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PLoS Genetics Jun 2024Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this...
Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, methods for genetically manipulating tardigrades have long been desired. Despite our prior success in somatic cell gene editing by microinjecting Cas9 ribonucleoproteins (RNPs) into the body cavity of tardigrades, the generation of gene-edited individuals remained elusive. In this study, employing an extremotolerant parthenogenetic tardigrade species, Ramazzottius varieornatus, we established conditions that led to the generation of gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRISPR (DIPA-CRISPR) technique employed in several insects, we simply injected a concentrated Cas9 RNP solution into the body cavity of parental females shortly before their initial oviposition. This approach yielded gene-edited G0 progeny. Notably, only a single allele was predominantly detected at the target locus for each G0 individual, indicative of homozygous mutations. By co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, we achieved the generation of homozygously knocked-in G0 progeny, and these edited alleles were inherited by G1/G2 progeny. This is the first example of heritable gene editing in the entire phylum of Tardigrada. This establishment of a straightforward method for generating homozygous knockout/knock-in individuals not only facilitates in vivo analyses of the molecular mechanisms underpinning extreme tolerance, but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades.
Topics: Animals; Tardigrada; CRISPR-Cas Systems; Gene Editing; Parthenogenesis; Homozygote; Female; Gene Knock-In Techniques; Gene Knockout Techniques; Alleles
PubMed: 38870088
DOI: 10.1371/journal.pgen.1011298 -
Animal Models and Experimental Medicine Jun 2024This study aimed to construct and characterize a humanized influenza mouse model expressing hST6GAL1.
BACKGROUND
This study aimed to construct and characterize a humanized influenza mouse model expressing hST6GAL1.
METHODS
Humanized fragments, consisting of the endothelial cell-specific K18 promoter, human ST6GAL1-encoding gene, and luciferase gene, were microinjected into the fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. The offspring were identified using PCR. Mice exhibiting elevated expression of the hST6GAL1 gene were selectively bred for propagation, and in vivo analysis was performed for screening. Expression of the humanized gene was tested by performing immunohistochemical (IHC) analysis. Hematologic and biochemical analyses using the whole blood and serum of humanized hST6GAL1 mice were performed.
RESULTS
Successful integration of the human ST6GAL1 gene into the mouse genome led to the overexpression of human SiaT ST6GAL1. Seven mice were identified as carrying copies of the humanized gene, and the in vivo analysis indicated that hST6GAL1 gene expression in positive mice mirrored influenza virus infection characteristics. The IHC results revealed that hST6GAL1 was expressed in the lungs of humanized mice. Moreover, the hematologic and biochemical parameters of the positive mice were within the normal range.
CONCLUSION
A humanized influenza mouse model expressing the hST6GAL1 gene was successfully established and characterized.
PubMed: 38859745
DOI: 10.1002/ame2.12449 -
Cureus May 2024Patterned hair loss (PHL) is a severe hair condition that affects both sexes. Mesotherapy is a treatment that involves microinjecting medications and/or vitamins into... (Review)
Review
Patterned hair loss (PHL) is a severe hair condition that affects both sexes. Mesotherapy is a treatment that involves microinjecting medications and/or vitamins into the middle layer of the skin. Mesotherapy reduces systemic adverse effects by delivering drugs directly to the hair follicle, increasing local bioavailability while lowering systemic exposure. Local side effects and reactions may develop due to mesotherapy. This study systematically evaluated the safety and efficacy of mesotherapy to minoxidil 5%, as well as addressing its limitations, dosing, and technique, with the intent of providing valuable trials and insights for clinicians and patients considering mesotherapy for improved androgenetic alopecia (AGA) outcomes. The literature search carried out by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria yielded 11 relevant studies from an initial pool of 18 articles. These studies covered various aspects of the role of mesotherapy and minoxidil in AGA, including techniques, complications, limitations, and outcomes. In conclusion, available trials and research on mesotherapy and minoxidil demonstrated excellent statistical significance and a high patient satisfaction rate, with the exception of two publications that took into account certain uncommon adverse effects of mesotherapy. However, recent research suggests that a mesotherapy method for alopecia with a low risk of side effects is effective.
PubMed: 38841017
DOI: 10.7759/cureus.59705 -
Molecular Brain Jun 2024Loss-of-function mutations in the progranulin (GRN) gene are an autosomal dominant cause of Frontotemporal Dementia (FTD). These mutations typically result in...
Loss-of-function mutations in the progranulin (GRN) gene are an autosomal dominant cause of Frontotemporal Dementia (FTD). These mutations typically result in haploinsufficiency of the progranulin protein. Grn mice provide a model for progranulin haploinsufficiency and develop FTD-like behavioral abnormalities by 9-10 months of age. In previous work, we demonstrated that Grn mice develop a low dominance phenotype in the tube test that is associated with reduced dendritic arborization of layer II/III pyramidal neurons in the prelimbic region of the medial prefrontal cortex (mPFC), a region key for social dominance behavior in the tube test assay. In this study, we investigated whether progranulin haploinsufficiency induced changes in dendritic spine density and morphology. Individual layer II/III pyramidal neurons in the prelimbic mPFC of 9-10 month old wild-type or Grn mice were targeted for iontophoretic microinjection of fluorescent dye, followed by high-resolution confocal microscopy and 3D reconstruction for morphometry analysis. Dendritic spine density in Grn mice was comparable to wild-type littermates, but the apical dendrites in Grn mice had a shift in the proportion of spine types, with fewer stubby spines and more thin spines. Additionally, apical dendrites of Grn mice had longer spines and smaller thin spine head diameter in comparison to wild-type littermates. These changes in spine morphology may contribute to altered circuit-level activity and social dominance deficits in Grn mice.
Topics: Animals; Dendritic Spines; Prefrontal Cortex; Haploinsufficiency; Progranulins; Mice; Pyramidal Cells; Male; Mice, Inbred C57BL
PubMed: 38840181
DOI: 10.1186/s13041-024-01095-5 -
IBRO Neuroscience Reports Jun 2024The precise cause of autism spectrum disorder (ASD) is not fully understood. Despite the involvement of glutamatergic dysregulation in autism, the specific contribution...
Effect of intrahippocampal microinjection of VU0155041, a positive allosteric modulator of mGluR4, on long term potentiation in a valproic acid-induced autistic male rat model.
The precise cause of autism spectrum disorder (ASD) is not fully understood. Despite the involvement of glutamatergic dysregulation in autism, the specific contribution of mGlu4 receptors to synaptic plasticity remains unclear. Using the positive allosteric modulator VU0155041, we aimed to restore long-term potentiation (LTP) in the perforant path-dentate gyrus (PP-DG) pathway in VPA-induced autistic rat model. High-frequency stimulation was applied to the PP-DG synapse to induce LTP, while the VU0155041 was administered into the DG. Unexpectedly, VU0155041 failed to alleviate the observed LTP reduction in VPA-exposed rats, further resulting in a significant decrease in population spike LTP. This unexpected outcome prompts discussion on the complex nature of mGlu4 receptor modulation, highlighting potential interference with physiological processes underlying synaptic plasticity.
PubMed: 38832089
DOI: 10.1016/j.ibneur.2024.05.005 -
Journal of Psychopharmacology (Oxford,... Jun 2024The highly selective 5-HT serotonin receptor "biased" agonists NLX-101 and NLX-204 display, like ketamine, potent and efficacious rapid-acting antidepressant (RAAD)...
The 5-HT1A receptor biased agonists, NLX-204 and NLX-101, like ketamine, elicit rapid-acting antidepressant activity in the rat chronic mild stress model via cortical mechanisms.
BACKGROUND
The highly selective 5-HT serotonin receptor "biased" agonists NLX-101 and NLX-204 display, like ketamine, potent and efficacious rapid-acting antidepressant (RAAD) activity in the rat chronic mild stress (CMS) model with systemic (i.p.) administration. They rapidly (within 1 day) reverse anhedonia (i.e., CMS-induced sucrose consumption deficit), attenuate working memory deficit (novel object recognition: NOR), and decrease anxiety behavior in the elevated-plus maze (EPM).
AIMS
Here, we sought to explore the contribution of prefrontal cortex (PFC) 5-HT receptor activation in the RAAD activity of NLX compounds.
RESULTS/OUTCOMES
In male Wistar rats, unilateral PFC microinjections of NLX-204 and NLX-101 (16 µg), like ketamine (10 µg), reproduced the effects of their systemic administration: they reversed CMS-induced sucrose consumption deficit, attenuated anxiety (EPM), and reduced working memory deficits (NOR). In addition, unilateral PFC microinjections of the selective 5-HT antagonist, WAY-100,635 (2 µg), attenuated the beneficial effects of systemic NLX-204 and NLX-101 (0.16 mg/kg i.p.) in the sucrose intake and NOR models, indicating that these compounds exert their RAAD activity specifically through activation of PFC 5-HT receptors.
CONCLUSIONS/INTERPRETATION
These data indicate that 5-HT receptor biased agonists share with ketamine a common neuroanatomical site for RAAD activity, which can be obtained not only by targeting glutamatergic/NMDA neurotransmission (ketamine's primary mechanism of action) but also by activating 5-HT receptors, as is the case for the NLX compounds. The present observations also reinforce the notion that biased agonism at 5-HT receptors constitutes a promising strategy to achieve RAAD effects, with additional benefits against cognitive deficits and anxiety in depressed patients, without ketamine's troublesome side effects.
PubMed: 38825869
DOI: 10.1177/02698811241254832 -
Heliyon May 2024Peroxiredoxin 1 (Prx1) is an antioxidant protein that may promote the carcinogenesis in oral leukoplakia (OLK). To investigate the effect of Prx1 on the oral mucosal...
Peroxiredoxin 1 (Prx1) is an antioxidant protein that may promote the carcinogenesis in oral leukoplakia (OLK). To investigate the effect of Prx1 on the oral mucosal epithelium of OLK, we generated a Prx1 conditional knockout (cKO) mouse model. The mRNA and gRNA were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technique. An infusion cloning method was used to construct a homologous recombination vector. To obtain the F0 generation mice, fertilized eggs of C57BL/6J mice were microinjected with Cas9 mRNA, gRNA, and a donor vector. Polymerase chain reaction (PCR) amplification and sequencing were used to identify F1 generation mice. Using the cyclization recombination-enzyme-locus of the X-overP1 (Cre-loxP) system, we created a Prx1 cKO mouse model, and the effectiveness of the knockout was confirmed through immunohistochemistry. We examined the influence of Prx1 knockout on the occurrence of OLK in mice by constructing a model of tongue mucosa carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO). Prx1 modification was present in the F1 generation, as evidenced by PCR amplification and sequencing. Prx1: Cre + mice exhibited normal growth and fertility. Immunohistochemical analysis revealed that tongue epithelial cells in Prx1: Cre + mice displayed a distinct deletion of Prx1. An examination of the heart, liver, spleen, lung, and kidney tissues revealed no visible histological changes. Histological analysis showed a reduction in the occurrence of the malignant transformation of OLK in the tongue tissues of Prx1: Cre + mice. Ki67 immunostaining showed that Prx1 knockout significantly inhibited cell proliferation in the tongue epithelial. Our research developed a conditional knockout mouse model for Prx1. The obtained results provide insights into the function of Prx1 in the development of oral cancer and emphasize its potential as a therapeutic target for precancerous oral lesions.
PubMed: 38818156
DOI: 10.1016/j.heliyon.2024.e31227 -
Scientific Reports May 2024Dysfunction of subcortical D2-like dopamine receptors (DRs) can lead to positive symptoms of schizophrenia, and their analog, the increased locomotor activity in...
Dysfunction of subcortical D2-like dopamine receptors (DRs) can lead to positive symptoms of schizophrenia, and their analog, the increased locomotor activity in schizophrenia model MAM-E17 rats. The ventral pallidum (VP) is a limbic structure containing DRs. The DR antagonist sulpiride is a widespread antipsychotic drug, which can alleviate positive symptoms in human patients. However, it is still not known how sulpiride can influence positive symptoms via VP DRs. We hypothesize that the microinjection of sulpiride into the VP can normalize hyperactivity in MAM-E17 rats. In addition, recently, we showed that the microinjection of sulpirid into the VP induces place preference in neurotypical rats. Thus, we aimed to test whether intra-VP sulpiride can also have a rewarding effect in MAM-E17 rats. Therefore, open field-based conditioned place preference (CPP) test was applied in neurotypical (SAL-E17) and MAM-E17 schizophrenia model rats to test locomotor activity and the potential locomotor-reducing and rewarding effects of sulpiride. Sulpiride was microinjected bilaterally in three different doses into the VP, and the controls received only vehicle. The results of the present study demonstrated that the increased locomotor activity of the MAM-E17 rats was caused by habituation disturbance. Accordingly, larger doses of sulpiride in the VP reduce the positive symptom-analog habituation disturbance of the MAM-E17 animals. Furthermore, we showed that the largest dose of sulpiride administered into the VP induced CPP in the SAL-E17 animals but not in the MAM-E17 animals. These findings revealed that VP DRs play an important role in the formation of positive symptom-like habituation disturbances in MAM-E17 rats.
Topics: Animals; Sulpiride; Schizophrenia; Antipsychotic Agents; Rats; Basal Forebrain; Disease Models, Animal; Male; Microinjections; Habituation, Psychophysiologic; Locomotion; Receptors, Dopamine D2
PubMed: 38811614
DOI: 10.1038/s41598-024-63059-y -
Heliyon May 2024The reserve of glycogen is essential for embryonic development. In oviparous fish, egg is an isolated system after egg laying with all the required energy deposits by...
The reserve of glycogen is essential for embryonic development. In oviparous fish, egg is an isolated system after egg laying with all the required energy deposits by their mothers. However, the key regulated factor mediates the storage of maternal glycogen reserve which support for embryogenesis in the offspring is largely unknown. Glycogen synthase (GYS) is a central enzyme for glycogen synthesis. In our previous study, we generated a knockout zebrafish line, showed an embryonic developmental defect in F generation. In this study, firstly we determined that the was maternal origin by backcrossing the F mutant with wildtype lines. PAS staining and glycogen content measurement showed that glycogen reserve was reduced both in ovaries and embryos in the mutant group compared to wildtypes. Free glucose measurement analysis showed a 50 % of reduction in mutant embryos compared to wildtype embryos at 24 hpf; showed an approximal 50 % of reduction in mutant adults compared to wildtypes. Microinjection of 2-NBDG in embryos and comparison of fluorescent signal demonstrated that glucose uptake ability was decreased in the mutant embryos, indicating an impaired glucose metabolism. Untargeted metabolomics analysis then was employed and revealed that key modified metabolites enriched into vitamin B pathway, carbohydrate and unsaturated fatty acid pathways. These results demonstrated that played a role on glycogen metabolism, involved into the maternal glycogen reserve which essentially contribute to embryonic development.
PubMed: 38803914
DOI: 10.1016/j.heliyon.2024.e31149 -
STAR Protocols Jun 2024During development, the zebrafish embryo relies on its yolk sac as a nutrient source. Here, we present a protocol for modifying the free fatty acid (FFA) and...
During development, the zebrafish embryo relies on its yolk sac as a nutrient source. Here, we present a protocol for modifying the free fatty acid (FFA) and triacylglycerol (TAG) content of the zebrafish yolk sac by microinjection. We describe steps for needle and injection mold preparation, FFA and TAG solution preparation, and microinjection. This protocol can elucidate how excesses of FFA and TAG affect development and modify the transcriptome of zebrafish embryos. For complete details on the use and execution of this protocol, please refer to Konadu et al. .
Topics: Animals; Zebrafish; Microinjections; Triglycerides; Fatty Acids, Nonesterified; Embryo, Nonmammalian; Yolk Sac
PubMed: 38795351
DOI: 10.1016/j.xpro.2024.103086