Functional suppression of Ripk1 blocks the NF-κB signaling pathway and induces neuron autophagy after traumatic brain injury.

Traumatic brain injury (TBI), known as intracranial injury, has been a serious threat to human health. Evidence exists indicating that autophagy and inflammatory responses contribute to secondary brain injury after TBI. Notably, receptor-interacting protein kinase 1 (Ripk1) exerts an important role in cell autophagy. Therefore, this study aims to explore the effect of Ripk1 on neuron autophagy and apoptosis in TBI. Initially, blood samples of patients with TBI and healthy persons were collected to detect the expression of Ripk1, nuclear factor-kappa B (NF-κB), and NF-kB inhibitor α (IKBα). Then rat models with TBI were successfully established and, respectively, treated with shRNA targeting Ripk1 (sh-Ripk1), Ripk1 overexpression plasmid (oe-Ripk1), or IKKα inhibitor (BAY 11-7082). Subsequently, reverse transcription quantitative polymerase chain reaction and Western blot analysis were conducted to detect the expression of Ripk1, IKBα, NF-κB signaling pathway-, and apoptosis-related factors. Enzyme-linked immunosorbent assay was used to detect the expression of inflammatory cytokines. Compared with healthy persons, the expression of Ripk1, NF-κB and IKBα in blood of TBI patients was significantly upregulated. After silencing of Ripk1 or inhibition of the NF-κB signaling pathway, the expression of IL-1ß, IL-6, TNF-α, Bax, and cleaved-caspase-3 was downregulated, and the expression of Bcl-2, ATG5, and LC3II/LC3I was upregulated. Furthermore, neuron injury and apoptosis were notably reduced and neuron autophagy increased significantly by Ripk1 downregulation or IKKα inhibitor. Ripk1 overexpression contributed to activation of NF-κB signaling pathway, whereby aggravating TBI-induced damage. Silencing Ripk1 suppresses TBI by inhibiting inflammation and promoting autophagy of neurons via inhibition of NF-κB signaling pathway.

CCR9 AND CCR7 are overexpressed in CD4- CD8- thymocytes of myasthenia gravis patients.

INTRODUCTION: Chemokine CC motif receptors 9 and 7 (CCR9 and CCR7) play a major role in the migration of T-cell precursors to the thymus to initiate T thymopoiesis. However, their role in development of T-cells in myasthenia gravis (MG) patients has not been fully elucidated. METHODS: Expression and distribution of CCR9+ and CCR7+ cells were detected by flow cytometry and immunofluorescence. Real-time polymerase chain reaction was used to check the adhesion molecules on CD4- CD8- double-negative (DN) thymocytes. RESULTS: CCR9 and CCR7 expression by DN thymocytes increased in the MG thymus; the levels of CCR9, CCR7, interleukin-7R mRNA increased, and CXCR4 levels decreased compared with levels in the non-MG thymus. More CCR7 and CCR9 double-positive (DP) thymocytes were gathered near the subcapsular region in MG thymus. CONCLUSIONS: Enhanced expression of CCR9 and CCR7 may complicate the differentiation of DP thymocytes from the DN stage in MG thymus. Muscle Nerve, 2016 Muscle Nerve 55: 84-90, 2017.

[Over-expression of CCL21 up-regulates the antigen presentation-related genes of CK8/18 positive thymic epithelial cells in patients with myasthenia gravis].

OBJECTIVE: To identify the distribution of chemokine (C-C motif) ligand 21 (CCL21) in the thymus of patients with myasthenia gravis (MG) and explore the effects of up-regulation of CCL21 on the expressions of antigen presentation-related genes in cytokeratin 8/18 (CK8/18) positive thymic epithelial cells (TECs) after transfected with CCL21 genes. METHODS: The expressions and distributions of CK8/18 and CCL21 in the thymus tissue of MG patients were detected by immunohistochemistry. The mRNA levels of CCL21, CCL19 and their receptor chemokine (C-C motif) receptor 7 (CCR7) in the thymus tissue of MG patients were determined by real-time quantitative PCR (qRT-PCR). Primary cultured CK8/18⁺ TECs were transfected with pCMV-CCL21, and the relative mRNA expressions of function-associated genes (CD80, ICAM-1, CD86, HLA-DR, HLA-A) in CK8/18⁺ TECs before and after the transfection were investigated by qRT-PCR. RESULTS: Immunohistochemical results showed that the number of CK8/18 positive cells in the hyperplastic thymus tissues of MG patients was significantly more than that in the normal controls, and the protein expression of CCL21 was also much higher in the hyperplastic thymus tissues. The qRT-PCR showed that the expressions of CCL21 and CCR7 mRNA increased significantly in hyperplastic thymus tissues of MG patients compared with those in normal controls, while there was no difference in the expression of CCL19. Furthermore, CK8/18 positive cells were found mainly located in cortico-medullary junction and medulla area. The relative mRNA expression levels of HLA-A, HLA-DR, ICAM and CD80 rose significantly in CK8/18⁺ TECs after transfected with pCMV-CCL21. CONCLUSION: The over-expression of CCL21 could increased the expressions of antigen presentation-related genes in CK8/18⁺ TECs in MG patients.

Nanostructured biosensors built with layer-by-layer electrostatic assembly of hemoglobin and Fe3O4@Pt nanoparticles.

This paper reports layer-by-layer (LBL) films fabricated with hemoglobin and core-shell nanoparticles with Fe(3)O(4) as the core covered by Pt (Fe(3)O(4)@Pt) and their applications in biosensing. The characterization of {Hb/Fe(3)O(4)@Pt}(n) LBL films at different layers revealed that the formation of films is step-by-step and uniform. Meanwhile, at glassy carbon electrodes modified with {Hb/Fe(3)O(4)@Pt}(n) film at different layers there was a pair of well-defined and nearly reversible peaks in cyclic voltammetry (CV). CV results indicated that the electroactivity of the structure with four bilayers was the best. The {Hb/Fe(3)O(4)@Pt}(4) film modified electrode could be used to detect H(2)O(2) and nitrite with the linear range from 0.125 µM to 0.16 mM for H(2)O(2) and 1.5 µM to 0.12 mM for nitrite as well as the detection limits of 0.03 µM for H(2)O(2) and 0.29 µM for nitrite (S/N=3). The biosensors also exhibited good reproducibility, high selectivity, and long-term stability. Our investigation showed that the strategy taking advantages of Fe(3)O(4)@Pt and LBL assembly is ideal for direct electrochemistry of redox proteins as well as the sensitive and stable mediator-free biosensors.

Mitochondrial transporter ATP binding cassette mitochondrial erythroid is a novel gene required for cardiac recovery after ischemia/reperfusion.

BACKGROUND: Oxidative stress and mitochondrial dysfunction are central mediators of cardiac dysfunction after ischemia/reperfusion. ATP binding cassette mitochondrial erythroid (ABC-me; ABCB10; mABC2) is a mitochondrial transporter highly induced during erythroid differentiation and predominantly expressed in bone marrow, liver, and heart. Until now, ABC-me function in heart was unknown. Several lines of evidence demonstrate that the yeast ortholog of ABC-me protects against increased oxidative stress. Therefore, ABC-me is a potential modulator of the outcome of ischemia/reperfusion in the heart. METHODS AND RESULTS: Mice harboring 1 functional allele of ABC-me (ABC-me(+/-)) were generated by replacing ABC-me exons 2 and 3 with a neomycin resistance cassette. Cardiac function was assessed with Langendorff perfusion and echocardiography. Under basal conditions, ABC-me(+/-) mice had normal heart structure, hemodynamic function, mitochondrial respiration, and oxidative status. However, after ischemia/reperfusion, the recovery of hemodynamic function was reduced by 50% in ABC-me(+/-) hearts as a result of impairments in both systolic and diastolic function. This reduction was associated with impaired mitochondrial bioenergetic function and with oxidative damage to both mitochondrial lipids and sarcoplasmic reticulum calcium ATPase after reperfusion. Treatment of ABC-me(+/-) hearts with the superoxide dismutase/catalase mimetic EUK-207 prevented oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and restored mitochondrial and cardiac function to wild-type levels after reperfusion. CONCLUSIONS: Inactivation of 1 allele of ABC-me increases the susceptibility to oxidative stress induced by ischemia/reperfusion, leading to increased oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and to impaired functional recovery. Thus, ABC-me is a novel gene that determines the ability to tolerate cardiac ischemia/reperfusion.