Biology subjects

Gopalakrishnan, R.

Publications and source records attributed to Gopalakrishnan, R..

6 recordsLinked to original sources

bioRxiv2025

Enhanced delivery of lipid nanoparticle-based immunotherapy by modulating the tumor tissue stiffness using ultrasound-activated nanobubbles

Tumors often exhibit an extracellular matrix with elevated stiffness due to excessive accumulation and crosslinking of proteins, particularly collagen. This elevated stiffness acts as a physical barrier, impeding the infiltration of immune cells and the effective delivery of various immunotherapeutic agents, such as lipid nanoparticle-based RNA therapeutics. Here, we investigate the ability of ultrasound-activated nanobubbles (US-NBs) to increase the permeability and immunogenicity of tumors. Our results show that US-NBs physically remodel the tumor tissue by decreasing its stiffness by 60% five days after a single treatment. US-NB-treated tumors display randomly oriented collagen with a 5.47-fold lower deposition compared to untreated tumors. This leads to the effective delivery and widespread distribution of lipid nanoparticles (LNPs) in the tumor. When LNPs are assisted by US-NB, they have higher gene-transfection across pan-immune cells relative to LNPs alone. Notably, US-NB enables LNPs to genetically modify T cells directly in vivo. By effectively engaging both arms of the immune system, US-NB-assisted LNPs enhance the tumor immunogenicity and infiltration of cytotoxic cells by 4-fold when compared to LNPs alone. These results indicate that gentle mechanical stimulation of the tumor using US-NB offers a promising strategy to augment the delivery and efficacy of existing immunotherapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/648512v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@da7fborg.highwire.dtl.DTLVardef@8f5f27org.highwire.dtl.DTLVardef@b002f6org.highwire.dtl.DTLVardef@19265da_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Robust prediction of relative binding energies for protein-protein complex mutations using free energy perturbation calculations

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/590325v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c5e607org.highwire.dtl.DTLVardef@1810ee5org.highwire.dtl.DTLVardef@1f8f1acorg.highwire.dtl.DTLVardef@c28053_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIReliable calculation of relative binding free energy changes for most protein mutations to within [~]1 kcal/mol. C_LIO_LIAutomated Protein FEP+ Groups treatment of alternate protonation states for titratable residues. C_LIO_LIApplication of FEP+ methodology to "real-world" protein design projects. C_LI

biophysics↗

bioRxiv2022

Suppressor mutations that make the essential transcription factor Spn1/Iws1 dispensable in Saccharomyces cerevisiae

Spn1/Iws1 is an essential eukaryotic transcription elongation factor that is conserved from yeast to humans as an integral member of the RNA polymerase II elongation complex. Several studies have shown that Spn1 functions as a histone chaperone to control transcription, RNA splicing, genome stability, and histone modifications. However, the precise role of Spn1 is not understood, and there is little understanding of why it is essential for viability. To address these issues, we have isolated eight suppressor mutations that bypass the essential requirement for Spn1 in Saccharomyces cerevisiae. Unexpectedly, the suppressors identify several functionally distinct complexes and activities, including the histone chaperone FACT, the histone methyltransferase Set2, the Rpd3S histone deacetylase complex, the histone acetyltransferase Rtt109, the nucleosome remodeler Chd1, and a member of the SAGA co-activator complex, Sgf73. The identification of these distinct groups suggests that there are multiple ways in which Spn1 bypass can occur, including changes in histone acetylation and alterations of other histone chaperones. Thus, Spn1 may function to overcome repressive chromatin by multiple mechanisms during transcription. Our results suggest that bypassing a subset of these functions allows viability in the absence of Spn1.

molecular biology↗

bioRxiv2021

The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions

The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5 ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.

molecular biology↗

bioRxiv2020

Detection of 'relict' western lineage of citrus tristeza virus virulent genotype in declining Arunachal Wakro orange

Eastern Himalayan foothills are known to have optimal agro-climatic conditions for production of quality citrus fruits including oranges. Among the citrus growing regions of eastern Himalayas, Wakro in the far eastern state of Arunachal Pradesh is known for its superior quality oranges, popular as the Wakro orange or the Arunachal orange, which has been included in Geographical Indication Registry by the Government of India. However, during last few years, Arunachal orange orchards have been experiencing severe infestation of aphid associated with rapid decline disease, causing catastrophe to the farmers as well as state economy. Therefore, in 2015, an intensive survey of severely affected orange orchards was carried out to investigate the aetiological factors of citrus decline. RNA samples extracted from leaf and aphid specimens collected from Wakro orchards were subjected to CTV detection through 3-UTR specific RT-PCR. Subsequently, ORF1 and CP genetic regions were amplified and clonal-sequencing was performed. Although, BLAST search showed close homology of the present sequences with other virulent genotype VT sequences, detailed phylogenetic analysis demonstrated affinity and clustering of present sequences with VT sequences belonging to the western lineage. This finding is considerably distinct from CTV sequences reported from citrus growing orchards in India and other neighbouring countries. Additionally, low diversity of CP gene sequences, recombination patterns and presence of sequence segments identical to the present ones in other CTV genotypes was also revealed. Collectively, these observations indicate pristine and primeval nature of present CTV sequences, corroborating well with the proposed origin of CTV in this part of the globe. We here report our finding of western lineage of CTV virulent genotype VT, which is distinct from CTV molecular epidemiology in other parts of India and discuss the implications of these findings.

microbiology↗

bioRxiv2018

A conserved genetic interaction between Spt6 and Set2 regulates H3K36 methylation

The transcription elongation factor Spt6 and the H3K36 methyltransferase Set2 are both required for H3K36 methylation and transcriptional fidelity in Saccharomyces cerevisiae. By selecting for suppressors of a transcriptional defect in an spt6 mutant, we have isolated dominant SET2 mutations (SET2sup mutations) in a region encoding a proposed autoinhibitory domain. The SET2sup mutations suppress the H3K36 methylation defect in the spt6 mutant, as well as in other mutants that impair H3K36 methylation. ChIP-seq studies demonstrate that the H3K36 methylation defect in the spt6 mutant, as well as its suppression by a SET2sup mutation, occur at a step following the recruitment of Set2 to chromatin. Other experiments show that a similar genetic relationship between Spt6 and Set2 exists in Schizosaccharomyces pombe. Taken together, our results suggest a conserved mechanism by which the Set2 autoinhibitory domain requires multiple interactions to ensure that H3K36 methylation occurs specifically on actively transcribed chromatin.

genetics↗