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Biomedicine & Pharmacotherapy =... Jun 2024Ischemic heart disease invariably leads to devastating damage to human health. Nicotinamide ribose (NR), as one of the precursors of NAD synthesis, has been discovered...
Ischemic heart disease invariably leads to devastating damage to human health. Nicotinamide ribose (NR), as one of the precursors of NAD synthesis, has been discovered to exert a protective role in various neurological and cardiovascular disorders. Our findings demonstrated that pretreatment with 200 mg/kg NR for 3 h significantly reduced myocardial infarct area, decreased levels of CK-MB and LDH in serum, and improved cardiac function in the rats during myocardial ischemia-reperfusion (I/R) injury. Meanwhile, 0.5 mM NR also effectively increased the viability and decreased the LDH release of H9c2 cells during OGD/R. We had provided evidence that NR pretreatment could decrease mitochondrial reactive oxygen species (mtROS) production and MDA content, and enhance SOD activity, thereby mitigating mitochondrial damage and inhibiting apoptosis during myocardial I/R injury. Further investigations revealed that NR increased NAD content and upregulated SIRT3 protein expression in myocardium. Through using of SIRT3 small interfering RNA and the SIRT3 deacetylase activity inhibitor 3-TYP, we had confirmed that the cardioprotective effect of NR on cardiomyocytes was largely dependent on the inhibition of mitochondrial oxidative stress via SIRT3-SOD2 axis. Overall, our study suggested that exogenous supplementation with NR mitigated mitochondrial damage and inhibited apoptosis during myocardial I/R injury by reducing mitochondrial oxidative stress via SIRT3-SOD2-mtROS pathway.
Topics: Animals; Myocardial Reperfusion Injury; Sirtuin 3; Signal Transduction; Male; Niacinamide; Superoxide Dismutase; Rats, Sprague-Dawley; Rats; Apoptosis; Oxidative Stress; Pyridinium Compounds; Myocytes, Cardiac; Reactive Oxygen Species; Cell Line; Cardiotonic Agents; Sirtuins
PubMed: 38703508
DOI: 10.1016/j.biopha.2024.116689 -
Regenerative Therapy Dec 2024Brachial plexus injury (BPI) with motor neurons (MNs) damage still remain poor recovery in preclinical research and clinical therapy, while cell-based therapy approaches...
Brachial plexus injury (BPI) with motor neurons (MNs) damage still remain poor recovery in preclinical research and clinical therapy, while cell-based therapy approaches emerged as novel strategies. Previous work of rat skin precursor-derived Schwann cells (SKP-SCs) provided substantial foundation for repairing peripheral nerve injury (PNI). Given that, our present work focused on exploring the repair efficacy and possible mechanisms of SKP-SCs implantation on rat BPI combined with neurorrhaphy post-neurotomy. Results indicated the significant locomotive and sensory function recovery, with improved morphological remodeling of regenerated nerves and angiogenesis, as well as amelioration of target muscles atrophy and motor endplate degeneration. Besides, MNs could restore from oxygen-glucose-deprivation (OGD) injury upon SKP-SCs-sourced secretome treatment, implying the underlying paracrine mechanisms. Moreover, rat cytokine array assay detected 67 cytokines from SKP-SC-secretome, and bioinformatic analyses of screened 32 cytokines presented multiple functional clusters covering diverse cell types, including inflammatory cells, Schwann cells, vascular endothelial cells (VECs), neurons, and SKP-SCs themselves, relating distinct biological processes to nerve regeneration. Especially, a panel of hypoxia-responsive cytokines (HRCK), can participate into multicellular biological process regulation for permissive regeneration milieu, which underscored the benefits of SKP-SCs and sourced secretome, facilitating the chorus of nerve regenerative microenvironment. Furthermore, platelet-derived growth factor-AA (PDGF-AA) and vascular endothelial growth factor-A (VEGF-A) were outstanding cytokines involved with nerve regenerative microenvironment regulating, with significantly elevated mRNA expression level in hypoxia-responsive SKP-SCs. Altogether, through recapitulating the implanted SKP-SCs and derived secretome as niche sensor and paracrine transmitters respectively, HRCK would be further excavated as molecular underpinning of the neural recuperative mechanizations for efficient cell therapy; meanwhile, the analysis paradigm in this study validated and anticipated the actions and mechanisms of SKP-SCs on traumatic BPI repair, and was beneficial to identify promising bioactive molecule cocktail and signaling targets for cell-free therapy strategy on neural repair and regeneration.
PubMed: 38694448
DOI: 10.1016/j.reth.2024.04.002 -
Metallomics : Integrated Biometal... May 2024Eukaryotic DNA codes not only for proteins but contains a wealth of information required for accurate splicing of messenger RNA precursors and inclusion of...
Eukaryotic DNA codes not only for proteins but contains a wealth of information required for accurate splicing of messenger RNA precursors and inclusion of constitutively or alternatively spliced exons in mature transcripts. This "auxiliary" splicing code has been characterized as exonic splicing enhancers and silencers (ESE and ESS). The exact interplay between protein and splicing codes is, however, poorly understood. Here, we show that exons encoding copper-coordinating amino acids in human cuproproteins lack ESEs and/or have an excess of ESSs, yet RNA sequencing and expressed sequence tags data show that they are more efficiently included in mature transcripts by the splicing machinery than average exons. Their largely constitutive inclusion in messenger RNA is facilitated by stronger splice sites, including polypyrimidine tracts, consistent with an important role of the surrounding intron architecture in ensuring high expression of metal-binding residues during evolution. ESE/ESS profiles of codons and entire exons that code for copper-coordinating residues were very similar to those encoding residues that coordinate zinc but markedly different from those that coordinate calcium. Together, these results reveal how the traditional and auxiliary splicing motifs responded to constraints of metal coordination in proteins.
Topics: Humans; Exons; Copper; RNA Splicing; Alternative Splicing; RNA, Messenger; Enhancer Elements, Genetic; Carrier Proteins
PubMed: 38692844
DOI: 10.1093/mtomcs/mfae023 -
Nucleic Acids Research Jun 2024Although DNA-PK inhibitors (DNA-PK-i) have been applied in clinical trials for cancer treatment, the biomarkers and mechanism of action of DNA-PK-i in tumor cell...
Although DNA-PK inhibitors (DNA-PK-i) have been applied in clinical trials for cancer treatment, the biomarkers and mechanism of action of DNA-PK-i in tumor cell suppression remain unclear. Here, we observed that a low dose of DNA-PK-i and PARP inhibitor (PARP-i) synthetically suppresses BRCA-deficient tumor cells without inducing DNA double-strand breaks (DSBs). Instead, we found that a fraction of DNA-PK localized inside of nucleoli, where we did not observe obvious DSBs. Moreover, the Ku proteins recognize pre-rRNA that facilitates DNA-PKcs autophosphorylation independent of DNA damage. Ribosomal proteins are also phosphorylated by DNA-PK, which regulates pre-rRNA biogenesis. In addition, DNA-PK-i acts together with PARP-i to suppress pre-rRNA biogenesis and tumor cell growth. Collectively, our studies reveal a DNA damage repair-independent role of DNA-PK-i in tumor suppression.
Topics: DNA Breaks, Double-Stranded; DNA-Activated Protein Kinase; Humans; DNA Repair; RNA Precursors; Cell Line, Tumor; Ku Autoantigen; Phosphorylation; Cell Nucleolus; Poly(ADP-ribose) Polymerase Inhibitors; BRCA1 Protein; RNA, Ribosomal; Animals; Ribosomal Proteins
PubMed: 38682589
DOI: 10.1093/nar/gkae316 -
Nucleic Acids Research Jun 2024MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that...
MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors' sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We here develop a novel strategy based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Leveraging the highly conserved Xenopus miR-181a family as proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting the apical region of pre-miRNAs achieve precursor-selective inhibition of mature miRNA-5p production. Furthermore, we extend the applicability of our approach to the human miR-16 family, illustrating its universality in targeting precursors generating identical miRNAs. Overall, our strategy enables efficient manipulation of miRNA expression, offering a powerful tool to dissect the functions of identical or highly similar miRNAs derived from different precursors within miRNA families.
Topics: MicroRNAs; Animals; Humans; Oligonucleotides; Oligonucleotides, Antisense; RNA Precursors; Xenopus
PubMed: 38676942
DOI: 10.1093/nar/gkae284 -
Applied Microbiology and Biotechnology Apr 2024As a kind of biosurfactants, iturin A has attracted people's wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high...
As a kind of biosurfactants, iturin A has attracted people's wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens. Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5'-UTRs of downstream genes (ituA, ituB, and ituC) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH, serC, and introducing ocD. Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. KEY POINTS: • Optimizing 5'-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far.
Topics: Bacillus amyloliquefaciens; Metabolic Engineering; Surface-Active Agents; Peptides, Cyclic; Promoter Regions, Genetic; Ligases
PubMed: 38676716
DOI: 10.1007/s00253-024-13083-9 -
Nutrients Apr 2024As gluten may trigger gastrointestinal disorders (GIDs), its presence or absence in the diet can change the diversity and proportion of gut microbiota. The effects of... (Randomized Controlled Trial)
Randomized Controlled Trial
As gluten may trigger gastrointestinal disorders (GIDs), its presence or absence in the diet can change the diversity and proportion of gut microbiota. The effects of gluten after six weeks of a double-blind, placebo-controlled intervention with a gluten-free diet (GFD) were studied in participants with GIDs suffering from migraines and atopic dermatitis (n = 46). Clinical biomarkers, digestive symptoms, stool, the Migraine Disability Assessment questionnaire, and zonulin levels were analyzed. Next-generation sequencing was used to amplify the 16S rRNA gene of bacteria and the internal transcribed spacer (ITS) regions of fungi. The GFD increased Chao1 fungal diversity after the intervention, while the fungal composition showed no changes. Bacterial diversity and composition remained stable, but a positive association between bacterial and fungal Chao1 diversity and a negative association between Dothideomycetes and were observed. GIDs decreased in both groups and migraines improved in the placebo group. Our findings may aid the development of GID treatment strategies.
Topics: Humans; Migraine Disorders; Gastrointestinal Microbiome; Female; Male; Gastrointestinal Diseases; Adult; Double-Blind Method; Glutens; Middle Aged; Diet, Gluten-Free; Dermatitis, Atopic; Feces; Bacteria; Fungi; RNA, Ribosomal, 16S; Protein Precursors; Haptoglobins
PubMed: 38674918
DOI: 10.3390/nu16081228 -
International Journal of Molecular... Apr 2024Acute myeloid leukemia (AML) is a hematological malignancy that is characterized by an expansion of immature myeloid precursors. Despite therapeutic advances, the...
Acute myeloid leukemia (AML) is a hematological malignancy that is characterized by an expansion of immature myeloid precursors. Despite therapeutic advances, the prognosis of AML patients remains poor and there is a need for the evaluation of promising therapeutic candidates to treat the disease. The objective of this study was to evaluate the efficacy of duocarmycin Stable A (DSA) in AML cells in vitro. We hypothesized that DSA would induce DNA damage in the form of DNA double-strand breaks (DSBs) and exert cytotoxic effects on AML cells within the picomolar range. Human AML cell lines Molm-14 and HL-60 were used to perform 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), DNA DSBs, cell cycle, 5-ethynyl-2-deoxyuridine (EdU), colony formation unit (CFU), Annexin V, RNA sequencing and other assays described in this study. Our results showed that DSA induced DNA DSBs, induced cell cycle arrest at the G2M phase, reduced proliferation and increased apoptosis in AML cells. Additionally, RNA sequencing results showed that DSA regulates genes that are associated with cellular processes such as DNA repair, G2M checkpoint and apoptosis. These results suggest that DSA is efficacious in AML cells and is therefore a promising potential therapeutic candidate that can be further evaluated for the treatment of AML.
Topics: Humans; Apoptosis; Leukemia, Myeloid, Acute; Cell Proliferation; Duocarmycins; Cell Line, Tumor; DNA Breaks, Double-Stranded; HL-60 Cells; Antineoplastic Agents; Cell Cycle Checkpoints; DNA Damage
PubMed: 38673926
DOI: 10.3390/ijms25084342 -
International Journal of Molecular... Apr 2024Pre-mRNA splicing plays a key role in the regulation of gene expression. Recent discoveries suggest that defects in pre-mRNA splicing, resulting from the dysfunction of...
Pre-mRNA splicing plays a key role in the regulation of gene expression. Recent discoveries suggest that defects in pre-mRNA splicing, resulting from the dysfunction of certain splicing factors, can impact the expression of genes crucial for genome surveillance mechanisms, including those involved in cellular response to DNA damage. In this study, we analyzed how cells with a non-functional spliceosome-associated Gpl1-Gih35-Wdr83 complex respond to DNA damage. Additionally, we investigated the role of this complex in regulating the splicing of factors involved in DNA damage repair. Our findings reveal that the deletion of any component within the Gpl1-Gih35-Wdr83 complex leads to a significant accumulation of unspliced pre-mRNAs of DNA repair factors. Consequently, mutant cells lacking this complex exhibit increased sensitivity to DNA-damaging agents. These results highlight the importance of the Gpl1-Gih35-Wdr83 complex in regulating the expression of DNA repair factors, thereby protecting the stability of the genome following DNA damage.
Topics: DNA Damage; DNA Repair; Gene Expression Regulation, Fungal; RNA Precursors; RNA Splicing; RNA Splicing Factors; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Spliceosomes; Multiprotein Complexes
PubMed: 38673778
DOI: 10.3390/ijms25084192 -
International Journal of Molecular... Apr 2024The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not...
The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar ( × ) lines modified in their DXS activity. Single leaves were dynamically labeled with CO in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.
Topics: Populus; Erythritol; Sugar Phosphates; Transferases; Hemiterpenes; Butadienes; Plant Leaves; Plant Proteins; Gene Expression Regulation, Plant; Pentanes; Plants, Genetically Modified
PubMed: 38673766
DOI: 10.3390/ijms25084181