-
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 -
Methods in Molecular Biology (Clifton,... 2024Ion channels comprise one of the largest targets for drug development and treatment and have been a subject of enduring fascination since first discovered in the 1950s....
Ion channels comprise one of the largest targets for drug development and treatment and have been a subject of enduring fascination since first discovered in the 1950s. Over the past decades, thousands of publications have explored the cellular biology and molecular physiology of these proteins, and many channel structures have been determined since the late 1990s. Trying to connect the dots between ion channel function and structure, voltage clamp fluorometry (VCF) emerges as a powerful tool because it allows monitoring of the conformational rearrangements underlying the different functional states of the channel. This technique represents an elegant harmonization of molecular biology, electrophysiology, and fluorescence. In the following chapter, we will provide a concise guide to performing VCF on Xenopus laevis oocytes using the two-electrode voltage clamp (TEVC) modality. This is the most widely used configuration on Xenopus oocytes for its relative simplicity and demonstrated success in a number of different ion channels utilizing a variety of attached labels.
Topics: Animals; Patch-Clamp Techniques; Fluorometry; Xenopus laevis; Oocytes; Ion Channels; Ion Channel Gating
PubMed: 38856899
DOI: 10.1007/978-1-0716-3818-7_8 -
Biochemical Genetics Jun 2024The objective of the study is to investigate how miR-146b-5p might contribute to the etiology of HSCR. The study investigated the expression levels of miRNA, mRNA, and...
The objective of the study is to investigate how miR-146b-5p might contribute to the etiology of HSCR. The study investigated the expression levels of miRNA, mRNA, and proteins in colon tissues obtained from the HSCR and control groups. The role of miR-146b-5p in cell proliferation and migration was studied in vitro. The interaction between miR-146b-5p and RET was validated through a dual-luciferase reporter experiment. To assess the impact of miR-146b-5p on the development of the enteric nervous system, zebrafish embryos were micro-injected with either miR-146b-5p mimics or negative control, followed by subsequent evaluation. Compared to the control group, miR-146b-5p expression levels in the spastic region of HSCR were significantly increased. In vitro, miR-146b-5p prevented cell migration and proliferation by targeting RET pathway. In zebrafish, miR-146b-5p negatively regulates the migration of neural crest cells through a reduction in RET expression. Overexpression of miR-146b-5p hinders the development of mature neurons by decreasing RET expression. Additionally, the aberrant phenotypes induced by miR-146b-5p were partially ameliorated when RET mRNA was co-injected. By targeting RET in HSCR patients, aberrant expression of miR-146b-5p may play a unique role in the etiology of the disease and be involved in enteric nervous system development.
PubMed: 38844639
DOI: 10.1007/s10528-024-10852-z -
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 -
Journal of Assisted Reproduction and... Jun 2024This study examined SSC proliferation on an epididymosome-enriched decellularized testicular matrix (DTM) hydrogel and spermatogenesis induction in azoospermic mice.
The effect of epididymosomes on the development of frozen-thawed mouse spermatogonial stem cells after culture in a decellularized testicular scaffold and transplantation into azoospermic mice.
PURPOSE
This study examined SSC proliferation on an epididymosome-enriched decellularized testicular matrix (DTM) hydrogel and spermatogenesis induction in azoospermic mice.
METHODS
Epididymosomes were extracted and characterized using SEM and western blotting. After cryopreservation, thawed SSCs were cultured in a hydrogel-based three-dimensional (3D) culture containing 10 ng/mL GDNF or 20 µg/mL epididymosomes. SSCs were assessed using the MTT assay, flow cytometry, and qRT-PCR after two weeks of culture. The isolated SSCs were microinjected into the efferent ducts of busulfan-treated mice. DiI-labeled SSCs were followed, and cell homing was assessed after two weeks. After 8 weeks, the testes were evaluated using morphometric studies and immunohistochemistry.
RESULTS
The expression of PLZF, TGF-β, and miR-10b did not increase statistically significantly in the 3D + GDNF and 3D + epididymosome groups compared to the 3D group. Among the groups, the GDNF-treated group exhibited the highest expression of miR-21 (*P < 0.05). Caspase-3 expression was lower in the epididymosome-treated group than in the other groups (***P < 0.001). Compared to the 3D and negative control groups, the 3D + epididymosomes and 3D + GDNF groups showed an increase in spermatogenic cells. Immunohistochemical results confirmed the growth and differentiation of spermatogonial cells into spermatids in the treatment groups.
CONCLUSION
The DTM hydrogel containing 20 µg/mL epididymosomes or 10 ng/mL GDNF is a novel and safe culture system that can support SSC proliferation in vitro to obtain adequate SSCs for transplantation success. It could be a novel therapeutic agent that could recover deregulated SSCs in azoospermic patients.
PubMed: 38839698
DOI: 10.1007/s10815-024-03157-y -
Brain, Behavior, and Immunity Jun 2024It is widely believed that the activation of the central dopamine (DA) system is crucial to the rewarding effects of methamphetamine (METH) and to the behavioral...
It is widely believed that the activation of the central dopamine (DA) system is crucial to the rewarding effects of methamphetamine (METH) and to the behavioral outcomes of METH use disorder. It was reported that METH exposure induced gasdermin D (GSDMD)-dependent pyroptosis in rats. The membrane pore formation caused by METH-induced pyroptosis may also contribute to the overflow of DA into the extracellular space and subsequently increase the DA levels in the brain. The present study firstly investigated whether the membrane pore information induced by GSDMD-dependent pyroptosis was associated with the increased DA levels in the ventral tegmental area (VAT) and nucleus accumbens (NAc) of rats self-administering METH and SY-SH5Y cells treated by METH. Subsequently, the effect of pore formation blockade or genetic inhibition of GSDMD on the reinforcing and motivational effect of METH was determined in rats, using the animal model of METH self-administration (SA). METH exposure significantly increased the activity of NLRP1/Cas-1/GSDMD pathway and the presence of pyroptosis, accompanied by the significantly increased DA levels in VTA and NAc. Moreover, intraperitoneal injections of disulfiram (DSF) or microinjection of rAAV-shGSDMD into VTA/NAc significantly reduced the reinforcing and motivational effect of METH, accompanied by the decreased level of DA in VTA and NAc. The results provided novel evidence that METH-induced pyroptosis could increase DA release in VTA and NAc via the NLRP1/Cas-1/GSDMD pathway. Additionally, membrane pores or GSDMD blockade could significantly reduce the reinforcing and motivational effect of METH. In conclusion, blocking GSDMD and membrane pore formation could be a promising potential target for the development of agents to treat METH use disorder.
PubMed: 38834156
DOI: 10.1016/j.bbi.2024.05.040 -
Journal of Psychiatric Research May 2024Numerous findings confirm that the metabotropic glutamate receptors (mGluRs) are involved in the conditioned place preference (CPP) induced by morphine. Here we focused...
Numerous findings confirm that the metabotropic glutamate receptors (mGluRs) are involved in the conditioned place preference (CPP) induced by morphine. Here we focused on the role of mGluR5 in the nucleus accumbens (NAc) as a main site of glutamate action on the rewarding effects of morphine. Firstly, we investigated the effects of intra-NAc administrating mGluR5 antagonist 3-((2-Methyl-1,3-thiazol-4-yl) ethynyl) pyridine hydrochloride (MTEP; 1, 3, and 10 μg/μl saline) on the extinction and the reinstatement phase of morphine CPP. Moreover, to determine the downstream signaling cascades of mGluR5 in morphine CPP, the protein levels of stromal interaction molecules (STIM1 and 2) in the NAc and hippocampus (HPC) were measured by western blotting. The behavioral data indicated that the mGluR5 blockade by MTEP at the high doses of 3 and 10 μg facilitated the extinction of morphine-induced CPP and attenuated the reinstatement to morphine in extinguished rats. Molecular results showed that the morphine led to increased levels of STIM proteins in the HPC and increased the level of STIM1 without affecting STIM2 in the NAc. Furthermore, intra-NAc microinjection of MTEP (10 μg) in the reinstatement phase decreased STIM1 in the NAc and HPC and reduced the STIM2 in the HPC. Collectively, our data show that morphine could facilitate brain reward function in part by increasing glutamate-mediated transmission through activation of mGluR5 and modulation of STIM proteins. Therefore, these results highlight the therapeutic potential of mGluR5 antagonists in morphine use disorder.
PubMed: 38833749
DOI: 10.1016/j.jpsychires.2024.05.047 -
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 Neuroendocrinology Jun 2024Reproduction in all mammalian species depends on the growth and maturation of ovarian follicles, that is, folliculogenesis. Follicular development can culminate with the...
Reproduction in all mammalian species depends on the growth and maturation of ovarian follicles, that is, folliculogenesis. Follicular development can culminate with the rupture of mature follicles and the consequent expulsion of their oocytes (ovulation) or in atresia, characterized by the arrest of development and eventual degeneration. These processes are regulated by different neuroendocrine signals arising at different hypothalamic nuclei, including the suprachiasmatic nucleus (SCN). In the later, the activation of muscarinic receptors (mAChRs) and nicotinic receptors (nAChRs) by acetylcholine is essential for the regulation of the pre-ovulatory signals that stimulate the rupture of mature follicles. To evaluate the participation of the nAChRs in the SCN throughout the oestrous cycle in the regulation of the hypothalamic-pituitary-ovarian axis. For this purpose, 90-day-old adult female rats in metoestrus, dioestrus, proestrus or oestrus were microinjected into the left- or right-SCN with 0.3 μL of saline solution as vehicle or with 0.225 μg of mecamylamine (Mec), a non-selective antagonist of the nicotinic receptors, diluted in 0.3 μL of vehicle. The animals were sacrificed when they presented vaginal cornification, indicative of oestrus stage, and the effects of the unilateral pharmacological blockade of the nAChRs in the SCN on follicular development, ovulation and secretion of oestradiol and follicle-stimulating hormone (FSH) were evaluated. The microinjection of Mec decreased the serum levels of FSH, which resulted in a lower number of growing and healthy follicles and an increase in atresia. The higher percentage of atresia in pre-ovulatory follicles was related to a decrease in the number of ova shed and abnormalities in oestradiol secretion. We also detected asymmetric responses between the left and right treatments that depended on the stage of the oestrous cycle. The present results allow us to suggest that during all the stages of the oestrous cycle, cholinergic signals that act on the nAChRs in the SCN are pivotal to modulate the secretion of gonadotropins and hence the physiology of the ovaries. Further research is needed to determine if such signals are generated by the cholinergic neurons in the SCN or by cholinergic afferents to the SCN.
PubMed: 38826071
DOI: 10.1111/jne.13421