肝硬化原发性肝癌直径<1 cm超声造影表现及其与血清AFU、AFP-L3、GPC3、TSGF、GP73水平相关性研究

摘要 目的：探讨肝硬化原发性肝癌（PHC）直径<1cm超声造影（CEUS）表现及其与血清α-L-岩藻糖苷酶（AFU）、甲胎蛋白异质体-L3（AFP-L3）、磷脂酰肌醇蛋白聚糖-3（GPC3）、肿瘤特异生长因子（TSGF）、高尔基体糖蛋白（GP73）水平相关性。方法：选取2018年1月-2022年8月于湖北省襄阳市中医院收治的肝硬化PHC直径<1 cm患者44例,根据术后病理结果分为高分化组、中分化组和低分化组。所有患者术前均完善CEUS和血清AFU、AFP-L3、GPC3、TSGF、GP73水平检查。比较三组CEUS表现、定量时间-强度曲线（TIC）分析、血清AFU、AFP-L3、GPC3、TSGF、GP73水平。采用Spearman相关性分析肝硬化PHC直径<1 cm患者的CEUS表现与血清AFU、AFP-L3、GPC3、TSGF、GP73水平的相关性。结果：44例肝硬化PHC直径<1 cm患者的CEUS表现均为肝内单发病灶,呈圆形或类圆形,病灶边界清晰,周围可见声晕。不同分化程度肝硬化PHC直径<1 cm患者在动脉期、门脉期和延迟期的CEUS表现上差异均无统计学意义（P＞0.05）。高分化组、中分化组和低分化组的达峰时间、廓清时间和峰值加速时间逐渐减少,差异有统计学意义（P＜0.05）。而高分化组、中分化组和低分化组的峰值强度增加率逐渐增加,差异有统计学意义（P＜0.05）。高分化组、中分化组和低分化组的增强时间对比差异无统计学意义（P＞0.05）。高分化组、中分化组和低分化组血清AFU、AFP-L3、GPC3、TSGF、GP73水平逐渐升高,差异有统计学意义（P＜0.05）。Spearman相关性分析显示,达峰时间、廓清时间和峰值加速时间与血清AFU、AFP-L3、GPC3、TSGF、GP73水平呈负相关（P＜0.05）;峰值强度增加率与血清AFU、AFP-L3、GPC3、TSGF、GP73水平呈正相关（P＜0.05）。结论：肝硬化PHC直径<1 cm患者的CEUS表现均为肝内单发病灶,呈圆形或类圆形,病灶边界清晰,周围可见声晕。CEUS表现和血清AFU、AFP-L3、GPC3、TSGF、GP73水平具有相关性,两者可辅助鉴别肝硬化PHC直径<1 cm的不同分化程度。     

A cell-based high-throughput screen identifies tyrphostin AG 879 as an inhibitor of animal cell phospholipid and fatty acid biosynthesis

Inhibition of animal cell phospholipid biosynthesis has been proposed for anticancer and antiviral therapies. Using CHOK1 derived cell lines, we have developed and used a cell-based high-throughput procedure to screen a 1280 compound, small molecule library for inhibitors of phospholipid biosynthesis. We identified tyrphostin AG 879 (AG879), which inhibited phospholipid biosynthesis by 85–90% at a concentration of 10 μM, displaying an IC₅₀ of 1–3 μM. The synthesis of all phospholipid head group classes was heavily affected. Fatty acid biosynthesis was also dramatically inhibited (90%). AG879 inhibited phospholipid biosynthesis in all additional cell lines tested, including MDCK, HUH7, Vero, and HeLa cell lines. In CHO cells, AG879 was cytostatic; cells survived for at least four days during exposure and were able to divide following its removal. AG879 is an inhibitor of receptor tyrosine kinases (RTK) and inhibitors of signaling pathways known to be activated by RTK's also inhibited phospholipid biosynthesis. We speculate that inhibition of RTK by AG879 results in an inhibition of fatty acid biosynthesis with a resulting decrease in phospholipid biosynthesis and that AG879's effect on fatty acid synthesis and/or phospholipid biosynthesis may contribute to its known capacity as an effective antiviral/anticancer agent.     

The inhibitor profiling of the caspase family of proteases using substrate-derived peptide glyoxals

A series of substrate-based α-keto-β-aldehyde (glyoxal) sequences have been synthesised and evaluated as inhibitors of the caspase family of cysteine proteases. A number of potent inhibitor sequences have been identified. For example, a palmitic acid containing sequence pal-Tyr-Val-Ala-Asp-glyoxal was demonstrated to be an extremely effective inhibitor of caspase-1, inhibiting not only the action of the protease against synthetic fluorogenic substrates (K_i = 0.3 nM) but also blocking its processing of pro-interleukin-1beta (pro-IL-1β). In addition, the peptide Ac-Asp-Glu-Val-Asp-glyoxal, which is based on the consensus cleavage sequence for caspase-3, is a potent inhibitor of this protease (K_i = 0.26 nM) yet only functions as a comparatively modest inhibitor of caspase-1 (K_i = 451 nM). Potent inhibitor sequences were also identified for caspases-6 and -8. However, the degree of discrimination between the family members is limited. The ability of Ac-Asp-Glu-Val-Asp-glyoxal to block caspase-3 like activity in whole cells and to delay the development of apoptosis was assessed. When tested against caspase-3 like activity in cell lysates, Ac-Asp-Glu-Val-Asp-glyoxal displayed effective inhibition similar to that observed against recombinant caspase-3. Treatment of whole cells with this potent caspase-3 inhibitor was however, not sufficient to significantly stall the development of apoptosis in-vitro.     

Evaluation of known and novel inhibitors of Orai1-mediated store operated Ca2+ entry in MDA-MB-231 breast cancer cells using a Fluorescence Imaging Plate Reader assay

The Orai1 Ca²⁺ permeable ion channel is an important component of store operated Ca²⁺ entry (SOCE) in cells. It’s over-expression in basal molecular subtype breast cancers has been linked with poor prognosis, making it a potential target for drug development. We pharmacologically characterised a number of reported inhibitors of SOCE in MDA-MB-231 breast cancer cells using a convenient Fluorescence Imaging Plate Reader (FLIPR) assay, and show that the rank order of their potencies in this assay is the same as those reported in a wide range of published assays. The assay was also used in a screening project seeking novel inhibitors. Following a broad literature survey of classes of calcium channel inhibitors we used simplified ligand structures to query the ZINC on-line database, and following two iterations of refinement selected a novel Orai1-selective dichlorophenyltriazole hit compound. Analogues of this were synthesized and evaluated in the FLIPR assay to develop structure–activity relationships (SAR) for the three domains of the hit; triazole (head), dichlorophenyl (body) and substituted phenyl (tail). For this series, the results suggested the need for a lipophilic tail domain and an out-of-plane twist between the body and tail domains.     

Evaluation of urinary biomarkers of oxidative/nitrosative stress in adolescents and adults with Down syndrome

Urinary biomarkers of oxidative stress have been little studied in adults with Down syndrome (DS), usually no more than two biomarkers have been measured in the population studied and controversial results are reported in literature. Thus, we aimed to assess a set of oxidative and nitrosative stress biomarkers in urine samples of adolescents and adults with DS, with and without hypothyroidism, which comprise: 8-hydroxy-2′-deoxyguanosine (8-OHdG), isoprostane 15-F_2t-IsoP, thiobarbituric acid-reacting substances (TBARS), advanced glycation end products (AGEs), dityrosine (diTyr), hydrogen peroxide (H₂O₂) and nitrite/nitrate (NOx). Fluorimetric and spectrophotometric assays were performed in DS (n = 78), some of them taking levothyroxine for hypothyroidism (n = 24), and in their healthy age-matched controls (n = 65). We found that levels of AGEs, diTyr, H₂O₂ and NOx are increased in DS patients in any or in all age groups, whereas Cr levels were lower in DS than in controls in all age groups. Besides, correlations with age in DS were positive for diTyr and negative for Cr, TBARS, 15-F_2t-IsoP and NOx. We also found lower levels of Cr from 15 to 19 years, higher levels of TBARS and AGEs from 20 to 40 years and higher levels of diTyr from 15 to 40 years in DS patients receiving levothyroxine than in DS without hypothyroidism diagnosed. We conclude that AGEs, diTyr, H₂O₂ and NOx could be used as oxidative stress biomarkers in DS in contrast to 8-OHdG, 15-F_2t-IsoP and TBARS, at least with the methods used. However, renal impairment could occur in DS and Cr adjustment may bias the results, particularly in hypothyroid patients.     

Epigallocatechin gallate counteracts oxidative stress in docosahexaenoxic acid-treated myocytes

Skeletal muscle is a key organ of mammalian energy metabolism, and its mitochondria are multifunction organelles that are targets of dietary bioactive compounds. The goal of this work was to examine the regulation of mitochondrial dynamics, functionality and cell energy parameters using docosahexaenoic acid (DHA), epigallocatechin gallate (EGCG) and a combination of both in L6 myocytes. Compounds (at 25 μM) were incubated for 4 h. Cells cultured with DHA displayed less oxygen consumption with higher ADP/ATP ratio levels concomitant with downregulation of Cox and Ant1 gene expression. The disruption of energetic homeostasis by DHA, increases intracellular reactive oxygen species (ROS) levels and decreases mitochondrial membrane potential. The defence mechanism to counteract the excess of ROS production was by the upregulation of Ucp2, Ucp3 and MnSod gene expression. Moreover myocytes cultured with DHA had a higher mitochondrial mass with a higher proportion of large and elongated mitochondria, whereas the fission genes Drp1 and Fiss1 and the fusion gene Mfn2 were downregulated. In myocytes co-incubated with DHA and EGCG, ROS levels and the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio were similar to untreated myocytes and the decrease of oxygen consumption, higher mitochondrial mass and the overexpression of Ucp2 and Ucp3 genes were similar to the DHA-treated cells with also a higher amount of mitochondrial deoxyribonucleic acid (DNA), and reduced Drp1 and Fiss1 gene expression levels. In conclusion the addition of EGCG to DHA returned the cells to the control conditions in terms of mitochondrial morphology, energy and redox status, which were unbalanced in the DHA-treated myocytes.     

AFP、AFU、β_2-MG、CA199联合检测对原发性肝癌的早期诊断价值

目的：探讨原发性肝癌患者血清甲胎蛋白（AFP）、а-L岩藻糖苷酶（AFU）、β2-微球蛋白（β2-MG）、糖类抗原-199（CA199）的含量及其联合检测对原发性肝癌的早期诊断价值。方法：选择56例原发性肝癌患者、60名肝炎肝硬化患者和60名健康对照作为研究对象,分别应用比值法和化学发光法、生化法检测其血清AFP、AFU、β2-MG、CA199的含量。结果：原发性肝癌患者血清AFP、AFU、β2-MG、CA199含量均显着高于肝炎肝硬化组及健康对照组,差异均有统计学意义（P均〈0.05）;联合检测AFP、AFU、β2-MG、CA199四种肿瘤标志物,其阳性率达（85.7%）明显高于AFP（53.6%）、AFU（55.4%）、β2-MG（48.2%）和CA199（42.9%）单项检测组（P均〈0.05）;且AFP、AFU、β2-MG、CA199四种肿瘤标志物联合检测的敏感性均高于单一检测指标,差异有统计学意义（P〈0.05）,但其特异性显著低于AFU、β2-MG单项检测（P〈0.05）。结论：联合检测血清AFP、AFU、β2-MG、CA199含量可以提高对原发性肝癌的阳性诊断率,对诊断及鉴别诊断原发性肝癌具有重要意义。     

AFU, AFP, GP73和GPC3联合检测对原发性肝癌的诊断价值

目的:研究α-L-岩藻糖苷酶(AFU)、甲种胎儿球蛋白(AFP)、高尔基体蛋白73(GP73)和磷脂酰肌醇蛋白聚糖3(GPC3)联合检测对原发性肝癌的诊断价值。方法:选择2014年1月~2016年5月在我院进行诊治的原发性肝癌患者90例为肝癌组,同期住院诊治的肝硬化患者60例为肝硬化组,以及同期在我院体检健康者60例为对照组。比较三组的AFU、AFP、GP73和GPC3水平和阳性检出率,并观察AFU、AFP、GP73和GPC3单独检测和联合检测对原发性肝癌的诊断效率。结果:肝癌组和肝硬化组的AFU、AFP、GP73和GPC3水平均明显高于对照组(P0.05),且肝癌组明显高于肝硬化组(P0.05);肝癌组和肝硬化组的AFU、AFP、GP73和GPC3阳性率均明显高于对照组(P0.05),且肝癌组明显高于肝硬化组(P0.05);AFU、AFP、GP73和GPC3联合检测对原发性肝癌的特异性、敏感性、阳性预测值及阴性预测值均明显高于单独检测和三项指标联合检测(P0.05)。结论:AFU、AFP、GP73和GPC3联合检测可以提高对原发性肝癌的检出率,具有较高的临床应用价值。     

Inhibition of thrombin,an unexplored function of retinoic acid

Retinoic acid, a derivative of vitamin A, is known to possess in vivo anti-inflammatory, anti-platelet and fibrinolytic activities. We have investigated the in vitro thrombin and platelet aggregation inhibitory activities of vitamin A (retinol) and its derivatives, retinoic acid and retinaldehyde. The thrombin enzymatic assay was performed fluorimetrically to assess the inhibition of thrombin (Sigma and plasma). Retinoic acid, retinaldehyde and retinol exhibited potent inhibition of thrombin, with IC₅₀ values of 67μg/ml, 74μg/ml and 152μg/ml, respectively for the inhibition of thrombin (Sigma); and 49μg/ml, 74μg/ml and 178μg/ml, respectively for the inhibition of thrombin (plasma). Amongst vitamin A and its derivatives, retinoic acid showed the highest inhibition of both the forms of thrombin. Vitamin A and its derivatives also displayed remarkable inhibition of platelet aggregation. This is the first report of vitamin A and its derivatives showing inhibition of thrombin and platelet aggregation in vitro.     

Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects

Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix α-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix α-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization.