Suction blister fluid as potential body fluid for biomarker proteins

Early diagnosis is important for effective disease management. Measurement of biomarkers present at the local level of the skin could be advantageous in facilitating the diagnostic process. The analysis of the proteome of suction blister fluid, representative for the interstitial fluid of the skin, is therefore a desirable first step in the search for potential biomarkers involved in biological pathways of particular diseases. Here, we describe a global analysis of the suction blister fluid proteome as potential body fluid for biomarker proteins. The suction blister fluid proteome was compared with a serum proteome analyzed using identical protocols. By using stringent criteria allowing less than 1% false positive identifications, we were able to detect, using identical experimental conditions and amount of starting material, 401 proteins in suction blister fluid and 240 proteins in serum. As a major result of our analysis we construct a prejudiced list of 34 proteins, relatively highly and uniquely detected in suction blister fluid as compared to serum, with established and putative characteristics as biomarkers. We conclude that suction blister fluid might potentially serve as a good alternative biomarker body fluid for diseases that involve the skin.     

Nitrogen-doped carbon dots as a fluorescent probe for the highly sensitive detection of bilirubin and cell imaging

Nitrogen-doped carbon dots (NCDs) with bright blue fluorescence were constructed by a hydrothermal method using sucrose and l- proline as raw materials. The NCDs were characterized by transmitted electron microscopy, X-ray diffraction, Fourier-transform infrared spectrometry, X-ray photoelectron spectroscopy, and ultraviolet-visible absorption and fluorescence spectroscopy to investigate the morphology, elemental composition, and optical properties. The NCDs had good water solubility, high dispersibility with an average diameter of only 1.7 nm, and satisfactory optical properties with a fluorescence quantum yield of 23.4%. The NCDs were employed for the detection of bilirubin. A good linear response of the NCDs in the range 0.35–9.78 μM was obtained for bilirubin with a detection limit of 33 nM. The NCDs were also applied to the analysis of real samples, serum and urine, with a recovery of 95.34% to 104.66%. The low cytotoxicity and good biocompatibility of the NCDs were indicated by an MTT assay and cell imaging of HeLa cells. Compared with other detection systems, using NCDs for bilirubin detection was a facile and efficient method with good selectivity and sensitivity.     

淀粉样蛋白A、D-二聚体、肌酸激酶同工酶联合检测对川崎病患儿冠状动脉损伤的诊断价值分析

摘要 目的：探讨血清淀粉样蛋白A(SAA)、D-二聚体(D-D)、肌酸激酶同工酶(CK-MB)联合检测对川崎病患儿冠状动脉损伤(CAL)的诊断价值。方法：选取2018年9月～2021年5月我院收治的80例川崎病患儿,根据是否合并CAL分为CAL组(n=34)和NCAL组(n=46)。收集患儿基础资料,并检测SAA、D-D、CK-MB水平。多因素Logistic回归分析川崎病患儿CAL影响因素,受试者工作特征（ROC）曲线分析血清SAA、D-D、CK-MB水平对川崎病患儿CAL的诊断价值。结果：与NCAL组比较,CAL组C反应蛋白(CRP)、红细胞沉降率(ESR)、SAA、D-D、CK-MB水平升高(P＜0.05)。多因素Logistic回归分析显示,CRP、ESR、SAA、D-D、CK-MB为川崎病患儿CAL独立影响因素(P＜0.05)。SAA、D-D、CK-MB、三项联合诊断川崎病患儿CAL的曲线下面积（AUC）分别为0.661、0.687、0.746、0.799,联合应用的诊断效能最高。结论：血清SAA、D-D、CK-MB是川崎病患儿CAL独立影响因素,且联合检测以上指标可辅助诊断川崎病患儿CAL。     

不同剂量右美托咪定静脉维持对重型颅脑损伤患者术后生命体征、免疫功能和血清神经细胞因子的影响

摘要 目的：观察不同剂量右美托咪定静脉维持在重型颅脑损伤患者中的应用价值。方法：选取2018年9月~2020年9月期间江苏大学附属宜兴市人民医院麻醉与重症医学科接收的重型颅脑损伤患者96例,根据随机数字表法分为三组：A组（右美托咪定剂量为0.3μg/kg?h）、B组（右美托咪定剂量为0.5 μg/kg?h）和C组（右美托咪定剂量为0.7 μg/kg?h）,各32例。观察三组患者不同时间点的生命体征、免疫功能、镇静镇痛情况、血清神经细胞因子,记录三组不良反应发生情况。结果：B组、C组术后24 h、术后72 h的心率（HR）、呼吸频率（RR）、平均动脉压（MAP）低于A组（P<0.05）。B组、C组术后24 h、术后72 h的Ramsay镇静评分、视觉模拟评分法（VAS）评分低于A组（P<0.05）。B组、C组术后24 h、术后72 h的CD3⁺、CD4⁺/CD8⁺高于A组（P<0.05）。B组、C组术后24 h、术后72 h的神经元特异性烯醇化酶（NSE）、中枢神经特异性蛋白（S100β）低于A组（P<0.05）。C组的不良反应总发生率高于A组、B组（P<0.05）。结论：重型颅脑损伤患者术中给予右美托咪定剂量为0.5 μg/kg?h、0.7 μg/kg?h维持,可有效维持患者生命体征平稳,促进患者免疫功能和血清神经细胞因子水平改善,但0.7 μg/kg?h剂量的右美托咪定使用后不良反应发生率相对更高。     

Expression of inflammatory cytokines in mesenchymal stromal cells is sensitive to culture conditions and simple cell manipulations

Background

Mesenchymal stromal cells (MSCs) can be used in several clinical applications. While MSCs are frequently cultured in fetal bovine serum for in vitro experimentation, human serum supplements are required for cells to be used in patients. Here we show how different human serum supplements and in vitro manipulations used during the cell culture impact on MSC proliferation rate and expression of inflammatory molecules.

Anti-cancer study and whey protein complexation of new lanthanum(III) complex with the aim of achieving bioactive anticancer metal-based drugs

In this study, a new lanthanum (III)-amino acid complex utilizing cysteine has been synthesized and characterized. The anticancer activities of the prepared La(III) complex against MCF-7 cell lines were studied. Results of MTT assay showed that at all three incubation times, the cytotoxic effect of prepared La(III) complex on MCF-7 breast cancer cell lines displays a time- and dose-dependent inhibitory effects. The interactions of the La(III) complex with two whey proteins (bovine serum albumin, BSA, and Bovine β-lactoglobulin, βLG) have been explored by using spectroscopic and molecular dicking methods. The obtained results indicated that La(III) complex strongly quenched the fluorescence of two carrier proteins in static quenching mode and also, BSA hah stronger binding affinity toward studied complex than βLG whit binding constant values of K_{BSA-La?Complex}?～?0.11?×?10⁴ M^?1 and K_{βLG-La?Complex}?～?0.63?×?10³ M^?1 at 300 K. The thermodynamic parameters revealed the contribution of hydrogen bond and Vander Waals interactions in both systems. The distances of the La(III) complex whit whey proteins were calculated using Förster energy transfer theory and proved existence of the energy transfer between two proteins and prepared La(III) complex with a high probability. FT-IR and UV–Vis absorption measurements indicated that the binding of the La(III) to BSA and βLG may induce conformational and micro-environmental changes of the proteins. The docking results indicate that the La(III) complex bind to residues located in the site II of BSA and second site of βLG.

Communicated by Ramaswamy H. Sarma     

Contribution of serum albumin to the transport of orally administered L-tryptophan into liver of rats with L-tryptophan depletion

Summary The role of serum albumin in the transport of orally administered L-tryptophan (Trp) into rat tissues was examined using analbuminemic and Sprague-Dawley (SD) rats with and without a-methyl-DL-tryptophan (AMT)-induced Trp depletion. Trp was orally administered to rats 16h after AMT or 0.85% NaCl administration, when liver tryptophan 2,3-dioxygenase and protein synthetic activities in AMT-treated rats were similar to those of 0.85% NaCl-treated rats. After oral Trp administration, regardless of the presence or absence of Trp depletion, free serum Trp concentrations were similar in the analbuminemic and SD rats, while total serum Trp concentrations were lower in analbuminemic rats than in SD rats. Although liver, brain, and muscle Trp concentrations after oral Trp administration under Trp depletion were lower in analbuminemic rats than in SD rats, the ratio of the liver Trp concentration in analbuminemic rats to that in SD rats was smaller than that of the brain or muscle Trp concentration. These results suggest that variations in serum albumin levels could affect the transport of orally administered Trp into the liver of rats with Trp depletion.     

Antiapoptotic role of heme oxygenase (HO) and the potential of HO as a target in anticancer treatment

Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes oxidative degradation of heme to form biliverdin, carbon monoxide (CO), and free iron. Biliverdin is subsequently reduced to bilirubin by the enzyme biliverdin reductase. Increasing evidence has indicated the critical role of HO-1 in cytoprotection and more diverse biological functions. Induction of HO-1 by various chemical inducers that are primarily cell stress inducers or by HO-1 gene transfection confers a protective capacity to cultured cells as well as to cells in several in vivo animal models. In addition, HO-1-deficient mice exhibit a significant increase in susceptibility to tissue injury. The cytoprotective action of HO-1 seems to be mainly a function of the antiapoptotic effects of the enzyme. HO-1 is believed to exert this antiapoptotic action by multiple mechanisms: (a) decreased intracellular pro-oxidant levels, (b) increased bilirubin levels, and (c) elevated CO production. CO may produce an antiapoptotic effect by inhibiting both expression of p53 and release of mitochondrial cytochrome c. HO-1 may also be a target in antitumor therapy because the growth of most tumors depends on HO-1. Our preliminary studies with an HO inhibitor showed a promising antitumor effect. This preliminary work warrants continued investigation for possible novel anticancer chemotherapy.     

Recombinant human serum amyloid P component from Pichia pastoris: production and characterization

Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris. SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag. The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted. Treatment of the cell paste with EDTA increased the yield further by about 30%. The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence. Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa. Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP. r-SAP bound to antibodies produced against h-SAP. Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination. These results indicate that r-SAP produced in P. pastoris has the same biological activity as purified h-SAP.     

Biliverdin reductase from the liver of Atlantic salmon (Salmo salar)

Biliverdin reductase was characterized and purified from the liver of Atlantic salmon (Salmo salar) using a novel enzymatic staining method. The properties of the enzyme are quite different from those of mammals. The purified enzyme is a monomeric protein with a molecular weight of approximately 68 kD and an isoelectric point of around 3.8. The enzyme can utilize both NADH and NADPH as coenzyme, but the kinetic properties of the NADH-dependent and the NADPH-dependent enzyme activities are different: K _m value for biliverdin IX is 0.6 M in the NADPH system, while it is 6.8 M in the NADH system. Both enzyme activities are inhibited by excess biliverdin IX, but the NADPH-dependent enzyme activity is far more susceptible. The optimum pH for activity is 5.5 with NADPH and 6.0 with NADH. The optimum reaction temperature is 35°C.