ethnomedicine for cardiovascular ailments in the Indian subcontinent for centuries. Studies using hemodynamic, ROS scavenging and anti-inflammatory parameters in animal models have shown its anti-atherogenic, hypotensive, inotropic, anti-inflammatory effects. However, details analysis on its effects on established molecular and cell biological markers are a prerequisite for its wider acceptance to the medical community.

Proteomic analysis of the protective effects of aqueous bark extract of Terminalia arjuna (Roxb.) on isoproterenol-induced cardiac hypertrophy in rats.

Present study was designed to investigate the role of acemannan acetyl group in murine radioprotection. Acemannan, extracted from Aloe vera gel, has molecular weight of 1.02 × 10 Da and mainly composed of mannose (84.9%), glucose (7.2%), and galactose (3.9%), with backbone of (1→4)-linked mannose and glucose. Acemannan was over-acetylated and deacetylated to investigate the role of acetyl group. Acetylation enhances acemannan viscosity and thermal stability. Free radicals scavenging and Fenton reaction inhibition was mediated by acemannan acetyl and hydroxyl group respectively. Native or over-acetylated or deacetylated acemannan pre-treatment to mice has shown to reduce the γ-radiation-induced oxidative damage, and hematopoietic injuries by free radical scavenging and microphage activation (secretes pro-hematopoietic factors through TLR-4) respectively. Over-acetylated acemannan has stronger effects on immunomodulation/radioprotection. In summary, acemannan acetyl-group modulates immune system, while hydroxyl-group participate in free radical scavenging, and present finding can be employed in food and pharma industries for enhancing polysaccharide bioactivity.

Role of acemannan O-acetyl group in murine radioprotection.

Aqueous extract of the bark of  (TA) is used by a large population in the Indian subcontinent for treating various cardiovascular conditions. Animal experiments have shown its anti-atherogenic, anti-hypertensive, and anti-inflammatory effects. It has several bioactive ingredients with hemodynamic, ROS scavenging, and anti-inflammatory properties. Earlier we have done limited proteomic and transcriptomic analysis to show its efficacy in ameliorating cardiac hypertrophy induced by isoproterenol (ISO) in rats. In the present study we have used high-throughput sequencing of the mRNA from control and treated rat heart to further establish its efficacy. ISO (5 mg/kg/day s.c.) was administered in male adult rats for 14 days to induce cardiac hypertrophy. Standardized aqueous extract TA bark extract was administered orally. Total RNA were isolated from control, ISO, ISO + TA, and TA treated rat hearts and subjected to high throughput sequence analysis. The modulations of the transcript levels were then subjected to bio-informatics analyses using established software. Treatment with ISO downregulated 1,129 genes and upregulated 204 others. Pre-treatment with the TA bark extracts markedly restored that expression pattern with only 97 genes upregulated and 85 genes downregulated. The TA alone group had only 88 upregulated and 26 downregulated genes. The overall profile of expression in ISO + TA and TA alone groups closely matched with the control group. The genes that were modulated included those involved in metabolism, activation of receptors and cell signaling, and cardiovascular and other diseases. Networks associated with those genes included those involved in angiogenesis, extracellular matrix organization, integrin binding, inflammation, drug metabolism, redox metabolism, oxidative phosphorylation, and organization of myofibril. Overlaying of the networks in ISO and ISO_TA group showed that those activated in ISO group were mostly absent in ISO_TA and TA group, suggesting a global effect of the TA extracts. This study for the first time reveals that TA partially or completely restores the gene regulatory network perturbed by ISO treatment in rat heart; signifying its efficacy in checking ISO-induced cardiac hypertrophy.

Transcriptomic Validation of the Protective Effects of Aqueous Bark Extract of  (Roxb.) on Isoproterenol-Induced Cardiac Hypertrophy in Rats.

Mental health in old age is of great concern due to the increased incidence of neurological and psychiatric diseases. With age, probability of cognitive and behavioral deficits increase and the prognosis deteriorates. Although a few in vitro studies have reported that flavonoid fisetin is beneficial for healthy aging, its effect on deteriorating mental health with aging in vivo is very limited and poorly understood. The brain aging is physiologically characterized by electroencephalograph (EEG) wave frequency, power, and distribution. Brain oscillatory waves from neural tissue get altered by various sensory-cognitive inputs. Besides, the fast-wave α(8-12 Hz)- and β(12-28 Hz)-oscillations are associated with coordination and indeed deal with complex behavioral performances. Therefore, the effect of fisetin supplementation on age-associated EEG mean cortical spectral power in α- and β-oscillations, multi-unit activity (MUA) count were studied in vivo which was not addressed so far. Besides, age-associated cognitive and behavioral alterations were also studied. The relative spectral power of α and β declined along with the MUA count in aged rats compared to young. However, supplementing fisetin for four weeks has improved relative α-power, β-power, and MUA count in aged rats. Also, fisetin supplemented aged rats showed significantly improved cognitive and behavioral performances than aged controls. These findings demonstrated the relative cortical spectral power in α-, β-oscillations, and MUA count change in aged rats and that some of these changes and behavioral alterations may be partly as a result of oxidative stress, which was prevented significantly in fisetin supplemented aged rats. Thus, fisetin boosted mental health in the aged animals.

Fisetin prevents the aging-associated decline in relative spectral power of α, β and linked MUA in the cortex and behavioral alterations.

Precise gene editing techniques are paramount to gain deeper insights into the biological processes such as host-parasite interactions, drug resistance mechanisms, and gene-function relationships. Discovery of CRISPR-Cas9 system has spearheaded mechanistic understanding of protozoan parasite biology as evident from the number of reports in the last decade. Here, we have described the use of CRISPR-Cas9 in understanding the biology of medically important protozoan parasites such as Plasmodium, Leishmania, Trypanosoma, Babesia and Trichomonas. In spite of intrinsic difficulties in genome editing in these protozoan parasites, CRISPR-Cas9 has acted as a catalyst for faster generation of desired transgenic parasites. Modifications in the CRISPR-Cas9 system for improving the efficiency have been useful in better understanding the molecular mechanisms associated with repair of double strand breaks in the parasites. Moreover, improvement in reagents used for CRISPR mediated gene editing have been instrumental in addressing the issue of non-specificity and toxicity for therapeutic use. These application-based modifications may help in further increasing the efficiency of gene editing in protozoan parasites.

CRISPRing protozoan parasites to better understand the biology of diseases.

Calcium ions are used as important signals during various physiological processes. In malaria parasites,  spp., calcium dependent protein kinases (CDPKs) have acquired the unique ability to sense and transduce calcium signals at various critical steps during the lifecycle, either through phosphorylation of downstream substrates or mediating formation of high molecular weight protein complexes. Calcium signaling cascades establish important crosstalk events with signaling pathways mediated by other secondary messengers such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). CDPKs play critical roles at various important physiological steps during parasite development in vertebrates and mosquitoes. They are also important for transmission of the parasite between the two hosts. Combined with the fact that CDPKs are not present in humans, they continue to be pursued as important targets for development of anti-malarial drugs.

CDPKs: The critical decoders of calcium signal at various stages of malaria parasite development.

The protein-protein interaction (PPI) network of the Mediator complex is very tightly regulated and depends on different developmental and environmental cues. Here, we present an interactive platform for comparative analysis of the Mediator subunits from humans, baker's yeast Saccharomyces cerevisiae, and model plant Arabidopsis thaliana in a user-friendly web-interface database called MediatorWeb. MediatorWeb provides an interface to visualize and analyze the PPI network of Mediator subunits. The database facilitates downloading the untargeted and unweighted network of Mediator complex, its submodules, and individual Mediator subunits to better visualize the importance of individual Mediator subunits or their submodules. Further, MediatorWeb offers network visualization of the Mediator complex and interacting proteins that are functionally annotated. This feature provides clues to understand functions of Mediator subunits in different processes. In an additional tab, MediatorWeb provides quick access to secondary and tertiary structures, as well as residue-level contact information for Mediator subunits in each of the three model organisms. Another useful feature of MediatorWeb is detection of interologs based on orthologous analyses, which can provide clues to understand the functions of Mediator complex in less explored kingdoms. Thus, MediatorWeb and its features can help the user to understand the role of Mediator complex and its subunits in the transcription regulation of gene expression.

MediatorWeb: a protein-protein interaction network database for the RNA polymerase II Mediator complex.

Conditional regulation is a highly beneficial system for studying the function of essential genes in Plasmodium falciparum and dimerizable Cre recombinase (DiCre) is a recently adapted conditional regulation system suitable for this purpose. In the DiCre system, two inactive fragments of Cre are reconstituted to form a functionally active enzyme in the presence of rapamycin. Different loci have been targeted to generate parasite lines that express the DiCre enzyme. Here, we have used marker-free CRISPR-Cas9 gene editing to integrate the DiCre cassette in a redundant cg6 locus. We have shown the utility of the newly generated ∆cg6DC4 parasites in mediating robust, rapid, and highly specific excision of exogenously encoded gfp sequence. The ∆cg6DC4 parasites are also capable of conditional excision of an endogenous parasite gene, PF3D7_1246000. Conditional deletion of PF3D7_1246000 did not cause any inhibition in the asexual proliferation of the parasites. Furthermore, the health and morphology of the mutant parasites were comparable to that of the control parasites in Giemsa smears. The availability of another stable DiCre parasite strain competent for conditional excision of target genes will expedite functional characterization and validation of novel drug and vaccine targets against malaria.

Article	Year
Norepinephrine-induced apoptotic and hypertrophic responses in H9c2 cardiac myoblasts are characterized by different repertoire of reactive oxygen species generation. Redox biology\| PubMed ID: 26070033	2015
Emerging targets for radioprotection and radiosensitization in radiotherapy. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine\| PubMed ID: 27318945	2016
Proteomic analysis of the protective effects of aqueous bark extract of Terminalia arjuna (Roxb.) on isoproterenol-induced cardiac hypertrophy in rats. Journal of ethnopharmacology\| PubMed ID: 28063919	2017
Role of acemannan O-acetyl group in murine radioprotection. Carbohydrate polymers\| PubMed ID: 30600029	2019
Transcriptomic Validation of the Protective Effects of Aqueous Bark Extract of (Roxb.) on Isoproterenol-Induced Cardiac Hypertrophy in Rats. Frontiers in pharmacology\| PubMed ID: 31920643	2019
Fisetin prevents the aging-associated decline in relative spectral power of α, β and linked MUA in the cortex and behavioral alterations. Experimental gerontology\| PubMed ID: 32592831	2020
CRISPRing protozoan parasites to better understand the biology of diseases. Progress in molecular biology and translational science\| PubMed ID: 33934837	2021
CDPKs: The critical decoders of calcium signal at various stages of malaria parasite development. Computational and structural biotechnology journal\| PubMed ID: 34589185	2021
MediatorWeb: a protein-protein interaction network database for the RNA polymerase II Mediator complex. The FEBS journal\| PubMed ID: 38975839	2024
Generation of a new DiCre expressing parasite strain for functional characterization of Plasmodium falciparum genes in blood stages. Scientific reports\| PubMed ID: 39402380	2024

Manish Sharma

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