-
Proceedings of the National Academy of... Oct 2023Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many...
Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many disorders including cardiac arrhythmias. Prior work revealed association of mRNAs encoding cardiac Na1.5 () and hERG1 (), but the functional significance of this association was not established. Here, we provide a more comprehensive picture of , , , and transcripts collectively copurifying with nascent hERG1, Na1.5, Ca1.2, or KCNQ1 channel proteins. Single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence reveals that the channel proteins are synthesized predominantly as heterotypic pairs from discrete molecules of mRNA, not as larger cotranslational complexes. Puromycin disrupted colocalization of mRNA with its encoded protein, as expected, but remarkably also pairwise mRNA association, suggesting that transcript association relies on intact translational machinery or the presence of the nascent protein. Targeted depletion of by specific shRNA resulted in concomitant reduction of all associated mRNAs, with a corresponding reduction in the encoded channel currents. This co-knockdown effect, originally described for and , thus appears to be a general phenomenon among transcripts encoding functionally related proteins. In multielectrode array recordings, proarrhythmic behavior arose when I was reduced by the selective blocker dofetilide at IC concentrations, but not when equivalent reductions were mediated by shRNA, suggesting that co-knockdown mitigates proarrhythmic behavior expected from the selective reduction of a single channel species. We propose that coordinated, cotranslational association of functionally related ion channel mRNAs confers electrical stability by co-regulating complementary ion channels in macromolecular complexes.
Topics: Humans; KCNQ1 Potassium Channel; ERG1 Potassium Channel; In Situ Hybridization, Fluorescence; Arrhythmias, Cardiac; RNA, Messenger; RNA, Small Interfering; NAV1.5 Voltage-Gated Sodium Channel
PubMed: 37816059
DOI: 10.1073/pnas.2305295120 -
Journal of Biological Rhythms Feb 2024Circadian-paced biological processes are key to physiology and required for metabolic, immunologic, and cardiovascular homeostasis. Core circadian clock components are...
Circadian-paced biological processes are key to physiology and required for metabolic, immunologic, and cardiovascular homeostasis. Core circadian clock components are transcription factors whose half-life is precisely regulated, thereby controlling the intrinsic cellular circadian clock. Genetic disruption of molecular clock components generally leads to marked pathological events phenotypically affecting behavior and multiple aspects of physiology. Using a transcriptional signature similarity approach, we identified anti-cancer protein synthesis inhibitors as potent modulators of the cardiomyocyte molecular clock. Eukaryotic protein translation inhibitors, ranging from translation initiation (rocaglates, 4-EGI1, etc.) to ribosomal elongation inhibitors (homoharringtonine, puromycin, etc.), were found to potently ablate protein abundance of REV-ERBα, a repressive nuclear receptor and component of the molecular clock. These inhibitory effects were observed both in vitro and in vivo and could be extended to PER2, another component of the molecular clock. Taken together, our observations suggest that the activity spectrum of protein synthesis inhibitors, whose clinical use is contemplated not only in cancers but also in viral infections, must be extended to circadian rhythm disruption, with potential beneficial or iatrogenic effects upon acute or prolonged administration.
Topics: Circadian Clocks; Circadian Rhythm; Protein Synthesis Inhibitors; Nuclear Receptor Subfamily 1, Group D, Member 1; Heart
PubMed: 37872767
DOI: 10.1177/07487304231202561 -
Discover Oncology May 2024Breast cancer is a prevalent malignant tumor among women with an increasing incidence rate annually. Breast cancer stem cells (BCSCs) are integral in impeding tumor...
Breast cancer is a prevalent malignant tumor among women with an increasing incidence rate annually. Breast cancer stem cells (BCSCs) are integral in impeding tumor advancement and addressing drug resistance. Bestatin serves as an adjuvant chemotherapy, triggering apoptosis in cancer cells. In this study, the effects of bestatin on sorted BCSCs from breast cancer cell lines have been studied. Our results indicated that bestatin inhibits the migration and proliferation of breast cancer cells by reducing the stemness of BCSCs both in vitro and in vivo. Puromycin-sensitive aminopeptidase is implicated in the process through the regulation of cell cycle, resulting in heightened cell apoptosis and diminished cell proliferation of BCSCs. Our study suggest that targeting cancer stem cell may offer a promising approach in breast cancer treatment, presenting noval therapeutic strategies for patients with breast cancer.
PubMed: 38814491
DOI: 10.1007/s12672-024-01063-4 -
Cold Spring Harbor Protocols Jun 2024The selection of mosquito transgenic larvae using a nonfluorescent approach can be advantageous to reserve fluorophores for downstream applications, such as...
The selection of mosquito transgenic larvae using a nonfluorescent approach can be advantageous to reserve fluorophores for downstream applications, such as immunostaining or for the study of promoter activity by cloning a fluorescence reporter gene under the control of that promoter. We previously reported that puromycin selection is efficient in transgenic or larvae expressing an selection marker. A concentration of puromycin of >10 µg/mL is lethal for larvae, unless they carry the resistance gene, conferring them resistance to puromycin concentrations of 25-80 µg/mL. A drawback of this fully dominant selection marker is that, unlike with fluorescence markers, homozygous transgenics cannot be distinguished from heterozygotes. Here, we outline the procedure for selecting puromycin-resistant transgenic larvae.
Topics: Animals; Puromycin; Larva; Animals, Genetically Modified; Anopheles; Selection, Genetic
PubMed: 37696571
DOI: 10.1101/pdb.prot108308 -
Seminars in Nuclear Medicine Sep 2023It is important to constantly monitor developments in the preclinical imaging arena of infection. Firstly, novel radiopharmaceuticals with the correct characteristics... (Review)
Review
It is important to constantly monitor developments in the preclinical imaging arena of infection. Firstly, novel radiopharmaceuticals with the correct characteristics must be identified to funnel into the clinic. Secondly, it must be evaluated if enough innovative research is being done and adequate resources are geared towards the development of radiopharmaceuticals that could feed into the Nuclear Medicine Clinic in the near future. It is proposed that the ideal infection imaging agent will involve PET combined with CT but more ideally MRI. The radiopharmaceuticals currently presented in preclinical literature have a wide selection of vectors and targets. Ionic formulations of PET-radionuclides such CuCl and GaCl are evaluated for bacterial infection imaging. Many small molecule based radiopharmaceuticals are being investigated with the most prominent targets being cell wall synthesis, maltodextrin transport (such as [F]F-maltotriose), siderophores (bacterial and fungal infections), the folate synthesis pathway (such as [F]F-PABA) and protein synthesis (radiolabelled puromycin). Mycobacterial specific antibiotics, antifungals and antiviral agents are also under investigation as infection imaging agents. Peptide based radiopharmaceuticals are developed for bacterial, fungal and viral infections. The radiopharmaceutical development could even react quickly enough on a pandemic to develop a SARS-CoV-2 imaging agent in a timely fashion ([Cu]Cu-NOTA-EK1). New immuno-PET agents for the imaging of viruses have recently been published, specifically for HIV persistence but also for SARS-CoV2. A very promising antifungal immuno-PET agent (hJ5F) is also considered. Future technologies could include the application of aptamers and bacteriophages and even going as far as the design of theranostic infection. Another possibility would be the application of nanobodies for immuno-PET applications. Standardization and optimization of the preclinical evaluation of radiopharmaceuticals could enhance clinical translation and reduce time spent in pursuing less than optimal candidates.
Topics: Humans; Radiopharmaceuticals; RNA, Viral; COVID-19; SARS-CoV-2; Positron-Emission Tomography
PubMed: 37012169
DOI: 10.1053/j.semnuclmed.2023.03.001 -
Molecular Biotechnology Aug 2023We attempted to construct a myeloid leukemia cell strain for stable overexpression and knock-down of miR-217 and explored the possible mechanism underlying miR-217 in...
We attempted to construct a myeloid leukemia cell strain for stable overexpression and knock-down of miR-217 and explored the possible mechanism underlying miR-217 in chronic myeloid leukemia (CML). MiR-217 overexpression and the knock-down lentiviral vector with puromycin resistance were constructed and packaged within recombinant lentivirus. Stably transfected K562 cells were obtained through puromycin screening, and the qPCR assay detected the relative expression of the target gene. The proliferation, apoptosis, and methylation level of PER2 within cultured cells were detected using the CCK-8 assay, flow cytometry, and TaqMan real‑time fluorescence quantitative methylation-specific PCR. qPCR and Western blot detected the expression of miR-217-related genes within the constructed K562 cell model. Colony PCR and sequencing proved that recombinant lentivirus expression vectors pSE16 and pSE17 were correctly constructed. The lentivirus titer was 2.95 × 10 and 2.61 × 10 IU/mL. The miR-217 expression level was high in pSE5316-K562 cells, and that of the miR-217 sponge was high in pSE5317-K562 cells. Overexpressed miR-217 could inhibit the K562 cell proliferation and induce apoptosis. Inhibition of miR-217 enhanced the expression of DNMT3A, decreased the PER2 expression, and elevated the degree of PER2 methylation. The miR-217 overexpression and knock-down of the K562 cell line were successfully constructed, providing a tool for further exploring the miR-217 mechanism in CML. DNMT3A could be the molecular target of miR-217 by regulating PER2 gene methylation and getting involved with the occurrence and development of CML.
Topics: Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; K562 Cells; Lentivirus; Cells, Cultured; Apoptosis; Cell Proliferation; MicroRNAs; Cell Line, Tumor
PubMed: 36495416
DOI: 10.1007/s12033-022-00615-9 -
Pharmacology & Therapeutics Dec 2023During carcinogenesis, neoplastic cells accumulate mutations in genes important for cellular homeostasis, producing defective proteins. Viral infections occur when viral... (Review)
Review
During carcinogenesis, neoplastic cells accumulate mutations in genes important for cellular homeostasis, producing defective proteins. Viral infections occur when viral capsid proteins bind to the host cell receptor, allowing the virus to enter the cells. In both cases, proteins play important roles in cancer development and viral infection, so these targets can be exploited to develop alternative treatments. mRNA display technology is a very powerful tool for the development of peptides capable of acting on specific targets in neoplastic cells or on viral capsid proteins. mRNA display technology allows the selection and evolution of peptides with desired functional properties from libraries of many nucleic acid variants. Among other advantages of this technology, the use of flexizymes allows the production of peptides with unnatural amino acid residues, which can enhance the activity of these molecules. From target immobilization, peptides with greater specificity for the targets of interest are generated during the selection rounds. Herein, we will explore the use of mRNA display technology for the development of active peptides after successive rounds of selection, using proteins present in neoplastic cells and viral particles as targets.
Topics: Humans; Capsid Proteins; RNA, Messenger; Peptides; Mutation; Neoplasms
PubMed: 37952905
DOI: 10.1016/j.pharmthera.2023.108559 -
International Journal of Biological... Feb 2024It is generally believed that the regulation of gene expression involves protein translation occurring before RNA transcription. Therefore, it is crucial to investigate... (Review)
Review
It is generally believed that the regulation of gene expression involves protein translation occurring before RNA transcription. Therefore, it is crucial to investigate protein translation and its regulation. Recent advancements in biological sciences, particularly in the field of omics, have revolutionized protein translation research. These studies not only help characterize changes in protein translation during specific biological or pathological processes but also have significant implications in disease prevention and treatment. In this review, we summarize the latest methods in ribosome-based translation omics. We specifically focus on the application of fluorescence imaging technology and omics technology in studying overall protein translation. Additionally, we analyze the advantages, disadvantages, and application of these experimental methods, aiming to provide valuable insights and references to researchers studying translation.
Topics: Protein Biosynthesis; RNA, Messenger; Ribosomes
PubMed: 38171441
DOI: 10.1016/j.ijbiomac.2023.129150 -
Scientific Reports Apr 2024The modification of the surgical polypropylene mesh and the polytetrafluoroethylene vascular prosthesis with cecropin A (small peptide) and puromycin (aminonucleoside)...
The modification of the surgical polypropylene mesh and the polytetrafluoroethylene vascular prosthesis with cecropin A (small peptide) and puromycin (aminonucleoside) yielded very stable preparations of modified biomaterials. The main emphasis was placed on analyses of their antimicrobial activity and potential immunomodulatory and non-cytotoxic properties towards the CCD841 CoTr model cell line. Cecropin A did not significantly affect the viability or proliferation of the CCD 841 CoTr cells, regardless of its soluble or immobilized form. In contrast, puromycin did not induce a significant decrease in the cell viability or proliferation in the immobilized form but significantly decreased cell viability and proliferation when administered in the soluble form. The covalent immobilization of these two molecules on the surface of biomaterials resulted in stable preparations that were able to inhibit the multiplication of Staphylococcus aureus and S. epidermidis strains. It was also found that the preparations induced the production of cytokines involved in antibacterial protection mechanisms and stimulated the immune response. The key regulator of this activity may be related to TLR4, a receptor recognizing bacterial LPS. In the present study, these factors were produced not only in the conditions of LPS stimulation but also in the absence of LPS, which indicates that cecropin A- and puromycin-modified biomaterials may upregulate pathways leading to humoral antibacterial immune response.
Topics: Biocompatible Materials; Lipopolysaccharides; Anti-Infective Agents; Anti-Bacterial Agents; Polymers; Staphylococcus epidermidis; Puromycin
PubMed: 38580807
DOI: 10.1038/s41598-024-58730-3 -
Micromachines Feb 2024We developed a 3D glomeruli tissue chip for glomerulonephritis (GN) testing, featuring a gravity-driven glomerular filtration barrier (GFB) with human podocytes and...
We developed a 3D glomeruli tissue chip for glomerulonephritis (GN) testing, featuring a gravity-driven glomerular filtration barrier (GFB) with human podocytes and endothelial cells with a bidirectional flow in the bottom channel. Using puromycin-induced GN, we observed decreased cell viability, increased albumin permeability, and reduced WT1 and nephrin compared to the normal GFB. Tacrolimus restored cell viability, reduced albumin permeability, and increased WT1 expression. Using serum from five membranous nephropathy (MN) patients, we created MN models using a GFB-mimicking chip. A notable decline in cell viability was observed in the serum-induced MN1 and MN2 models. However, tacrolimus restored it. Albumin permeability was reduced in the MN1, MN2, and MN5 models by tacrolimus treatment. MN1 displayed the best clinical response to tacrolimus, exhibiting increased expression of WT1 in chip-based evaluations after tacrolimus treatment. We successfully evaluated the efficacy of tacrolimus using puromycin-induced and serum-induced GN models on a chip that mimicked the structure and function of the GFB. The GFB-mimicking chip holds promise as a personalized platform for assessing drug efficacy using patient serum samples.
PubMed: 38542564
DOI: 10.3390/mi15030317