Biology subjects

Ratnayake, S.

Publications and source records attributed to Ratnayake, S..

8 recordsLinked to original sources

bioRxiv2025

A simple liquid 3D cell culture paradigm models oxidative mitochondrial metabolism of epithelial breast cancer cells with relevance for lung metastases.

Three-dimensional (3D) cell culture systems have emerged as powerful tools for modeling tumor biology in ex vivo settings. However, the diverse array of available 3D culture methods presents challenges in selecting the most appropriate model for specific research questions. This study provides a comparative analysis of breast cancer cells (SUM149, IBC-3, and MDA-MB-468) in the mammosphere culture (SphC) model or an "emboli" culture (EmC) model, which enrich for cancer stem cells and epithelial features, respectively. The EmC model, designed originally for inflammatory breast cancer, is characterized by media viscosity and mechanical rocking of the culture vessel. Notably, cells in EmC showed a distinct and durable reduction in cell proliferation ex vivo while demonstrating increased capacity to establish experimental lung metastases in vivo. Ultrastructural quantitative analysis of electron microscopy images suggested that cells in EmC acquire nuclear and mitochondrial features that resemble those of tumor tissue. Proteomics, single-cell transcriptomics, and metabolic flux analyses showed that cells in EmC and SphC favor mitochondrial oxidative metabolism (OXPHOS) and glycolysis, respectively. EmC rendered cells hypersensitive to OXPHOS inhibition, but more resistant to oxidative stress. Several genes associated with lung metastasis, including ID1, were specifically enriched in EmC. Given the emerging role of OXPHOS in cancer cell survival during dissemination and as established metastases, we propose that the EmC paradigm is a suitable ex vivo model to study signaling pathways relevant for tumor tissue and to assess drug sensitivities and resistance mechanisms of metastatic breast cancer cells ex vivo. SIGNIFICANCEThis study provides an in-depth characterization of a resource-efficient yet powerful 3D culture paradigm to improve the physiological relevance of ex vivo approaches. Applicable to epithelial cancers, this model offers a platform to accelerate the discovery of physiologically relevant signaling pathways and specific cancer cell vulnerabilities.

cancer biology↗

bioRxiv2024

TRAIL-induced cytokine production via NFKB2 pathway promotes neutrophil chemotaxis and immune suppression in triple negative breast cancers

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic that induces apoptosis in cancer cells while sparing the non-malignant cells in preclinical models. However, its efficacy in clinical trials has been limited, suggesting unknown modulatory mechanisms responsible for the lack of TRAIL activity in patients. Here, we hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer (TNBC) cells that alter the immune milieu. To test this, we performed an RNAseq analysis of MDA-MB-231 cells treated with TRAIL, followed by validation in additional TNBC cell lines. TRAIL significantly induces expression of multiple cytokines such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to modify neutrophil function. Mechanistically, the induction of these cytokines was predominantly mediated by death receptor 5, caspase 8 (but not caspase 8 enzymatic activity), and the non-canonical NFKB2 pathway. The cytokines produced by the TRAIL-treated TNBC cells enhanced chemotaxis of healthy human donor isolated neutrophils. In vivo, TRAIL treated TNBC murine xenograft tumors showed activation of the NFKB2 pathway, elevated production of CXCLs and IL-6, and increased neutrophil recruitment into the tumors. Moreover, donor isolated neutrophils preincubated in supernatants from TRAIL-treated TNBC cells exhibited impaired cytotoxic effect against TNBC cells. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC cells revealed increased expression of inflammatory cytokines, immune modulatory genes, immune checkpoint genes, and genes implicated in delayed neutrophil apoptosis. Functional studies with these neutrophils confirmed their suppressive effect on T cell proliferation and an increase in Treg suppressive phenotype. Collectively, our study demonstrates a novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and immune suppression.

cancer biology↗

bioRxiv2024

A transposable element insertion in IAA16 interrupts normal splicing and generates a novel dicamba resistance allele in Bassia scoparia

A dicamba-resistant population of kochia (Bassia scoparia) identified in Colorado, USA in 2012 was used to generate a synthetic mapping population that segregated for dicamba resistance. Linkage mapping associating dicamba injury with genotype derived from restriction-site-associated DNA sequencing identified a single locus in the kochia genome associated with resistance on chromosome 4. A mutant version of Auxin/Indole-3-Acetic Acid 16 (AUX/IAA16; a gene previously implicated in dicamba resistance in kochia) was found near the middle of this locus in resistant plants. Long read sequencing of dicamba-resistant plants identified a recently inserted Ty1/Copia retrotransposon near the beginning of the second exon of AUX/IAA16, leading to disruption of normal splicing. A molecular marker for this insertion allows for rapid detection of resistance. Stable transgenic lines of Arabidopsis thaliana ectopically expressing the mutant and wildtype alleles of AUX/IAA16 were developed. Arabidopsis thaliana plants expressing the mutant AUX/IAA16 allele grew shorter roots on control media. However, transgenic root growth was less inhibited on media containing either dicamba (5 M) or IAA (0.5 M) when compared to non-transgenic plants or those expressing the wildtype allele of AUX/IAA16. In vitro assays indicate reduced binding affinity and more rapid dissociation of the mutant AUX/IAA with TIR1 in the presence of several auxins, and protein modeling suggests the substitution of the glycine residue in the degron domain of AUX/IAA16 is especially important for resistance. A fitness cost associated with the mutant allele of AUX/IAA16 has implications for resistance evolution and management of kochia populations with this resistance mechanism. SignificanceAuxin mimics are amongst the most important herbicides in modern agriculture. Evolution of weeds that are resistant to these herbicides threatens sustainable crop production. Understanding the basis of auxin herbicide resistance informs the development of improved weed control technologies. Additionally, auxin-resistant mutations and their pleotropic effects help us understand auxin perception and signalling. We describe a transposable element insertion within an herbicide target site gene that alters splicing and reduces synthetic and natural auxin perception.

plant biology↗

bioRxiv2024

The ESCRT protein CHMP5 promotes T cell leukemia by controlling BRD4-p300-dependent transcription

Oncogene activity rewires cellular transcription, creating new transcription networks to which cancer cells become addicted, by mechanisms that are still poorly understood. Using human and mouse models of T cell acute lymphoblastic leukemia (T-ALL), we identify an essential nuclear role for CHMP5, a cytoplasmic endosomal sorting complex required for transport (ESCRT) protein, in establishing and maintaining the T-ALL transcriptional program. Nuclear CHMP5 promoted the T-ALL gene program by augmenting recruitment of the co-activator BRD4 by the histone acetyl transferase p300 selectively at enhancers and super-enhancers, an interaction that potentiated H3K27 acetylation at these regulatory enhancers. Consequently, loss of CHMP5 diminished BRD4 occupancy at enhancers and super-enhancers and impaired RNA polymerase II pause release, which resulted in downregulation of key T-ALL genes, notably MYC. Reinforcing its importance in T-ALL pathogenesis, CHMP5 deficiency mitigated chemoresistance in human T-ALL cells and abrogated T-ALL induction by oncogenic NOTCH1 in vivo. Thus, the ESCRT protein CHMP5 is an essential positive regulator of the transcriptional machinery promoting T-ALL disease. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/577409v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@191e10dorg.highwire.dtl.DTLVardef@4e5093org.highwire.dtl.DTLVardef@18c0a08org.highwire.dtl.DTLVardef@e6a887_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIIdentification of a nuclear role for the cytosolic ESCRT protein CHMP5 in transcription C_LIO_LICHMP5 mediates BRD4-dependent Pol II pause release and transcription of T-ALL genes C_LIO_LIP300-BRD4 induced enhancer and super-enhancer H3K27 acetylation requires CHMP5 C_LIO_LICHMP5 depletion mitigates chemoresistance and abrogates T-ALL initiation in vivo C_LI

cancer biology↗

bioRxiv2022

Gene regulatory network inference in soybean upon infection by Phytophthora sojae

Phytophthora sojae is a soil-borne oomycete and the causal agent of Phytophthora root and stem rot (PRR) in soybean (Glycine max [L.] Merrill). Yield losses attributed to P. sojae are devastating in disease-conducive environments, with global estimates surpassing 1.1 million tonnes annually. Historically, management of PRR has entailed host genetic resistance (both vertical and horizontal) complemented by disease-suppressive cultural practices (e.g., oomicide application). However, the vast expansion of complex and/or diverse P. sojae pathotypes necessitates developing novel technologies to attenuate PRR in field environments. Therefore, the objective of the present study was to couple high-throughput sequencing data and deep learning to elucidate molecular features in soybean following infection by P. sojae. In doing so, we generated transcriptomes to identify differentially expressed genes (DEGs) during compatible and incompatible interactions with P. sojae and a mock inoculation. The expression data were then used to select two defense-related transcription factors (TFs) belonging to WRKY and RAV families. DNA Affinity Purification and sequencing (DAP-seq) data were obtained for each TF, providing putative DNA binding sites in the soybean genome. These bound sites were used to train Deep Neural Networks with convolutional and recurrent layers to predict new target sites of WRKY and RAV family members in the DEG set. Moreover, we leveraged publicly available Arabidopsis (Arabidopsis thaliana) DAP-seq data for five TF families enriched in our transcriptome analysis to train similar models. These Arabidopsis data-based models were used for cross-species TF binding site prediction on soybean. Finally, we created a gene regulatory network depicting TF-target gene interactions that orchestrate an immune response against P. sojae. Information herein provides novel insight into molecular plant-pathogen interaction and may prove useful in developing soybean cultivars with more durable resistance to P. sojae. Author SummaryGlobal food security is threatened continually by plant pathogens. One approach to circumvent these disease-causing agents entails understanding how hosts balance primary growth and defense upon pathogen perception. Molecular signatures of perception-rendered defense may be leveraged subsequently to develop resistant/tolerant crop plants. Additionally, evidence suggests that the plant immune system is characterized by tuning primary and secondary metabolic activity via transcription factor-mediated transcriptional reprogramming. Therefore, we investigated transcription factor-target gene interactions in soybean upon infection by compatible and incompatible races of Phytophthora sojae. Through transcriptome analysis, we found that the interactions elicited vast, overlapping transcriptional responses and identified overrepresented, defense-related transcription factor families. We then generated/acquired DNA-protein interactome data for the most represented transcription factor families in the transcriptome analysis and trained deep learning-based models to predict novel transcription factor targets. Transcription factor/target gene metrics were used to construct a gene regulatory network with prioritized components. We identified hub transcription factors belonging to WRKY and ERF families, the majority of which function in response to various biotic and abiotic stressors. These findings propose novel regulators in the soybean defense response to Phytophthora sojae and provide an avenue for the investigation of transcription factor-target gene interactions in plants.

plant biology↗

bioRxiv2022

Evidence for the digital programming of cellular differentiation: characterization of binary switches in lineage-determining transcription factors

Previous studies of the murine Ly49 and human KIR gene clusters have revealed a role for bidirectional promoters in the control of variegated gene expression. Whether or not competing promoters control other instances of cell fate determination remains an outstanding question. Although divergent transcripts within 300 bp are found in ~6% of human genes, an analysis of human transcription factor (TF) genes (1640) revealed that 33% possess stable divergent transcripts with a transcription start site (TSS) less than 300 bp upstream, with many separated by less than 30 bp, indicating an enrichment for potential binary switches in TFs. We have performed a detailed examination of putative bidirectional promoter switches in three lineage-determining TF genes: AHR, GATA3, and ROR{gamma}T. These genes also contain additional pairs of opposing promoters that would prevent simultaneous transcription of sense and antisense, and thus may represent simple on/off switches rather than probabilistic switches. In situ RNA hybridization of human tissues revealed mutually exclusive expression of sense versus antisense transcription, indicating switching between stable sense or antisense transcriptional states. Single-cell RNAseq confirmed the separate sense/antisense states, and revealed the identity of cells with active switch elements. Differential gene expression analysis of cells with antisense switch transcripts revealed an enrichment for genes found in immature/stem cells and a lack of genes associated with terminally differentiated cells. Taken together, these data indicate that there is a digital component to the differentiation program mediated by binary promoter switches in lineage-determining transcription factors.

developmental biology↗

bioRxiv2021

Single cell profiling of the Timp gene family

Tissue inhibitor of metalloproteinases (TIMPs/Timps) are an endogenous family of widely expressed matrisome-associated proteins that were initially identified as inhibitors of matrix metalloproteinase activity (Metzincin family proteases). Consequently, TIMPs are often considered simply as protease inhibitors by many investigators. However, an evolving list of new metalloproteinase-independent functions for TIMP family members suggests that this concept is outdated. These novel TIMP functions include direct agonism/antagonism of multiple transmembrane receptors, as well as functional interactions with matrisome targets. While the family was fully identified over two decades ago, there has yet to be an in-depth study describing the expression of TIMPs in normal tissues of adult mammals. An understanding of the tissues and cell-types that express TIMPs 1 through 4, in both normal and disease states are important to contextualize the growing functional capabilities of TIMP proteins, which are often dismissed as non-canonical. Using publicly available single cell RNA sequencing data from the Tabula Muris Consortium, we analyzed approximately 100,000 murine cells across eighteen tissues from non-diseased organs, representing seventy-three annotated cell types, to define the diversity in Timp gene expression across healthy tissues. We describe the unique expression profiles across tissues and organ-specific cell types that all four Timp genes display. Within annotated cell-types, we identify clear and discrete cluster-specific patterns of Timp expression, particularly in cells of stromal and endothelial origins. RNA in-situ hybridization across four organs expands on the scRNA sequencing analysis, revealing novel compartments associated with individual Timp expression. These analyses provide evidence of the biological significance of Timp expression in the identified cell sub-types, which are consistent with novel roles in normal tissue homeostasis and changing roles in disease progression. This understanding of the tissues, specific cell types and microenvironment conditions in which Timp genes are expressed adds important physiological context to the growing array of novel functions for TIMP proteins.

cell biology↗

bioRxiv2017

BLAST-based validation of metagenomic sequence assignments

When performing bioforensic casework, it is important to be able to reliably detect the presence of a particular organism in a metagenomic sample, even if the organism is only present in a trace amount. For this task, it is common to use a sequence classification program that determines the taxonomic affiliation of individual sequence reads by comparing them to reference database sequences. As metagenomic data sets often consist of millions or billions of reads that need to be compared to reference databases containing millions of sequences, such sequence classification programs typically use search heuristics and databases with reduced sequence diversity to speed up the analysis, which can lead to incorrect assignments. Thus, in a bioforensic setting where correct assignments are paramount, assignments of interest made by \"first-pass\" classifiers should be confirmed using the most precise methods and comprehensive databases available. In this study we present a O_SCPLOWBLASTC_SCPLOW-based method for validating the assignments made by less precise sequence classification programs, with optimal parameters for filtering of O_SCPLOWBLASTC_SCPLOW results determined via simulation of sequence reads from genomes of interest, and we apply the method to the detection of four pathogenic organisms. The software implementing the method is open source and freely available.

bioinformatics↗