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Frontiers in Microbiology 2024Small RNA (sRNA) molecules, a class of non-coding RNAs, have emerged as pivotal players in the regulation of gene expression and cellular processes. and other... (Review)
Review
Small RNA (sRNA) molecules, a class of non-coding RNAs, have emerged as pivotal players in the regulation of gene expression and cellular processes. and other pathogenic mycobacteria produce diverse small RNA species that modulate bacterial physiology and pathogenesis. Recent advances in RNA sequencing have enabled identification of novel small RNAs and characterization of their regulatory functions. This review discusses the multifaceted roles of bacterial small RNAs, covering their biogenesis, classification, and functional diversity. Small RNAs (sRNAs) play pivotal roles in orchestrating diverse cellular processes, ranging from gene silencing to epigenetic modifications, across a broad spectrum of organisms. While traditionally associated with eukaryotic systems, recent research has unveiled their presence and significance within bacterial domains as well. Unlike their eukaryotic counterparts, which primarily function within the context of RNA interference (RNAi) pathways, bacterial sRNAs predominantly act through base-pairing interactions with target mRNAs, leading to post-transcriptional regulation. This fundamental distinction underscores the necessity of elucidating the unique roles and regulatory mechanisms of bacterial sRNAs in bacterial adaptation and survival. By doing these myriad functions, they regulate bacterial growth, metabolism, virulence, and drug resistance. In , apart from having various roles in the bacillus itself, small RNA molecules have emerged as key regulators of gene expression and mediators of host-pathogen interactions. Understanding sRNA regulatory networks in mycobacteria can drive our understanding of significant role they play in regulating virulence and adaptation to the host environment. Detailed functional characterization of Mtb sRNAs at the host-pathogen interface is required to fully elucidate the complex sRNA-mediated gene regulatory networks deployed by Mtb, to manipulate the host. A deeper understanding of this aspect could pave the development of novel diagnostic and therapeutic strategies for tuberculosis.
PubMed: 38903780
DOI: 10.3389/fmicb.2024.1399280 -
Journal of Nanobiotechnology Jun 2024Breast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the...
BACKGROUND
Breast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the human epidermal growth factor receptor 2 (HER2). A quantitative evaluation of HER2 levels is essential for a correct diagnosis, selection of the most appropriate therapeutic strategy and monitoring the response to therapy.
RESULTS
In this paper, we propose the synergistic use of SERS and Raman technologies for the identification of HER2 expressing cells and its accurate assessment. To this end, we selected SKBR3 and MDA-MB-468 breast cancer cell lines, which have the highest and lowest HER2 expression, respectively, and MCF10A, a non-tumorigenic cell line from normal breast epithelium for comparison. The combined approach provides a quantitative estimate of HER2 expression and visualization of its distribution on the membrane at single cell level, clearly identifying cancer cells. Moreover, it provides a more comprehensive picture of the investigated cells disclosing a metabolic signature represented by an elevated content of proteins and aromatic amino acids. We further support these data by silencing the HER2 gene in SKBR3 cells, using the RNA interference technology, generating stable clones further analysed with the same combined methodology. Significant changes in HER2 expression are detected at single cell level before and after HER2 silencing and the HER2 status correlates with variations of fatty acids and downstream signalling molecule contents in the context of the general metabolic rewiring occurring in cancer cells. Specifically, HER2 silencing does reduce the growth ability but not the lipid metabolism that, instead, increases, suggesting that higher fatty acids biosynthesis and metabolism can occur independently of the proliferating potential tied to HER2 overexpression.
CONCLUSIONS
Our results clearly demonstrate the efficacy of the combined SERS and Raman approach to definitely pose a correct diagnosis, further supported by the data obtained by the HER2 gene silencing. Furthermore, they pave the way to a new approach to monitor the efficacy of pharmacologic treatments with the aim to tailor personalized therapies and optimize patients' outcome.
Topics: Humans; Spectrum Analysis, Raman; Receptor, ErbB-2; Breast Neoplasms; Cell Line, Tumor; Female; Gene Silencing; Metal Nanoparticles
PubMed: 38902746
DOI: 10.1186/s12951-024-02600-7 -
Signal Transduction and Targeted Therapy Jun 2024This study aimed to develop a pan-genotypic and multifunctional small interfering RNA (siRNA) against hepatitis B virus (HBV) with an efficient delivery system for...
This study aimed to develop a pan-genotypic and multifunctional small interfering RNA (siRNA) against hepatitis B virus (HBV) with an efficient delivery system for treating chronic hepatitis B (CHB), and explore combined RNA interference (RNAi) and immune modulatory modalities for better viral control. Twenty synthetic siRNAs targeting consensus motifs distributed across the whole HBV genome were designed and evaluated. The lipid nanoparticle (LNP) formulation was optimized by adopting HO-PEG-DMG lipid and modifying the molar ratio of traditional polyethylene glycol (PEG) lipid in LNP prescriptions. The efficacy and safety of this formulation in delivering siHBV (tLNP/siHBV) along with the mouse IL-2 (mIL-2) mRNA (tLNP/siHBVIL2) were evaluated in the rAAV-HBV1.3 mouse model. A siRNA combination (terms "siHBV") with a genotypic coverage of 98.55% was selected, chemically modified, and encapsulated within an optimized LNP (tLNP) of high efficacy and security to fabricate a therapeutic formulation for CHB. The results revealed that tLNP/siHBV significantly reduced the expression of viral antigens and DNA (up to 3log reduction; vs PBS) in dose- and time-dependent manners at single-dose or multi-dose frequencies, with satisfactory safety profiles. Further studies showed that tLNP/siHBVIL2 enables additive antigenic and immune control of the virus, via introducing potent HBsAg clearance through RNAi and triggering strong HBV-specific CD4 and CD8 T cell responses by expressed mIL-2 protein. By adopting tLNP as nucleic acid nanocarriers, the co-delivery of siHBV and mIL-2 mRNA enables synergistic antigenic and immune control of HBV, thus offering a promising translational therapeutic strategy for treating CHB.
Topics: Animals; Mice; Hepatitis B virus; Interleukin-2; Humans; RNA, Small Interfering; Nanoparticles; RNA, Messenger; Hepatitis B, Chronic; RNA Interference; Hepatitis B; RNAi Therapeutics; Liposomes
PubMed: 38902241
DOI: 10.1038/s41392-024-01871-8 -
MBio Jun 2024Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising...
Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes , , and ) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP levels derepress the regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP toxicity is alleviated by deletion of , the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating () allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of hyper-repressed expression in phosphate-replete cells; suppressed the derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious lncRNA termination); and delayed induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the and promoters, but not for the promoters that drive the synthesis of the -repressive lncRNAs. Transcription profiling of ∆ and () cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast genes , , and by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic alleles derepress the genes via the action of IP as an agonist of precocious lncRNA 3'-processing/termination. IP toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP levels on termination. In this study, a forward genetic screen revealed that IP toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of -activating transcription factor Pho7 to its binding sites in the mRNA promoters.
PubMed: 38899862
DOI: 10.1128/mbio.01252-24 -
Journal of Nanobiotechnology Jun 2024Tumor-associated macrophages (TAMs) are a promising target for cancer immunotherapy, but delivering therapeutic agents to TAMs within the tumor microenvironment (TME) is...
Tumor-associated macrophages (TAMs) are a promising target for cancer immunotherapy, but delivering therapeutic agents to TAMs within the tumor microenvironment (TME) is challenging. In this study, a photosensitive, dual-targeting nanoparticle system (M.RGD@Cr-CTS-siYTHDF1 NPs) was developed. The structure includes a shell of DSPE-modified RGD peptides targeting integrin receptors on tumor cells and carboxymethyl mannose targeting CD206 receptors on macrophages, with a core of chitosan adsorbing m6A reading protein YTHDF1 siRNA and chromium nanoparticles (Cr NPs). The approach is specifically designed to target TAM and cancer cells, utilizing the photothermal effect of Cr NPs to disrupt the TME and deliver siYTHDF1 to TAM. In experiments with tumor-bearing mice, M.RGD@Cr-CTS-siYTHDF1 NPs, when exposed to laser irradiation, effectively killed tumor cells, disrupted the TME, delivered siYTHDF1 to TAMs, silenced the YTHDF1 gene, and shifted the STAT3-STAT1 equilibrium by reducing STAT3 and enhancing STAT1 expression. This reprogramming of TAMs towards an anti-tumor phenotype led to a pro-immunogenic TME state. The strategy also suppressed immunosuppressive IL-10 production, increased expression of immunostimulatory factors (IL-12 and IFN-γ), boosted CD8 + T cell infiltration and M1-type TAMs, and reduced Tregs and M2-type TAMs within the TME. In conclusion, the dual-targeting M.RGD@Cr-CTS-siYTHDF1 NPs, integrating dual-targeting capabilities with photothermal therapy (PTT) and RNA interference, offer a promising approach for molecular targeted cancer immunotherapy with potential for clinical application.
Topics: Animals; Mice; Immunotherapy; RNA, Small Interfering; Humans; Liver Neoplasms; Cell Line, Tumor; Tumor Microenvironment; Tumor-Associated Macrophages; RNA-Binding Proteins; Nanoparticles; Metal Nanoparticles; Photosensitizing Agents
PubMed: 38898486
DOI: 10.1186/s12951-024-02612-3 -
Plant Cell Reports Jun 2024Recently published high-quality reference genome assemblies indicate that, in addition to RDR1-deficiency, the loss of several key RNA silencing-associated genes may...
Recently published high-quality reference genome assemblies indicate that, in addition to RDR1-deficiency, the loss of several key RNA silencing-associated genes may contribute to the hypersusceptibility of Nicotiana benthamiana to viruses.
Topics: Nicotiana; RNA Interference; Plant Diseases; Plant Viruses; Plant Proteins; Genes, Plant; Gene Expression Regulation, Plant
PubMed: 38898307
DOI: 10.1007/s00299-024-03262-3 -
Scientific Reports Jun 2024Legumin A is a seed storage protein that provides nutrients for seed germination. The purpose of this study was to describe the structure and expression pattern of the...
Legumin A is a seed storage protein that provides nutrients for seed germination. The purpose of this study was to describe the structure and expression pattern of the EuLEGA gene in Eucommia ulmoides Oliver (E. ulmoides) and to infer its functional role. The 1287 bp coding sequence of the EuLEGA CDS of the EuLEGA gene, encoding a protein containing 428 amino acid residues, was cloned. The structure predicted that the protein belonged to the RmlC (deoxythymidine diphosphates, dTDP)-4-dehydrorhamnose 3,5-epimerase)-like cupin conserved domain family, which contains both RmlC, a key enzyme for the synthesis of rhamnose and legumin A. The overexpression (OE) vector of the EuLEGA gene was constructed and genetically transformed into tobacco and E. ulmoides; the RNA interference (RNAi) vector of the EuLEGA gene was constructed and genetically transformed into E. ulmoides; and the contents of legumin A and rhamnose were detected. The results showed that the EuLEGA gene could significantly increase the content of legumin A in transgenic tobacco leaves and transgenic E. ulmoides regenerative buds, and the OE of this gene in E. ulmoides could promote an increase in rhamnose content. RNAi caused a significant decrease in the legumin A content in the regenerated buds of E. ulmoides. These was a significant increase in legumin A in the transgenic tobacco seeds, and these results indicate that the expression of the EuLEGA gene is closely related to the accumulation of legumin A. Subcellular localization studies revealed that EuLEGA is localized to the cytoplasm with the vacuolar membrane. Analysis of the EuLEGA gene expression data revealed that the expression level of the EuLEGA gene in the samaras was significantly greater than that in the leaves and stems. In addition, the study also demonstrated that GA can upregulate the expression levels of the EuLEGA gene, while ABA and MeJA can downregulate its expression levels.
Topics: Cloning, Molecular; Plants, Genetically Modified; Eucommiaceae; Gene Expression Regulation, Plant; Plant Proteins; Legumins; Nicotiana; Rhamnose; RNA Interference
PubMed: 38898092
DOI: 10.1038/s41598-024-65020-5 -
The Journal of Biological Chemistry Jun 2024Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer, and is the major autoantigen in membranous...
Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer, and is the major autoantigen in membranous nephropathy (MN), a rare but severe autoimmune kidney disease. A soluble form of PLA2R1 has been detected in mouse and human serum. It is likely produced by proteolytic shedding of membrane-bound PLA2R1 but the mechanism is unknown. Here, we show that human PLA2R1 is cleaved by A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 in HEK293 cells, mouse embryonic fibroblasts and human podocytes. By combining site-directed mutagenesis and sequencing, we determined the exact cleavage site within the extracellular juxtamembrane stalk of human PLA2R1. Orthologs and paralogs of PLA2R1 are also shed. By using pharmacological inhibitors and genetic approaches with RNA interference and knock-out cellular models, we identified a major role of ADAM10 in the constitutive shedding of PLA2R1, and a dual role of ADAM10 and ADAM17 in the stimulated shedding. We did not observe evidence for cleavage by β- or γ-secretase, suggesting that PLA2R1 may not be a substrate for Regulated Intramembrane Proteolysis. PLA2R1 shedding occurs constitutively and can be triggered by the calcium ionophore ionomycin, the protein kinase C inducer PMA, cytokines and lipopolysaccharides, in vitro and in vivo. Altogether, our results show that PLA2R1 is a novel substrate for ADAM10 and ADAM17, producing a soluble form that is increased in inflammatory conditions and likely exerts various functions in physiological and pathophysiological conditions including inflammation, cancer and MN.
PubMed: 38897568
DOI: 10.1016/j.jbc.2024.107480 -
Biomedicine & Pharmacotherapy =... Jun 2024Burkitt's lymphoma (BL) is a rare and highly aggressive B-cell non-Hodgkin lymphoma. Although the outcomes of patients with BL have greatly improved, options for...
Burkitt's lymphoma (BL) is a rare and highly aggressive B-cell non-Hodgkin lymphoma. Although the outcomes of patients with BL have greatly improved, options for patients with relapsed and refractory BL are limited. Therefore, there is an urgent need to improve BL therapeutics and to develop novel drugs with reduced toxicity. In this study, we demonstrated that enolase 1 (ENO1) is a potential novel drug target for BL treatment. We determined that ENO1 was aberrantly upregulated in BL, which was closely related to its invasiveness and poor clinical outcomes. Furthermore, using RNA interference, we demonstrated that ENO1 depletion significantly inhibited cell proliferation and invasion both in vitro and in vivo. Mechanistically, we established that ENO1 knockdown suppressed the PI3K-AKT and epithelial-mesenchymal transition (EMT) signaling pathways by reducing plasminogen (PLG) recruitment, plasmin (PL) generation, and TGF-β1 activation. Addition of activated TGF-β1 protein to the culture medium of shENO1 cells reversed the inhibitory effects on cell proliferation and invasion, as well as those on the PI3K-AKT and EMT signaling pathways. Notably, our research led to the discovery of a novel ENO1-PLG interaction inhibitor, Ciwujianoside E (L-06). L-06 effectively disrupts the interaction between ENO1 and PLG, consequently reducing PL generation and suppressing TGF-β1 activation. In both in vitro and in vivo experiments, L-06 exerted impressive antitumor effects. In summary, our study elucidated the critical role of ENO1 in BL cell proliferation and invasion and introduced a novel ENO1 inhibitor, which holds promise for improving the treatment of patients with BL in the future.
PubMed: 38897160
DOI: 10.1016/j.biopha.2024.116970 -
Frontiers in Plant Science 2024Plant viruses cause substantial losses in crop yield and quality; therefore, devising new, robust strategies to counter viral infections has important implications for...
Plant viruses cause substantial losses in crop yield and quality; therefore, devising new, robust strategies to counter viral infections has important implications for agriculture. Virus inhibitory protein endoplasmic reticulum-associated interferon-inducible (Viperin) proteins are conserved antiviral proteins. Here, we identified a set of Viperin and Viperin-like proteins from multiple species and tested whether they could interfere with RNA viruses . Our data from transient and stable overexpression of these proteins in reveal varying levels of interference against the RNA viruses tobacco mosaic virus (TMV), turnip mosaic virus (TuMV), and potato virus x (PVX). Harnessing the potential of these proteins represents a novel avenue in plant antiviral approaches, offering a broader and more effective spectrum for application in plant biotechnology and agriculture. Identifying these proteins opens new avenues for engineering a broad range of resistance to protect crop plants against viral pathogens.
PubMed: 38895613
DOI: 10.3389/fpls.2024.1385169