Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes

Introduction: In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination. Method: EVs released from three different kinds of cell lines: HMC-1, TF-1 and BV-2 were isolated using two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer®. Results: RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF-1-derived vesicles, as these cells do not express CD9. Conclusions: Our results demonstrate that centrifugation-based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and morphological characteristics, but they are indistinguishable using CD63-coated beads for flow cytometry analysis

Microguards and micromessengers of the genome

The regulation of gene expression is of fundamental importance to maintain organismal function and integrity and requires a multifaceted and highly ordered sequence of events. The cyclic nature of gene expression is known as ‘transcription dynamics’. Disruption or perturbation of these dynamics can result in significant fitness costs arising from genome instability, accelerated ageing and disease. We review recent research that supports the idea that an important new role for small RNAs, particularly microRNAs (miRNAs), is in protecting the genome against short-term transcriptional fluctuations, in a process we term ‘microguarding’. An additional emerging role for miRNAs is as ‘micromessengers’—through alteration of gene expression in target cells to which they are trafficked within microvesicles. We describe the scant but emerging evidence that miRNAs can be moved between different cells, individuals and even species, to exert biologically significant responses. With these two new roles, miRNAs have the potential to protect against deleterious gene expression variation from perturbation and to themselves perturb the expression of genes in target cells. These interactions between cells will frequently be subject to conflicts of interest when they occur between unrelated cells that lack a coincidence of fitness interests. Hence, there is the potential for miRNAs to represent both a means to resolve conflicts of interest, as well as instigate them. We conclude by exploring this conflict hypothesis, by describing some of the initial evidence consistent with it and proposing new ideas for future research into this exciting topic

Rule-based modeling of biochemical systems with BioNetGen

Totowa, NJ. Please cite this article when referencing BioNetGen in future publications. Rule-based modeling involves the representation of molecules as structured objects and molecular interactions as rules for transforming the attributes of these objects. The approach is notable in that it allows one to systematically incorporate site-specific details about proteinprotein interactions into a model for the dynamics of a signal-transduction system, but the method has other applications as well, such as following the fates of individual carbon atoms in metabolic reactions. The consequences of protein-protein interactions are difficult to specify and track with a conventional modeling approach because of the large number of protein phosphoforms and protein complexes that these interactions potentially generate. Here, we focus on how a rule-based model is specified in the BioNetGen language (BNGL) and how a model specification is analyzed using the BioNetGen software tool. We also discuss new developments in rule-based modeling that should enable the construction and analyses of comprehensive models for signal transduction pathways and similarly large-scale models for other biochemical systems. Key Words: Computational systems biology; mathematical modeling; combinatorial complexity; software; formal languages; stochastic simulation; ordinary differential equations; protein-protein interactions; signal transduction; metabolic networks. 1

Large oncosomes contain distinct protein cargo and represent a separate functional class of tumor-derived extracellular vesicles

Large oncosomes (LO) are atypically large (1-10 mu m diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrep (TM)) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.1182Ysciescopu

An approach for the identification of targets specific to bone metastasis using cancer genes interactome and gene ontology analysis

Metastasis is one of the most enigmatic aspects of cancer pathogenesis and is a major cause of cancer-associated mortality. Secondary bone cancer (SBC) is a complex disease caused by metastasis of tumor cells from their primary site and is characterized by intricate interplay of molecular interactions. Identification of targets for multifactorial diseases such as SBC, the most frequent complication of breast and prostate cancers, is a challenge. Towards achieving our aim of identification of targets specific to SBC, we constructed a 'Cancer Genes Network', a representative protein interactome of cancer genes. Using graph theoretical methods, we obtained a set of key genes that are relevant for generic mechanisms of cancers and have a role in biological essentiality. We also compiled a curated dataset of 391 SBC genes from published literature which serves as a basis of ontological correlates of secondary bone cancer. Building on these results, we implement a strategy based on generic cancer genes, SBC genes and gene ontology enrichment method, to obtain a set of targets that are specific to bone metastasis. Through this study, we present an approach for probing one of the major complications in cancers, namely, metastasis. The results on genes that play generic roles in cancer phenotype, obtained by network analysis of 'Cancer Genes Network', have broader implications in understanding the role of molecular regulators in mechanisms of cancers. Specifically, our study provides a set of potential targets that are of ontological and regulatory relevance to secondary bone cancer.Comment: 54 pages (19 pages main text; 11 Figures; 26 pages of supplementary information). Revised after critical reviews. Accepted for Publication in PLoS ON

Ameliorative Effect of Plant Growth Promoting Rhizobacteria Enhancing micronutrient uptake of Groundnut (Arachis hypogaea. L).

Groundnut (Arachis hypogaea L.) is a premier oil seed crop in India occupying 45 per cent of total oil seed production. Though India leads both in area and production of groundnut, the country ranks eight in productivity due to fertilizer management and erratic response of the crop in terms of yield. Innovative agriculture is an immediate need of today’s world that has increased tremendously in its population. Hence, we need to increase the agricultural produce substantially with the help of plant growth promoting rhizobacteria. A pot experiment was undertaken under botanical garden in Annamalai university, with three bacterial strains (Rhizobium sp.), (Pseudomonas sp.) and (Bacillus sp.), applied in our experiment.  The treatment involving consortium inoculation with Rhizobium + Pseudomonas + Bacillus recorded highest values of Mg, Zn and Cu can improve nutrient uptake with a shoot portion of groundnut when compared to the root portion

Sustainable cultivation practices and market trends of Indian sandalwood: A comprehensive review

Indian sandalwood, renowned for its aromatic heartwood and essential oil, holds a revered status in traditional practices and modern industries. Its essential oil, celebrated for its exquisite fragrance, is a staple in perfumery, cosmetics and traditional medicine due to its antimicrobial, anti-inflammatory and anti-cancer properties.Culturally, sandalwood is deeply embedded in Indian traditions, where it is used in religious ceremonies and for crafting sacred artifacts symbolizing purity and serenity. From an economic perspective, the high demand for sandalwood, coupled with its slow growth rate, presents significant challenges in its cultivation and conservation. This review addresses the over-exploitation of natural sandalwood resources, emphasizing the need for sustainable cultivation practices to ensure its future availability. such practices involve enhancing genetic strains, adapting to climate change and promoting biodiversity through agroforestry systems that provide multiple income streams for farmers. The review also highlights the critical challenges in cultivating sandalwood, such as its speciesspecific climatic requirements and susceptibility to pests and diseases. These challenges are compounded by the economic risks associated with the high value of sandalwood and its long maturity period, which can deter investment and conservation efforts. Technological advancements play a pivotal role in addressing these challenges, with innovations in biotechnology and precision agriculture enhancing yields and sustainability. The review advocates for a balanced approach that meets commercial demands while preserving ecological and cultural integrity. It calls for continued research, international cooperation and adaptive management strategies to secure a sustainable future for Indian Sandalwood cultivation and trade. Additionally, the review emphasizes the need for integrated approaches combining traditional knowledge with modern scientific and technological advances to optimize sandalwood cultivation

Growth factor stimulation of cardiomyocytes induces changes in the transcriptional contents of secreted exosomes

Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in targets cells. We recently reported that cultured cardiomyocytes are able to release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the transcriptional content of the released exosomes.Exosomes were isolated from media collected from cultured cardiomyocytes (HL-1) with or without growth factor treatment (TGF-β2 and PDGF-BB), with a series of differential centrifugations, including preparative ultracentrifugation and separation with a sucrose gradient. The exosomes were characterized with dynamic light scattering (DLS), electron microscopy (EM) and Western blot and analyzed with Illumina whole genome microarray gene expression.The exosomes were rounded in shape and had an average size of 50–90 nm in diameter with no difference between treatment groups. Analysis of the mRNA content in repeated experiments conclusively revealed 505 transcripts in the control group, 562 in the TGF-β2-treated group and 300 in the PDGF-BB-treated group. Common transcripts (217) were found in all 3 groups.We show that the mode of stimulation of parental cells affects the characteristics of exosomes released. Hence, there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated, or not stimulated, with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system. To access the supplementary material to this article, please see Supplementary files under Article Tools online

A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells

Betulinic acid (BA), a member of pentacyclic triterpenes has shown important biological activities like anti-bacterial, anti-malarial, anti-inflammatory and most interestingly anticancer property. To overcome its poor aqueous solubility and low bioavailability, structural modifications of its functional groups are made to generate novel lead(s) having better efficacy and less toxicity than the parent compound. BA analogue, 2c was found most potent inhibitor of colon cancer cell line, HT-29 cells with IC50 value 14.9 μM which is significantly lower than standard drug 5-fluorouracil as well as parent compound, Betulinic acid. We have studied another mode of PCD, autophagy which is one of the important constituent of cellular catabolic system as well as we also studied proteasomal degradation pathway to investigate whole catabolic pathway after exploration of 2c on HT-29 cells. Mechanism of autophagic cell death was studied using fluorescent dye like acridine orange (AO) and monodansylcadaverin (MDC) staining by using fluorescence microscopy. Various autophagic protein expression levels were determined by Western Blotting, qRT-PCR and Immunostaining. Confocal Laser Scanning Microscopy (CLSM) was used to study the colocalization of various autophagic proteins. These were accompanied by formation of autophagic vacuoles as revealed by FACS and transmission electron microscopy (TEM). Proteasomal degradation pathway was studied by proteasome-Glo™ assay systems using luminometer.The formation of autophagic vacuoles in HT-29 cells after 2c treatment was determined by fluorescence staining – confirming the occurrence of autophagy. In addition, 2c was found to alter expression levels of different autophagic proteins like Beclin-1, Atg 5, Atg 7, Atg 5-Atg 12, LC3B and autophagic adapter protein, p62. Furthermore we found the formation of autophagolysosome by colocalization of LAMP-1 with LC3B, LC3B with Lysosome, p62 with lysosome. Finally, as proteasomal degradation pathway downregulated after 2c treatment colocalization of ubiquitin with lysosome and LC3B with p62 was studied to confirm that protein degradation in autophagy induced HT-29 cells follows autolysosomal pathway. In summary, betulinic acid analogue, 2c was able to induce autophagy in HT-29 cells and as proteasomal degradation pathway downregulated after 2c treatment so protein degradation in autophagy induced HT-29 cell