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International Journal of Molecular... May 2024MicroRNAs (miRNAs) regulate approximately one-third of all human genes. The dysregulation of miRNAs has been implicated in the development of numerous human diseases,...
MicroRNAs (miRNAs) regulate approximately one-third of all human genes. The dysregulation of miRNAs has been implicated in the development of numerous human diseases, including cancers. In our investigation focusing on altering specific miRNA expression in human pancreatic cancer cells, we encountered an interesting finding. While two expression vector designs effectively enhanced miR-708 levels, they were unable to elevate mature forms of miR-29b, -1290, -2467, and -6831 in pancreatic cancer cell lines. This finding was also observed in a panel of other non-pancreatic cancer cell lines, suggesting that miRNA processing efficiency was cell line specific. Using a step-by-step approach in each step of miRNA processing, we ruled out alternative strand selection by the RISC complex and transcriptional interference at the primary miRNA (pri-miRNA) level. DROSHA processing and pri-miRNA export from the nucleus also appeared to be occurring normally. We observed precursor (pre-miRNA) accumulation only in cell lines where mature miRNA expression was not achieved, suggesting that the block was occurring at the pre-miRNA stage. To further confirm this, synthetic pre-miRNA mimics that bypass DICER processing were processed into mature miRNAs in all cases. This study has demonstrated the distinct behaviours of different miRNAs with the same vector in the same cell line, the same miRNA between the two vector designs, and with the same miRNA across different cell lines. We identified a stable vector pre-miRNA processing block. Our findings on the structural and sequence differences between successful and non-successful vector designs could help to inform future chimeric miRNA design strategies and act as a guide to other researchers on the intricate processing dynamics that can impact vector efficiency. Our research confirms the potential of miRNA mimics to surmount some of these complexities.
Topics: MicroRNAs; Humans; Pancreatic Neoplasms; RNA Processing, Post-Transcriptional; Cell Line, Tumor; Ribonuclease III; Gene Expression Regulation, Neoplastic; Transfection; RNA Precursors; Animals
PubMed: 38891854
DOI: 10.3390/ijms25115666 -
Journal of Agricultural and Food... Jul 2024Beyond the key bitter compound kaempferol 3--(2‴--sinapoyl-β-d-sophoroside) previously described in the literature (), eight further bitter and astringent-tasting...
Beyond the key bitter compound kaempferol 3--(2‴--sinapoyl-β-d-sophoroside) previously described in the literature (), eight further bitter and astringent-tasting kaempferol glucosides (-) have been identified in rapeseed protein isolates ( L.). The bitterness and astringency of these taste-active substances have been described with taste threshold concentrations ranging from 3.3 to 531.7 and 0.3 to 66.4 μmol/L, respectively, as determined by human sensory experiments. In this study, the impact of and kaempferol 3--β-d-glucopyranoside () on TAS2R-linked proton secretion by HGT-1 cells was analyzed by quantification of the intracellular proton index. mRNA levels of bitter receptors TAS2R3, 4, 5, 13, 30, 31, 39, 40, 43, 45, 46, 50 and TAS2R8 were increased after treatment with compounds and . Using quantitative UHPLC-MS/MS measurements, the concentrations of - were determined in rapeseed/canola seeds and their corresponding protein isolates. Depending on the sample material, compounds , , and - exceeded dose over threshold (DoT) factors above one for both bitterness and astringency in selected protein isolates. In addition, an increase in the key bitter compound during industrial protein production (apart from enrichment) was observed, allowing the identification of the potential precursor of to be kaempferol 3--(2‴--sinapoyl-β-d-sophoroside)-7--β-d-glucopyranoside (). These results may contribute to the production of less bitter and astringent rapeseed protein isolates through the optimization of breeding and postharvest downstream processing.
Topics: Humans; Receptors, G-Protein-Coupled; Kaempferols; Brassica napus; Taste; Plant Proteins; Glycosides; Plant Extracts; Seeds; Brassica rapa
PubMed: 38888424
DOI: 10.1021/acs.jafc.4c02342 -
DifferentialRegulation: a Bayesian hierarchical approach to identify differentially regulated genes.Biostatistics (Oxford, England) Jun 2024Although transcriptomics data is typically used to analyze mature spliced mRNA, recent attention has focused on jointly investigating spliced and unspliced (or...
Although transcriptomics data is typically used to analyze mature spliced mRNA, recent attention has focused on jointly investigating spliced and unspliced (or precursor-) mRNA, which can be used to study gene regulation and changes in gene expression production. Nonetheless, most methods for spliced/unspliced inference (such as RNA velocity tools) focus on individual samples, and rarely allow comparisons between groups of samples (e.g. healthy vs. diseased). Furthermore, this kind of inference is challenging, because spliced and unspliced mRNA abundance is characterized by a high degree of quantification uncertainty, due to the prevalence of multi-mapping reads, ie reads compatible with multiple transcripts (or genes), and/or with both their spliced and unspliced versions. Here, we present DifferentialRegulation, a Bayesian hierarchical method to discover changes between experimental conditions with respect to the relative abundance of unspliced mRNA (over the total mRNA). We model the quantification uncertainty via a latent variable approach, where reads are allocated to their gene/transcript of origin, and to the respective splice version. We designed several benchmarks where our approach shows good performance, in terms of sensitivity and error control, vs. state-of-the-art competitors. Importantly, our tool is flexible, and works with both bulk and single-cell RNA-sequencing data. DifferentialRegulation is distributed as a Bioconductor R package.
PubMed: 38887902
DOI: 10.1093/biostatistics/kxae017 -
CNS Neuroscience & Therapeutics Jun 2024Islet cell autoantigen 1 (ICA1) is involved in autoimmune diseases and may affect synaptic plasticity as a neurotransmitter. Databases related to Alzheimer's disease...
AIMS
Islet cell autoantigen 1 (ICA1) is involved in autoimmune diseases and may affect synaptic plasticity as a neurotransmitter. Databases related to Alzheimer's disease (AD) have shown decreased ICA1 expression in patients with AD. However, the role of ICA1 in AD remains unclear. Here, we report that ICA1 expression is decreased in the brains of patients with AD and an AD mouse model.
RESULTS
The ICA1 increased the expression of amyloid precursor protein (APP), disintegrin and metalloprotease 10 (ADAM10), and disintegrin and metalloprotease 17 (ADAM17), but did not affect protein half-life or mRNA levels. Transcriptome sequencing analysis showed that ICA1 regulates the G protein-coupled receptor signaling pathway. The overexpression of ICA1 increased PKCα protein levels and phosphorylation.
CONCLUSION
Our results demonstrated that ICA1 shifts APP processing to non-amyloid pathways by regulating the PICK1-PKCα signaling pathway. Thus, this study suggests that ICA1 is a novel target for the treatment of AD.
Topics: Amyloid beta-Protein Precursor; Animals; Protein Kinase C-alpha; Signal Transduction; Humans; Alzheimer Disease; Mice; Carrier Proteins; Nuclear Proteins; Male; Mice, Transgenic; Female; Mice, Inbred C57BL; Amyloid Precursor Protein Secretases; Brain; Cell Cycle Proteins
PubMed: 38884369
DOI: 10.1111/cns.14754 -
Developmental Biology Jun 2024Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors...
Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors responsible for PGC development are synthesized during early oogenesis and assembled into the germ plasm. During early embryonic development, germ plasm is inherited by a few cells, leading to the formation of PGCs. While germline development has been extensively studied, how components of the germ plasm regulate PGC development is not fully understood. Here, we report that Dzip1 is dynamically expressed in vertebrate germline and is a novel component of the germ plasm in Xenopus and zebrafish. Knockdown of Dzip1 impairs PGC development in Xenopus embryos. At the molecular level, Dzip1 physically interacts with Dazl, an evolutionarily conserved RNA-binding protein that plays a multifaced role during germline development. We further showed that the sequence between amino acid residues 282 and 550 of Dzip1 is responsible for binding to Dazl. Disruption of the binding between Dzip1 and Dazl leads to defective PGC development. Taken together, our results presented here demonstrate that Dzip1 is dynamically expressed in the vertebrate germline and plays a novel function during Xenopus PGC development.
PubMed: 38880277
DOI: 10.1016/j.ydbio.2024.06.003 -
Developmental Biology Jun 2024In chordates, the central nervous system arises from precursors that have distinct developmental and transcriptional trajectories. Anterior nervous systems are...
In chordates, the central nervous system arises from precursors that have distinct developmental and transcriptional trajectories. Anterior nervous systems are ontogenically associated with ectodermal lineages while posterior nervous systems are associated with mesoderm. Taking advantage of the well-documented cell lineage of ascidian embryos, we asked to what extent the transcriptional states of the different neural lineages become similar during the course of progressive lineage restriction. We performed single-cell RNA sequencing (scRNA-seq) analyses on hand-dissected neural precursor cells of the two distinct lineages, together with those of their sister cell lineages, with a high temporal resolution covering five successive cell cycles from the 16-cell to neural plate stages. A transcription factor binding site enrichment analysis of neural specific genes at the neural plate stage revealed limited evidence for shared transcriptional control between the two neural lineages, consistent with their different ontogenies. Nevertheless, PCA analysis and hierarchical clustering showed that, by neural plate stages, the two neural lineages cluster together. Consistent with this, we identified a set of genes enriched in both neural lineages at the neural plate stage, including miR-124, Celf3.a, Zic.r-b, and Ets1/2. Altogether, the current study has revealed genome-wide transcriptional dynamics of neural progenitor cells of two distinct developmental origins. Our scRNA-seq dataset is unique and provides a valuable resource for future analyses, enabling a precise temporal resolution of cell types not previously described from dissociated embryos.
PubMed: 38878991
DOI: 10.1016/j.ydbio.2024.06.005 -
BMC Cancer Jun 2024Checkpoint inhibitors (CPIs) are widely used in cancer treatment, with transformative impacts on survival. They nonetheless carry a significant risk of toxicity in the... (Observational Study)
Observational Study
BACKGROUND
Checkpoint inhibitors (CPIs) are widely used in cancer treatment, with transformative impacts on survival. They nonetheless carry a significant risk of toxicity in the form of immune-related adverse events (IrAEs), which may be sustained and life-altering. IrAEs may require high-dose and/or prolonged steroid use and represent a significant healthcare burden. They mimic immune-mediated inflammatory diseases (IMIDs) but understanding of their pathogenesis is limited. The MEDALLION project aims to determine targetable mechanisms of immune dysregulation in IrAE development, employing an immune monitoring approach to determine changes in circulating and tissue resident cells of CPI recipients who do/do not develop them and assessing the contribution of the microbiome in parallel.
METHODS
MEDALLION is a non-randomised longitudinal cohort study aiming to recruit 66 cancer patient recipients of anti-PD1/PD-L1, anti-CTLA-4 or combination therapy. Eligible participants include those with malignant melanoma in the adjuvant or metastatic setting, mesothelioma and non-small cell lung carcinoma (NSCLC) treated in the metastatic setting. Comprehensive clinical evaluation is carried out alongside blood, skin swab and stool sampling at the time of CPI initiation (baseline) and during subsequent routine hospital visits on 6 occasions over a 10-month follow-up period. It is conservatively anticipated that one third of enrolled patients will experience a "significant IrAE" (SirAE), defined according to pre-determined criteria specific to the affected tissue/organ system. Those developing such toxicity may optionally undergo a biopsy of affected tissue where appropriate, otherwise being managed according to standard of care. Peripheral blood mononuclear cells will be analysed using multi-parameter flow cytometry to investigate immune subsets, their activation status and cytokine profiles. Stool samples and skin swabs will undergo DNA extraction for 16 S ribosomal RNA (rRNA) sequencing and internal transcribed spacer (ITS) gene sequencing to determine bacterial and fungal microbiome diversity, respectively, including species associated with toxicity. Stored tissue biopsies will be available for in situ and single-cell transcriptomic evaluation. Analysis will focus on the identification of biological predictors and precursors of SirAEs.
DISCUSSION
The pathogenesis of IrAEs will be assessed through the MEDALLION cohort, with the potential to develop tools for their prediction and/or strategies for targeted prevention or treatment.
TRIAL REGISTRATION
The study was registered on 18/09/2023 in the ISRCTN registry (43,419,676).
Topics: Humans; Immune Checkpoint Inhibitors; Neoplasms; Longitudinal Studies; Immunotherapy; Cohort Studies; Monitoring, Immunologic; Melanoma
PubMed: 38877461
DOI: 10.1186/s12885-024-12468-3 -
Genome Biology and Evolution Jun 2024In flowering plants, euchromatic transposons are transcriptionally silenced by RNA-directed DNA Methylation, a small RNA-guided de novo methylation pathway. RNA-directed...
In flowering plants, euchromatic transposons are transcriptionally silenced by RNA-directed DNA Methylation, a small RNA-guided de novo methylation pathway. RNA-directed DNA Methylation requires the activity of the RNA Polymerases IV and V, which produce small RNA precursors and noncoding targets of small RNAs, respectively. These polymerases are distinguished from Polymerase II by multiple plant-specific paralogous subunits. Most RNA-directed DNA Methylation components are present in all land plants, and some have been found in the charophytic green algae, a paraphyletic group that is sister to land plants. However, the evolutionary origin of key RNA-directed DNA Methylation components, including the two largest subunits of Polymerase IV and Polymerase V, remains unclear. Here, we show that multiple lineages of charophytic green algae encode a single-copy precursor of the largest subunits of Polymerase IV and Polymerase V, resolving the two presumed duplications in this gene family. We further demonstrate the presence of a Polymerase V-like C-terminal domain, suggesting that the earliest form of RNA-directed DNA Methylation utilized a single Polymerase V-like polymerase. Finally, we reveal that charophytic green algae encode a single CLSY/DRD1-type chromatin remodeling protein, further supporting the presence of a single specialized polymerase in charophytic green algae.
Topics: DNA-Directed RNA Polymerases; Evolution, Molecular; DNA Methylation; Phylogeny; Charophyceae; Plant Proteins; Chlorophyta; Protein Subunits
PubMed: 38874416
DOI: 10.1093/gbe/evae119 -
Mathematical Biosciences and... Mar 2024B-cell acute lymphoblastic leukemia (B-ALL) is a malignant blood disorder, particularly detrimental to children and adolescents, with recurrent or unresponsive cases...
B-cell acute lymphoblastic leukemia (B-ALL) is a malignant blood disorder, particularly detrimental to children and adolescents, with recurrent or unresponsive cases contributing significantly to cancer-associated fatalities. IKBKE, associated with innate immunity, tumor promotion, and drug resistance, remains poorly understood in the context of B-ALL. Thus, this research aimed to explore the impact of the IKBKE inhibitor MCCK1 on B-ALL cells. The study encompassed diverse experiments, including clinical samples, in vitro and in vivo investigations. Quantitative real-time fluorescence PCR and protein blotting revealed heightened IKBKE mRNA and protein expression in B-ALL patients. Subsequent in vitro experiments with B-ALL cell lines demonstrated that MCCK1 treatment resulted in reduced cell viability and survival rates, with flow cytometry indicating cell cycle arrest. In vivo experiments using B-ALL mouse tumor models substantiated MCCK1's efficacy in impeding tumor proliferation. These findings collectively suggest that IKBKE, found to be elevated in B-ALL patients, may serve as a promising drug target, with MCCK1 demonstrating potential for inducing apoptosis in B-ALL cells both in vitro and in vivo.
Topics: Animals; Humans; Mice; I-kappa B Kinase; Cell Line, Tumor; Apoptosis; Cell Proliferation; Female; Cell Survival; Male; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Child; Adolescent; Xenograft Model Antitumor Assays; Protein Kinase Inhibitors
PubMed: 38872531
DOI: 10.3934/mbe.2024228 -
Nature Immunology Jun 2024Natural killer (NK) cells traffic through the blood and mount cytolytic and interferon-γ (IFNγ)-focused responses to intracellular pathogens and tumors. Type 1 innate...
Natural killer (NK) cells traffic through the blood and mount cytolytic and interferon-γ (IFNγ)-focused responses to intracellular pathogens and tumors. Type 1 innate lymphoid cells (ILC1s) also produce type 1 cytokines but reside in tissues and are not cytotoxic. Whether these differences reflect discrete lineages or distinct states of a common cell type is not understood. Using single-cell RNA sequencing and flow cytometry, we focused on populations of TCF7 cells that contained precursors for NK cells and ILC1s and identified a subset of bone marrow lineage-negative NK receptor-negative cells that expressed the transcription factor Eomes, termed EomesNK cells. Transfer of EomesNK cells into Rag2Il2rg recipients generated functional NK cells capable of preventing metastatic disease. By contrast, transfer of PLZF ILC precursors generated a mixture of ILC1s, ILC2s and ILC3s that lacked cytotoxic potential. These findings identified EomesNK cells as the bone marrow precursor to classical NK cells and demonstrated that the NK and ILC1 lineages diverged early during development.
PubMed: 38871999
DOI: 10.1038/s41590-024-01861-6