Role of LGL1 in cerebellar primordium of embryonic mice.

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.

RhoGDIα regulates spermatogenesis through Rac1/cofilin/F-actin signaling.

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.

Hadh deficiency induced oligoasthenoteratozoospermia through the TNF-α/Bcl-2 pathway in male mice.

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.

Activation of Rictor/mTORC2 signaling acts as a pivotal strategy to protect against sensorineural hearing loss.

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.

Simultaneous Study of Anti-Ferroptosis and Antioxidant Mechanisms of Butein and (S)-Butin.

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 (PTIO•)-trapping assay, and α,α-diphenyl-ß-picrylhydrazyl radical (DPPH•)-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.

Role of CCR7 on dendritic cell­mediated immune tolerance in the airways of allergy­induced asthmatic rats.

Dendritic cells (DCs) have an important role in initiating and maintaining the immune inflammatory response in allergic asthma, and CC chemokine receptor 7 (CCR7) is directly involved in the pathogenesis of DC­ and T cell­mediated allergic asthma. The present study aimed to investigate the effects of CCR7 on DC­mediated immune tolerance in allergic asthma. In the present study, bone marrow­derived DCs were transfected with an adenovirus encoding the rat CCR7 gene or a short hairpin RNA targeting CCR7 (sh­CCR7). Rats injected with DCs overexpressing CCR7 or presenting CCR7 knockdown were examined. After the rats were injected with DCs via the tail vein, bronchoalveolar lavage fluid was collected to assess its cellular composition. The protein expression levels of CCR7 in DCs were determined using immunohistochemistry and western blot analysis. The protein expression levels of interferon­Î³ (IFN­Î³), interleukin­4 (IL­4), IL­10, IL­12, transforming growth factor­ß (TGF­ß) and immunoglobulin E (IgE) were determined by ELISA. Compared with the control group, the protein expression level of CCR7 was significantly higher in the CCR7 overexpression group and significantly lower in sh­CCR7 group. Similarly, the number of DCs was higher in the CCR7 overexpression group and lower in the sh­CCR7 group. The protein expression levels of IL­10 and TGF­ß were significantly lower in the CCR7 overexpression group and higher in the sh­CCR7 group. In addition, the expression levels of IL­4, IL­12, IFN­Î³ and IgE were higher in the CCR7 overexpression group and lower in the sh­CCR7 group. The present results suggested that the role of cytokines and IgE in immune inflammation and immune tolerance in allergic asthma may be associated with the expression level of CCR7 in DCs, suggesting that CCR7 may serve a role in DC­mediated immune tolerance in allergic asthma.

Expression and pathological significance of CC chemokine receptor 7 and its ligands in the airway of asthmatic rats exposed to cigarette smoke.

BACKGROUND: Cigarette smoking aggravates the symptoms of asthma, leading to the rapid decline of lung function. Dendritic cells (DCs) and lymphocytes are considered initiating and promoting factors for the airway inflammation reactions of asthma. In addition, activation of CC chemokine receptor 7 (CCR7) by chemokine (C-C motif) ligand (CCL) 19 and 21 promotes DCs and T cells migration to lymphoid tissues during inflammation. We aimed to examine how cigarette smoke affects the expression of CCR7 in the lungs of asthmatic rats and explore the signaling mechanism linking CCR7 expression to exacerbation of symptoms. METHODS: Forty Wistar rats were randomized to four groups: control, asthma, smoke exposure, and asthma with smoke exposure groups. A rat asthma model was established by intraperitoneal ovalbumin injection. CCR7 expression was examined with immunohistochemistry and western blotting. The number of airway DCs was determined by OX62 immunohistochemistry. Interferon (INF)-γ, interleukin (IL)-4, CCL19, and CCL21 expression levels in blood and bronchioalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assays (ELISAs). RESULTS: Tissue CCR7 expression, peripheral blood and BALF CCL19 and CCL21 concentrations, and the number of airway DCs were significantly higher in the asthma with smoke exposure group than the asthma group (P<0.01). In addition, INF-γ expression was decreased and IL-4 increased in the asthma and asthma with smoke exposure groups compared with the control group (P<0.01), and in the asthma with smoke exposure group compared with the asthma group (P<0.01). Expression of CCR7 correlated negatively with INF-γ expression in peripheral blood and BALF (P<0.01), and positively with the airway DCs and IL-4 expression in the peripheral blood and BALF (P<0.01). CONCLUSIONS: Cigarette smoking may aggravate asthma symptoms by attenuating immunity, possibly through CCR7-mediated DCs aggregation in lung tissue.

Loss of liver kinase B1 causes planar polarity defects in cochlear hair cells in mice.

The tumor suppressor gene liver kinase B1 (LKB1), also called STK11, encodes a serine/threonine kinase. LKB1 plays crucial roles in cell differentiation, proliferation, and polarity. In this study, LKB1 conditional knockout mice (LKB1Pax2 CKO mice) were generated using Pax2-Cre mice to investigate the function of LKB1 in inner ear hair cells during early embryonic period. LKB1Pax2 CKO mice died perinatally. Immunofluorescence and scanning electron microscopy revealed that stereociliary bundles in LKB1Pax2 CKO mice were clustered and misoriented, respectively. Moreover, ectopic distribution of kinocilium bundles resulting from abnormal migration of kinocilium was observed in the mutant mice. The orientation of stereociliary bundles and the migration of kinocilia are critical indicators of planar cell polarity (PCP) of hair cells. LKB1 deficiency in LKB1Pax2 CKO mice thus disrupted hair cell planar polarity during embryonic development. Our results suggest that LKB1 is required in PCP formation in cochlear hair cells in mice.

Accelerated hepatocellular carcinoma development in CUL4B transgenic mice.

Cullin 4B (CUL4B) is a component of the Cullin 4B-Ring E3 ligase (CRL4B) complex that functions in proteolysis and in epigenetic regulation. CUL4B possesses tumor-promoting properties and is markedly upregulated in many types of human cancers. To determine the role of CUL4B in liver tumorigenesis, we generated transgenic mice that expressed human CUL4B in livers and other tissues and evaluated the development of spontaneous and chemically-induced hepatocellular carcinomas. We observed that CUL4B transgenic mice spontaneously developed liver tumors at a high incidence at old ages and exhibited enhanced DEN-induced hepatocarcinogenesis. There was a high proliferation rate in the livers of CUL4B transgenic mice that was accompanied by increased levels of Cdk1, Cdk4 and cyclin D1 and decreased level of p16. The transgenic mice also exhibited increased compensatory proliferation after DEN-induced liver injury, which was accompanied by activation of Akt, Erk, p38 and NF-κB. We also found that Prdx3 was downregulated and that DEN induced a higher level of reactive oxygen species in the livers of transgenic mice. Together, our results demonstrate a critical role of CUL4B in hepatocarcinogenesis in mice.

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

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.

Comprehensive evaluation and clinical implementation of commercially available Monte Carlo dose calculation algorithm.

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.

[Microsomal epoxide hydrolase gene polymorphism and susceptibility to chronic obstructive pulmonary disease in Han nationality of North China].

OBJECTIVE: We investigated whether polymorphism in gene for microsomal epoxide hydrolase (mEH) has any bearing on individual susceptibility to the development of chronic obstructive pulmonary disease. METHOD: The genotypes of 55 patients with COPD and 52 healthy smoking control subjects were tested with polymerase chain reaction followed by restriction fragment length polymorphism for mEH gene. RESULT: The frequency of polymorphic genotypes of mEH showed no difference between the COPD group and the control group. In COPD group mEH exon 3 homozygous wild-type, heterozygote and homozygous mutant was 27.3%, 27.3% and 45.5% respectively and exon 4 homozygous wild-type, heterozygote and homozygous mutant was 72.7%, 18.2% and 9.1% respectively. CONCLUSION: Genetic polymorphism in mEH is not associated with development of COPD in Han nationality of North China.