Monitoring the activation of neuronal nitric oxide synthase in brain tissue and cells with a potentiometric immunosensor

An all solid state potentiometric immunosensor (ASPI) has been developed to study the activation process of neuronal nitric oxide synthase (nNOS), the enzyme involved in the synthesis of nitric oxide generated under physiological conditions. At first, an all solid state H⁺-selective ISE was fabricated with the carboxylated poly(vinyl chloride) (PVC-COOH) film containing H⁺ ionophore, antibody was then immobilized on the polymer layer. The immunocomplex formation was detected by monitoring pH change due to interaction between urease labeled secondary antibody and antigen. Experimental parameters such as the amount of phosphorylated nNOS immobilized on the electrode surface and pH responses due to the antibody–antigen reaction were studied in detail. The calibration plot of the potentiometric potential vs. phosphorylated nNOS concentration exhibited a linear relationship in the range of 3.4–340.0 μg/ml. The calibration sensitivity of the phosphorylated nNOS immunosensor was −0.073 ± 0.002 mV/μg ml⁻¹. The detection limit of nNOS was determined to be 0.2 μg/ml based on five-time measurements (95% confidence level, k = 3, n = 5). The reliability of the immunosensor was examined with rat brain tissues as well as neuronal cells, and the results shown were good, implying a promising approach for a novel electrochemical immunosensor platform with potential applications to clinical diagnosis.     

Apigenin enhances the cytotoxic effects of tumor necrosis factor-related apoptosis-inducing ligand in human rheumatoid arthritis fibroblast-like synoviocytes

Activated rheumatoid arthritis (RA) fibroblast-like synoviocytes (RAFLSs) play a central role in both initiating and driving RA. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been documented to induce apoptosis only in a small proportion of RAFLSs, which is followed by an induction of proliferation in surviving cells. Apigenin, a chemopreventive bioflavonoid, exhibits proapoptotic activity in many types of cells. In the present study, we sought to determine whether apigenin could enhance the cytotoxic effect of TRAIL on activated RAFLSs. Human RAFLSs isolated from patients with RA were treated with TRAIL (1 nM), apigenin (20 μM), or their combination, and subjected to apoptosis analysis after a 24-h incubation and proliferation analysis after a 72-h incubation. Apoptosis assay revealed that TRAIL or apigenin alone induced a marked apoptosis in RAFLS and their combination yielded a synergistic increase in RAFLS apoptosis. Immunoblotting analysis of apoptosis regulators demonstrated that combined treatment with apigenin increased caspase-3 expression and activity and decreased the Bcl-2/Bax ratio relative to treatment with TRAIL alone. The presence of apigenin significantly restrained TRAIL-induced RAFLS proliferation, coupled with restoration of the expression of two cell-cycle inhibitors p21 and p27. Moreover, the combination with apigenin blunted TRAIL-induced activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Our data collectively demonstrate that apigenin sensitizes RAFLS to TRAIL-induced apoptosis and counteracts TRAIL-dependent RAFLS proliferation, which is likely mediated through inactivation of PI3-K/Akt signaling pathway.     

Construction of a high density integrated genetic map for cucumber (Cucumis sativus L.)

The high-density consensus map was constructed based on the GY14 × PI 183967 map from an inter-subspecific cross and the extended S94 × S06 map from an intra-subspecific cross. The consensus map was composed of 1,369 loci, including 1,152 SSR loci, 192 SRAP loci, 21 SCAR loci and one STS locus as well as three gene loci of fruit external quality traits in seven chromosomes, and spanned 700.5 cM, of which 682.7 cM (97.5%) were covered by SSR markers. The average genetic distance and physical interval between loci were 0.51 cM and ~268 kbp, respectively. Additionally, the physical position of the sequence-associated markers aligned along the assembled cucumber genome sequence established a relationship between genetic maps and cucumber genome sequence and to a great extent validated the order of markers in individual maps and consensus map. This consensus map with a high marker density and well-ordered markers is a saturated and reliable linkage map for genetic analysis of cucumber or the Cucurbitaceae family of plants.     

Modification of alternative splicing in bovine somatic cell nuclear transfer embryos using engineered CRISPR-Cas13d

Animal cloning can be achieved by somatic cell nuclear transfer(SCNT), but the resulting live birth rate is relatively low. We previously improved the efficiency of bovine SCNT by exogenous melatonin treatment or by overexpression of lysine-specific demethylase 4D(KDM4D) and 4E(KDM4E). In this study, we revealed abundant alternative splicing(AS) transitions during fertilization and embryonic genome activation, and demonstrated abnormal AS in bovine SCNT embryos compared with in vitro fertilized ...     

黄孢原毛平革菌抗营养阻遏产漆酶诱变育种及其产酶特性

【目的】筛选能抗营养阻遏产漆酶的黄孢原毛平革菌,论证其产漆酶的确定性及抗营养阻遏产木质素酶的可行性,为白腐菌产酶代谢调控、木质素降解机理的研究奠定基础。【方法】利用重复紫外诱变法,以愈创木酚富氮鉴别培养基筛选目标菌株;比较不同营养条件下菌体生长与产酶动力学差异研究产酶营养调控机理;通过热处理、排除锰离子和加入过氧化氢酶等不同措施论证黄孢原平毛平革菌能否产生漆酶。【结果】3种不同方法均证实选育到的pcR5305和pcR5324菌株在限氮与富氮条件下均能产生漆酶,pcR5305和pcR5324在限氮条件下产漆酶分别达到203.5、187.6 U/L;在富氮条件下为220.6、183.9 U/L,而原菌株pc530在两种条件下都基本不产生漆酶。二菌株产漆酶调控方式不同,pcR5305漆酶产生与菌体生长同步,而pcR5324漆酶产生却受营养氮阻遏。二菌株同时具有抗营养阻遏高产木质素过氧化物酶(LiP)和锰过氧化物酶(MnP)(分别为LiP 1343.2、MnP 252.2 U/L;LiP 1169.5、MnP 172.4 U/L)的能力。【结论】筛选到的黄孢原毛平革菌变异菌株能产漆酶,同时表现了抗营养阻遏产漆酶、木质素过氧化物酶和锰过氧化物酶的能力,具有重要的生产应用与理论研究价值,为白腐菌产酶代谢调控机理研究提供了原始菌株并奠定了良好的基础。     

Genetic co-transfer of CCR7 ligands enhances immunity and prolongs survival against virulent challenge of pseudorabies virus

The CC chemokine receptor 7 (CCR7) and cognate CCR7 ligands, CCL19 and CCL21, help establish microenvironments in lymphoid tissue that can facilitate encounters between naive T cells and mature dendritic cells (DCs). This study was conducted to determine if CCR7 ligands can augment the immunogenicity of a DNA vaccine that expresses glycoprotein B (gB) of the pseudorabies virus (PrV). The genetic co-transfer of CCR7 ligands along with a PrV DNA vaccine increased the levels of serum PrV-specific immunoglobulin (Ig) G by 2- to 2.5-fold. In addition, the level of PrV-specific IgG2a isotype was significantly enhanced by co-injection of CCR7 ligand DNA, which indicates that CCR7 ligand biases the humoral immunity toward the Th1-type pattern. The co-injection of CCR7 ligand DNA consistently enhanced the level of Th1-type cytokines (IL-2 and IFN-gamma) produced by stimulated immune cells when compared with a group that was vaccinated with the PrV DNA vaccine. Also, the genetic co-transfer of CCR7 ligand DNAs with PrV DNA vaccine provided prolonged survival against a virulent challenge by PrV. Moreover, the co-administration of CCR7 ligand DNA increased the number of mature DCs into the secondary lymphoid tissues, which appeared to enhance the proliferation of PrV-immune CD4(+) T cells. Taken together, these findings indicate that CCR7 ligands are an attractive adjuvant for a PrV DNA vaccine that can offer protective immunity against the PrV.     

Long-Term Clinical Outcome of Internal Globus Pallidus Deep Brain Stimulation for Dystonia

Background

GPi (Internal globus pallidus) DBS (deep brain stimulation) is recognized as a safe, reliable, reversible and adjustable treatment in patients with medically refractory dystonia.

Objectives

This report describes the long-term clinical outcome of 36 patients implanted with GPi DBS at the Neurosurgery Department of Seoul National University Hospital.

Methods

Nine patients with a known genetic cause, 12 patients with acquired dystonia, and 15 patients with isolated dystonia without a known genetic cause were included. When categorized by phenomenology, 29 patients had generalized, 5 patients had segmental, and 2 patients had multifocal dystonia. Patients were assessed preoperatively and at defined follow-up examinations postoperatively, using the Burke-Fahn-Marsden dystonia rating scale (BFMDRS) for movement and functional disability assessment. The mean follow-up duration was 47 months (range, 12–84)

Results

The mean movement scores significantly decreased from 44.88 points preoperatively to 26.45 points at 60-month follow up (N = 19, P = 0.006). The mean disability score was also decreased over time, from 11.54 points preoperatively to 8.26 points at 60-month follow up, despite no statistical significance (N = 19, P = 0.073). When analyzed the movement and disability improvement rates at 12-month follow up point, no significant difference was noted according to etiology, disease duration, age at surgery, age of onset, and phenomenology. However, the patients with DYT-1 dystonia and isolated dystonia without a known genetic cause showed marked improvement.

Conclusions

GPi DBS is a safe and efficient therapeutic method for treatment of dystonia patients to improve both movement and disability. However, this study has some limitations caused by the retrospective design with small sample size in a single-center.