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Chemical Science Jun 2024Methylation of microRNAs (miRNAs) is a post-transcriptional modification that affects miRNA activity by altering the specificity of miRNAs to target mRNAs. Abnormal...
Methylation of microRNAs (miRNAs) is a post-transcriptional modification that affects miRNA activity by altering the specificity of miRNAs to target mRNAs. Abnormal methylation of miRNAs in cancer suggests their potential as a tumor marker. However, the traditional methylated miRNA detection mainly includes mass spectrometry, sequencing and others; complex procedures and reliance on large instruments greatly limit their application in point-of-care testing (POCT). Based on this, we developed DNAzyme-RCA-based gold nanoparticle (AuNP) colorimetric and lateral flow dipstick (LFD) assays to achieve convenient detection of exosomal 5-methylcytosine miRNA-21 (m5C-miRNA-21) for the first time. The two assays achieved specific recognition and linear amplification of m5C-miRNA-21 through the DNAzyme triggered RCA reaction and color output with low background interference through AuNP aggregation induced by base complementary pairing. The lowest concentration of m5C-miRNA-21 visible to the naked eye of the two assays can reach 1 pM and 0.1 pM, respectively. Detection of exosomal m5C-miRNA-21 in clinical blood samples showed that the expression level of m5C-miRNA-21 in colorectal cancer patients was significantly higher than that in healthy individuals. This approach not only demonstrates a new strategy for the detection of colorectal cancer but also provides a reference for the development of novel diagnostic tools for other miRNA methylation-related diseases.
PubMed: 38903234
DOI: 10.1039/d4sc02648a -
Nature Communications Jun 2024Mitochondria require an extensive proteome to maintain a variety of metabolic reactions, and changes in cellular demand depend on rapid adaptation of the mitochondrial...
Mitochondria require an extensive proteome to maintain a variety of metabolic reactions, and changes in cellular demand depend on rapid adaptation of the mitochondrial protein composition. The TOM complex, the organellar entry gate for mitochondrial precursors in the outer membrane, is a target for cytosolic kinases to modulate protein influx. DYRK1A phosphorylation of the carrier import receptor TOM70 at Ser91 enables its efficient docking and thus transfer of precursor proteins to the TOM complex. Here, we probe TOM70 phosphorylation in molecular detail and find that TOM70 is not a CK2 target nor import receptor for MIC19 as previously suggested. Instead, we identify TOM20 as a MIC19 import receptor and show off-target inhibition of the DYRK1A-TOM70 axis with the clinically used CK2 inhibitor CX4945 which activates TOM20-dependent import pathways. Taken together, modulation of DYRK1A signalling adapts the central mitochondrial protein entry gate via synchronization of TOM70- and TOM20-dependent import pathways for metabolic rewiring. Thus, DYRK1A emerges as a cytosolic surveillance kinase to regulate and fine-tune mitochondrial protein biogenesis.
Topics: Dyrk Kinases; Protein-Tyrosine Kinases; Protein Serine-Threonine Kinases; Mitochondria; Mitochondrial Precursor Protein Import Complex Proteins; Signal Transduction; Humans; Mitochondrial Membrane Transport Proteins; Phosphorylation; Protein Transport; HEK293 Cells; HeLa Cells; Mitochondrial Proteins
PubMed: 38902238
DOI: 10.1038/s41467-024-49611-4 -
Proceedings of the National Academy of... Jun 2024The photoinduced all-trans to 13-cis isomerization of the retinal Schiff base represents the ultrafast first step in the reaction cycle of bacteriorhodopsin (BR)....
The photoinduced all-trans to 13-cis isomerization of the retinal Schiff base represents the ultrafast first step in the reaction cycle of bacteriorhodopsin (BR). Extensive experimental and theoretical work has addressed excited-state dynamics and isomerization via a conical intersection with the ground state. In conflicting molecular pictures, the excited state potential energy surface has been modeled as a pure S[Formula: see text] state that intersects with the ground state, or in a 3-state picture involving the S[Formula: see text] and S[Formula: see text] states. Here, the photoexcited system passes two crossing regions to return to the ground state. The electric dipole moment of the Schiff base in the S[Formula: see text] and S[Formula: see text] state differs strongly and, thus, its measurement allows for assessing the character of the excited-state potential. We apply the method of ultrafast terahertz (THz) Stark spectroscopy to measure electric dipole changes of wild-type BR and a BR D85T mutant upon electronic excitation. A fully reversible transient broadening and spectral shift of electronic absorption is induced by a picosecond THz field of several megavolts/cm and mapped by a 120-fs optical probe pulse. For both BR variants, we derive a moderate electric dipole change of 5 [Formula: see text] 1 Debye, which is markedly smaller than predicted for a neat S[Formula: see text]-character of the excited state. In contrast, S[Formula: see text]-admixture and temporal averaging of excited-state dynamics over the probe pulse duration gives a dipole change in line with experiment. Our results support a picture of electronic and nuclear dynamics governed by the interaction of S[Formula: see text] and S[Formula: see text] states in a 3-state model.
Topics: Bacteriorhodopsins; Retinaldehyde; Terahertz Spectroscopy; Schiff Bases; Halobacterium salinarum; Isomerism
PubMed: 38900801
DOI: 10.1073/pnas.2319676121 -
Nature Communications Jun 2024The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and...
The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and biotechnological importance. Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how these introns recognize small molecules through their conserved active site. These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression. Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily-conserved RNA core of splicing machines can recognize small molecules specifically, our work provides a solid basis for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers.
Topics: RNA Splicing; Spliceosomes; Humans; Catalytic Domain; Introns; Small Molecule Libraries
PubMed: 38898052
DOI: 10.1038/s41467-024-48697-0 -
Scientific Reports Jun 2024This study probes the utility of biomarkers for microsatellite instability (MSI) detection and elucidates the molecular dynamics propelling colorectal cancer (CRC)...
This study probes the utility of biomarkers for microsatellite instability (MSI) detection and elucidates the molecular dynamics propelling colorectal cancer (CRC) progression. We synthesized a primer panel targeting 725 MSI loci, informed by The Cancer Genome Atlas (TCGA) and ancillary databases, to construct an amplicon library for next-generation sequencing (NGS). K-means clustering facilitated the distillation of 8 prime MSI loci, including activin A receptor type 2A (ACVR2A). Subsequently, we explored ACVR2A's influence on CRC advancement through in vivo tumor experiments and hematoxylin-eosin (HE) staining. Transwell assays gauged ACVR2A's role in CRC cell migration and invasion, while colony formation assays appraised cell proliferation. Western blotting illuminated the impact of ACVR2A suppression on CRC's PI3K/AKT/mTOR pathway protein expressions under hypoxia. Additionally, ACVR2A's influence on CRC-induced angiogenesis was quantified via angiogenesis assays. K-means clustering of NGS data pinpointed 32 MSI loci specific to tumor and DNA mismatch repair deficiency (dMMR) tissues. ACVR2A emerged as a pivotal biomarker, discerning MSI-H tissues with 90.97% sensitivity. A curated 8-loci set demonstrated 100% sensitivity and specificity for MSI-H detection in CRC. In vitro analyses corroborated ACVR2A's critical role, revealing its suppression of CRC proliferation, migration, and invasion. Moreover, ACVR2A inhibition under CRC-induced hypoxia markedly escalated MMP3, CyclinA, CyclinD1, and HIF1α protein expressions, alongside angiogenesis, by triggering the PI3K/AKT/mTOR cascade. The 8-loci ensemble stands as the optimal marker for MSI-H identification in CRC. ACVR2A, a central element within this group, deters CRC progression, while its suppression amplifies PI3K/AKT/mTOR signaling and angiogenesis under hypoxic stress.
Topics: Colorectal Neoplasms; Humans; Microsatellite Instability; Activin Receptors, Type II; Disease Progression; Animals; Cell Movement; Mice; Cell Line, Tumor; Cell Proliferation; Biomarkers, Tumor; Signal Transduction; Male; High-Throughput Nucleotide Sequencing; Female; Proto-Oncogene Proteins c-akt
PubMed: 38898042
DOI: 10.1038/s41598-024-62753-1 -
Hearing Research Jun 2024Hearing impairment is the most prevalent sensory disease in humans and can have dramatic effects on the development, and preservation, of our cognitive abilities and... (Review)
Review
Hearing impairment is the most prevalent sensory disease in humans and can have dramatic effects on the development, and preservation, of our cognitive abilities and social interactions. Currently 20 % of the world's population suffer from a form of hearing impairment; this is predicted to rise to 25 % by 2050. Despite this staggering disease load, and the vast damage it inflicts on the social, medical and economic fabric of humankind, our ability to predict, or prevent, the loss of hearing is very poor indeed. We here make the case for a paradigm shift in our approach to studying deafness. By exploiting more forcefully the molecular-genetic conservation between human hearing and hearing in morphologically distinct models, such as the fruit fly Drosophila melanogaster, we believe, a deeper understanding of hearing and deafness can be achieved. An understanding that moves beyond the surface of the 'deafness genes' to probe the underlying bedrock of hearing, which is shared across taxa, and partly shared across modalities. When it comes to understanding the workings (and failings) of human sensory function, a simple fruit fly has a lot to offer and a fly eye might sometimes be a powerful model for a human ear. Particularly the use of fly avatars, in which specific molecular (genetic or proteomic) states of humans (e.g. specific patients) are experimentally reproduced, in order to study the corresponding molecular mechanisms (e.g. specific diseases) in a controlled yet naturalistic environment, is a tool that promises multiple unprecedented insights. The use of the fly - and fly avatars - would benefit humans and will help enhance the power of other scientific models, such as the mouse.
PubMed: 38896942
DOI: 10.1016/j.heares.2024.109047 -
RSC Advances Jun 2024Tracking hypoxic environments and changes in oxygen levels contribute to the elucidation of pathological mechanisms. In this study, we attempted to design molecular...
Tracking hypoxic environments and changes in oxygen levels contribute to the elucidation of pathological mechanisms. In this study, we attempted to design molecular probes that can be activated to show fluorescence under hypoxic conditions and that can move to specific cell organelles. Considering that azide groups were selectively reduced to primary amines by reductases under hypoxic conditions, we prepared Hoechst and fluorophore Cy-5 derivatives with azide groups (Hoechst-N and Cy-N) as hypoxia probes. Hoechst-N and Cy-N showed weak fluorescence, but once activated in the cytosol of hypoxic cells, they exhibited robust fluorescence and then moved to their target organelles, the cell nucleus and mitochondria. In addition, when these probes were administered to the cells in the proper sequence, each probe was activated in response to the intracellular oxygen concentration at that point and exhibited oxygen concentration-dependent fluorescence at the target organelle. By measuring the fluorescence intensity of the cell nucleus and mitochondria, we successfully traced the history of changes in intracellular oxygen levels. Thus, we achieved tracking and recording of oxygen status in the cells.
PubMed: 38895527
DOI: 10.1039/d4ra01625d -
PNAS Nexus Jun 2024Plasmalogens are glycerophospholipids with a vinyl ether linkage at the sn-1 position of the glycerol backbone. Despite being suggested as antioxidants due to the high...
Plasmalogens are glycerophospholipids with a vinyl ether linkage at the sn-1 position of the glycerol backbone. Despite being suggested as antioxidants due to the high reactivity of their vinyl ether groups with reactive oxygen species, our study reveals the generation of subsequent reactive oxygen and electrophilic lipid species from oxidized plasmalogen intermediates. By conducting a comprehensive analysis of the oxidation products by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), we demonstrate that singlet molecular oxygen [O (Δ)] reacts with the vinyl ether bond, producing hydroperoxyacetal as a major primary product (97%) together with minor quantities of dioxetane (3%). Furthermore, we show that these primary oxidized intermediates are capable of further generating reactive species including excited triplet carbonyls and O (Δ) as well as electrophilic phospholipid and fatty aldehyde species as secondary reaction products. The generation of excited triplet carbonyls from dioxetane thermal decomposition was confirmed by light emission measurements in the visible region using dibromoanthracene as a triplet enhancer. Moreover, O (Δ) generation from dioxetane and hydroperoxyacetal was evidenced by detection of near-infrared light emission at 1,270 nm and chemical trapping experiments. Additionally, we have thoroughly characterized alpha-beta unsaturated phospholipid and fatty aldehydes by LC-HRMS analysis using two probes that specifically react with aldehydes and alpha-beta unsaturated carbonyls. Overall, our findings demonstrate the generation of excited molecules and electrophilic lipid species from oxidized plasmalogen species unveiling the potential prooxidant nature of plasmalogen-oxidized products.
PubMed: 38894877
DOI: 10.1093/pnasnexus/pgae216 -
Frontiers in Genetics 2024This study aimed to evaluate the efficacy of α-thalassemia gene testing as a part of an antenatal intervention program over a 10-year period.
OBJECTIVE
This study aimed to evaluate the efficacy of α-thalassemia gene testing as a part of an antenatal intervention program over a 10-year period.
METHODS
All patients underwent α-thalassemia gene testing, which included the analysis of three types of deletions and mutations. Rare α-thalassemia gene testing was performed using Sanger sequencing, multiplex ligation-dependent probe amplification, and sequencing techniques. Prenatal diagnosis was performed in high-risk couples using chorionic villus sampling or amniocentesis.
RESULTS
From 2010 to 2019, among the 91,852 patients examined, α-thalassemia mutations were identified in 41.78% of patients. The most frequent α gene mutation was--, followed by--. Two rare α-thalassemia gene mutations at -- and --, were also observed. A total of 2,235 high-risk couples were identified, of which 562 were affected, including three with the--/-- genotype and one with the--/-- genotype. Additionally, prenatal diagnosis revealed four cases of fetal anemia and/or mild edema, along with two cases of severe fetal edema. Chromosome and gene chip results were normal. Thalassemia gene testing showed an αα/αα genotype in four patients with anemia and/or mild edema, while two patients with severe fetal edema had one--/αα genotype and one--/-- genotype. Using the cut-off points of 74.6 fL and 24.4 pg as criteria for identifying α-thalassemia carriers and HbH disease, the detection rate of missed diagnoses in high-risk couples is consistent with national guidelines for standards, potentially saving 10,217,700 ¥.
CONCLUSION
Routine molecular testing for α-thalassemia in high-risk prenatal populations effectively prevented severe α-thalassemia births. Despite the high cost, the cutoff points proposed by this study suggest that implementing screening using a new parameter has the potential to reduce current expenses.
PubMed: 38894721
DOI: 10.3389/fgene.2024.1416047 -
Molecules (Basel, Switzerland) Jun 2024Spinal muscular atrophy (SMA) is a severe neuromuscular disorder that is caused by mutations in the survival motor neuron 1 () gene, hindering the production of... (Review)
Review
Spinal muscular atrophy (SMA) is a severe neuromuscular disorder that is caused by mutations in the survival motor neuron 1 () gene, hindering the production of functional survival motor neuron (SMN) proteins. Antisense oligonucleotides (ASOs), a versatile DNA-like drug, are adept at binding to target RNA to prevent translation or promote alternative splicing. Nusinersen is an FDA-approved ASO for the treatment of SMA. It effectively promotes alternative splicing in pre-mRNA transcribed from the gene, an analog of the gene, to produce a greater amount of full-length SMN protein, to compensate for the loss of functional protein translated from . Despite its efficacy in ameliorating SMA symptoms, the cellular uptake of these ASOs is suboptimal, and their inability to penetrate the CNS necessitates invasive lumbar punctures. Cell-penetrating peptides (CPPs), which can be conjugated to ASOs, represent a promising approach to improve the efficiency of these treatments for SMA and have the potential to transverse the blood-brain barrier to circumvent the need for intrusive intrathecal injections and their associated adverse effects. This review provides a comprehensive analysis of ASO therapies, their application for the treatment of SMA, and the encouraging potential of CPPs as delivery systems to improve ASO uptake and overall efficiency.
Topics: Cell-Penetrating Peptides; Humans; Muscular Atrophy, Spinal; Oligonucleotides, Antisense; Animals; Oligonucleotides; Survival of Motor Neuron 2 Protein; Survival of Motor Neuron 1 Protein; Blood-Brain Barrier
PubMed: 38893532
DOI: 10.3390/molecules29112658