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Biology subjects

Pant, P.

Publications and source records attributed to Pant, P..

8 recordsLinked to original sources

Higher frequency of transition mutation over transversion mutation in genomes: evidence from the binding energy calculation of base pairs using DFT

Base substitution mutations such as transition (ti) and transversion (tv) in organisms are major driving force in molecular evolution. In this study, different possible types of base pairing that can cause ti and tv were investigated using the density functional theory (DFT) method. The chemical structures of bases as well as base pairs were optimized using B3LYP hybrid functional along with 6-31G(d,p) basis set. We performed single point energy calculation of all optimized species using the same functional but combined with higher diffuse and polarized basis set i.e. 6-311++G(d,p) to get more refined energy of all species. The binding energy of various base pairs was calculated considering basis set superposition error (BSSE) as well as without BSSE. The binding energy of the base pairs leading to ti was found to be more stable than that of the base pairs leading to tv. This was interesting considering the observations in organisms that tis are more frequent than tvs. Among the base pairs leading to the same ti, G(keto): T (enol) base pair was found to be more stable than A(imino):C(amino) base pair. This theoretical study of binding energy of different base pairs using the DFT method has provided additional evidences in support to the biological observations of a higher transition rate than transversion in genomes.

molecular biology↗

Comparative Transcriptomics of Arabidopsis, Medicago, Brachypodium and Setaria species during Phosphorus limitation

Translating biological knowledge from Arabidopsis to crop species is important to advance agriculture and secure food production in the face of dwindling fertilizer resources, biotic and abiotic stresses. However, it is often not trivial to identify functional homologs (orthologs) of Arabidopsis genes in crops. Combining sequence and expression data can improve the correct prediction of orthologs. Here, we conducted a large-scale RNA sequencing based transcriptomics study of Arabidopsis, Medicago, Brachypodium and Setaria grown side-by-side in Phosphorus (P)-sufficient and P-limited conditions to generate comparable transcriptomics datasets. Comparison of top 200 P-limitation induced genes in Arabidopsis revealed that [~]80% of these genes have identifiable close homologs in the other three species but only [~]50% retain their P-limitation response in the legume and grasses. Most of the hallmark genes of the P-starvation response were found conserved in all four species. This study reveals many known, novel, unannotated, conserved and species-specific regulations of the transcriptional P-starvation response. Identification and experimental verification of expressologs by independent RT-qPCR for P-limitation marker genes in Prunus showed the usefulness of comparative transcriptomics in pinpointing the functional orthologs in diverse crop species. This study provides an unprecedented resource for functional genomics and translational research to create P-efficient crops. HIGHLIGHTComparative transcriptomics reveals novel, known, conserved and specific transcriptome responding to Phosphorus limitation in Arabidopsis, Medicago, Brachypodium and Setaria to facilitate translational research in crops.

molecular biology↗

The long non-coding RNA NEAT1 regulates the transcriptional landscape of cardiomyocytes

Long non-coding RNAs (lncRNAs) play a crucial role in fine-tuning the gene expression. In the present study, we identified that the lncRNA NEAT1 is upregulated in failing murine hearts and in human hypertrophic cardiomyopathies. Further investigations demonstrated that NEAT1 expression is regulated by calcium dependent NFAT (nuclear factor of activated T cells). Overexpression of NEAT1 led to increased cardiomyocyte size and elevated expression of cardiac stress markers while its depletion had the opposite effect. Through transcriptomic analysis in NEAT1-KO cells, we demonstrated the cis-regulatory role of NEAT1, wherein it maintains the transcription of neighboring genes by interacting with the coactivator p300. Additionally, NEAT1 depletion resulted in decreased expression of GRK2 (G protein-coupled receptor kinase 2), a key player in the development of cardiac hypertrophy, suggesting that NEAT1 contributes to the development of cardiac hypertrophy by regulating GRK2 expression. Consistent with this, Neat1-/- mice are resistant to {beta}-adrenergic stimulation. Overall, our study provides enough evidence that NEAT1 is a calcium-regulated, pro-hypertrophic lncRNA that exerts significant control over the transcriptional landscape of cardiomyocytes during heart failure.

cell biology↗

Musashi-2 causes cardiac hypertrophy and heart failure by inducing mitochondrial dysfunction through destabilizing Cluh and Smyd1 mRNA

Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 are known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains entirely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 was significantly enriched in the hearts cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified, Msi2 isoforms 1, 4, and 5 and two novel putative isoforms labeled as Msi2 isoforms 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 was also found to be significantly increased in a pressure-overload model of cardiac hypertrophy. To validate the hypertrophic effects, we selected isoforms 4 and 7 due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, and transmembrane potential measurement assays increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream targets of Msi2. Overexpression of Cluh or Smyd1 inhibited Msi2-induced hypertrophy and mitochondrial dysfunction in cardiomyocytes. Collectively, we show that Msi2 induces hypertrophy, mitochondrial dysfunction, and heart failure.

cell biology↗

Zika virus E protein alters blood-brain barrier by modulating brain microvascular endothelial cell and astrocyte functions.

Neurotropic viruses can cross the otherwise dynamically regulated blood-brain barrier (BBB) and affect the brain cells. Zika virus (ZIKV) is an enveloped neurotropic Flavivirus known to cause severe neurological complications, such as encephalitis and foetal microcephaly. In the present study, we used human brain microvascular endothelial cells (hBMECs) and human progenitor derived astrocytes to form a physiologically relevant BBB model. We used this model to investigate the effects of ZIKV envelope (E) protein on properties of cells comprising the BBB. E protein is the principal viral protein involved in interaction with host cell surface receptors, facilitating the viral entry. Our findings show that ZIKV E protein results in activation of both hBMECs and astrocytes. hBMECs showed reduced expression of endothelial junction proteins - ZO-1, Occludin and VE-Cadherin, which are crucial in establishing and maintaining the BBB. As a result, ZIKV E protein triggered alteration in BBB integrity and permeability. We also found upregulation of genes involved in leukocyte recruitment along with increased proinflammatory chemokines and cytokines upon exposure to E protein. Furthermore, E protein resulted in astrogliosis as seen by increased expression of GFAP and Vimentin. Both BBB cell types exhibited inflammatory response following exposure to E protein which may influence viral access into the central nervous system (CNS), resulting in infection of other CNS cells. Overall, our study provides valuable insights into the transient changes that occur at the site of BBB upon ZIKV infection.

neuroscience↗

Differentially accessible Cdc4 phospho-degrons regulate Ctf19CCAN kinetochore subunit stability in mitosis

Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. How effective, yet strictly centromere-dependent kinetochore assembly is coupled to cell cycle progression is incompletely understood. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-Phase and targets the protein for degradation. Binding of the Mtw1 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature-sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic to cells, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits protects against ectopic kinetochore assembly and contributes to mitotic checkpoint silencing. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.

cell biology↗

NAChRDB: A Web Resource of Structure-Function Annotations to Unravel the Allostery of Nicotinic Acetylcholine Receptors

SummaryDue to their paramount importance, near-ubiquitous presence, and complex nature, nicotinic acetylcholine receptors (nAChRs) have remained the focus of intensive research for over 50 years. The vast amount of knowledge accumulated on the topic has become extremely difficult to navigate. NAChRDB addresses this challenge by providing web-based, real-time access to curated residue-level functional annotations of neuromuscular nAChRs with interactive 3D visualization and sequence alignment. NAChRDB provides systematic access to experimental observations and predictions from computational studies reported in the literature or performed specifically to complement current knowledge, which allows new findings to be interpreted in a more holistic context, both from a structural and a functional perspective. NAChRDB aims to serve as an invaluable resource for identifying gaps in knowledge and for guiding discovery through structural and molecular biology experiments, especially when exploring the allosteric mechanisms underlying neuromuscular nAChR function and pathology. Availability and ImplementationNAChRDB is freely available online, with a self-explanatory interface and useful tool tips (https://crocodile.ncbr.muni.cz/Apps/NAChRDB/). No installation or user registration is required. NAChRDB content is stored in .json format, queried using Python, and rendered in browser using Javascript and WebGL (LiteMol). NAChRDB is highly responsive and accessible through any modern Internet browser on desktop and mobile devices. Contactjkoca@ceitec.cz Supplementary informationSupplementary data are available online.

bioinformatics↗

Charge-perturbation dynamics - a new avenue towards in silico protein folding

Molecular dynamics (MD) has greatly contributed to understanding and predicting the way proteins fold. However, the time-scale and complexity of folding are not accessible via classical MD. Furthermore, efficient folding pipelines involving enhanced MD techniques are not routinely accessible. We aimed to determine whether perturbing the electrostatic component of the MD force field can help expedite folding simulations. We developed charge-perturbation dynamics (CPD), an MD-based simulation approach that involves periodically perturbing the atomic charges to values non-native to the MD force field. CPD obtains suitable sampling via multiple iterations in which a classical MD segment (with native charges) is followed by a very short segment of perturbed MD (using the same force field and conditions, but with non-native charges); subsequently, partially folded intermediates are refined via a longer segment of classical MD. Among the partially folded structures from low-energy regions of the free-energy landscape sampled, the lowest-energy conformer with high root-mean-square deviation to the starting structure and low radius of gyration is defined as the folded structure. Upon benchmark testing, we found that medium-length peptides such as an alanine-based pentadecapeptide, an amyloid-{beta} peptide, and the tryptophan-cage mini-protein can fold starting from their extended linear structure in under 45 ns of CPD (total simulation time), versus over 100 ns of classical MD. CPD not only achieved folding close to the desired conformation but also sampled key intermediates along the folding pathway without prior knowledge of the folding mechanism or final folded structure. Our findings confirmed that perturbing the electrostatic component of the classical MD force field can help expedite folding simulations without changing the MD algorithm or using expensive computing architectures. CPD can be employed to probe the folding dynamics of known, putative, or planned peptides, as well as to improve sampling in more advanced simulations or to guide further experiments.\n\nAuthor summaryFolding represents the process by which proteins assemble into biologically active conformations. While computational techniques such as molecular dynamics (MD) have provided invaluable insight into protein folding, efficient folding pipelines are not routinely accessible. In MD, the behavior of the studied molecule is simulated under the concerted action of multiple forces described by mathematical functions employing optimized parameters. Using non-native parameters effectively perturbs the MD force field. We show that this can be exploited to help expedite folding simulations. Specifically, we developed charge-perturbation dynamics (CPD), an MD-based simulation approach that involves periodically perturbing the force field by using non-native atomic charges. For folding medium-length peptides such as the tryptophan-cage mini-protein starting from the extended linear structure, CPD is much faster than other MD-based approaches while using the same software, hardware, and know-how required for running classical MD simulations. Furthermore, CPD not only achieves folding close to the desired conformation but also samples key intermediates along the folding pathway without prior knowledge of the folding mechanism or final folded structure. CPD can be employed to probe the folding dynamics of known, putative, or planned peptides, as well as to generate different conformations that can guide further experiments or more advanced simulations.

bioinformatics↗