RESUMO
The Food and Drug Administrationapproved drug sirolimus, which inhibits mechanistic target of rapamycin (mTOR), is the leading candidate for targeting aging in rodents and humans. We previously demonstrated that sirolimus could treat ARHL in mice. In this study, we further demonstrate that sirolimus protects mice against cocaine-induced hearing loss. However, using efficacy and safety tests, we discovered that mice developed substantial hearing loss when administered high doses of sirolimus. Using pharmacological and genetic interventions in murine models, we demonstrate that the inactivation of mTORC2 is the major driver underlying hearing loss. Mechanistically, mTORC2 exerts its effects primarily through phosphorylating in the AKT/PKB signaling pathway, and ablation of P53 activity greatly attenuated the severity of the hearing phenotype in mTORC2-deficient mice. We also found that the selective activation of mTORC2 could protect mice from acoustic trauma and cisplatin-induced ototoxicity. Thus, in this study, we discover a function of mTORC2 and suggest that its therapeutic activation could represent a potentially effective and promising strategy to prevent sensorineural hearing loss. More importantly, we elucidate the side effects of sirolimus and provide an evaluation criterion for the rational use of this drug in a clinical setting.
Assuntos
Perda Auditiva Neurossensorial/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Transdução de Sinais , Animais , Modelos Animais de Doenças , Perda Auditiva Neurossensorial/induzido quimicamente , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/prevenção & controle , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Sirolimo/efeitos adversos , Sirolimo/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The process of spermatogenesis is a complex and delicate process that is still not fully understood. In this study, we examined the role of fatty acid oxidase 3-hydroxy acyl CoA dehydrogenase (HADH) in maintaining normal spermatogenesis in mice. In male mice, ablation of the Hadh gene using CRISPR/Cas9 technology arrested spermatocyte meiosis, increased multinucleated giant germ cells and vacuoles in seminiferous tubules, and accompanied with acrosomal dysplasia. Hadh-/- male mice showed the typical features of oligoasthenoteratozoospermia (OAT), including decreased sperm concentration and motility and increased sperm abnormalities. Next, we explored the molecular events in the testes of the mutant mice. We found fatty acids accumulated in the testis of Hadh-/- mice. And also, inflammatory factors TNF-α, IL-1ß, and IL-6 were significantly increased, apoptosis-related protein Bcl-2 was decreased, and Bax and cleaved-Caspase3 were increased in Hadh-/- male mice testis. After using etanercept, a specific inhibitor of TNF-α, testis injury caused by Hadh knockout was significantly alleviated, the sperm quality and motility were improved, and germ cell apoptosis was reduced. So our study demonstrated that Hadh deletion caused an increase in fatty acids. The accumulated fatty acids further induced testicular inflammation and germ cell apoptosis through the TNF-α/Bcl-2 signaling pathway, finally resulting in OAT in the Hadh-/- mice. Inhibiting TNF-α may be used as a new treatment approach for testicular inflammation and OAT.
Assuntos
3-Hidroxiacil-CoA Desidrogenase , Astenozoospermia , Infertilidade Masculina , Oligospermia , Animais , Masculino , Camundongos , Astenozoospermia/genética , Astenozoospermia/metabolismo , Ácidos Graxos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Inflamação/genética , Inflamação/metabolismo , Oligospermia/genética , Oligospermia/metabolismo , Sêmen/metabolismo , Espermatócitos/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , 3-Hidroxiacil-CoA Desidrogenase/deficiência , 3-Hidroxiacil-CoA Desidrogenase/genética , 3-Hidroxiacil-CoA Desidrogenase/metabolismo , Genes bcl-2/genética , Genes bcl-2/fisiologiaRESUMO
Lipocalin family members, LCN8 and LCN9, are specifically expressed in the initial segment of mouse caput epididymis. However, the biological functions of the molecules in vivo are yet to be clarified. In this study, CRISPR/Cas9 technology was used to generate Lcn8 and Lcn9 knockout mice, respectively. Lcn8-/- and Lcn9-/- male mice showed normal spermatogenesis and fertility. In the cauda epididymis of Lcn8-/- male mice, morphologically abnormal sperm was increased significantly, the proportion of progressive motility sperm was decreased, the proportion of immobilized sperm was elevated, and the sperm spontaneous acrosome reaction (AR) frequency was increased. Conversely, the knockout of Lcn9 did not have any effect on the ratio of morphologically abnormal sperm, sperm motility, and sperm spontaneous AR frequencies. These results demonstrated the role of LCN8 in maintaining the sperm quality in the epididymis, and suggested that the deficiency of LCN8 leads to epididymal sperm maturation defects.
Assuntos
Epididimo/patologia , Lipocalinas/metabolismo , Maturação do Esperma/fisiologia , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Fertilidade , Masculino , Camundongos Endogâmicos C57BL , Espermatogênese , EspermatozoidesRESUMO
Cdc14a is an evolutionarily conserved dual-specific protein phosphatase, and it plays different roles in different organisms. Cdc14a mutations in human have been reported to cause male infertility, while the specific role of Cdc14a in regulation of the male reproductive system remains elusive. In the present study, we established a knockout mouse model to study the function of Cdc14a in male reproductive system. Cdc14a-/- male mice were subfertile and they could only produce very few offspring. The number of sperm was decreased, the sperm motility was impaired, and the proportion of sperm with abnormal morphology was elevated in Cdc14a-/- mice. When we mated Cdc14a-/- male mice with wild-type (WT) female mice, fertilized eggs could be found in female fallopian tubes, however, the majority of these embryos died during development. Some empty spaces were observed in seminiferous tubule of Cdc14a-/- testes. Compared with WT male mice, the proportions of pachytene spermatocytes were increased and germ cells stained with γH2ax were decreased in Cdc14a-/- male mice, indicating that knockout of Cdc14a inhibited meiotic initiation. Subsequently, we analyzed the expression levels of some substrate proteins of Cdc14a, including Cdc25a, Wee1, and PR-Set7, and compared those with WT testes, in which the expression levels of these proteins were significantly increased in Cdc14a-/- testes. Our results revealed that Cdc14a-/- male mice are highly subfertile, and Cdc14a is essential for normal spermatogenesis and sperm function.
Assuntos
Infertilidade Masculina/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Maturação do Esperma/fisiologia , Motilidade dos Espermatozoides/fisiologia , Animais , Infertilidade Masculina/genética , Infertilidade Masculina/fisiopatologia , Masculino , Camundongos Knockout , Espermatócitos/metabolismo , Espermatogênese/genética , Espermatozoides/citologia , Testículo/metabolismo , Testículo/patologiaRESUMO
To elucidate the mechanism of anti-ferroptosis and examine structural optimization in natural phenolics, cellular and chemical assays were performed with 2'-hydroxy chalcone butein and dihydroflavone (S)-butin. C11-BODIPY staining and flow cytometric assays suggest that butein more effectively inhibits ferroptosis in erastin-treated bone marrow-derived mesenchymal stem cells than (S)-butin. Butein also exhibited higher antioxidant percentages than (S)-butin in five antioxidant assays: linoleic acid emulsion assay, Fe3+-reducing antioxidant power assay, Cu2+-reducing antioxidant power assay, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIOâ¢)-trapping assay, and α,α-diphenyl-ß-picrylhydrazyl radical (DPPHâ¢)-trapping assay. Their reaction products with DPPH⢠were further analyzed using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS). Butein and (S)-butin produced a butein 5,5-dimer (m/z 542, 271, 253, 225, 135, and 91) and a (S)-butin 5',5'-dimer (m/z 542, 389, 269, 253, and 151), respectively. Interestingly, butein forms a cross dimer with (S)-butin (m/z 542, 523, 433, 419, 415, 406, and 375). Therefore, we conclude that butein and (S)-butin exert anti-ferroptotic action via an antioxidant pathway (especially the hydrogen atom transfer pathway). Following this pathway, butein and (S)-butin yield both self-dimers and cross dimers. Butein displays superior antioxidant or anti-ferroptosis action to (S)-butin. This can be attributed the decrease in π-π conjugation in butein due to saturation of its α,ß-double bond and loss of its 2'-hydroxy group upon biocatalytical isomerization.
Assuntos
Antioxidantes/farmacologia , Chalconas/química , Chalconas/farmacologia , Ferroptose/efeitos dos fármacos , Doenças Neurodegenerativas/tratamento farmacológico , Antioxidantes/química , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Chalcona/análogos & derivados , Chalcona/química , Cromatografia Líquida de Alta Pressão , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: To investigate the current status of antibiotic use for very and extremely low birth weight (VLBW/ELBW) infants in neonatal intensive care units (NICUs) of Hunan Province. METHODS: The use of antibiotics was investigated in multiple level 3 NICUs of Hunan Province for VLBW and ELBW infants born between January, 2017 and December, 2017. RESULTS: The clinical data of 1â442 VLBW/ELBW infants were collected from 24 NICUs in 2017. The median antibiotic use duration was 17 days (range: 0-86 days), accounting for 53.0% of the total length of hospital stay. The highest duration of antibiotic use was up to 91.4% of the total length of hospital stay, with the lowest at 14.6%. In 16 out of 24 NICUs, the antibiotic use duration was accounted for more than 50.0% of the hospitalization days. There were 113 cases with positive bacterial culture grown in blood or cerebrospinal fluid, making the positive rate of overall bacterial culture as 7.84%. The positive rate of bacterial culture in different NICUs was significantly different from 0% to 14.9%. The common isolated bacterial pathogens Klebsiella pneumoniae was 29 cases (25.7%); Escherichia coli 12 cases (10.6%); Staphylococcus aureus 3 cases (2.7%). The most commonly used antibiotics were third-generation of cephalosporins, accounting for 41.00% of the total antibiotics, followed by penicillins, accounting for 32.10%, and followed by carbapenems, accounting for 13.15%. The proportion of antibiotic use time was negatively correlated with birth weight Z-score and the change in weight Z-score between birth and hospital discharge (rs=-0.095, -0.151 respectively, P<0.01), positively correlated with death/withdrawal of care (rs=0.196, P<0.01). CONCLUSIONS: Antibiotics used for VLBW/ELBW infants in NICUs of Hunan Province are obviously prolonged in many NICUs. The proportion of routine use of third-generation of cephalosporins and carbapenems antibiotics is high among the NICUs.
Assuntos
Recém-Nascido de Peso Extremamente Baixo ao Nascer , Antibacterianos , Peso ao Nascer , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Inquéritos e QuestionáriosRESUMO
SLC26A4 gene mutations lead to Pendred syndrome and non-syndromic hearing loss (DFNB4). The mouse model is well used to study the pathology of Pendred syndrome, however, mice with different Slc26a4 mutations exhibit different phenotypes, and these mice have severe deafness and inner ear malformations that are not imitated less severely Human phenotype. In this study, we generated a knock-in mouse model of Pendred syndrome with Slc26a4 L236P mutation to mimic the most common mutation found in human. Some L236P mice were observed to have significant vestibular dysfunction including torticollis and circling, the giant otoconia and destruction of the otoconial membrane was observed in L236P mice. Unlike other profoundly deafness in Slc26a4 mouse model, L236P mice present mild to profound hearing loss, consistent with the hearing threshold, inner ear hair cells also lost from slight to significant. Together, these data demonstrate that the L236P mouse phenotype is more similar to the human phenotype and should be used as a tool for further research into the human Pendred syndrome.
Assuntos
Bócio Nodular/genética , Perda Auditiva Neurossensorial/genética , Transportadores de Sulfato/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Modelos Animais de Doenças , Orelha Interna/fisiopatologia , Orelha Interna/ultraestrutura , Técnicas de Introdução de Genes , Bócio Nodular/patologia , Bócio Nodular/fisiopatologia , Células Ciliadas Auditivas/ultraestrutura , Perda Auditiva Neurossensorial/patologia , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Mutação de Sentido Incorreto , Fenótipo , Transportadores de Sulfato/fisiologiaRESUMO
The stimulatory heterotrimeric Gs protein alpha subunit (Gsα) is a ubiquitous guanine nucleotide-binding protein that regulates the intracellular cAMP signaling pathway and consequently participates in a wide range of biological events. In the reproductive system, despite Gsα being associated with oocyte meiotic arrest in vitro, the exact role of Gsα in female fertility in vivo remains largely unknown. Here, we generated oocyte-specific Gsα knockout mice by using the Cre/LoxP system. We observed that the deletion of Gsα caused complete female infertility. Exclusion of post-implantation abnormalities, oogenesis, fertilization, and early embryo development was subsequently monitored; meiosis in Gsα-deficient oocytes precociously resumed in only 43% of antral follicles from mutant mice, indicating that alteration of meiotic pause was not the key factor in infertility. Ovulation process and number were normal, but the rate of morphological abnormal oocytes was apparently increased; spindle organization, fertilization, and early embryo development were impaired. Furthermore, the level of ROS (reactive oxygen species) and the mitochondrial aggregation increased, and antioxidant glutathione (GSH) content, ATP level, mtDNA copy number, and mitochondrial membrane potential decreased in Gsα-deficient oocytes. GV oocytes from mutant mice showed early-stage apoptosis. Meanwhile, the Gsα knockout-induced decline in oocyte quality and low developmental potential was partially rescued by antioxidant supplementation. To sum up, our results are the first to reveal that the profile of Gsα oocyte-specific deletion caused female infertility in vivo, and oxidative stress plays an important role in this event.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Oócitos/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/metabolismo , Apoptose/fisiologia , Desenvolvimento Embrionário/fisiologia , Meiose/fisiologia , Camundongos Transgênicos , Mitocôndrias/metabolismoRESUMO
Myo3a is expressed in cochlear hair cells and retinal cells and is responsible for human recessive hereditary nonsyndromic deafness (DFNB30). To investigate the mechanism of DFNB30-type deafness, we established a mouse model of Myo3a kinase domain Y137C mutation by using CRISPR/Cas9 system. No difference in hearing between 2-month-old Myo3a mutant mice and wild-type mice was observed. The hearing threshold of the ≥6-month-old mutant mice was significantly elevated compared with that of the wild-type mice. We observed degeneration in the inner ear hair cells of 6-month-old Myo3a mutant mice, and the degeneration became more severe at the age of 12 months. We also found structural abnormality in the cochlear hair cell stereocilia. Our results showed that Myo3a is essential for normal hearing by maintaining the intact structure of hair cell stereocilia, and the kinase domain plays a critical role in the normal functions of Myo3a. This mouse line is an excellent model for studying DFNB30-type deafness in humans.
Assuntos
Células Ciliadas Auditivas Internas/patologia , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Cadeias Pesadas de Miosina/genética , Miosina Tipo III/genética , Estimulação Acústica , Animais , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Técnicas de Introdução de Genes , Células Ciliadas Auditivas Internas/ultraestrutura , Masculino , MutaçãoRESUMO
In mammals, embryonic development are highly regulated morphogenetic processes that are tightly controlled by genetic elements. Failure of any one of these processes can result in embryonic malformation. The lysyl oxidase (LOX) family genes are closely related to human diseases. In this study, we investigated the essential role of lysyl oxidase-like 3 (LOXL3), a member of the LOX family, in embryonic development. Mice lacking LOXL3 exhibited perinatal lethality, and the deletion of the Loxl3 gene led to impaired development of the palate shelves, abnormalities in the cartilage primordia of the thoracic vertebrae and mild alveolar shrinkage. We found that the obvious decrease of collagen cross-links in palate and spine that was induced by the lack of LOXL3 resulted in cleft palate and spinal deformity. Thus, we provide critical in vivo evidence that LOXL3 is indispensable for mouse palatogenesis and vertebral column development. The Loxl3 gene may be a candidate disease gene resulting in cleft palate and spinal deformity.
Assuntos
Aminoácido Oxirredutases/fisiologia , Fissura Palatina/embriologia , Desenvolvimento Embrionário/fisiologia , Coluna Vertebral/anormalidades , Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/genética , Animais , Aorta/patologia , Colágeno/metabolismo , Anormalidades Craniofaciais/genética , Desmosina/metabolismo , Diafragma/patologia , Olho/metabolismo , Olho/patologia , Marcação de Genes , Hidroxiprolina/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Palato/metabolismo , Vértebras Torácicas/embriologia , Traqueia/patologiaRESUMO
MYH14 is a member of the myosin family, which has been implicated in many motile processes such as ion-channel gating, organelle translocation, and the cytoskeleton rearrangement. Mutations in MYH14 lead to a DFNA4-type hearing impairment. Further evidence also shows that MYH14 is a candidate noise-induced hearing loss (NIHL) susceptible gene. However, the specific roles of MYH14 in auditory function and NIHL are not fully understood. In the present study, we used CRISPR/Cas9 technology to establish a Myh14 knockout mice line in CBA/CaJ background (now referred to as Myh14-/- mice) and clarify the role of MYH14 in the cochlea and NIHL. We found that Myh14-/- mice did not exhibit significant hearing loss until five months of age. In addition, Myh14-/- mice were more vulnerable to high intensity noise compared to control mice. More significant outer hair cell loss was observed in Myh14-/- mice than in wild type controls after acoustic trauma. Our findings suggest that Myh14 may play a beneficial role in the protection of the cochlea after acoustic overstimulation in CBA/CaJ mice.
Assuntos
Limiar Auditivo/fisiologia , Cóclea/fisiopatologia , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Perda Auditiva Provocada por Ruído/fisiopatologia , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/metabolismo , Animais , Genótipo , Perda Auditiva Provocada por Ruído/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Cadeias Pesadas de Miosina/deficiência , Miosina Tipo II/deficiênciaRESUMO
Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs.
Assuntos
Cerebelo/metabolismo , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição PAX2/metabolismo , Células de Purkinje/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/genética , Diferenciação Celular/genética , Proliferação de Células , Cerebelo/embriologia , Cerebelo/crescimento & desenvolvimento , Proteínas do Citoesqueleto , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Proteínas de Homeodomínio/genética , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fator de Transcrição PAX2/genética , Células de Purkinje/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genéticaRESUMO
AIM: This study aims to investigate the expression levels of elastin and lysyl oxidase (LOX) family members in the urogenital tissues of natural aging mice and accelerated ovarian aging mice. METHOD: Uteri, vaginas and bladders were harvested from 18-month-old female mice and accelerated ovarian aging mice developed by chemotherapeutic agents. Untreated 3-month-old female mice were used as controls. The expression levels of elastin and LOX family members were determined by real-time polymerase chain reaction and western blot. RESULTS: Compared with untreated young female mice, the expression of elastin and LOX family members significantly decreased both in natural aging mice and accelerated ovarian aging mice. CONCLUSION: Aging is a high-risk factor for pelvic floor disorders. The failure of elastic fiber synthesis and assembly due to the decline in expression levels of elastin and LOX family members during aging may explain the molecular mechanism causing pelvic floor disorders.
Assuntos
Envelhecimento , Regulação para Baixo , Elastina/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Bexiga Urinária/metabolismo , Útero/metabolismo , Vagina/metabolismo , Animais , Elastina/genética , Feminino , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos Endogâmicos , Proteína-Lisina 6-Oxidase/genética , Bexiga Urinária/crescimento & desenvolvimento , Útero/crescimento & desenvolvimento , Vagina/crescimento & desenvolvimentoRESUMO
Lgl1 protein plays a critical role in neurodevelopment, including hippocampus, olfactory bulb, and Purkinje cell. However, the specific mechanism of LGL1 function in the midbrain remains elusive. In this study, we generated Lgl1 conditional knockout mice using Pax2-Cre, which is expressed in the midbrain, and examined the functions of Lgl1 in the midbrain. Histological analysis exhibited abnormal midbrain development characterized by enlarged ventricular aqueduct and thinning tectum cortex. Lgl1 deletion caused excessive proliferation and heightened apoptosis of neural progenitor cells in the tectum of LP cko mice. BrdU labeling studies demonstrated abnormal neuronal migration. Immunofluorescence analysis of Nestin demonstrated an irregular and clustered distribution of glial cell fibers, with the adhesion junction marker N-cadherin employed for immunofluorescent labeling, unveiling abnormal epithelial connections within the tectum of LP cko mice. The current findings suggest that the deletion of Lgl1 leads to the disruption of the expression pattern of N-cadherin, resulting in abnormal development of the midbrain.
RESUMO
Lethal giant larvae 1 (LGL1) is originally recognized as a tumor suppressor, implicated in maintaining cell polarity in Drosophila and mammalian cells. Cell polarity plays a crucial role in tumorigenesis. We previously established Pax2-LGL1 -/- conditional knockout mice but did not focus on the tumorigenesis in cerebellar primordium. HE staining was used to detect the morphological structure of the cerebellar primordium during early embryonic development in Pax2-LGL1 -/- mice. Immunofluorescence assays were used to detect the expression of polar molecules. TUNEL staining assessed tissue apoptosis. Our findings reveal that deletion of LGL1 leads to the emergence of neuroblastoma-like tissues within the cerebellum primordium during early embryogenesis. This outcome can be attributed to alterations in expression patterns of polar molecules Cdc42 and ß-catenin following early deletion of LGL1, resulting in loss of cell polarity among neuroepithelial cells and subsequent formation of tumor-like tissues. However, further histological examination demonstrated that these tumor-like tissues disappear from embryonic day 15.5 onwards within the cerebellar primordium of Pax2-LGL1 -/- mice due to apoptosis-mediated cellular compensation. Our data emphasize the importance of LGL1 in maintaining neuroepithelial cell polarity and reveal a novel role for LGL1 in regulating tumorigenesis and ablation in the cerebellar primordium.
Assuntos
Apoptose , Cerebelo , Glicoproteínas , Animais , Camundongos , Carcinogênese , Cerebelo/metabolismo , Glicoproteínas/metabolismo , Mamíferos/metabolismo , Camundongos KnockoutRESUMO
BACKGROUND: This study aimed to explore the causal relationship between age at first birth (AFB) and depression. METHODS: Using the univariable Mendelian randomization (UVMR) and multivariable Mendelian randomization (MVMR) methods to examine the potential correlation between age at first birth (AFB) and major depressive disorder and postpartum depression. A public database was used to obtain the genome-wide association studies (GWAS) summary data. We put inverse-variance-weighted (IVW) as the primary method in Mendelian randomization (MR) analysis and used sensitivity analysis to confirm the robustness of our result. RESULTS: We found a significant causal association between AFB and major depressive disorder by using the IVW algorithm (odd ratio [OR] 0.826; 95% confidence interval [CI] 0.793 - 0.861; P = 4.51 × 10- 20). MR-Egger, weighted median, simple mode and weighted mode method concluded the same result (P < 0.05). During the sensitivity analysis, the heterogeneity test (Q-value = 55.061, df = 48, P = 2.81 × 10- 01, I2 = 12.82%) and the leave-one-out plot analysis confirmed the stability of the results. The outcomes of the pleiotropy test (MR-Egger intercept = 8.932 × 10- 3. SE = 6.909 × 10- 3. P = 2.02 × 10- 01) and MR_PRESSO global test (P = 2.03 × 10- 01) indicated there is no pleiotropy. CONCLUSION: There is solid evidence that a higher age at first birth is associated with a lower risk of major depressive disorder.
Assuntos
Transtorno Depressivo Maior , Estudo de Associação Genômica Ampla , Análise da Randomização Mendeliana , Humanos , Feminino , Transtorno Depressivo Maior/genética , Depressão Pós-Parto/genética , Idade Materna , Polimorfismo de Nucleotídeo ÚnicoRESUMO
HMG (high mobility group) proteins are a diverse family of nonhistone chromosomal proteins that interact with DNA and a wide range of transcriptional regulators to regulate the structural architecture of DNA. HMGXB4 (also known as HMG2L1) is an HMG protein family member that contains a single HMG box domain. Our previous studies have demonstrated that HMGXB4 suppresses smooth muscle differentiation and exacerbates endotoxemia by promoting a systemic inflammatory response in mice. However, the expression of Hmgxb4 in vivo has not fully examined. Herein, we generated a mouse model that harbors a gene trap in the form of a lacZ gene insertion into the Hmgxb4 gene. This mouse enables the visualization of endogenous HMGXB4 expression in different tissues via staining for the ß-galactosidase activity of LacZ which is under the control of the endogenous Hmgxb4 gene promoter. We found that HMGXB4 is widely expressed in mouse tissues and is a nuclear protein. Furthermore, the Hmgxb4 gene trap mice exhibit normal cardiac function and blood pressure. Measurement of ß-galactosidase activity in the Hmgxb4 gene trap mice demonstrated that the arterial injury significantly induces Hmgxb4 expression. In summary, the Hmgxb4 gene trap reporter mouse described here provides a valuable tool to examine the expression level of endogenous Hmgxb4 in both physiological and pathological settings in vivo.
Assuntos
Proteínas de Grupo de Alta Mobilidade , Camundongos Endogâmicos C57BL , Animais , Masculino , Camundongos , beta-Galactosidase/metabolismo , beta-Galactosidase/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Óperon Lac/genética , Camundongos Transgênicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
A commercial electron Monte Carlo (eMC) dose calculation algorithm has become available in Eclipse treatment planning system. The purpose of this work was to evaluate the eMC algorithm and investigate the clinical implementation of this system. The beam modeling of the eMC algorithm was performed for beam energies of 6, 9, 12, 16, and 20 MeV for a Varian Trilogy and all available applicator sizes in the Eclipse treatment planning system. The accuracy of the eMC algorithm was evaluated in a homogeneous water phantom, solid water phantoms containing lung and bone materials, and an anthropomorphic phantom. In addition, dose calculation accuracy was compared between pencil beam (PB) and eMC algorithms in the same treatment planning system for heterogeneous phantoms. The overall agreement between eMC calculations and measurements was within 3%/2 mm, while the PB algorithm had large errors (up to 25%) in predicting dose distributions in the presence of inhomogeneities such as bone and lung. The clinical implementation of the eMC algorithm was investigated by performing treatment planning for 15 patients with lesions in the head and neck, breast, chest wall, and sternum. The dose distributions were calculated using PB and eMC algorithms with no smoothing and all three levels of 3D Gaussian smoothing for comparison. Based on a routine electron beam therapy prescription method, the number of eMC calculated monitor units (MUs) was found to increase with increased 3D Gaussian smoothing levels. 3D Gaussian smoothing greatly improved the visual usability of dose distributions and produced better target coverage. Differences of calculated MUs and dose distributions between eMC and PB algorithms could be significant when oblique beam incidence, surface irregularities, and heterogeneous tissues were present in the treatment plans. In our patient cases, monitor unit differences of up to 7% were observed between PB and eMC algorithms. Monitor unit calculations were also preformed based on point-dose prescription. The eMC algorithm calculation was characterized by deeper penetration in the low-density regions, such as lung and air cavities. As a result, the mean dose in the low-density regions was underestimated using PB algorithm. The eMC computation time ranged from 5 min to 66 min on a single 2.66 GHz desktop, which is comparable with PB algorithm calculation time for the same resolution level.
Assuntos
Algoritmos , Método de Monte Carlo , Radiometria/métodos , Planejamento da Radioterapia Assistida por Computador/métodos , Radioterapia Conformacional/métodos , Validação de Programas de Computador , Software , Indústrias , Dosagem Radioterapêutica , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Peroxisomes are involved in the regulation of various pathological processes. Peroxisomal biogenesis factor 5 (PEX5), which plays an essential role in peroxisomal biogenesis, is critical for reactive oxygen species (ROS) accumulation. However, its underlying functions in spermatogenesis have not yet been identified. Pex5 was deleted by crossing Stra8-Cre mice with Pex5flox/flox mice before the onset of meiosis. The morphology of testes and epididymides, spermatogenesis function, and fertility in both wild type (WT) and Pex5-/- mice were analysed by haematoxylin and eosin (HE) and immunofluorescent staining. Mechanism of PEX5 affecting peroxisomes and spermatogenesis were validated by Western blot and transmission electron microscopy (TEM). Transcriptome RNA sequencing (RNA-seq) was used to profile the dysregulated genes in testes from WT and Pex5-/- mice on postnatal day (P) 35. The adult Pex5 knockout male mice were completely sterile with no mature sperm production. Loss of Pex5 in spermatocytes resulted in multinucleated giant cell formation, meiotic arrest, abnormal tubulin expression, and deformed acrosome formation. Furthermore, Pex5 deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene stage. Impaired peroxisome function in Pex5 knockout mice induced ROS redundancy, which in turn led to an increase in germ cell apoptosis and a decline in autophagy. Pex5 regulates ROS during meiosis and is essential for spermatogenesis and male fertility in mice.
Assuntos
Infertilidade , Sêmen , Animais , Masculino , Camundongos , Infertilidade/metabolismo , Meiose , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Espermatócitos/metabolismo , Espermatogênese/genética , Testículo/metabolismoRESUMO
Spermatogenesis is an extremely complex process, and any obstruction can cause male infertility. RhoGDIα has been identified as a risk of male sterility. In this study, we generate RhoGDIα knockout mice, and find that the males have severely low fertility. The testes from RhoGDIα-/- mice are smaller than that in WT mice. The numbers of spermatogonia and spermatocytes are decreased in RhoGDIα-/- testis. Spermatogenesis is compromised, and spermatocyte meiosis is arrested at zygotene stage in RhoGDIα-/- mice. Acrosome dysplasia is also observed in sperms of the mutant mice. At the molecular level, RhoGDIα deficiency activate the LIMK/cofilin signaling pathway, inhibiting F-actin depolymerization, impairing testis and inducing low fertility in mouse. In addition, the treatment of RhoGDIα-/- mice with Rac1 inhibitor NSC23766 alleviate testis injury and improve sperm quality by inhibiting the LIMK/cofilin/F-actin pathway during spermatogenesis. Together, these findings reveal a previously unrecognized RhoGDIα/Rac1/F-actin-dependent mechanism involved in spermatogenesis and male fertility.