~（60）Co－γ射线诱变柠檬酸产生菌黑曲霉Co9－6的研究

本试验采用￣（６０）Ｃｏ－γ射线对柠檬酸产生菌黑曲霉Ｃｏ９－６进行辐射,经两次处理后选育出Ｌ１２１７和Ｌ８０１两株优良柠檬酸产生菌。中试结果表明菌株Ｌ１２１７较Ｌ８０１更优：产酸率较菌株Ｃｏ９－６提高１７．６％、发酵周期缩短１３．４％、对糖转化率提高１３．３％。     

Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION? sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.     

Cell death induced by Pteris semipinnata L. is associated with p53 and oxidant stress in gastric cancer cells

In this study, we demonstrated that Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) had stronger cytotoxicity against MKN-45, a gastric cancer cell line bearing wild-type p53 than MKN-28, another gastric cancer cell line containing missense mutation in p53. The rapid increase of ROS level was involved in the mechanism of cytotoxicity. Classical features of apoptosis induced by 5F were observed in MKN-45 cells only or more significant in MKN-45 cells than MKN-28 cells. Translocation of Bax from cytosol to mitochondria, reduction of delta psi m and DNA fragmentation were induced by 5F in the p53-dependent manner. We conclude that the expression of Bax and its downstream molecules requires the presentation of a wild-type p53 in the cells treated by 5F.     

miR-455-5p靶向SOCS3抑制RSV感染致气道上皮炎症反应的机制研究

摘要 目的：揭示miR-455-5p对呼吸道合胞病毒（RSV）感染致气道上皮细胞炎症反应的作用机制。方法：qRT-PCR检测30例健康体检儿童（健康组）、RSV感染轻症组（n=41）和重症组（n=31）患儿血清miR-455-5p水平。将16HBE细胞分为Control组、NC-agomir组、miR-455-5p-agomir组、NC-antagomir组、miR-455-5p-antagomir组。使用Lipofectamine 3000转染16HBE细胞后培养48 h后,分为Blank组、NC组（转染了NC-agomir的细胞）、RSV+NC-agomir组、RSV+miR-455-5p-agomir组,用RSV病毒液感染RSV+NC-agomir组和RSV+miR-455-5p-agomir组16HBE细胞,Blank组和NC组16HBE细胞正常培养。用CCK-8法和EdU法检测细胞增殖、TUNEL法检测细胞凋亡、ELISA法检测上清液中TNF-α、IL-6、IL-8水平,qRT-PCR检测miR-455-5p和SOCS3的mRNA水平,Western blot检测SOCS3、IFN-α、STAT1、STAT2、p-STAT1和p-STAT2的蛋白水平。结果：与健康组相比,轻症组和重症组患儿的血清miR-455-5p水平降低（P<0.05）。与轻症组相比,重症组的血清miR-455-5p水平降低（P<0.05）。与Control组和NC-agomir组相比,miR-455-5p-agomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率升高（P<0.05）,TUNEL阳性率降低（P<0.05）,上清液中的TNF-α、IL-6和IL-8水平降低（P<0.05）,SOCS3 mRNA和蛋白水平降低（P<0.05）,IFN-α蛋白、STAT1和STAT2磷酸化水平升高（P<0.05）。与Control组和NC-antagomir组相比,miR-455-5p-antagomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率降低（P<0.05）,TUNEL阳性率升高（P<0.05）,上清液中的TNF-α、IL-6和IL-8水平升高（P<0.05）,SOCS3 mRNA和蛋白水平升高（P<0.05）,IFN-α蛋白、STAT1和STAT2磷酸化水平降低（P<0.05）。与Blank组和NC组相比,RSV+NC-agomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率降低（P<0.05）,TUNEL阳性率升高（P<0.05）,上清液中的TNF-α、IL-6和IL-8水平升高（P<0.05）,SOCS3 mRNA和蛋白水平升高（P<0.05）,IFN-α蛋白、STAT1和STAT2磷酸化水平降低（P<0.05）。与RSV+NC-agomir组相比,RSV+miR-455-5p-agomir组的miR-455-5p水平、相对细胞活力和EdU阳性率升高（P<0.05）,TUNEL阳性率降低（P<0.05）,上清液中的TNF-α、IL-6和IL-8水平降低（P<0.05）,SOCS3 mRNA和蛋白水平降低（P<0.05）,IFN-α蛋白、STAT1和STAT2磷酸化水平升高（P<0.05）。结论：miR-455-5p在RSV感染患儿血清中下调,上调miR-455-5p通过抑制SOCS3的转录和表达从而激活RSV感染的16HBE细胞中IFN-α介导的抗病毒反应。     

血清微小RNA-141-3p、微小RNA-150-5p与鼻咽癌患者临床病理特征和放疗敏感性的关系研究

摘要 目的：探讨血清微小核糖核酸（miRNA）-141-3p、miR-150-5p与鼻咽癌（NPC）患者临床病理特征和放疗敏感性的关系。方法：收集2019年1月～2022年7月在我院接受放疗的92例NPC患者为NPC组,根据放疗疗效分为抵抗组和敏感组,另选取同期80名在我院体检的健康志愿者为对照组。比较对照组、NPC组血清miR-141-3p、miR-150-5p表达。分析NPC患者血清miR-141-3p、miR-150-5p表达与临床病理特征的关系。利用单因素和多因素Logistic回归分析NPC患者放疗抵抗的影响因素。结果：与对照组比较,NPC组血清miR-141-3p表达升高,miR-150-5p表达降低（P＜0.05）。NPC患者血清miR-141-3p、miR-150-5p表达在不同分化程度、TNM分期和淋巴结转移中比较有差异（P＜0.05）。92例NPC患者放疗抵抗发生率为22.83%（21/92）。单因素分析显示,抵抗组TNM分期Ⅲ～Ⅳa期和miR-141-3p ≥ 2.60比例高于敏感组,miR-150-5p ≥ 0.80比例低于敏感组（P＜0.05）。多因素Logistic回归分析显示,miR-141-3p ≥ 2.60为NPC患者放疗抵抗的独立危险因素,miR-150-5p ≥ 0.80为独立保护因素（P＜0.05）。结论：NPC患者血清miR-141-3p高表达,miR-150-5p低表达,与分化程度、TNM分期、淋巴结转移和放疗敏感性有关,有望成为NPC患者放疗抵抗的评价指标。     

甲状腺乳头状癌组织微小RNA-93-5p、微小RNA-98-5p表达与临床病理特征和增殖、侵袭基因表达的关系研究

摘要 目的：研究甲状腺乳头状癌（PTC）组织微小核糖核酸-93-5p（miR-93-5p）、微小RNA-98-5p（miR-98-5p）表达与临床病理特征和增殖、侵袭基因表达的关系。方法：选取2020年10月到2023年10月在广东省中医院行手术切除的PTC患者153例作为研究对象,收集术中切除的癌组织以及癌旁组织。检测并比较癌组织与癌旁组织miR-93-5p、miR-98-5p及增殖基因、侵袭基因mRNA表达水平,分析miR-93-5p及miR-98-5p表达与PTC患者临床病理特征的关系。利用Pearson法分析miR-93-5p、miR-98-5p表达水平与增殖基因、侵袭基因mRNA表达的相关性。结果：癌组织的miR-93-5p表达水平较癌旁组织更高,miR-98-5p表达水平较癌旁组织更低（P<0.05）。miR-93-5p高表达PTC患者TNM分期Ⅲ~Ⅳ期、有淋巴结转移及低分化的比例较miR-93-5p低表达PTC患者更高（P<0.05）。miR-98-5p低表达PTC患者TNM分期Ⅲ~Ⅳ期、有淋巴结转移及低分化的比例较miR-98-5p高表达PTC患者更高（P<0.05）。癌组织的增殖基因程序性细胞死亡因子4（PDCD4）及蛋白磷酸酶4调节亚基 （PP4R1）水平较癌旁组织更低（P<0.05）,侵袭基因金属蛋白酶解离素9（ADAM9）及Bcl-6共抑制因子样蛋白（BCORL1）水平较癌旁组织更高（P<0.05）。Pearson法分析结果显示,miR-93-5p表达与增殖基因PDCD4及PP4R1表达水平呈负相关,与侵袭基因ADAM9及BCORL1表达水平呈正相关。miR-98-5p表达水平与增殖基因PDCD4及PP4R1水平呈正相关,与侵袭基因ADAM9及BCORL1表达水平呈负相关。结论：PTC患者癌组织miR-93-5p表达升高,miR-98-5p表达降低,与TNM分期、淋巴结转移及分化程度等临床病理特征有关,还可促进PTC癌细胞增殖、侵袭。     

Application of ultra-high magnetic field for saccharide molecules: 1H NMR spectra of 6-O-alpha-D-glucopyranosyl-cyclomaltoheptaose and -cyclomaltohexaose

1H NMR spectra of G1-alpha-CD and G1-beta-CD were recorded using a spectrometer equipped with a 21.6 T magnet. An ultra-high magnetic field was effective for detecting 1H NMR signals with a small difference in chemical shifts. Introducing a glucosyl group onto CDs as a branch caused deformation of equilibrated 1H signals of cyclodextrin. Particularly, 1H signals in branched glucose were shifted greatly.     

Regulation of Na/K/Cl cotransport in vascular smooth muscle cells

The regulation of Na/K/Cl cotransport was investigated in vascular smooth muscle cells. That a Na/K/Cl cotransport system exists was established by the finding that the ouabain insensitive K influx was sensitive to the "loop" diuretic bumetanide. Furthermore, bumetanide sensitive K influx was dependent upon the presence of both Na and Cl in the extracellular milieu. Bumetanide sensitive K influx was inhibited by agents which elevate cellular cyclic AMP levels, and to a lesser extent by agents which elevate cellular cyclic GMP levels. When serum, EGF or TPA was added, bumetanide sensitive K influx was enhanced. These results suggest that vascular smooth muscle cells have a ouabain insensitive, bumetanide sensitive Na/K/Cl cotransport system which is stimulated by serum, EGF or TPA and inhibited by cAMP or cGMP.     

Absolute configuration of eremophilanoids by vibrational circular dichroism

The absolute configurations (AC) of natural occurring 6-hydroxyeuryopsin (1), of its acetyl derivative 2, and of eremophilanolide 8 were confirmed by comparison of the experimental vibrational circular dichroism (VCD) spectra with theoretical curves generated from density functional theory (DFT) calculations. Initial analyses were carried out using a Monte Carlo searching with the MMFF94 molecular mechanics force field. All MMFF94 conformers were further optimized using DFT at the B3LYP/6-31G(d) level of theory, followed by calculations of their vibrational frequencies at the B3LYP/6-31G(d,p); the VCD spectra of 2 and 8 were also calculated at the B3PW91/DGDZVP level of theory. Good agreement between theoretical and experimental VCD curves unambiguously verified the 4S,5R,6S absolute configuration for 1 and 2, and the 1S,4S,5R,6S,8S,10S configuration for 8.     

MiR-423-5p inhibition alleviates cardiomyocyte apoptosis and mitochondrial dysfunction caused by hypoxia/reoxygenation through activation of the wnt/β-catenin signaling pathway via targeting MYBL2

MicroRNA (miR) plays an integral role in cardiovascular diseases. M-iR-423-5p is aberrantly expressed in patients with myocardial infarction and heart failure. The aim of the present study was to study the roles and mechanisms of miR-423-5p in hypoxia/reoxygenation (H/R) mediated cardiomyocytes injury. H9C2 cells were transfected with negative control, miR-423-5p mimic, and inhibitor for 48 hr, followed by exposed to H/R condition. Cell apoptosis rate, caspase 3/7 activities, Bax and cleaved-caspase 3 (c-caspase 3) protein levels were assayed by flow cytometry, Caspase-Glo 3/7 Assay kit, western blot analysis, respectively. Furthermore, the mitochondrial membrane potential, adenosine triphosphate (ATP) content, reactive oxygen species (ROS) production, and Drp1 expression were also investigated. Furthermore, the dual-luciferase reporter assay was used to evaluate the relationship between miR-423-5p and Myb-related protein B (MYBL2). The roles of miR-423-5p in wnt/β-catenin were assessed by western blot analysis. The results revealed that H/R triggered miR-423-5p expression. Overexpression of miR-423-5p promoted cardiomyocyte apoptosis, enhanced the activities of caspase 3/7, upregulated the expression of Bax and c-caspase 3. miR-423-5p upregulation caused the loss of mitochondrial membrane potential and the reduction of ATP content, the augment of ROS production and Drp1 expression. However, the opposite trends were observed upon suppression of miR-423-5p. In addition, miR-423-5p could target the 3′ untranslated region of MYBL2. miR-423-5p depletion led to the activation of the wnt/β-catenin signaling pathway via targeting MYBL2. Knockdown of MYBL2 was obviously reversed the roles of miR-423-5p in apoptosis and mitochondrial dysfunction. Taken together, miR-423-5p suppression reduced H/R-induced cardiomyocytes injury through activation of the wnt/β-catenin signaling pathway via targeting MYBL2 in cardiomyocytes.