Targeting Mitochondrial Complex I Deficiency in MPP+/MPTP-induced Parkinson's Disease Cell Culture and Mouse Models by Transducing Yeast NDI1 Gene.

BACKGROUND: MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), original found in synthetic heroin, causes Parkinson's disease (PD) in human through its metabolite MPP+ by inhibiting complex I of mitochondrial respiratory chain in dopaminergic neurons. This study explored whether yeast internal NADH-quinone oxidoreductase (NDI1) has therapeutic effects in MPTP- induced PD models by functionally compensating for the impaired complex I. MPP+-treated SH-SY5Y cells and MPTP-treated mice were used as the PD cell culture and mouse models respectively. The recombinant NDI1 lentivirus was transduced into SH-SY5Y cells, or the recombinant NDI1 adeno-associated virus (rAAV5-NDI1) was injected into substantia nigra pars compacta (SNpc) of mice. RESULTS: The study in vitro showed NDI1 prevented MPP+-induced change in cell morphology and decreased cell viability, mitochondrial coupling efficiency, complex I-dependent oxygen consumption, and mitochondria-derived ATP. The study in vivo revealed that rAAV-NDI1 injection significantly improved the motor ability and exploration behavior of MPTP-induced PD mice. Accordingly, NDI1 notably improved dopaminergic neuron survival, reduced the inflammatory response, and significantly increased the dopamine content in striatum and complex I activity in substantia nigra. CONCLUSIONS: NDI1 compensates for the defective complex I in MPP+/MPTP-induced models, and vastly alleviates MPTP-induced toxic effect on dopaminergic neurons. Our study may provide a basis for gene therapy of sporadic PD with defective complex I caused by MPTP-like substance.

Sequence-Guided Localization of DNA Hybridization Enables Highly Selective and Robust Genotyping.

Genetic variations are always related to human diseases or susceptibility to therapies. Nucleic acid probes that precisely distinguish closely related sequences become an indispensable requisite both in research and clinical applications. Here, a Sequence-guided DNA LOCalization for leaKless DNA detection (SeqLOCK) is introduced as a technique for DNA hybridization, where the intended targets carrying distinct "guiding sequences" act selectively on the probes. In silicon modeling, experimental results reveal considerable agreement (R2  = 0.9228) that SeqLOCK is capable of preserving high discrimination capacity at an extraordinarily wide range of target concentrations. Furthermore, SeqLOCK reveals high robustness to various solution conditions and can be directly adapted to nucleic acid amplification techniques (e.g., polymerase chain reaction) without the need for laborious pre-treatments. Benefiting from the low hybridization leakage of SeqLOCK, three distinct variations with a clinically relevant mutation frequency under the background of genomic DNA can be discriminated simultaneously. This work establishes a reliable nucleic acid hybridization strategy that offers great potential for constructing robust and programmable systems for molecular sensing and computing.

Gene therapy of yeast NDI1 on mitochondrial complex I dysfunction in rotenone-induced Parkinson's disease models in vitro and vivo.

PURPOSE: Parkinson's disease (PD) is the second most common neurodegenerative disease without cure or effective treatment. This study explores whether the yeast internal NADH-quinone oxidoreductase (NDI1) can functionally replace the defective mammalian mitochondrial complex I, which may provide a gene therapy strategy for treating sporadic PD caused by mitochondrial complex I dysfunction. METHOD: Recombinant lentivirus expressing NDI1 was transduced into SH-SY5Y cells, or recombinant adeno-associated virus type 5 expressing NDI1 was transduced into the right substantia nigra pars compacta (SNpc) of mouse. PD cell and mouse models were established by rotenone treatment. The therapeutic effects of NDI1 on rotenone-induced PD models in vitro and vivo were assessed in neurobehavior, neuropathology, and mitochondrial functions, by using the apomorphine-induced rotation test, immunohistochemistry, immunofluorescence, western blot, complex I enzyme activity determination, oxygen consumption detection, ATP content determination and ROS measurement. RESULTS: NDI1 was expressed and localized in mitochondria in SH-SY5Y cells. NDI1 resisted rotenone-induced changes in cell morphology, loss of cell viability, accumulation of α-synuclein and pS129 α-synuclein, mitochondrial ROS production and mitochondria-mediated apoptosis. The basal and maximal oxygen consumption, mitochondrial coupling efficiency, basal and oligomycin-sensitive ATP and complex I activity in cell model were significantly increased in rotenone + NDI1 group compared to rotenone + vector group. NDI1 was efficiently expressed in dopaminergic neurons in the right SNpc without obvious adverse effects. The rotation number to the right side (NDI1-treated side) was significantly increased compared to that to the left side (untreated side) in mouse model. The number of viable dopaminergic neurons, the expression of tyrosine hydroxylase, total and maximal oxygen consumption, mitochondrial coupling efficiency and complex I enzyme activity in right substantia nigra, and the content of dopamine in right striatum were significantly increased in rotenone + NDI1 group compared to rotenone + vector group. CONCLUSION: Yeast NDI1 can rescue the defect of oxidative phosphorylation in rotenone-induced PD cell and mouse models, and ameliorate neurobehavioral and neuropathological damages. The results may provide a basis for the yeast NDI1 gene therapy of sporadic PD caused by mitochondrial complex I dysfunction.

Pt and black phosphorus co-modified flower-like WS2 composites for fast NO2 gas detection at low temperature.

Incomplete recovery, baseline drift, and a long response time have been impeding the practical applications of transition metal dichalcogenide (TMD)-based gas sensors. Here, we report WS2 sensors with significantly improved gas recovery, rapid response, and negligible baseline drift by the incorporation of black phosphorus (BP) as well as the decoration of Pt to detect NO2 for the first time. Compared to bare WS2, the BP-WS2 sensors show higher sensitivity, better repeatability, and more excellent selectivity towards NO2 at the optimal operating temperature of 50 °C. Furthermore, the optimized 30%BP-WS2/Pt sensors exhibit a continuous enhancement in the recovery level and sensitivity with negligible baseline drift. The 30%BP-WS2/Pt sensor also exhibits a shorter response time of 28 s than 49.5 s for its counterpart WS2 sensor towards 32 ppm NO2. The enhanced sensing properties are primarily due to the combined effects of more adsorption sites provided by BP, the spill-over effect of Pt catalysis, and the WS2/BP heterostructure. Therefore, the Pt-decorated 30%BP-WS2 sensor exhibits prominent gas-sensing properties of high gas sensitivity, a low detection limit of 100 ppb, good selectivity, and fast response. Our strategy provides a new route for designing and optimizing TMD-based gas sensors with excellent gas-sensing performance.

PSMD14 is a novel prognostic marker and therapeutic target in osteosarcoma.

BACKGROUND: Osteosarcoma is a bone tumor that is characterized by high malignancy and a high mortality rate, and that originates from primitive osteoblastic mesenchymal cells and is most common in rapidly growing long bones. PSMD14, also known as RPN11 or POH1, is a member of the JAMM isopeptidase family, which is able to remove the substrate protein ubiquitination label, thereby regulating the stability and function of the substrate protein. In this study, we explored the expression and potential biological significance of the PSMD14 deubiquitinating enzyme in osteosarcoma. METHODS: Immunohistochemical methods were used to detect the expression of PSMD14 in biopsies of 91 osteosarcoma patients, and the specimens were classified into high and low PSMD14 expression groups. The correlation between PSMD14 expression and clinical indicators and prognosis was compared.SiRNA was used to downregulate PSMD14 in two osteosarcoma cell lines (HOS and SJSA-1), and the effects of downregulation of PSMD14 on the viability, proliferation, and invasion ability of osteosarcoma cells were analyzed. RESULTS: We identified significant differences in recurrence, metastasis, and survival time of the osteosarcoma patients on the basis of PSMD14 expression. High expression of PSMD14 in osteosarcoma patients was associated with a low survival rate and high risk of metastasis and recurrence. Down-regulation of PSMD14 inhibited the viability, proliferation, and invasiveness of osteosarcoma cell lines. CONCLUSIONS: PSMD14 may be a new prognostic marker and therapeutic target for osteosarcoma.

Comparison of the effect of rotenone and 1­methyl­4­phenyl­1,2,3,6­tetrahydropyridine on inducing chronic Parkinson's disease in mouse models.

Animal models for Parkinson's disease (PD) are very useful in understanding the pathogenesis of PD and screening for new therapeutic approaches. The present study compared two commonly used neurotoxin­induced mouse models of chronic PD to guide model selection, explore the pathogenesis and mechanisms underlying PD and develop effective treatments. The chronic PD mouse models were established via treatment with rotenone or 1­methyl­4­phenyl­1,2,3,6-tetrahydropyridine (MPTP) for 6 weeks. The effects of rotenone and MPTP in the mice were compared by assessing neurobehavior, neuropathology and mitochondrial function through the use of the pole, rotarod and open field tests, immunohistochemistry for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), ionized calcium­binding adapter molecule 1 (Iba­1), neuronal nuclear antigen (NeuN) and (p)S129 α­synuclein, immunofluorescence for GFAP, Iba­1 and NeuN, western blotting for TH, oxygen consumption, complex I enzyme activity. The locomotor activity, motor coordination and exploratory behavior in both rotenone and MPTP groups were significantly lower compared with the control group. However, behavioral tests were no significant differences between the two groups. In the MPTP group, the loss of dopaminergic (DA) neurons in the substantia nigra (SN) pars compacta, the reduction of the tyrosine hydroxylase content in the SN and striatum and the astrocyte proliferation and microglial activation in the SN were more significant compared with the rotenone group. Notably, mitochondrial­dependent oxygen consumption and complex I enzyme activity in the SN were significantly reduced in the rotenone group compared with the MPTP group. In addition, Lewy bodies were present only in SN neurons in the rotenone group. Although no significant differences in neurobehavior were observed between the two mouse models, the MPTP model reproduced the pathological features of PD more precisely in terms of the loss of DA neurons, decreased dopamine levels and neuroinflammation in the SN. On the other hand, the rotenone model was more suitable for studying the role of mitochondrial dysfunction (deficient complex I activity) and Lewy body formation in the SN, which is a characteristic pathological feature of PD. The results indicated that MPTP and rotenone PD models have advantages and disadvantages, therefore one or both should be selected based on the purpose of the study.

The Third Generation Anti-HER2 Chimeric Antigen Receptor Mouse T Cells Alone or Together With Anti-PD1 Antibody Inhibits the Growth of Mouse Breast Tumor Cells Expressing HER2 in vitro and in Immune Competent Mice.

Chimeric Antigen Receptor (CAR)-T cells have great efficacy against CD19+ leukemia but little success for solid tumors. This study explored the effectiveness of third generation anti-HER2 CAR-T cells alone or in combination with anti-PD1 antibody on breast tumor cells expressing HER2 in vitro and in immune competent mouse model. The PDL1-positive mouse mammary tumor cell line 4T1 engineered to express luciferase and human HER2 was used as the target cell line (4T1-Luc-HER2). Anti-HER2 CAR-T cells were generated by transducing mouse spleen T cells with recombinant lentiviruses. ELISA analysis showed that IL-2 and IFN-γ secretion was increased in CAR-T cells co-cultured with the target cells, and the secretion of these two cytokines was increased further with the addition of anti-PD1 antibody. Lactate dehydrogenase assay revealed that CAR-T cells displayed a potent cytotoxicity against the target cells, and the addition of anti-PD1 antibody further enhanced the cytotoxicity. At the effector: target ratio of 16:1, cytotoxicity was 39.8% with CAR-T cells alone, and increased to 49.5% with the addition of anti-PD1 antibody. In immune competent syngeneic mouse model, CAR-T cells were found to be present in tumor stroma, inhibited tumor growth and increased tumor apoptosis significantly. Addition of anti-PD1 antibody further enhanced these anti-tumor activities. Twenty-one days after treatment, tumor weight was reduced by 50.0% and 73.3% in CAR-T group and CAR-T plus anti-PD1 group compared with blank T group. Our results indicate that anti-PD1 antibody can greatly increase the efficacy of anti-HER2 CAR-T against HER2-positive solid tumors.