Selective separation and enrichment of peptides for MS analysis using the microspheres composed of Fe3O4@nSiO2 core and perpendicularly aligned mesoporous SiO2 shell

In this work, we report the development of a novel enrichment protocol for peptides by using the microspheres composed of Fe₃O₄@nSiO₂ Core and perpendicularly aligned mesoporous SiO₂ shell (designated Fe₃O₄@nSiO₂@mSiO₂). The Fe₃O₄@nSiO₂@mSiO₂ microspheres possess useful magnetic responsivity which makes the process of enrichment fast and convenient. The highly ordered nanoscale pores (2 nm) and high‐surface areas of the microspheres were demonstrated to have good size‐exclusion effect for the adsorption of peptides. An increase of S/N ratio over 100 times could be achieved by using the microspheres to enrich a standard peptide, and the application of the microspheres to enrich universal peptides was performed by using myoglobin tryptic digest solution. The enrichment efficiency of re‐used Fe₃O₄@nSiO₂@mSiO₂ microspheres was also studied. Large‐scale enrichment of endogenous peptides in rat brain extract was achieved by the microspheres. Automated nano‐LC‐ESI‐MS/MS was applied to analyze the sample after enrichment, and 60 unique peptides were identified in total. The facile and low‐cost synthesis as well as the convenient and efficient enrichment process of the novel Fe₃O₄@nSiO₂@mSiO₂ microspheres makes it a promising candidate for selectively isolation and enrichment of endogenous peptides from complex biological samples.     

低温脂肪酶的产酶条件优化及其酶学性质

利用单因素筛选和正交试验对Burkholderia sp. SYBC LIP-Y发酵产酶的液体培养基和发酵条件进行了优化,其优化配方为：可溶性淀粉10 g/L、牛肉膏15 g/L、NaNO3 0.252 g/L、橄榄油40ml/L、Triton x-100 10ml/L、初始pH 7.5、接种量10%(V/V),脂肪酶酶活达到85.23U/ml,是优化前的3.63倍。通过对双水相纯化得到的脂肪酶进行酶学性质研究,确定该酶反应的最适pH为10.0,最适温度为30℃,40℃下保温60min酶活性还有80%以上,该脂肪酶为低温脂肪酶,热稳定性好,具有一定的耐醇性,应用前景广阔。     

神经网络提高肝细胞癌磁共振波谱诊断正确率

通过评价31磷磁共振波谱(31Phosphorus Magnetic Resonance Spectroscopy,31P-MRS)来辨别三种诊断类型:肝细胞癌,正常肝和肝硬化。运用反向传输神经网络(BP)和径向基函数神经网络(RBF)分析31P-MRS数据,分别建立神经网络模型,进行肝细胞癌的诊断分类以期提高识别率。实验结果证明,应用神经网络模型后,31P-MR波谱对活体肝细胞癌的诊断正确率从89.47%提高到97.3%,且BP更优于RBF。     

Evaluation of the MTT lymphocyte proliferation assay for the diagnosis of neurocysticercosis

Neurocysticercosis (NCC) is caused by the larval form of the pork tapeworm Taenia solium when lodged in the central nervous system (CNS). Clinical diagnosis of NCC is complicated due to its polymorphic manifestations with no specific signs or symptoms. A wide range of serological assays and neuroimaging modalities are used for its diagnosis. The aim of the present study was to evaluate the MTT assay for the diagnosis of NCC and to determine its sensitivity, specificity and accuracy. MTT assay was based upon the cellular reduction of the tetrazolium salt by the proliferating cells and quantification of the colored product. Total 59 patients with NCC-related active epilepsy (AE), 30 with AE other than NCC (disease controls) and 64 healthy volunteers were enrolled for the study. Lymphocytes were freshly isolated from the enrolled subjects and cultured on cyst fluid antigen coated tissue culture plates. MTT assay was performed according to the standard protocol. The mean values of proliferation index (PI) with cyst fluid antigens were 2.13 ± 0.72, 0.622 ± 0.31 and 0.71 ± 0.36 for NCC patients, disease controls and healthy volunteers respectively. PI values for NCC patients were higher than the cut-off value (mean of controls + 2 standard deviations; 1.31). The sensitivity, specificity and accuracy of the MTT assay for the diagnosis of NCC were 87.93%, 94.68% and 91.5% respectively. For single cyst infection the sensitivity of the assay was found to be 86.4%. The present study shows that MTT is an adaptable technique which can be used for diagnosis of NCC.     

Accumulation of Eu chelates in cells expressing or not P-glycoprotein: Implications for blood-brain barrier crossing

Alzheimer’s disease (AD) is the most commonly form of dementia in the elderly. The development of molecules able to detect biomarkers characteristic of AD is critical to its understanding and treatment. However, such molecules must be able to pass blood-brain barrier (BBB) which is a major impediment to the entry of many therapeutic drugs into the brain. Such a limitation applies to the development of magnetic resonance imaging molecular neuroimaging agents using biomarkers of AD-like β-amyloid deposits, as the common extracellular contrast agents (CAs) are not able to cross an intact BBB. In this work, we have studied the ability of a series of simple Eu³⁺ complexes to enter cells overexpressing or not the ABCB1 (P-gp or P-glycoprotein) protein, which is expressed at the BBB and in human embryonic astrocytes. The intracellular uptake of the Eu³⁺ complexes of linear and macrocyclic polyaminocarboxylate ligands with different charges and lipophilicities was followed by atomic absorption spectrometry. Based on biochemical argument, we propose that lipophilic contrast agents can be efficiently taken up by cells and accumulate inside mitochondria when they are positively charged. The important point is that they are not P-gp substrates, which is one of the major obstacles for them to cross the BBB.     

An immunoaffinity liquid chromatography-tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum

An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitation of the zinc endopeptidase matrix metalloproteinase 9 (MMP-9) from mouse serum. Sample preparation for the assay included magnetic bead-based enrichment using an MMP-9 antibody and was performed in a 96-well plate format using a liquid-handling robotic platform. The surrogate peptide GSPLQGPFLTAR derived from MMP-9 by trypsin digestion was monitored using an on-line capillary flow trap-release chromatography setup incorporating a series of trap columns (C18, strong cation exchange, and another C18) prior to nanoflow chromatography and nanospray ionization with selected reaction monitoring (SRM) detection. The assay was fit-for-purpose validated and found to be accurate (<15% interbatch relative error) and precise (<15% interbatch coefficient of variation) across a range from 0.03 to 7.3 nM mouse MMP-9. Finally, the method was employed to measure MMP-9 concentrations in 30 naïve mouse serum samples, and results were compared with those obtained by an immunoassay.     

海洋菌W11产中温淀粉酶的酶学特性

本研究从威海文登海域筛选获得一株产淀粉酶海洋菌W11,初步鉴定为弧菌,并探讨pH、温度、无机离子对淀粉酶活性和稳定性的影响及该酶底物浓度效应和Km值。结果表明:在pH7.5左右酶活性最高,pH在4.0～7.5范围内体现较强的稳定性。最适酶解温度为55℃,酶液在60℃以下有较好的热稳定性;Ba2+、Mn2+对淀粉酶有激活作用,而Cu2+、Mg2+、Zn2+则抑制淀粉酶活性,表观Km值为0.973mg/mL。海洋菌W11所产的中温淀粉酶保存温度范围较广、适应pH作用的范围广及稳定性较强,将有着广泛的应用潜力。     

Modeling Harvest Intensity of Sooty Shearwater Chicks by Rakiura Māori in New Zealand

Abstract: Cultural evidence suggests that sooty shearwater (Puffinus griseus) chicks have been harvested by Rakiura Māori on islands in southern New Zealand since prehistoric times. Concerns exist that modern harvests may be impacting sooty shearwater abundance. We modeled human-related and ecological determinants of harvest (total no. of individuals harvested) of sooty shearwater chicks on 11 islands and examined the relationship between shearwater abundance and harvesting rates (chicks/hr) and harvester behavior throughout the harvesting season. Models best explaining variation in harvest between harvesting areas (manu), for both the early and late parts of the harvesting season, included harvester-days (included in all models with change in deviance information criteria [ΔDIC], ΔDIC < 8.36 and ΔDIC < 11.5, for the early and late periods, respectively). Other harvest determinants included shearwater density, size of the manu, and number of people helping harvesters (all included in the top 5 models within ΔDIC = 2.25 for the late period). Areas harvested by several families under a common-property harvesting system had higher harvest intensity for their size (24% points higher, 95% credible interval 11–36%) than those managed as an exclusive resource for one family. The slowest harvesters spent more time harvesting but on average only harvested 36% (95% credible interval 15–65%) and 34% (95% credible interval 12–63%) of the harvest taken by the fastest harvesters during the early and late periods, respectively. Our results highlight the possibility of elevated harvest intensity as the population of harvesters increases. However, our models suggested that a corresponding reduction in harvesting rate at low prey densities during the most productive period could potentially regulate harvest intensity. Future research will integrate these results into prospective shearwater demographic models to assess the utility of a range of harvesting strategies in ensuring harvest sustainability.     

毛竹林土壤酶活性变化的海拔效应

在毛竹分布南缘的中亚热带与南亚热带气候过渡区,选择土壤类型、坡度、坡向、经营水平等一致的3个海拔高度(低海拔90～120m、中海拔360～400m、高海拔700～780m)毛竹林,对其土壤酶活性和物理、化学性质进行了测定。磷酸酶、蔗糖酶和脲酶活性分别为0.031～0.042、0.104～0.146和0.017～0.039mg·g-1.h-1,中海拔是3种酶活性显著变化的转折点。蛋白酶活性为0.248～0.259mg·g-1.h-1,随海拔的升高酶活性缓慢提高。过氧化氢酶活性为0.097～0.143mg·g-1.h-1,随海拔的升高酶活性显著上升。不同海拔高度毛竹林土壤物理性质对土壤酶活性无显著影响。土壤有机质、全氮、碱解氮含量与土壤酶活性呈显著或极显著正相关,pH值对土壤酶活性具正效应,海拔梯度上的土壤全磷、全钾、速磷、速钾含量对土壤酶活性影响不显著。不同种类土壤酶活性间呈显著或极显著相关,酶促反应具专一性和共同性特点。     

磁共振成像弥散张量成像参数联合血清NSE、Lp-PLA2在脑梗死患者的诊断和预后不良风险评估中的应用价值

摘要 目的：探讨磁共振成像（MRI）弥散张量成像（DTI）参数联合血清神经元特异性烯醇化酶（NSE）、脂蛋白相关磷脂酶A2（Lp-PLA2）在脑梗死患者的诊断和预后不良风险评估中的应用价值。方法：选择2021年3月至2022年9月吉林大学中日联谊医院收治的106例脑梗死患者作为脑梗死组，另选同期62例体检健康志愿者作为对照组，比较两组DTI参数，血清NSE和Lp-PLA2水平。脑梗死组出院90d后，采用改良Rankin量表（mRS）进行预后评估，分为预后良好组与预后不良组，并比较两组上述指标水平。受试者工作特征（ROC）曲线分析DTI参数联合血清NSE、Lp-PLA2诊断脑梗死和预测脑梗死患者预后的价值。结果：脑梗死组表观弥散系数（ADC）值、部分各向异性指数（FA）值低于对照组（P＜0.05），血清NSE、Lp-PLA2水平高于对照组（P＜0.05）。预后不良组FA值、ADC值低于预后良好组（P＜0.05），血清NSE、Lp-PLA2水平高于预后良好组（P＜0.05）。联合FA值、ADC值、NSE和Lp-PLA2诊断脑梗死以及预测脑梗死患者预后不良的曲线下面积（AUC）分别为0.852、0.874，均高于各因素单独诊断和预测。结论：脑梗死DTI参数FA值、ADC值降低，血清NSE、Lp-PLA2水平增高，联合DTI参数和血清NSE、Lp-PLA2检测在脑梗死诊断和预后预测中具有较高价值。