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Cells Jun 2020Hspb8 is a member of the small heat shock protein (sHSP) family. Its expression is known to be upregulated under heat shock. This protein interacts with different...
Hspb8 is a member of the small heat shock protein (sHSP) family. Its expression is known to be upregulated under heat shock. This protein interacts with different partners and can, therefore, be involved in various processes relevant to tissue integrity and functioning. In humans, mutations in the gene encoding Hspb8 can lead to the development of various diseases such as myopathies and neuropathies. In our study, we aimed to perform an in-depth characterization of zebrafish Hspb8 during zebrafish development. We applied techniques such as RT-qPCR, Western blot, immunofluorescence, co-immunoprecipitation, LC-MS, and morpholino-mediated knockdown. We broadened the knowledge regarding zebrafish expression during development under normal and heat shock conditions as well as its tissue- and subcellular-specific localization. A co-IP analysis allowed us to conclude that zebrafish Hspb8 can interact with proteins such as Bag3 and Hsc70, which are crucial for formation of an autophagy-inducing complex. We also demonstrated that morpholino-mediated knockdown has an impact on zebrafish embryos' morphology, muscle ultrastructure, and motility behavior. Our research provides a valuable resource for the potential use of the zebrafish as a model for studying pathological conditions associated with disorders.
Topics: Animals; Autophagy; Heat-Shock Proteins; Molecular Chaperones; Zebrafish
PubMed: 32604890
DOI: 10.3390/cells9061562 -
ACS Omega May 2022The strategies for nucleic acid sensing based on nucleic acid hybridization between the target sequence and the capture probe sequence are considered to be largely... (Review)
Review
The strategies for nucleic acid sensing based on nucleic acid hybridization between the target sequence and the capture probe sequence are considered to be largely successful as far as detection of a specific target of known sequence is concerned. However, when compared with other complementary methods, like direct sequencing, a number of results are still found to be either "false positives" or "false negatives". This suggests that modifications in these strategies are necessary to make them more accurate. In this minireview, we propose that one way toward improvement could be replacement of the DNA capture probes with the xeno nucleic acid or XNA capture probes. This is because the XNAs, especially the locked nucleic acid, the peptide nucleic acid, and the morpholino, have shown better single nucleobase mismatch discrimination capacity than the DNA capture probes, indicating their capacity for more precise detection of nucleic acid sequences, which is beneficial for detection of gene stretches having point mutations. Keeping the current trend in mind, this minireview will include the recent developments in nanoscale, fluorescent label-free applications, and present the cases where the XNA probes show clear advantages over the DNA probes.
PubMed: 35571783
DOI: 10.1021/acsomega.2c00581 -
International Journal of Molecular... Mar 2023Sphingolipidoses are inborn errors of metabolism due to the pathogenic mutation of genes that encode for lysosomal enzymes, transporters, or enzyme cofactors that... (Review)
Review
Sphingolipidoses are inborn errors of metabolism due to the pathogenic mutation of genes that encode for lysosomal enzymes, transporters, or enzyme cofactors that participate in the sphingolipid catabolism. They represent a subgroup of lysosomal storage diseases characterized by the gradual lysosomal accumulation of the substrate(s) of the defective proteins. The clinical presentation of patients affected by sphingolipid storage disorders ranges from a mild progression for some juvenile- or adult-onset forms to severe/fatal infantile forms. Despite significant therapeutic achievements, novel strategies are required at basic, clinical, and translational levels to improve patient outcomes. On these bases, the development of in vivo models is crucial for a better understanding of the pathogenesis of sphingolipidoses and for the development of efficacious therapeutic strategies. The teleost zebrafish () has emerged as a useful platform to model several human genetic diseases owing to the high grade of genome conservation between human and zebrafish, combined with precise genome editing and the ease of manipulation. In addition, lipidomic studies have allowed the identification in zebrafish of all of the main classes of lipids present in mammals, supporting the possibility to model diseases of the lipidic metabolism in this animal species with the advantage of using mammalian lipid databases for data processing. This review highlights the use of zebrafish as an innovative model system to gain novel insights into the pathogenesis of sphingolipidoses, with possible implications for the identification of more efficacious therapeutic approaches.
Topics: Animals; Humans; Zebrafish; Sphingolipids; Sphingolipidoses; Lysosomal Storage Diseases; Models, Biological; Mammals
PubMed: 36902174
DOI: 10.3390/ijms24054747 -
Methods in Cell Biology 2022Electrophoresis is one of the most important analytical technologies for characterization of macromolecules and their interactions. Among them, native gel...
Electrophoresis is one of the most important analytical technologies for characterization of macromolecules and their interactions. Among them, native gel electrophoresis is used to analyze the macromolecules in the native structure. It differs in principle and information from those obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) or blue native polyacrylamide gel electrophoresis (BN-PAGE). SDS-PAGE is carried out in the presence of strong denaturant, SDS, while BN-PAGE is done in the presence of negatively charged dye, e.g., Coomassie brilliant blue, G-250. Here, we describe native gel electrophoresis using agarose gel and a buffer at pH 6.1 composed of histidine and 2-(N-morpholino) ethanesulfonic acid. First, a protocol for vertical and horizontal formats of agarose native gel electrophoresis is described followed by different staining procedures. Then, various examples obtained using the developed procedure will be shown to demonstrate how the technology can be applied to specific cases and the advantages or caveats of the present technology.
Topics: Electrophoresis, Polyacrylamide Gel; Sepharose
PubMed: 35623712
DOI: 10.1016/bs.mcb.2021.12.030 -
Pediatric Pulmonology Oct 2020Neuromuscular cardiopulmonary medicine is entering a new and exciting phase, with studies that assess the respiratory effect of emerging genetic and molecular therapies.... (Review)
Review
Neuromuscular cardiopulmonary medicine is entering a new and exciting phase, with studies that assess the respiratory effect of emerging genetic and molecular therapies. In this year's neuromuscular Year in Review, we focus on Duchenne muscular dystrophy (DMD), reviewing studies that evaluate the respiratory effect of eteplirsen, the cardiopulmonary effects of ataluren, and a study comparing the use of spironolactone with eplerenone for the treatment of DMD-related cardiomyopathy.
Topics: Cardiomyopathies; Humans; Morpholinos; Muscular Dystrophy, Duchenne; Oxadiazoles; Spironolactone
PubMed: 32691988
DOI: 10.1002/ppul.24970 -
Developmental Biology Oct 2022The impact of new technology can be appreciated by how broadly it is used. Investigators that previously relied only on pharmacological approaches or the use of...
The impact of new technology can be appreciated by how broadly it is used. Investigators that previously relied only on pharmacological approaches or the use of morpholino antisense oligonucleotide (MASO) technologies are now able to apply CRISPR-Cas9 to study biological problems in their model organism of choice much more effectively. The transitions to new CRISPR-based approaches could be enhanced, first, by standardized protocols and education in their applications. Here we summarize our results for optimizing the CRISPR-Cas9 technology in a sea urchin and a sea star, and provide advice on how to set up CRISPR-Cas9 experiments and interpret the results in echinoderms. Our goal through these protocols and sharing examples of success by other labs is to lower the activation barrier so that more laboratories can apply CRISPR-Cas9 technologies in these important animals.
Topics: Animals; CRISPR-Cas Systems; Gene Editing; Morpholinos; RNA, Guide, CRISPR-Cas Systems; Sea Urchins
PubMed: 35917936
DOI: 10.1016/j.ydbio.2022.07.008 -
Indian Pediatrics Feb 2020Synthetic Antisense oligonucleotides (ASOs) are novel and efficient laboratory tools to regulate the expression of specific genes, and have only recently come into... (Review)
Review
Synthetic Antisense oligonucleotides (ASOs) are novel and efficient laboratory tools to regulate the expression of specific genes, and have only recently come into clinical use. These are synthetic single-stranded DNA analogs, whose sequence is complementary to a target nucleotide and alter protein synthesis by several mechanisms. We herein provide a primer on the topic for pediatricians, as this group of drugs is likely to see many more drugs for previously incurable diseases.
Topics: Adolescent; Child; Child, Preschool; Humans; Infant; Morpholinos; Muscular Atrophy, Spinal; Muscular Dystrophy, Duchenne; Oligonucleotides; Oligonucleotides, Antisense; Randomized Controlled Trials as Topic
PubMed: 32060244
DOI: No ID Found -
European Journal of Cell Biology Jun 2023Spinal muscular atrophy (SMA), the most common genetic cause of infantile death, is caused by a mutation in the survival of motor neuron 1 gene (SMN1), leading to the... (Review)
Review
Spinal muscular atrophy (SMA), the most common genetic cause of infantile death, is caused by a mutation in the survival of motor neuron 1 gene (SMN1), leading to the death of motor neurons and progressive muscle weakness. SMN1 normally produces an essential protein called SMN. Although humans possess a paralogous gene called SMN2, ∼90% of the SMN it produces is non-functional. This is due to a mutation in SMN2 that causes the skipping of a required exon during splicing of the pre-mRNA. The first treatment for SMA, nusinersen (brand name Spinraza), was approved by the FDA in 2016 and by the EMU in 2017. Nusinersen is an antisense oligonucleotide-based therapy that alters the splicing of SMN2 to make functional full-length SMN protein. Despite the recent advancements in antisense oligonucleotide therapy and SMA treatment development, nusinersen is faced with a multitude of challenges, such as intracellular and systemic delivery. In recent years, the use of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) in antisense therapy has gained interest. These are antisense oligonucleotides conjugated to cell-penetrating peptides such as Pips and DG9, and they have the potential to address the challenges associated with delivery. This review focuses on the historic milestones, development, current challenges, and future perspectives of antisense therapy for SMA.
Topics: Humans; Oligonucleotides, Antisense; Muscular Atrophy, Spinal; Morpholinos; Motor Neurons; RNA Splicing
PubMed: 37295266
DOI: 10.1016/j.ejcb.2023.151326 -
Cells Oct 2023Antisense oligonucleotide-based (ASO) therapeutics have emerged as a promising strategy for the treatment of human disorders. Charge-neutral PMOs have promising... (Review)
Review
Antisense oligonucleotide-based (ASO) therapeutics have emerged as a promising strategy for the treatment of human disorders. Charge-neutral PMOs have promising biological and pharmacological properties for antisense applications. Despite their great potential, the efficient delivery of these therapeutic agents to target cells remains a major obstacle to their widespread use. Cellular uptake of naked PMO is poor. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular uptake and intracellular delivery of oligonucleotide-based drugs. Among these, the DG9 peptide has been identified as a versatile CPP with remarkable potential for enhancing the delivery of ASO-based therapeutics due to its unique structural features. Notably, in the context of phosphorodiamidate morpholino oligomers (PMOs), DG9 has shown promise in enhancing delivery while maintaining a favorable toxicity profile. A few studies have highlighted the potential of DG9-conjugated PMOs in DMD (Duchenne Muscular Dystrophy) and SMA (Spinal Muscular Atrophy), displaying significant exon skipping/inclusion and functional improvements in animal models. The article provides an overview of a detailed understanding of the challenges that ASOs face prior to reaching their targets and continued advances in methods to improve their delivery to target sites and cellular uptake, focusing on DG9, which aims to harness ASOs' full potential in precision medicine.
Topics: Animals; Humans; Oligonucleotides, Antisense; Cell-Penetrating Peptides; Oligonucleotides; Morpholinos; Muscular Dystrophy, Duchenne; Muscular Atrophy, Spinal
PubMed: 37830609
DOI: 10.3390/cells12192395 -
Current Protocols Feb 2023Phosphorodiamidate morpholino oligonucleotides (PMOs) are a successful class of antisense reagents that efficiently modulate gene expression. Because PMOs do not follow...
Phosphorodiamidate morpholino oligonucleotides (PMOs) are a successful class of antisense reagents that efficiently modulate gene expression. Because PMOs do not follow standard phosphoramidite chemistry, optimized synthetic protocols for these compounds are relatively scarce in the literature. This paper presents detailed protocols for synthesizing full-length PMOs using chlorophosphoramidate chemistry by manual solid-phase synthesis. We first describe the synthesis of Fmoc-protected morpholino hydroxyl monomers, and the corresponding chlorophosphoramidate monomers, from commercially available protected ribonucleosides. The new Fmoc chemistry necessitates the use of a milder base, such as N-ethylmorpholine (NEM), and coupling reagent, such as 5-(ethylthio)-1H-tetrazole (ETT), which are also tolerated for acid-sensitive trityl chemistry. These chlorophosphoramidate monomers are then employed for PMO synthesis in a manual solid-phase procedure using four sequential steps. The synthetic cycle for each nucleotide incorporation consists of (a) deblocking of the 3'-N protecting group using an acidic deblocking cocktail for trityl and base deblocking for Fmoc, (b) neutralization, (c) coupling in the presence of ETT and NEM, and (d) capping of the unreacted morpholine ring-amine. The method uses safe, stable, and inexpensive reagents, and the process is expected to be scalable. After full-length PMO synthesis and ammonia-mediated cleavage from the solid support and deprotection, a range of PMOs with different lengths can be obtained conveniently and efficiently with reproducible good yields. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of the novel Fmoc-protected morpholino monomers Basic Protocol 2: Synthesis of the phosphorylating reagent (N,N-dimethylphosphoramic dichloride) required for chlorophosphoramidate monomer synthesis Basic Protocol 3: Synthesis of chlorophosphoramidate monomers of Fmoc-protected morpholino monomers Basic Protocol 4: Solution-phase standardization of dimer and trimer PMO synthesis using Fmoc chemistry Basic Protocol 5: Solid-phase synthesis, purification, and characterization of full-length (25-mer) no-tail PMO using both trityl and Fmoc chemistry.
Topics: Morpholinos; Oligonucleotides, Antisense
PubMed: 36802170
DOI: 10.1002/cpz1.686