Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: Identification of novel mutations

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene.     

免疫印迹法检测牛海绵状脑病和羊瘙痒病

用大肠杆菌表达的牛朊病毒正常成熟蛋白 (BoPrPC)免疫新西兰白兔 ,获得了与朊病毒蛋白 (PrP)反应的抗体T1。根据致病型朊病毒 (PrP^SC)能抵抗蛋白酶消化的特性 ,用蛋白酶K消化脑组织提取物 ,以抗体T1进行免疫印迹反应 ,结果表明从接种羊瘙痒病朊病毒 2 6 3K的金黄地鼠脑组织提取物内检测到抗蛋白酶K消化的致病型PrPSC ,而正常金黄地鼠脑组织中没有抗蛋白酶消化的蛋白。以我国正常牛羊为材料 ,制备其脑组织提取物 ,用上述方法和抗体T1进行检测 ,结果没有发现抗蛋白酶K的任何蛋白存在 ,说明没有牛海绵状脑病和羊瘙痒病存在。用 1A8抗体也获得了同样的结果。这些结果表明可以用自制的抗血清检疫牛海绵状脑病和羊瘙痒病 ,防止其传入我国     

The “brave new world” of transmissible spongiform encephalopathy (infectious cerebral amyloidosis)

The story of transmissible human spongiform encephalopathy, from its origins to the present time, enjoys the commentary of a cast of characters from Shakespeare's imaginary island inThe Tempest, with a brief visit to the real island of Tasmania for a bird's eye view of the prion, and some concluding thoughts about the current state of research in the netherworlds of molecular biology and physical chemistry.     

Transmissible spongiform encephalopathy strain-associated diversity of N-terminal proteinase K cleavage sites of PrPSc from scrapie-infected and bovine spongiform encephalopathy-infected mice

Assessment of the different conformational states of the abnormal prion protein (PrP^Sc) in the CNS provides an established basis for distinguishing transmissible spongiform encephalopathy (TSE) strains. PrP^Sc conformers are variably resistant to N-terminal proteinase K (PK) digestion, and analysis of the consensus products (PrP^res) by immunoassay enables effective, but relatively low-resolution differentiation. Determination of the precise N-terminal amino acid profile (N-TAAP) of PrP^res presents a potential high-resolution means of TSE-strain typing, and thus of differential disease diagnosis. This approach was evaluated using individual mice affected by model scrapie (22A, ME7, 87V and 79A) and bovine spongiform encephalopathy (BSE) (301V) strains. Nano liquid chromatography–mass spectrometry (LC-MS) was used to determine PrP^res N-terminal tryptic digestion products. Four major N-terminal tryptic peptides were generated from all mouse TSE strains investigated, corresponding with predominant N-termination of PrP^res at G⁸¹, G⁸⁵, G⁸⁹ and G⁹¹. Both the mass spectrometric abundance of the individual peptides and the ratios of pairs of these peptides were evaluated as markers of conformation in relation to their potential for strain discrimination. The yield of peptides was significantly greater for BSE than scrapie strains and the relative quantities of particular peptide pairs differed between strains. Thus, whereas peptide G⁹¹–K¹⁰⁵ was a dominant peptide from 301V, this was not the case for other strains and, significantly, the ratio of peptides G⁹¹–K¹⁰⁵:G⁸⁹–K¹⁰⁵ was substantially higher for BSE-infected compared with scrapie-infected mice. These data support the potential of the N-TAAP approach for high-resolution TSE strain typing and differential diagnosis.     

新生儿缺氧缺血性脑病血清S-100B蛋白与NBNA评分动态监测的临床研究

目的：观察新生儿缺氧缺血性脑病（hypoxic-ischemicencephalopathy,HIE）血清S-100B蛋白的动态变化规律,探讨其在HIE早期诊断中的价值,以及其浓度变化与病情严重程度及预后的关系。同时研究围产期高危因素以及NBNA评分在HIE发生发展与预后中的作用。方法：30例住院正常新生儿作为对照组,于出生后采血,55例HIE患儿（HIE组）分别于出生后1天、2天、7天采血,采用酶联免疫吸附试验、双抗体夹心法检测。收集并分析两组围产期相关资料。HIE组并于采血同时进行NBNA评分。结果：（1）HIE患儿生后第一天与第二天血清S-100B蛋白浓度明显高于对照组（P〈0.05）,生后第七天轻度HIE与对照组比较没有统计意义,中、重度HIE与对照组比较有统计学意义。（2）生后第一天与第二天不同病情组HIE患儿NBNA评分相互比较差异具有统计学意义（P〈0.05）,第七天轻、中和重度患儿NBNA评分〈35分的患儿分别占33.3%,47.1%,100%。结论：动态监测HIE患儿血清S-100B蛋白浓度和NBNA评分的变化,对HIE的早期诊断,严重程度的判断以及预后的估计有重要意义。     

Proteome adaptations in Ethe1-deficient mice indicate a role in lipid catabolism and cytoskeleton organization via post-translational protein modifications

Hydrogen sulfide is a physiologically relevant signalling molecule. However, circulating levels of this highly biologically active substance have to be maintained within tightly controlled limits in order to avoid toxic side effects. In patients suffering from EE (ethylmalonic encephalopathy), a block in sulfide oxidation at the level of the SDO (sulfur dioxygenase) ETHE1 leads to severe dysfunctions in microcirculation and cellular energy metabolism. We used an Ethe1-deficient mouse model to investigate the effect of increased sulfide and persulfide concentrations on liver, kidney, muscle and brain proteomes. Major disturbances in post-translational protein modifications indicate that the mitochondrial sulfide oxidation pathway could have a crucial function during sulfide signalling most probably via the regulation of cysteine S-modifications. Our results confirm the involvement of sulfide in redox regulation and cytoskeleton dynamics. In addition, they suggest that sulfide signalling specifically regulates mitochondrial catabolism of FAs (fatty acids) and BCAAs (branched-chain amino acids). These findings are particularly relevant in the context of EE since they may explain major symptoms of the disease.     

Evolution of covered stents in the contemporary era: clinical application,materials and manufacturing strategies using nanotechnology

Endovascular stents have revolutionised the field of interventional cardiology. Despite their excellent clinical outcome complications associated with percutaneous stent implantation following the procedure have remained a major drawback in their widespread use. To overcome such limitations, a number of novel endovascular stents have emerged including a covered stent wrapped in a thin membrane sleeve. As well as prevention of complications associated with stenting, covered stents owing to their physical barrier are used as the treatment option of choice for trauma devices during emergency situations and to treat a number of pathological disease states. The aim of this review is to provide the reader with an overall objective outlook in the use of covered stents as a treatment option in a number of vascular complications and addresses their design and materials used in the manufacturing process. In addition, new strategies are highlighted and future prospects with the emergence of novel smart alloys for 3D scaffolds and the use of nanotechnology in the development of nanocomposite materials.     

Brain cortical amino acids measured by intracerebral dialysis in portacaval shunted rats

The extracellular amino acid content was measured in the parietal cortex in portacaval and sham operated rats, using the brain dialysis technique. The amino acid content of the perfusate was determined for 10 min before and during stimulation with potassium chloride. Basal levels of aspartate, glutamine, glycine, methionie, valine, phenylalanine and leucine were 2-to 6-fold higher in the PC-shunted as compared to the sham operated rats. For glutamate, taurine, and GABA no differences were observed between the two groups. After KCl stimulation the release of glutamate and GABA increased significantly in both groups. For GABA this rise was approximately twice as high in the PC-shunted rats (+300%,P<0.01) as in the sham operated rats (+150%,P<0.01 as compared to basal). In the sham operated, but not in the PC-shunted rats, methionine and valine levels rose significantly (+200%,P<0.05) and glutamine release decreased (–50%,P<0.05). These findings suggest that the brain metabolism of amino acids is altered after a portacaval shunt. This could in turn alter the neurotransmission and partly explain the low spontaneous motor activity seen in these animals.     

Glutathione system in erythrocytes and blood plasma in strokes and dyscirculatory encephalopathy

Dyscirculatory encephalopathy and mild ischemic stroke are characterized by solitary changes in components of glutathione metabolism. In moderate and severe ischemic strokes essential changes have been found. Changes in glutathione metabolism are also expressed in hemorrhagic stroke. The clearest increase was found in activities of glutathione peroxidase and glutathione transferase and in rare cases in activities of glutathione reductase and GSH concentration. The increase of enzymes activity was not found in patients with delayed onset of treatment (more than 3 days) and also in severe cases terminated by subsequent death of patients. Glutathione system is obviously important for tolerance to cerebral ischemia.     

Effects of ammonia onl-glutamate uptake in cultured astrocytes

The effect of ammonia onl-glutamate (L-GLU) uptake was examined in cultured astrocytes. Acute ammonia treatment (5–10 mM) enhanced L-[³H]GLU uptake by 20–42% by increasing the V_max; this persisted for 2 days and then started to decline. Ammonia, however, did not affect the uptake ofd-[³H]aspartate (D-ASP), a non-metabolizable analog of L-GLU, that uses the same transport carrier as L-GLU. Also, L-GLU uptake was not affected during the first 2 min of the assay. Thus, ammonia did not have an acute effect on L-GLU transport (translocation); rather, ammonia enhanced the accumulation or “trapping” of L-GLU or its by-products. Chronic ammonia treatment, on the other hand, inhibited L-GLU transport in astrocytes by ∼30–45% and this was due to a decrease in V_max, suggesting that the number of L-GLU transporters was decreased. This inhibitory effect was observed after 1 day of treatment and persisted for at least 7 days. The inhibition of L-GLU transport was partially reversible following removal of ammonia. The effects of ammonia on L-GLU transport and uptake may explain the abnormal L-GLU neurotransmission observed in hyperammonemia/hepatic encephalopathy, and the brain swelling associated with fulminant hepatic failure.