The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination.

The signaling adaptor MAVS forms prion-like aggregates to activate an innate antiviral immune response after viral infection. However, the molecular mechanisms that regulate MAVS aggregation are poorly understood. Here we identified TRIM31, an E3 ubiquitin ligase of the TRIM family of proteins, as a regulator of MAVS aggregation. TRIM31 was recruited to mitochondria after viral infection and specifically regulated antiviral signaling mediated by RLR pattern-recognition receptors. TRIM31-deficient mice were more susceptible to infection with RNA virus than were wild-type mice. TRIM31 interacted with MAVS and catalyzed the Lys63 (K63)-linked polyubiquitination of Lys10, Lys311 and Lys461 on MAVS. This modification promoted the formation of prion-like aggregates of MAVS after viral infection. Our findings reveal new insights in the molecular regulation of MAVS aggregation and the cellular antiviral response through TRIM31-mediated K63-linked polyubiquitination of MAVS.

A Peptide Derived from IKK-Interacting Protein Attenuates NF-κB Activation and Inflammation.

The IκB kinase (IKK) complex plays a vital role in regulating the NF-κB activation. Aberrant NF-κB activation is involved in various inflammatory diseases. Thus, targeting IKK activation is an ideal therapeutic strategy to cure and prevent inflammatory diseases related to NF-κB activation. In a previous study, we demonstrated that IKK-interacting protein (IKIP) inhibits the phosphorylation of IKKα/ß and the activation of NF-κB through disruption of the formation of IKK complex. In this study, we identified a 15-aa peptide derived from mouse IKIP (46-60 aa of IKIP), which specifically suppressed IKK activation and NF-κB targeted gene expression via disrupting the association of IKKß and NEMO. Importantly, administration of the peptide reduced LPS-induced acute inflammation and attenuated Zymosan-induced acute arthritis in mice. These findings suggest that this IKIP peptide may be a promising therapeutic reagent in the prevention and treatment of inflammatory diseases.

OTUD1 Regulates Antifungal Innate Immunity through Deubiquitination of CARD9.

CARD9 is an essential adaptor protein in antifungal innate immunity mediated by C-type lectin receptors. The activity of CARD9 is critically regulated by ubiquitination; however, the deubiquitinases involved in CARD9 regulation remain incompletely understood. In this study, we identified ovarian tumor deubiquitinase 1 (OTUD1) as an essential regulator of CARD9. OTUD1 directly interacted with CARD9 and cleaved polyubiquitin chains from CARD9, leading to the activation of the canonical NF-κB and MAPK pathway. OTUD1 deficiency impaired CARD9-mediated signaling and inhibited the proinflammatory cytokine production following fungal stimulation. Importantly, Otud1 -/- mice were more susceptible to fungal infection than wild-type mice in vivo. Collectively, our results identify OTUD1 as an essential regulatory component for the CARD9 signaling pathway and antifungal innate immunity through deubiquitinating CARD9.

Development of a prediction model for the risk of 30-day unplanned readmission in older patients with heart failure: A multicenter retrospective study.

BACKGROUND AND AIM: Heart failure (HF) imposes significant global health costs due to its high incidence, readmission, and mortality rate. Accurate assessment of readmission risk and precise interventions have become important measures to improve health for patients with HF. Therefore, this study aimed to develop a machine learning (ML) model to predict 30-day unplanned readmissions in older patients with HF. METHODS AND RESULTS: This study collected data on hospitalized older patients with HF from the medical data platform of Chongqing Medical University from January 1, 2012, to December 31, 2021. A total of 5 candidate algorithms were selected from 15 ML algorithms with excellent performance, which was evaluated by area under the operating characteristic curve (AUC) and accuracy. Then, the 5 candidate algorithms were hyperparameter tuned by 5-fold cross-validation grid search, and performance was evaluated by AUC, accuracy, sensitivity, specificity, and recall. Finally, an optimal ML model was constructed, and the predictive results were explained using the SHapley Additive exPlanations (SHAP) framework. A total of 14,843 older patients with HF were consecutively enrolled. CatBoost model was selected as the best prediction model, and AUC was 0.732, with 0.712 accuracy, 0.619 sensitivity, and 0.722 specificity. NT.proBNP, length of stay (LOS), triglycerides, blood phosphorus, blood potassium, and lactate dehydrogenase had the greatest effect on 30-day unplanned readmission in older patients with HF, according to SHAP results. CONCLUSIONS: The study developed a CatBoost model to predict the risk of unplanned 30-day special-cause readmission in older patients with HF, which showed more significant performance compared with the traditional logistic regression model.

A Comparative Study of Skin Changes in Different Species of Mice in Chronic Photoaging Models.

This study aimed to design a novel mouse model of chronic photoaging. We used three different species of mice (C57BL/6J, ICR, and KM) to create a chronic photoaging model of the skin. The irradiation time was gradually increased for 40 consecutive days. The skins of the mice were removed on day 41 and subjected to staining to observe them for morphological changes. Immunohistochemistry was used to detect tumor necrosis factor-α (TNF-α) and p53 expression; superoxide dismutase (SOD) and malondialdehyde (MDA) were measured as well. Compared with C57BL/J mice, which showed hyperpigmentation, the irradiated skin of ICR and KM mice showed more obvious skin thickening and photoaging changes of the collagen and elastic fibers. KM mice had higher levels of inflammation, oxidative stress, and senescent cells. Compared with the 5-month-old KM mice, the photoaging changes of the 9-month-old KM mice were more pronounced, the SOD values were lower, and the MDA values were higher. In summary, KM mice have higher levels of abnormal elastic fibers, inflammation, cellular senescence, and oxidative stress than ICR mice, and are more suitable for studies related to chronic skin photoaging. C57BL/6J mice were found to be suitable for studies related to skin pigmentation due to photoaging.

Continuous ZnO nanoparticle exposure induces melanoma-like skin lesions in epidermal barrier dysfunction model mice through anti-apoptotic effects mediated by the oxidative stress-activated NF-κB pathway.

BACKGROUND: Increasing interest in the hazardous properties of zinc oxide nanoparticles (ZnO NPs), commonly used as ultraviolet filters in sunscreen, has driven efforts to study the percutaneous application of ZnO NPs to diseased skin; however, in-depth studies of toxic effects on melanocytes under conditions of epidermal barrier dysfunction remain lacking. METHODS: Epidermal barrier dysfunction model mice were continuously exposed to a ZnO NP-containing suspension for 14 and 49 consecutive days in vivo. Melanoma-like change and molecular mechanisms were also verified in human epidermal melanocytes treated with 5.0 µg/ml ZnO NPs for 72 h in vitro. RESULTS: ZnO NP application for 14 and 49 consecutive days induced melanoma-like skin lesions, supported by pigmented appearance, markedly increased number of melanocytes in the epidermis and dermis, increased cells with irregular nuclei in the epidermis, recruited dendritic cells in the dermis and dysregulated expression of melanoma-associated gene Fkbp51, Trim63 and Tsp 1. ZnO NPs increased oxidative injury, inhibited apoptosis, and increased nuclear factor kappa B (NF-κB) p65 and Bcl-2 expression in melanocytes of skin with epidermal barrier dysfunction after continuously treated for 14 and 49 days. Exposure to 5.0 µg/ml ZnO NPs for 72 h increased cell viability, decreased apoptosis, and increased Fkbp51 expression in melanocytes, consistent with histological observations in vivo. The oxidative stress-mediated mechanism underlying the induction of anti-apoptotic effects was verified using the reactive oxygen species scavenger N-acetylcysteine. CONCLUSIONS: The entry of ZnO NPs into the stratum basale of skin with epidermal barrier dysfunction resulted in melanoma-like skin lesions and an anti-apoptotic effect induced by oxidative stress, activating the NF-κB pathway in melanocytes.

Neoadjuvant therapy and sentinel lymph node biopsy in HER2-positive breast cancer patients: results from the PEONY trial.

OBJECTIVE: The aim of the study is to evaluate the optimal timing of sentinel lymph node biopsy (SLNB) in patients with clinical negative axillary lymph nodes (ALNs) before neoadjuvant therapy (NAT) and the feasibility of SLNB substituting for ALN dissection in patients with positive ALNs who convert to node negative, for HER2-positive disease. METHODS: Patients receiving SLNB with dual tracer mapping in the PEONY trial were analyzed. RESULTS: For 80 patients with clinical negative ALNs, the node negative rate by pathology after NAT was 83.8%. SLNB was performed after NAT in 71 patients. The identification rate of sentinel lymph nodes (SLNs) was 100%. For patients with positive ALNs before NAT, the axillary pathologic complete response rate in the dual HER2 blockade arm was significantly higher than that in the single blockade arm (p = 0.002). SLNB was performed in 71 patients. The identification rate was 100% and the false-negative rate was 17.2%. The false-negative rates were 33.3%, 14.3%, and 0 when 1, 2, and more than 2 SLNs were detected. There was no false-negative case when more than 1 SLN and the clipped nodes were removed simultaneously. CONCLUSIONS: For clinical ALN negative patients, HER2-positive subtype is found to have high node negative rate by pathology and it is recommended to undergo SLNB after NAT. For patients with positive ALNs who convert to negative, the false-negative rate is high. Dual tracer mapping, more than 2 SLNs detected, more than 1 SLN identified plus the clips placed are the guarantees for lower false-negative rate.

mDia1 and Cdc42 Regulate Activin B-Induced Migration of Bone Marrow-Derived Mesenchymal Stromal Cells.

In a previous study, we have shown that Activin B is a potent chemoattractant for bone marrow-derived mesenchymal stromal cells (BMSCs). As such, the combination of Activin B and BMSCs significantly accelerated rat skin wound healing. In another study, we showed that RhoA activation plays a key role in Activin B-induced BMSC migration. However, the role of the immediate downstream effectors of RhoA in this process is unclear. Here, we demonstrated that mammalian homolog of Drosophila diaphanous-1 (mDia1), a downstream effector of RhoA, exerts a crucial function in Activin B-induced BMSC migration by promoting membrane ruffling, microtubule morphology, and adhesion signaling dynamics. Furthermore, we showed that Activin B does not change Rac1 activity but increases Cdc42 activity in BMSCs. Inactivation of Cdc42 inhibited Activin B-stimulated Golgi reorientation and the cell migration of BMSCs. Furthermore, knockdown of mDia1 affected Activin B-induced BMSC-mediated wound healing in vivo. In conclusion, this study demonstrated that the RhoA-mDia1 and Cdc42 pathways regulate Activin B-induced BMSC migration. This study may help to optimize clinical MSC-based transplantation strategies to promote skin wound healing. Stem Cells 2019;37:150-162.

Effect of iPSCs-derived keratinocytes on healing of full-thickness skin wounds in mice.

Induced pluripotent stem cells (iPSCs) provide new approaches for the management of severe skin wound healing due to their infinite proliferative capacity, pluripotency into multiple lineages, and important ethical acceptability. In this study, we aimed to differentiate iPSCs into keratinocytes and to observe the therapeutic effects of transplanted iPSCs-derived keratinocytes on wound healing in mice. Here, mouse iPSCs had been successfully differentiated into keratinocytes. Next, iPSCs-derived keratinocytes labeled by CSFE were injected directly into the full-thickness skin wound. Hematoxylin & Eosin, Masson's trichrome, EdU staining and immunohistochemical staining were performed to assess the effects of iPSCs-derived keratinocytes on wound healing. Our results showed that transplantation of iPSCs-derived keratinocytes into full-thickness skin wound site accelerated re-epithelialization and reduced scar formation. In addition, we found that conditioned medium of iPSCs-derived keratinocytes reduced the expression of α-SMA and COL1 and increased the expression of MMP1 in fibroblasts in vitro. Further mechanism studies show the TNF-α-induced activation of NF-κB is involved in the effect of conditioned medium of iPSCs-derived keratinocytes on fibroblasts. In conclusion, this study has shown that iPSCs-derived keratinocytes decrease the healing time by increasing the epithelization rate and reduce scarring, suggesting a possible new treatment for skin wound healing.

Activin B Stimulates Mouse Vibrissae Growth and Regulates Cell Proliferation and Cell Cycle Progression of Hair Matrix Cells through ERK Signaling.

Activins and their receptors play important roles in the control of hair follicle morphogenesis, but their role in vibrissae follicle growth remains unclear. To investigate the effect of Activin B on vibrissae follicles, the anagen induction assay and an in vitro vibrissae culture system were constructed. Hematoxylin and eosin staining were performed to determine the hair cycle stages. The 5-ethynyl-2'-deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) assays were used to examine the cell proliferation. Flow cytometry was used to detect the cell cycle phase. Inhibitors and Western blot analysis were used to investigate the signaling pathway induced by Activin B. As a result, we found that the vibrissae follicle growth was accelerated by 10 ng/mL Activin B in the anagen induction assay and in an organ culture model. 10 ng/mL Activin B promoted hair matrix cell proliferation in vivo and in vitro. Moreover, Activin B modulates hair matrix cell growth through the ERK⁻Elk1 signaling pathway, and Activin B accelerates hair matrix cell transition from the G1/G0 phase to the S phase through the ERK⁻Cyclin D1 signaling pathway. Taken together, these results demonstrated that Activin B may promote mouse vibrissae growth by stimulating hair matrix cell proliferation and cell cycle progression through ERK signaling.

Deletion of epithelial cell-specific Cdc42 leads to enamel hypermaturation in a conditional knockout mouse model.

Recent evidence suggests that GTPases Rho family plays an important role in tooth development; however, the role of Cdc42 in tooth development remains unclear. We aimed to investigate the function of Cdc42 in tooth development and amelogenesis. We generated an epithelial cell-specific K5-Cdc42 knockout (KO) mouse to evaluate post-eruption dental phenotypes using a K5-Cre driver line. This model overcomes the previously reported perinatal lethality. Tooth phenotypes were analyzed by micro X-ray, micro-computed tomography (CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), wear rate, shear strength, and a microhardness test. Enamel matrix protein expression was determined by immunohistochemistry. KO mice displayed a hypomaturation phenotype, including incisors that lacked yellow pigmentation and were abnormally white, rapid attrition of molars following eruption, and decreased micro-hardness and shearing strength. Micro-CT data revealed that of incisor and molar enamel volumes were smaller in the KO than in wild-type (WT) mice. SEM analysis showed that the enamel prism structure was disordered. In addition, HE staining indicated a remarkable difference in the ameloblast morphology and function between KO and WT mice, and immunohistochemistry showed increased expression of amelogenin, ameloblastin, matrix metallopeptidase 20, kallikrein-related peptidase 4 and amelotin in the KO mice teeth. Our results suggest epithelium cell-specific Cdc42 deletion leads to tooth hypomaturation and transformation of the enamel prism structure that is likely due to altered ameloblast morphology and the secretion of enamel matrix proteins and proteases. This is the first in vivo evidence suggesting that Cdc42 is essential for proper tooth development and amelogenesis.

Antioxidative Effect of Quetiapine on Acute Ultraviolet-B-Induced Skin and HaCaT Cell Damage.

Quetiapine is a new type of antipsychotic drug, with effective protection of pheochromocytoma PC12 cells from oxidative stress-induced apoptosis. Ultraviolet-B radiation can increase reactive oxygen species (ROS) production, resulting in significant inflammatory responses in damaged skin. Thus, the purpose of this study is to explore whether quetiapine protects the skin from intermediate-wave ultraviolet (UVB)-induced damage through antioxidant stress. In vivo, we found quetiapine treatment was able to significantly decrease skin thickness, erythema, and edema, as well as inflammation compared to control group. Moreover, quetiapine treatment increased the activities of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). In addition, it reduced the production of malondialdehyde (MDA), a kind of oxidized lipid. In vitro, we found that quetiapine blocked UVB-induced intracellular ROS generation and maintained the cell activity at a normal level. Furthermore, we tested the phosphorylation of p38 both in vivo and in vitro, and we found that quetiapine could inhibit phosphorylation of p38, which is caused by UVB irradiation. We concluded that quetiapine was able to relieve UVB-induced skin damage through its antioxidative properties. These effects might be associated with p38 MAPK signaling pathway.

[Research progress of mitochondrial fission and fusion related proteins].

Mitochondria are an essential component of multicellular life and play important roles in the health of the cells and the body. Mitochondria can produce energy by oxidative phosphorylation, mediate calcium and reactive oxygen signal transduction, and regulate cell apoptosis. Recent studies indicate that mitochondria continually change their shapes and distribution by fission and fusion, which are collectively termed mitochondrial dynamics. Mitochondrial dynamics play critical roles in maintaining mitochondrial function. This review focuses on the structure and biological functions of mitochondrial fission and fusion related proteins in mammal cells.

RhoA regulates Activin B-induced stress fiber formation and migration of bone marrow-derived mesenchymal stromal cell through distinct signaling.

BACKGROUND: In our previous study, Activin B induced actin stress fiber formation and cell migration in Bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. However, the underlying molecular mechanisms are not well studied. RhoA is recognized to play a critical role in the regulation of actomyosin cytoskeletal organization and cell migration. METHODS: Pull-down assay was performed to investigate the activity of RhoA. The dominant-negative mutants of RhoA (RhoA(N19)) was used to determine whether RhoA has a role in Activin B-induced cytoskeleton organization and cell migration in BMSCs. Cytoskeleton organization was examined by fluorescence Rhodamine-phalloidin staining, and cell migration by transwell and cell scratching assay. Western blot was carried out to investigate downstream signaling cascade of RhoA. Inhibitor and siRNAs were used to detect the role of downstream signaling in stress fiber formation and/or cell migration. RESULTS: RhoA was activated by Activin B in BMSCs. RhoA(N19) blocked Activin B-induced stress fiber formation and cell migration. ROCK inhibitor blocked Activin B-induced stress fiber formation but enhanced BMSCs migration. Activin B induced phosphorylation of LIMK2 and Cofilin, which was abolished by ROCK inhibition. Both of siRNA LIMK2 and siRNA Cofilin inhibited Activin B-induced stress fiber formation. CONCLUSIONS: RhoA regulates Activin B-induced stress fiber formation and migration of BMSCs. A RhoA-ROCK-LIMK2-Cofilin signaling node exists and regulates actin stress fiber formation. RhoA regulates Activin B-induced cell migration independent of ROCK. GENERAL SIGNIFICANCE: Better understanding of the molecular mechanisms of BMSCs migration will help optimize therapeutic strategy to target BMSCs at injured tissues.

Autoantibodies to NR2A Peptide of the Glutamate/NMDA Receptor in Patients with Seizure Disorders in Neuropsychiatric Systemic Lupus Erythematosus.

Objective. Seizure disorders are one of the most disabling, life-threatening, and the least understood syndromes associated with neuropsychiatric SLE (NPSLE). N-Methyl-D-aspartate (NMDA) receptors are a subgroup of the glutamate receptor family, whose NR2A subunit was found on neuronal cells (anti-NR2A) in NPSLE patients with different types of epilepsy. The present study was conducted to determine the serum levels of anti-NR2A antibodies in a large group of SLE patients, to investigate the possible correlation between the presence of the NR2A specific antibodies and NPSLE-related seizure disorders. Methods and Results. The study population consisted of 107 SLE patients and 43 age- and sex-matched healthy controls. 73 SLE patients had active disease. 36 of these had NPSLE. NMDA levels were measured by ELISA. Clinical and serological parameters were assessed according to routine procedures. The levels of anti-NR2A antibodies were significantly higher in NPSLE patients, compared with non-NPSLE patients and healthy controls. Furthermore, the levels of NPSLE in patients with seizure disorders were shown to be higher than in those with cognitive dysfunction and other CNS symptoms, however, without significance. Increase in serum anti-NR2A antibodies levels correlated to anti-dsDNA antibody and SLEDAI as well as complement levels. Conclusion. We suggest that anti-NR2A antibodies play a role in the pathogenesis of NPSLE with seizure disorders.

Oxytocin inhibits lipopolysaccharide-induced inflammation in microglial cells and attenuates microglial activation in lipopolysaccharide-treated mice.

BACKGROUND: Overactivated microglia is involved in various kinds of neurodegenerative diseases. Suppression of microglial overactivation has emerged as a novel strategy for treatment of neuroinflammation-based neurodegeneration. In the current study, anti-inflammatory effects of oxytocin (OT), which is a highly conserved nonapeptide with hormone and neurotransmitter properties, were investigated in vitro and in vivo. METHODS: BV-2 cells and primary microglia were pre-treated with OT (0.1, 1, and 10 µM) for 2 h followed by LPS treatment (500 ng/ml); microglial activation and pro-inflammatory mediators were measured by Western blot, RT-PCR, and immunofluorescence. The MAPK and NF-κB pathway proteins were assessed by Western blot. The intracellular calcium concentration ([Ca(2+)]i) was determined using Fluo2-/AM assay. Intranasal application of OT was pre-treated in BALB/C mice (adult male) followed by injected intraperitoneally with LPS (5 mg/kg). The effect of OT on LPS-induced microglial activation and pro-inflammatory mediators was measured by Western blot, RT-PCR, and immunofluorescence in vivo. RESULTS: Using the BV-2 microglial cell line and primary microglia, we found that OT pre-treatment significantly inhibited LPS-induced microglial activation and reduced subsequent release of pro-inflammatory factors. In addition, OT inhibited phosphorylation of ERK and p38 but not JNK MAPK in LPS-induced microglia. OT remarkably reduced the elevation of [Ca(2+)]i in LPS-stimulated BV-2 cells. Furthermore, a systemic LPS-treated acute inflammation murine brain model was used to study the suppressive effects of OT against neuroinflammation in vivo. We found that pre-treatment with OT showed marked attenuation of microglial activation and pro-inflammatory factor levels. CONCLUSIONS: Taken together, the present study demonstrated that OT possesses anti-neuroinflammatory activity and might serve as a potential therapeutic agent for treating neuroinflammatory diseases.

Palmitic acid affects proliferation and differentiation of neural stem cells in vitro.

High-lipid diet composed of saturated fatty acids (SFAs) has significant detrimental effects on brain homeostasis, and deleterious effects of SFAs on various cells have been well documented. However, the effects of SFAs on neural stem Cells (NSCs) function have not been fully explored. The aim of this study was to determine whether palmitic acid (PA) affected the proliferation and differentiation of murine-derived NSCs. The results showed that PA dose dependently suppressed viability of NSCs and was cytotoxic at high concentrations. The toxic levels of PA inhibited the proliferation of NSCs as shown by reduced bromodeoxyuridine labeling of NSCs, which is correlated with reactive oxygen species generation. Pretreatment of the cells with the antioxidant N-acetyl-L-cysteine inhibitor significantly attenuated the effects of PA on the proliferation of NSCs. Furthermore, nontoxic levels of PA promoted astrocytogenesis in the differentiated NSCs, associated with Stat3 activation and altered expression of serial of basic helix-loop-helix transcription factor genes. Altogether, our data have demonstrated that PA has a significant impact on proliferation and differentiation of NSCs in vitro and may be useful for elucidating the role of SFAs in regulating NSCs fate in physiological and pathological settings.