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Genes Jun 2023In vertebrates, the development of the inner ear is a delicate process, whereas its relating molecular pathways are still poorly understood. , an LIM domain-only...
BACKGROUND
In vertebrates, the development of the inner ear is a delicate process, whereas its relating molecular pathways are still poorly understood. , an LIM domain-only transcriptional regulator, is drawing an increasing amount of interest for its multiple roles regarding human embryonic development and the modulation of ototoxic side effects of cisplatin including cochlear apoptosis and hearing loss. The aim of the present study is to further explore the role of in zebrafish inner ear development and thus explore its functional role.
METHODS
The Spatial Transcript Omics DataBase was referred to in order to evaluate the expression of during the first 24 h of zebrafish development. In situ hybridization was applied to validate and extend the expression profile of to 3 days post-fertilization. The morpholino (MO) knockdown and CRISPR/Cas9 knockout (KO) of was applied. Morphological analyses of otic vesical, hair cells, statoacoustic ganglion and semicircular canals were conducted. The swimming pattern of KO and MO zebrafish was tracked. In situ hybridization was further applied to verify the expression of genes of the related pathways. Rescue of the phenotype was attempted by blockage of the bmp pathway via heat shock and injection of Dorsomorphin.
RESULTS
is constitutively expressed in the otic placode and otic vesicle during the early stages of zebrafish development. Knockdown and knockout of both induced smaller otocysts, less hair cells, immature statoacoustic ganglion and malformed semicircular canals. Abnormal swimming patterns could be observed in both MO and KO zebrafish. in preplacodal ectoderm patterning was downregulated. and expressions were found to be upregulated and extended in morphants, and blockage of the Bmp pathway partially rescued the vestibular defects.
CONCLUSIONS
We concluded that holds a regulative effect on the Bmp pathway and is required for the normal development of zebrafish inner ear. Our study pointed out the conservatism of in inner ear development between mammals and zebrafish as well as shed more light on the molecular mechanisms behind it. Further research is needed to distinguish the relationships between and the Bmp pathway, which may lead to diagnostic and therapeutic approaches towards human inner ear malformation.
Topics: Animals; Adaptor Proteins, Signal Transducing; Cochlea; Ear, Inner; Hair Cells, Auditory; LIM Domain Proteins; Transcription Factors; Zebrafish; Zebrafish Proteins
PubMed: 37510276
DOI: 10.3390/genes14071371 -
Scientific Reports Jul 2023Drug-resistant Plasmodium falciparum (Pf) infections are a major burden on the population and the healthcare system. The establishment of Pf resistance to most existing...
Drug-resistant Plasmodium falciparum (Pf) infections are a major burden on the population and the healthcare system. The establishment of Pf resistance to most existing antimalarial therapies has complicated the problem, and the emergence of resistance to artemisinin derivatives is even more concerning. It is increasingly difficult to cure malaria patients due to the limited availability of effective antimalarial drugs, resulting in an urgent need for more efficacious and affordable treatments to eradicate this disease. Herein, new nucleoside analogues including morpholino-nucleoside hybrids and thio-substituted nucleoside derivatives were prepared and evaluated for in vitro and in vivo antiparasitic activity that led a few hits especially nucleoside-thiopyranoside conjugates, which are highly effective against Pf3D7 and PfRKL-9 strains in submicromolar concentration. One adenosine derivative and four pyrimidine nucleoside analogues significantly reduced the parasite burden in mouse models infected with Plasmodium berghei ANKA. Importantly, no significant hemolysis and cytotoxicity towards human cell line (RAW) was observed for the hits, suggesting their safety profile. Preliminary research suggested that these thiosugar-nucleoside conjugates could be used to accelerate the antimalarial drug development pipeline and thus deserve further investigation.
Topics: Animals; Mice; Humans; Antimalarials; Nucleosides; Sugars; Plasmodium falciparum; Malaria; Malaria, Falciparum; Plasmodium berghei
PubMed: 37507429
DOI: 10.1038/s41598-023-39541-4 -
Proceedings of the National Academy of... Aug 2023Cells of vertebrate and invertebrate organisms express proteins specialized in membrane channel-based cell-cell communication that are absent in unicellular organisms....
Cells of vertebrate and invertebrate organisms express proteins specialized in membrane channel-based cell-cell communication that are absent in unicellular organisms. We recently described the prediction of some members of the large-pore channel family in kinetoplastids, consisting of proteins called unnexins, which share several structural features with innexin and pannexin proteins. Here, we demonstrated that the unnexin1 protein (Unx1) is delivered to the cell membrane, displaying a topology consisting of four transmembrane domains with C and N termini on the cytoplasmic side and form large-pore channels that are permeable to small molecules. Low extracellular Ca/Mg levels or extracellular alkalinization, but not mechanical stretching, increases channel activity. The Unx1 channel mediates the influx of Ca and does not form intercellular dye coupling between HeLa Unx1 transfected cells. Unx1 channel function was further evidenced by its ability to mediate ionic currents when expressed in oocytes. Downregulation of Unx1 mRNA with morpholine contains invasion. Phylogenetic analysis revealed the presence of Unx1 homologs in other protozoan parasites, suggesting a conserved function for these channel parasites in other protists. Our data demonstrate that Unx1 forms large-pore membrane channels, which may serve as a diffusional pathway for ions and small molecules that are likely to be metabolic substrates or waste products, and signaling autocrine and paracrine molecules that could be involved in cell invasion. As morpholinos-induced downregulation of Unx1 reduces the infectivity of trypomastigotes, the Unx1 channels might be an attractive target for developing trypanocide drugs.
Topics: Protein Subunits; Phylogeny; Cell Membrane; Cytoplasm; Morpholinos
PubMed: 37487087
DOI: 10.1073/pnas.2307898120 -
International Journal of Molecular... Jul 2023Melon is a recalcitrant plant for stable genetic transformation. Various protocols have been tried to improve melon transformation efficiency; however, it remains...
Melon is a recalcitrant plant for stable genetic transformation. Various protocols have been tried to improve melon transformation efficiency; however, it remains significantly low compared to other plants such as tomato. In this study, the primary focus was on the optimization of key parameters during the inoculation and co-culture steps of the genetic transformation protocol. Our results showed that immersing the explants in the inoculation medium for 20 min significantly enhanced transformation efficiency. During the co-culture step, the use of filer paper, 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), and a temperature of 24 °C significantly enhanced the melon transformation efficiency. Furthermore, the impact of different ethylene inhibitors and absorbers on the transformation efficiency of various melon varieties was explored. Our findings revealed that the use of these compounds led to a significant improvement in the transformation efficiency of the tested melon varieties. Subsequently, using our improved protocol and reporter-gene construct, diploid transgenic melons successfully generated. The efficiency of plant genetic transformation ranged from 3.73 to 4.83%. Expanding the scope of our investigation, the optimized protocol was applied to generate stable gene-edited melon lines using the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated cytosine base editor and obtained melon lines with editions (C-to-T and C-to-G) in the eukaryotic translation initiation factor 4E, gene. In conclusion, the optimized melon transformation protocol, along with the utilization of the CRISPR/Cas9-mediated cytosine base editor, provides a reliable framework for functional gene engineering in melon. These advancements hold significant promise for furthering genetic research and facilitating crop improvement in this economically important plant species.
Topics: Gene Editing; CRISPR-Cas Systems; Cucumis melo; Cucurbitaceae; Plants
PubMed: 37446368
DOI: 10.3390/ijms241311189 -
BMC Research Notes Jul 2023RNA extraction from Mycobacterium tuberculosis has been a historically challenging task for researchers due to the thick lipids associated with the cell wall of this...
RNA extraction from Mycobacterium tuberculosis has been a historically challenging task for researchers due to the thick lipids associated with the cell wall of this "notorious" pathogen that is responsible for Tuberculosis (TB) outbreaks. Several studies have successfully extracted RNA from M. tuberculosis using a Trizol reagent combined with organic solvents. Recently, our laboratory has successfully extracted high quality total RNA using a commercial kit from clinical strains belonging to F15/LAM4/KZN, Beijing and F11 strain families and H37Rv laboratory strain by exploiting high speed homogenizer for cell lysis and spin columns for RNA purification. The quality and integrity of the extracted RNA was analyzed and confirmed through the Nanodrop, Bioanalyzer and RNA 3-(N-morpholino) propanesulfonic acid (MOPS) gel electrophoresis. Furthermore, to confirm the integrity of small RNA (sRNA) molecules due to their vulnerability to degradation, the RNA samples were converted to cDNA and sRNAs were amplified and confirmed through PCR. This detailed RNA extraction protocol proposes to carve a new path into TB transcriptome research without the use of organic solvent for downstream purification steps while yielding high quality RNA that can be used to understand M. tuberculosis transcriptome regulation.
Topics: Humans; Mycobacterium tuberculosis; RNA; Tuberculosis; Polymerase Chain Reaction; Beijing
PubMed: 37443138
DOI: 10.1186/s13104-023-06424-w -
European Journal of Medicinal Chemistry Oct 2023Phenotypic screening of an in-house library of small molecule purine derivatives against Mycobacterium tuberculosis (Mtb) led to the identification of...
Phenotypic screening of an in-house library of small molecule purine derivatives against Mycobacterium tuberculosis (Mtb) led to the identification of 2-morpholino-7-(naphthalen-2-ylmethyl)-1,7-dihydro-6H-purin-6-one 10 as a potent antimycobacterial agent with MIC of 4 μM. Thorough structure-activity relationship studies revealed the importance of 7-(naphthalen-2-ylmethyl) substitution for antimycobacterial activity, yet opened the possibility of structural modifications at positions 2 and 6 of the purine core. As the result, optimized analogues with 6-amino or ethylamino substitution 56 and 64, respectively, were developed. These compounds showed strong in vitro antimycobacterial activity with MIC of 1 μM against Mtb HRv and against several clinically isolated drug-resistant strains, had limited toxicity to mammalian cell lines, medium clearance with respect to phase I metabolic deactivation (27 and 16.8 μL/min/mg), sufficient aqueous solubility (>90 μM) and high plasma stability. Interestingly, investigated purines, including compounds 56 and 64, lacked activity against a panel of Gram-negative and Gram-positive bacterial strains, indicating a specific mycobacterial molecular target. To investigate the mechanism of action, Mtb mutants resistant to hit compound 10 were isolated and their genomes were sequenced. Mutations were found in dprE1 (Rv3790), which encodes decaprenylphosphoryl-β-d-ribose oxidase DprE1, enzyme essential for the biosynthesis of arabinose, a vital component of the mycobacterial cell wall. Inhibition of DprE1 by 2,6-disubstituted 7-(naphthalen-2-ylmethyl)-7H-purines was proved using radiolabelling experiments in Mtb HRv in vitro. Finally, structure-binding relationships between selected purines and DprE1 using molecular modeling studies in tandem with molecular dynamic simulations revealed the key structural features for effective drug-target interaction.
Topics: Animals; Antitubercular Agents; Alcohol Oxidoreductases; Mycobacterium tuberculosis; Purines; Structure-Activity Relationship; Molecular Dynamics Simulation; Bacterial Proteins; Mammals
PubMed: 37421887
DOI: 10.1016/j.ejmech.2023.115611 -
International Journal of Biological... 2023Loss of function in transport protein particles (TRAPP) links a new set of emerging genetic disorders called "TRAPPopathies". One such disorder is NIBP syndrome,...
Loss of function in transport protein particles (TRAPP) links a new set of emerging genetic disorders called "TRAPPopathies". One such disorder is NIBP syndrome, characterized by microcephaly and intellectual disability, and caused by mutations of , a crucial and unique member of TRAPPII. To investigate the neural cellular/molecular mechanisms underlying microcephaly, we developed Nibp/Trappc9-deficient animal models using different techniques, including morpholino knockdown and CRISPR/Cas mutation in zebrafish and Cre/LoxP-mediated gene targeting in mice. Nibp/Trappc9 deficiency impaired the stability of the TRAPPII complex at actin filaments and microtubules of neurites and growth cones. This deficiency also impaired elongation and branching of neuronal dendrites and axons, without significant effects on neurite initiation or neural cell number/types in embryonic and adult brains. The positive correlation of TRAPPII stability and neurite elongation/branching suggests a potential role for TRAPPII in regulating neurite morphology. These results provide novel genetic/molecular evidence to define patients with a type of non-syndromic autosomal recessive intellectual disability and highlight the importance of developing therapeutic approaches targeting the TRAPPII complex to cure TRAPPopathies.
Topics: Animals; Mice; Intellectual Disability; Microcephaly; Neurites; Neurons; Zebrafish
PubMed: 37416774
DOI: 10.7150/ijbs.78489 -
Research Square May 2023Deoxyribozymes (DNAzymes) are evolved DNA sequences capable of catalyzing chemical reactions. The RNA cleaving 10-23 DNAzyme was the first DNAzyme to be evolved and...
Deoxyribozymes (DNAzymes) are evolved DNA sequences capable of catalyzing chemical reactions. The RNA cleaving 10-23 DNAzyme was the first DNAzyme to be evolved and possesses clinical and biotechnical applications as a biosensor and a knockdown agent. DNAzymes do not require the recruitment of other components to cleave RNA and can turnover, thus they have a distinct advantage over other knockdown methods (siRNA, CRISPR, morpholinos). Despite this, a lack of structural and mechanistic information has hindered the optimization and application of the 10-23 DNAzyme. Here, we report a 2.7 Å crystal structure of the RNA cleaving 10-23 DNAzyme in a homodimer conformation. Although proper coordination of the DNAzyme to substrate is observed along with intriguing patterns of bound magnesium ions, the dimer conformation likely does not capture the true catalytic form of the 10-23 DNAzyme.
PubMed: 37398199
DOI: 10.21203/rs.3.rs-2252941/v1 -
Pharmaceutics Jun 2023Strategies for gene and nucleic acid delivery to skeletal muscles have been extensively explored to treat Duchenne muscular dystrophy (DMD) and other neuromuscular...
Strategies for gene and nucleic acid delivery to skeletal muscles have been extensively explored to treat Duchenne muscular dystrophy (DMD) and other neuromuscular diseases. Of these, effective intravascular delivery of naked plasmid DNA (pDNA) and nucleic acids into muscles is an attractive approach, given the high capillary density in close contact with myofibers. We developed lipid-based nanobubbles (NBs) using polyethylene-glycol-modified liposomes and an echo-contrast gas and found that these NBs could improve tissue permeability by ultrasound (US)-induced cavitation. Herein, we delivered naked pDNA or antisense phosphorodiamidate morpholino oligomers (PMOs) into the regional hindlimb muscle via limb perfusion using NBs and US exposure. pDNA encoding the luciferase gene was injected with NBs via limb perfusion into normal mice with application of US. High luciferase activity was achieved in a wide area of the limb muscle. DMD model mice were administered PMOs, designed to skip the mutated exon 23 of the dystrophin gene, with NBs via intravenous limb perfusion, followed by US exposure. The number of dystrophin-positive fibers increased in the muscles of mdx mice. Combining NBs and US exposure, which can be widely delivered to the hind limb muscles via the limb vein, could be an effective therapeutic approach for DMD and other neuromuscular disorders.
PubMed: 37376113
DOI: 10.3390/pharmaceutics15061665 -
Jak2 and Jaw Muscles Are Required for Buccopharyngeal Membrane Perforation during Mouth Development.Journal of Developmental Biology May 2023The mouth is a central feature of our face, without which we could not eat, breathe, or communicate. A critical and early event in mouth formation is the creation of a...
The mouth is a central feature of our face, without which we could not eat, breathe, or communicate. A critical and early event in mouth formation is the creation of a "hole" which connects the digestive system and the external environment. This hole, which has also been called the primary or embryonic mouth in vertebrates, is initially covered by a 1-2 cell layer thick structure called the buccopharyngeal membrane. When the buccopharyngeal membrane does not rupture, it impairs early mouth functions and may also lead to further craniofacial malformations. Using a chemical screen in an animal model () and genetic data from humans, we determined that Janus kinase 2 (Jak2) has a role in buccopharyngeal membrane rupture. We have determined that decreased Jak2 function, using antisense morpholinos or a pharmacological antagonist, caused a persistent buccopharyngeal membrane as well as the loss of jaw muscles. Surprisingly, we observed that the jaw muscle compartments were connected to the oral epithelium that is continuous with the buccopharyngeal membrane. Severing such connections resulted in buccopharyngeal membrane buckling and persistence. We also noted puncta accumulation of F-actin, an indicator of tension, in the buccopharyngeal membrane during perforation. Taken together, the data has led us to a hypothesis that muscles are required to exert tension across the buccopharyngeal membrane, and such tension is necessary for its perforation.
PubMed: 37367478
DOI: 10.3390/jdb11020024