A high-throughput screening assay of ascorbate in brain samples

Ascorbate is a vital reductant/free radical scavenger in the CNS, whose content defines - to a large extent - the redox status and the antioxidant reserves. Quick, reliable and specific methods for its measurement in brain samples are highly desirable. We have developed a new high-throughput screening assay for measurements of ascorbate using a fluorescence plate-reader. This assay is based on a direct reaction of ascorbate with a nitroxide radical conjugated with a fluorogenic acridine moiety, 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl radical (AC-TEMPO), yielding fluorescent hydroxylamine product (AC-TEMPO-H). The reaction was monitored over time using fluorescence and electron spin resonance techniques. The appearance of fluorescent AC-TEMPO-H was linear within the range of 3.75-75 μM AscH⁻ in the sample (0.5-10 μM AscH⁻ in the well). Assay was validated with high performance liquid chromatography method. The concentration of ascorbate in murine tissue samples, including brain samples after traumatic brain injury and hemorrhagic shock, was measured.     

Contribution of SNRNP200 sequence variations to retinitis pigmentosa

Purpose

Mutations in the SNRNP200 gene have been reported to cause autosomal dominant retinitis pigmentosa (adRP). In this study, we evaluate the mutation profile of SNRNP200 in a cohort of southern Chinese RP patients.

Methods

Twenty adRP patients from 11 families and 165 index patients with non-syndromic RP with mixed inheritance patterns were screened for mutations in the mutation hotspots of SNRNP200. These included exons 12–16, 22–32, and 38–45, which covered the two helicase ATP-binding domains in DEAD-box and two sec-63 domains. The targeted regions were amplified by polymerase chain reaction and analyzed by direct DNA sequencing, followed by in silico analyses.

Results

Totally 26 variants were identified, 18 of which were novel. Three non-synonymous variants (p.C502R, p.R1779H and p.I698V) were found exclusively in patients. Two of them, p.C502R and p.R1779H, were each identified in one simplex RP patient, whereas p.I698V occurred in one patient with unknown inheritance pattern. All three residues are highly conserved in SNRNP200 orthologs. Nevertheless, only p.C502R and p.R1779H were predicted to affect protein function by in silico analyses, suggesting these two variants are likely to be disease-causing mutations. Notably, all mutations previously identified in other study populations were not detected in this study.

Conclusions

Our results reveal a distinct mutation profile of the SNRNP200 gene in a southern Chinese cohort of RP patients. The identification of two novel candidate mutations in two respective patients affirmed that SNRNP200 contributes to a proportion of overall RP.     

Vitamin E deficiency enhances pulmonary inflammatory response and oxidative stress induced by single-walled carbon nanotubes in C57BL/6 mice

Exposure of mice to single-walled carbon nanotubes (SWCNTs) induces an unusually robust pulmonary inflammatory response with an early onset of fibrosis, which is accompanied by oxidative stress and antioxidant depletion. The role of specific components of the antioxidant protective system, specifically vitamin E, the major lipid-soluble antioxidant, in the SWCNT-induced reactions has not been characterized. We used C57BL/6 mice, maintained on vitamin E-sufficient or vitamin E-deficient diets, to explore and compare the pulmonary inflammatory reactions to aspired SWCNTs. The vitamin E-deficient diet caused a 90-fold depletion of alpha-tocopherol in the lung tissue and resulted in a significant decline of other antioxidants (GSH, ascorbate) as well as accumulation of lipid peroxidation products. A greater decrease of pulmonary antioxidants was detected in SWCNT-treated vitamin E-deficient mice as compared to controls. Lowered levels of antioxidants in vitamin E-deficient mice were associated with a higher sensitivity to SWCNT-induced acute inflammation (total number of inflammatory cells, number of polymorphonuclear leukocytes, released LDH, total protein content and levels of pro-inflammatory cytokines, TNF-alpha and IL-6) and enhanced profibrotic responses (elevation of TGF-beta and collagen deposition). Exposure to SWCNTs markedly shifted the ratio of cleaved to full-length extracellular superoxide dismutase (EC-SOD). Given that pulmonary levels of vitamin E can be manipulated through diet, its effects on SWCNT-induced inflammation may be of practical importance in optimizing protective strategies.     

Treatment with a novel hemigramicidin-TEMPO conjugate prolongs survival in a rat model of lethal hemorrhagic shock

OBJECTIVE: We sought to develop a therapeutic agent that would permit prolongation of survival in rats subjected to lethal hemorrhagic shock (HS), even in the absence of resuscitation with asanguinous fluids or blood. METHODS AND RESULTS: We synthesized a series of compounds that consist of the electron scavenger and superoxide dismutase mimic, 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-NH2-TEMPO), conjugated to fragments and analogs of the membrane-active cyclopeptide antibiotic, gramicidin S. Using an in vivo assay, wherein isolated intestinal segments were loaded inside the lumen with various test compounds, we studied these compounds for their ability to prevent ileal mucosal barrier dysfunction induced by subjecting rats to profound HS for 2 hours. The most active compound in this assay, XJB-5-131, ameliorated peroxidation of the mitochondrial phospholipid, cardiolipin, in ileal mucosal samples from rats subjected to HS. XJB-5-131 also ameliorated HS-induced activation of the pro-apoptotic enzymes, caspases 3 and 7, in ileal mucosa. Intravenous treatment with XJB-5-131 (2 micromol/kg) significantly prolonged the survival of rats subjected to profound blood loss (33.5 mL/kg) despite administration of only a minimal volume of crystalloid solution (2.8 mL/kg) and the absence of blood transfusion. CONCLUSION: These data support the view that mitochondrially targeted electron acceptors and SOD mimics are potentially valuable therapeutics for the treatment of serious acute conditions, such as HS, which are associated with marked tissue ischemia.     

胎盘免疫调节因子对细胞因子诱导的杀伤细胞超微结构及免疫表型的调节作用

目的:通过电子显微镜、流式细胞仪观察经胎盘免疫调节因子处理的细胞因子诱导的杀伤细胞超微结构及免疫表型变化,了解胎盘免疫调节因子的免疫调节活性。方法:实验于2006-02/2007-01在广西医科大学医学科学实验中心重点实验室进行。实验材料:重组人干扰素γ和白细胞介素1α为Peprotech公司产品。重组人白细胞介素2和抗人CD3单克隆抗体为北京远策药业有限公司产品。实验方法:①胎盘免疫调节因子的制备:将新鲜经检人胎盘去筋膜,生理盐水冲净、剪碎匀浆,置-20℃48h后,反复冻融3次,用生理盐水透析,取透析外液,过虑除菌,分装,抽样做有关鉴定实验。②细胞因子诱导的杀伤细胞的制备:抽取健康自愿者静脉血20mL,用淋巴细胞分离液分离外周血单个核细胞,加RPMI1640培养液调整细胞的密度至1×109L-1,放于50mL培养瓶中培养。于当天加入1×106U/L干扰素γ,置于37℃、50mL/LCO2孵育箱中培养24h。次日将悬浮的细胞转移到6孔板培养中,分别加入终浓度为100mg/L的PHA、3×105U/L白细胞介素2、133μg/L抗CD3mAb及1×105U/L白细胞介素1α,每3d半量换液1次,同时补加白细胞介素2至3×105U/L。将培养14d细胞因子诱导的杀伤细胞以5×109L-1接种于24孔板,实验组中加入一定剂量的胎盘免疫调节因子诱导,对照组加入等量生理盐水,继续培养72h。③实验评估:制备电镜标本,观察细胞超微结构。采用流式细胞术测定细胞因子诱导的杀伤细胞CD4 、CD8 、CD3 CD4 、CD3 CD8 、CD25 、HLA-DR、CD3 CD56 、CD80 百分含量。结果:①细胞因子诱导的杀伤细胞表型分析:实验组CD4 、CD8 和CD3 CD4 明显低于对照组[实验组:(8.40±8.15)%,(33.10±4.61)%,(7.98±12.65%);对照组:(39.43±9.16)%,(58.90±11.35)%,(31.30±5.94)%,P<0.05];实验组CD3 CD8 、CD25 表达明显高于对照组[实验组:(30.50±2.56)%,(24.8±6.31)%;对照组:(15.90±7.35)%,(0.56±0.21)%,P<0.05]。②透视电镜下观察细胞因子诱导的杀伤细胞超微结构特征:与对照组相比,实验组细胞因子诱导的杀伤细胞线粒体深染,溶酶体增加,异染色质比例增加,微绒毛肿胀及增长等。结论:胎盘免疫调节因子能促使细胞因子诱导的杀伤细胞超微结构及细胞表型发生明显变化:细胞因子诱导的杀伤细胞线粒体深染,溶酶体增加,异染色质比例增加,微绒毛肿胀增长,CD3 CD8 、CD25 细胞百分比升高,CD4 、CD8 和CD3 CD4 细胞百分比降低。     

下背痛的评价及其患者生活质量的评价

目的:总结分析近10年国内外下背痛患者生活质量的研究状况,以期提高医疗过程中的注意力。资料来源:应用计算机检索Medline1997-01/2006-06与下背痛患者生活质量研究相关的文献,检索词“low back pain,quality of life”,限定文献语言种类为English。同时计算机检索清华全文数据库1998-01/2006-06相关下背痛患者生活质量研究的文献,检索词“下背痛,生活质量”,限定文献语言种类为中文。资料选择:对资料进行初审,选取包括下背痛患者生活质量研究的文献,开始查找全文。纳入标准:包含下背痛的诊断、评定、治疗以及下背痛的健康教育和社会支持等多方面文献,绝大多数文献都与生活质量研究有关。排除标准:重复研究,Meta分析,没有对照组的文献。资料提炼:共检索到52篇关于下背痛患者生活质量研究的文献,最终纳入30篇符合标准的文献。资料综合:生活质量涉及到生理、心理、社会、文化、经济以及精神等不同层面,是指个体在其所在文化、风俗习惯的背景下,由他生活的标准、理想追求的目标所决定的对他目前社会地位、生活状态的认识和满意程度。提高下背痛患者的生活质量,关系到患者的治疗效果。下背痛患者普遍存在躯体症状和抑郁的心理反应,两方面的表现都可能使患者的生活质量受到影响。在对下背痛患者的治疗中,不仅只关注躯体功能和疾病本身,同时还应注重心理功能和社会职能。结论:随着对下背痛的病因、预防、治疗及康复研究的进一步深入,下背痛困扰人类的现象在不久必将得到很大改善。     

新生兔破骨细胞的体外分离培养与功能鉴定

目的:选择新生兔为实验对象,观察体外培养破骨细胞形态学和生物学特征。方法:实验于2007-03/06在辽宁医学院生理实验室完成。采用改良Chambers法机械分离兔四肢长骨获得新生兔(出生24h内)的破骨细胞,并应用倒置显微镜、光镜观察其形态学特征。结果:①倒置显微镜观察结果:培养1d后,细胞贴壁生长,数量增多,第3天后,可见多核巨细胞,呈油煎蛋、长条形,漏斗形等。②光镜观察结果:苏木精-伊红染色可见破骨细胞胞浆丰满,呈浅粉色,内含许多空泡结构,核3～20个不等,核呈浅蓝色,每个核含一两个核仁,有时周围伸出伪足样胞浆突起;Giemsa染色核集中于细胞中央或散在于细胞周边,呈花篮状,核浅粉色,胞浆着色较浅,其中可见数个大小不等未着色空泡;TRAP染色胞浆呈酒红色颗粒,核蓝色,核仁一两个;甲苯胺蓝染色可见骨吸收陷窝呈蓝紫色,圆形,椭圆形或不规则形,边界清晰,其边缘反光性强。结论:应用改良Chambers法可成功获得新生兔破骨细胞,且具有骨吸收功能。     

中药女贞子中水溶性成分二种新裂环环烯醚萜甙的分离和鉴定

从木犀科植物女贞(Ligustrum lucidum Ait)干燥成熟果实女贞子中分离得到2个新的裂环环烯醚萜甙类化合物,经光谱及化学分析,鉴定其化学结构,并分别命名为特女贞甙(specnuezhenide,I)和女贞苦甙(nuezhengalaside,I)。     

Prognostic Prediction of Cancer Based on Radiomics Features of Diagnostic Imaging: The Performance of Machine Learning Strategies

Journal of Digital Imaging - Tumor phenotypes can be characterized by radiomics features extracted from images. However, the prediction accuracy is challenged by difficulties such as small sample...     

Achievement of glycaemic targets with weight loss and without hypoglycaemia in type 2 diabetes with the once-weekly glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonist tirzepatide: A post hoc analysis of the SURPASS-1 to -5 studies

Aim

To assess composite endpoints combining glycaemic control (HbA1c < 7.0%, ≤ 6.5% or < 5.7%) with weight loss (≥ 5%, ≥ 10% or ≥ 15%) and without hypoglycaemia with tirzepatide in type 2 diabetes (T2D).

Materials and Methods

Data from the phase 3 SURPASS programme were evaluated post hoc by trial. Participants with T2D were randomized to tirzepatide (5, 10 and 15 mg), placebo (SURPASS-1,5), semaglutide 1 mg (SURPASS-2) or titrated basal insulin (SURPASS-3,4). The proportions of participants achieving the composite endpoints were compared between tirzepatide and the respective comparator groups at week 40/52.

Results

The proportions of participants achieving an HbA1c value of less than 7.0% with 5% or more weight loss and without hypoglycaemia ranged from 43% to 82% with tirzepatide across the SURPASS-1 to -5 trials versus 4%-5% with placebo, 51% with semaglutide 1 mg and 5% with basal insulin (P < .001 vs. all comparators). The proportions of participants achieving an HbA1c value of less than 7.0% with 10% or more, or 15% or more weight loss and without hypoglycaemia were significantly higher with all tirzepatide doses versus comparators across trials (P < .001 or P < .05). Similar results were observed for all other combinations of endpoints with an HbA1c value of 6.5% or less, or less than 5.7%, with more tirzepatide-treated participants achieving these endpoints versus those in the comparator groups, including semaglutide.

Conclusions

Across the SURPASS-1 to -5 clinical trials, more tirzepatide-treated participants with T2D achieved clinically meaningful composite endpoints, which included reaching glycaemic targets with various degrees of weight loss and without hypoglycaemia, than those in the comparator groups.