双歧杆菌乳杆菌三联活菌片对幽门螺杆菌根治后消化道不良反应的改善作用

探讨加用双歧杆菌乳杆菌三联活菌片对临床四联疗法根除幽门螺杆菌(H.pylori)过程中患者消化道不良反应的改善作用。

选择2019年1月至2020年1月在昆明医科大学第二附属医院门诊就诊的100例H.pylori感染患者, 分为益生菌组和正常组, 各50例。全部患者通过四联疗法根除H.pylori后, 益生菌组患者继续加用双歧杆菌乳杆菌三联活菌片治疗, 对结果进行分析。

2组患者使用含有铋剂的四联疗法根除H.pylori后效果满意。益生菌组患者不良反应发生率低于正常组(25.5% vs 47.5%;χ²=11.023, P=0.001)。

益生菌可减轻四联疗法根除H.pylori后消化道不良反应, 根除H.pylori过程中加用益生菌的时机尚待进一步探讨。

     

高危型人乳头瘤病毒感染与阴道微生态及宫颈局部细胞免疫的相关性

探讨高危型人乳头瘤病毒(HR-HPV)感染与阴道微生态及宫颈局部细胞免疫的相关性, 为该类患者的治疗提供参考。

选择2018年1月至2020年1月我院收治的126例HPV感染者, 依据感染病毒类型分为非HR-HPV感染组(n=69)和HR-HPV感染组(n=57), 并纳入同期来我院体检的50名健康体检者作为对照组。分析HPV感染类型、患者阴道微生态状况及宫颈局部组织T淋巴细胞亚群水平。

患者中复合型与单一型HR-HPV感染率分别为15.8%、84.2%, 差异有统计学意义(χ²=5.324, P=0.047)。对照组、非HR-HPV感染组和HR-HPV感染组对象阴道微生态失调率分别为22.0%、34.8%、45.6%, 其中对照组对象阴道微生态失调率最低(χ²=5.638, P=0.048)。非HR-HPV感染组和HR-HPV感染组患者阴道病原菌检出率高于对照组(均P < 0.05)。非HR-HPV感染组和HR-HPV感染组患者宫颈局部T淋巴细胞亚群水平低于对照组(均P < 0.05)。

HPV33、16、18型为HPV感染主要类型, 会加重阴道微生态紊乱程度, 影响局部细胞免疫功能。检测患者HPV感染情况和宫颈局部细胞免疫功能可为患者临床治疗提供有效数据, 有利于治疗措施的制定。

     

妊娠期女性HPV感染及阴道微生态失衡对妊娠结局及新生儿的影响

探讨妊娠期女性人乳头瘤病毒（HPV）感染及阴道微生态失衡对妊娠结局及新生儿结局的影响,为该类患者的治疗提供参考。

选取2020年6月至2023年6月于我院产检的102例HPV阳性妊娠妇女（HPV阳性组）以及同期产检的78例HPV阴性妊娠妇女（HPV阴性组）为研究对象,于怀孕28～34周时,收集阴道分泌物评价阴道微生态状况;另根据微生态评价结果将HPV阳性组对象分为微生态正常组（n=26）和微生态失调组（n=76）;比较HPV阳性组与HPV阴性组对象阴道微生态情况、妊娠结局及新生儿结局,比较微生态正常组与微生态失调组对象妊娠结局及新生儿结局。

HPV阳性组和HPV阴性组对象滴虫性阴道炎（TV）和外阴阴道假丝酵母菌病（VVC）发生率、阴道清洁度比较差异均无统计学意义（χ²=1.520、0.678、0.111,均P＞0.05）,而阴道pH、细菌性阴道病（BV）发生率、阴道菌群密集度、阴道菌群多样性以及微生态失调发生率比较差异均有统计学意义（χ²=10.106、8.247、4.337、5.236、13.865,均P＜0.05）。HPV阳性组对象早产、宫内感染、产褥感染及产后出血发生率显著高于HPV阴性组（χ²=5.710、10.721、6.799、4.294,均P＜0.05）,而两组对象剖宫产率及胎膜早破发生率比较差异无统计学意义（χ²=1.067、0.666,均P＞0.05）。HPV阳性组新生儿感染发生率显著高于HPV阴性组（χ²=9.001,P＜0.05）,两组胎儿窘迫、新生儿窒息和胎儿宫内生长受限（FGR）发生率比较差异均无统计学意义（χ²=2.503、1.547、0.560,均P＞0.05）。微生态失调组对象早产发生率显著高于微生态正常组（χ²=4.130,P＜0.05）,而两组胎膜早破、宫内感染、产褥感染及产后出血发生率比较差异均无统计学意义（χ²=1.401、0.578、0.141、1.368,均P＞0.05）。微生态失调组与微生态正常组胎儿窘迫、新生儿窒息、FGR和新生儿感染发生率比较差异均无统计学意义（χ²=0.261、0.698、1.057、0.242,均P＞0.05）。

妊娠期HPV感染能引发阴道微生态失调,增加不良母婴结局发生风险。

     

早期肠内营养联合益生菌对急性呼吸窘迫综合征患者预后的影响

探讨早期肠内营养联合益生菌对急性呼吸窘迫综合征（ARDS）患者预后的影响,为该类患者的治疗提供参考。

回顾性分析2018年6月至2020年12月我院急诊ICU收治的86例中重度ARDS患者的临床资料,按随机数字法将患者分为观察组（n = 48例）和对照组（n = 38例）。观察组患者在早期肠内营养基础上联合益生菌治疗,对照组患者采用常规治疗+早期肠内营养进行治疗。统计两组患者性别、年龄、既往基础疾病、原发疾病（包括肺内和肺外）、APACHE Ⅱ评分、SOFA评分、氧合指数、机械通气天数、脱机拔管成功率、ICU以及总的住院天数、28 d病死率等情况。

观察组患者机械通气时间短于对照组[（10.34±2.16）d vs （14.63±3.27）d,P = 0.020]。观察组患者脱机拔管成功率高于对照组（70.83% vs 63.16%,P = 0.038）。观察组患者ICU住院时间短于对照组[（15.34±3.28）d vs （18.68±3.54）d,P = 0.030]。观察组患者28 d病死率低于对照组（22.92% vs 26.32%,P = 0.035）。相关性分析提示早期肠内营养联合益生菌与患者的机械通气天数（r = −0.489,P = 0.039）和病死率（r = −0.312,P = 0.042）均存在相关性。非条件Logistic回归分析显示早期肠内营养联合益生菌能够降低患者的死亡风险。

相比单一予以早期肠内营养,采用早期肠内营养联合益生菌治疗ARDS患者更有利于缩短患者机械通气时间,降低病死率,改善患者的预后。

     

防护细节管理在消毒供应中心微生物污染手术器械处理中的应用

探讨防护细节管理在消毒供应中心微生物污染手术器械处理中的应用价值。

随机选取我院做到防护细节管理的600件手术器械(A组)和未实施防护细节管理的600件手术器械(B组)作为研究对象, 对比实施防护细节管理对手术器械微生物污染的影响。比较实施前后器械无菌检验和微生物限度检验一次合格率、质量管理情况(回收、分类、清洗消毒、包装不合格情况)以及患者医院感染发生率变化情况。

A组器械无菌检验一次合格率为98.67%, B组器械其一次检验合格率为95.67%, 差异有统计学意义(χ²=9.953, P=0.001 6)。A组器械微生物限度检验一次合格率为99.33%, B组为95.17%(χ²=9.305, P=0.002 3)。A组器械回收不合格率为0.33%, B组不合格率为2.00%。A组器械分类不合格率为0.50%, B组为1.50%。A组器械清洗消毒不合格率为0.83%, 器械包装不合格率为0.33%;B组器械清洗消毒不合格率为1.83%, 手术器械包装不合格率1.33%。A组和B组器械导致的医院感染发生率分别为0.67%、1.67%。

防护细节管理在降低消毒供应中心手术器械微生物的污染, 提高管理质量以及降低医院感染发生率方面有重要价值。

     

生殖道GBS感染对妊娠晚期妇女阴道微生态环境及免疫因子的影响

探讨妊娠晚期生殖道B族溶血性链球菌（GBS）感染对阴道微生态环境及免疫因子的影响。

收集2021年7月至2022年7月本院76例GBS筛查阳性妊娠晚期妊娠妇女（GBS阳性组）,另选取同期GBS筛查阴性的76例妊娠晚期妊娠妇女（对照组）,比较2组阴道微生态情况、血清免疫炎症因子（IL-1β、IL-6）水平及妊娠结局;另根据阴道微生态评价结果将GBS阳性组妊娠妇女进一步分为微生态失调组（n=56）和微生态正常组（n=20）,比较2组妊娠结局。

GBS阳性组和对照组研究对象的阴道pH值、细菌性阴道病（BV）发生率、外阴阴道假丝酵母菌病（VVC）发生率、阴道菌群密集度、阴道菌群多样性及微生态失调发生率比较差异均有统计学意义（χ²=8.550、5.842、5.156、4.682、5.339、14.341,均P＜0.05）,2组研究对象滴虫性阴道炎发生率、阴道清洁度比较差异均无统计学意义（χ²=0.541、1.685,均P＞0.05）。GBS阳性组血清IL-1β、IL-6水平显著高于对照组（t=16.711、19.388,均P＜0.05）。GBS阳性组早产、产褥感染、胎儿窘迫及病理性黄疸发生率显著高于对照组（χ²=5.365、10.059、7.938、5.787,均P＜0.05）,2组研究对象胎膜早破、产后出血、新生儿窒息及新生儿肺炎发生率比较差异均无统计学意义（χ²=1.849、0.882、2.027、2.027,均P＞0.05）。微生态失调组胎膜早破、胎儿窘迫发生率显著高于微生态正常组（χ²=4.113、4.113,均P＜0.05）,2组研究对象早产、产褥感染、产后出血、新生儿窒息、病理性黄疸和新生儿肺炎发生率比较差异均无统计学意义（χ²=2.805、1.281、0.384、0.734、0.880、0.734,均P＞0.05）。

妊娠晚期GBS感染妊娠妇女易发生阴道微生态及炎症因子失调,增加不良妊娠结局发生风险。

     

乙型肝炎肝硬化腹水合并自发性细菌性腹膜炎患者肠道菌群特征

初步分析乙型肝炎肝硬化腹水合并自发性细菌性腹膜炎（SBP）患者肠道菌群特征,为该类患者的治疗提供参考。

选取我院符合纳入标准的乙型肝炎肝硬化腹水患者45例为研究对象,其中24例合并SBP的患者为SBP组、21例未合并SBP的患者为NSBP组,另选同期20例健康人为HC组。应用16S rDNA测序技术检测患者肠道菌群,并使用生物信息学方法分析各组对象肠道菌群变化。

SBP组、NSBP组及HC组相比,其肠道菌群的物种多样性及丰富度差异均无统计学意义（均P＜0.05）。PCoA分析显示,NSBP组与HC组患者肠道菌群结构相似,SBP组与其他两组相比菌群结构较为不同。与NSBP组相比,SBP组患者肠道菌群中变形菌门（Proteobacteria,5.45% vs 13.50%,U = 161.000,P = 0.038）、软壁菌门（Tenericutes,0.019% vs 0.073%,U = 162.000,P = 0.035）、埃希—志贺菌属（Escherichia-Shigella,0.24% vs 9.53%,U = 103.000,P = 0.001）、柠檬酸杆菌属（Citrobacter,LDA＞2）等丰度均增加;酸杆菌门（Acidobacteria,0.026% vs 0.015%,U = 152.000,P = 0.023）、罗氏菌属（Roseburia,2.55% vs 2.00%,U = 152.000,P = 0.023）、粪球菌属（Coprococcus,LDA＞2）等丰度下降。

乙型肝炎肝硬化腹水的发生发展与肠道菌群密切相关,在SBP患者肠道菌群中发现潜在病原体数量显著增加,并伴随有益细菌数量的减少。

     

经颅直流电刺激联合益生菌对脑卒中后认知功能障碍患者的作用

探讨经颅直流电刺激（tDCS）联合益生菌对脑卒中后认知功能障碍（PSCI）患者认知功能和肠道菌群的影响。

按随机数字表法将2020年6月至2021年6月我院收治的150例PSCI患者分为对照组和观察组,各75例。对照组患者给予tDCS治疗,观察组患者给予tDCS联合益生菌治疗。采用蒙特利尔认知功能评估量表（MoCA）和简易智力状态检查量表（MMSE）评估患者治疗前后的认知功能。采用荧光定量PCR法检测患者治疗前后肠道菌群数量变化。比较两组患者认知功能、肠道菌群、临床疗效以及不良反应发生情况。

治疗后,观察组患者肠道双歧杆菌和乳杆菌数量较治疗前显著升高,且双歧杆菌和乳杆菌数量显著高于对照组;而大肠埃希菌和肠球菌数量较治疗前显著降低,且大肠埃希菌和肠球菌数量显著低于对照组（均P＜0.05）。治疗后,两组患者MoCA和MMSE评分均升高,且观察组高于对照组（均P＜0.05）。观察组患者临床总有效率显著高于对照组（90.67% vs 78.67%,χ² = 12.482,P＜0.001）。对照组和观察组患者的不良反应发生情况比较差异无统计学意义（χ² = 0.150,P = 0.699）。

tDCS联合益生菌治疗能够有效改善PSCI患者的认知功能和肠道菌群状态,且不良反应少,安全系数高,值得临床推广。

     

益生菌联合布地奈德对支气管哮喘患儿的疗效及对外周血CD4+和CD8+细胞水平的影响

研究益生菌联合布地奈德对支气管哮喘患儿的疗效及对外周血CD4⁺和CD8⁺细胞水平的影响, 为该类患者的治疗提供参考。

选择2019年6月至2020年5月我院收治的92例支气管哮喘患儿为研究对象, 按照随机数字表法分为观察组(益生菌联合布地奈德治疗)和对照组(布地奈德治疗)各46例, 比较两组患儿疗效、临床症状消失时间、治疗前及治疗4周后T淋巴细胞(CD4⁺、CD8⁺)和肺功能[用力肺活量(FVC)、第1秒用力呼气容积(FEV1)、最大呼气峰流速(PEF)]水平。

治疗4周后, 两组患儿总有效率比较差异无统计学意义(93.48%vs 89.13%, χ²=0.137, P=0.711)。观察组患儿咳嗽消失时间、气促消失时间、哮鸣音消失时间、憋喘消失时间均显著低于对照组(均P < 0.05)。治疗4周后, 观察组患儿CD4⁺细胞水平显著低于治疗前及同期对照组, CD8⁺细胞、肺功能(FVC、FEV1、PEF)水平显著高于治疗前及同期对照组(均P < 0.05)。

益生菌联合布地奈德对支气管哮喘的疗效显著, 能够促进患儿临床症状恢复, 改善患儿肺功能, 下调外周血CD4⁺细胞水平, 上调CD8⁺细胞水平。

     

益生菌联合标准四联疗法对幽门螺杆菌感染再次治疗的疗效及其对炎症因子的影响

研究布拉酵母菌联合标准四联疗法对幽门螺杆菌(H.pylori)感染患者再次治疗的疗效及其对炎症因子的影响。

选取我院124例H.pylori感染患者, 按随机分配的方法分为A组和B组, 每组62例。A组患者给予阿莫西林胶囊1 000 mg/次, 2次/d; 呋喃唑酮100 mg/次, 2次/d; 雷贝拉唑40 mg/次, 2次/d; 胶体果胶铋干混悬剂150 mg/次, 4次/d, 疗程14 d。B组患者在A组治疗方案的基础上加用布拉酵母菌250 mg/次, 3次/d, 疗程14 d。在治疗过程中观察患者的不良反应发生情况。疗程结束完全停药4周后检测两组患者H.pylori根除及症状改善情况。对比治疗前后患者血清白介素-6(IL-6)、肿瘤坏死因子(TNF-α)、C-反应蛋白(CRP)水平。

观察组患者H.pylori的根除率、症状改善率和不良反应的发生率与对照组相比差异均有统计学意义(χ²=7.322、9.239、10.016, 均P < 0.05)。治疗后观察组患者血清IL-6、TNF-α、CRP水平较对照组降低(t=9.780、8.495、7.002, 均P < 0.05)。

布拉酵母菌联合标准四联疗法可提高H.pylori根除率, 改善患者症状, 降低不良反应发生率, 同时可降低血清炎症因子水平。

     

三种前处理在基质辅助激光解析电离飞行时间质谱鉴定假丝酵母菌属中的应用比较

目的 比较3种前处理方法在基质辅助激光解析电离飞行时间质谱(MALDI TOF MS)鉴定假丝酵母菌属中的结果可靠性。 方法 以ITS测序鉴定结果为金标准,对临床分离的66株假丝酵母分别采用传统直涂法、改良直涂法和甲酸-乙腈蛋白提取法进行前处理,MALDI TOF MS鉴定,比较3种方法的Biotyper Log值,分析质谱图的差异。 结果 传统直涂法、改良直涂法和甲酸-乙腈提取法对66株假丝酵母的属水平鉴定率分别为48.5%、50.0%和97.0%,Biotyper Log均值分别为1.628、1.674和2.010,其中甲酸-乙腈提取法对66株假丝酵母的种水平鉴定率为53.0%。甲酸-乙腈提取法得到的质谱图比另2种方法的质谱图离子峰更加密集,图像更复杂,鉴定结果可信度更高。 结论 甲酸-乙腈蛋白提取法对假丝酵母菌属的鉴定成功率和可靠性明显高于传统直涂法和改良直涂法,对临床假丝酵母菌病的准确诊断具有重要价值。     

Liquid chromatography-tandem mass spectrometric assays for salinomycin in mouse plasma, liver, brain and small intestinal contents and in OptiMEM cell culture medium

Fast and sensitive liquid chromatography-tandem mass spectrometric assays for the determination of salinomycin in mouse plasma, liver, brain and small intestinal contents and in OptiMEM cell culture medium, were developed and validated using simple sample pre-treatment procedures. Tissue samples were homogenized with phosphate buffered saline or, for high levels in liver, with human plasma. After addition of monensin as the internal standard to plasma, homogenate or culture medium and acetonitrile extraction for tissue and plasma, the diluted medium or the supernatant was directly injected into the isocratic chromatographic system using a polar embedded reversed-phase column and formic acid in water-acetonitrile as the eluent. The eluate was completely led into an electrospray interface with positive ionization and the analytes were quantified using triple quadrupole mass spectrometry. The assays were successfully validated in the ranges 10-2000 ng/ml for OptiMEM cell culture medium, 1-2000 ng/ml for plasma and 3-2000 ng/g in liver brain and small intestinal contents. At the lowest levels, the intra-day precisions were < or =9%, inter-day precisions were < or =14% and accuracies were between 91 and 112%. The analytes were chemically stable under all relevant conditions and the assays were applied in different in vitro transport studies and in pharmacokinetic and tissue distribution studies with salinomycin in mice.     

红色毛癣菌临床分离株的菌落形态和镜下结构特征再分析

目的探讨红色毛癣菌的菌落形态和镜下结构特征以及与感染部位的相关性。方法采用传统培养方法对192株红色毛癣菌进行表型分型,选取其中39株在28℃、30℃、35℃3种温度孵育6d、10d、14d时观察菌落形态和生长速度。结果192株红色毛癣菌共分离出3种表型：绒毛型、沟纹型、粉末型（或颗粒型）。在相同培养基上,28℃、30℃时菌落生长速度无显著差异（P〈0.05）,均快于35℃（P〈0.05）。在相同温度时,菌落在SDA上的生长速度快于PDA培养基。在28℃、30℃时菌落形态比较稳定,在35℃时变异较大。菌落在PDA培养基上产孢丰富,镜下显示有较多的大、小分生孢子,而在SDA培养基上只有少量的小分生孢子,几乎见不到大分生孢子。各种浅部感染均以绒毛型为主,绒毛型在手足癣中占比例最高,沟纹型在体股癣中所占比例较高,粉末型在甲癣中占的比例较高。结论红色毛癣菌的菌落形态与培养基和培养温度有关,其表型和镜下结构与感染部位均有一定的相关性。     

In vitro propagation and mass scale multiplication of a critically endangered epiphytic orchid, Gastrochilus calceolaris (Buch.-Ham ex J.E.Sm.) D.Don. using immature seeds

In vitro asymbiotic seed germination potential of its immature seeds (36 weeks after pollination) of G. calceolaris was successfully tested on three different agar gelled nutrient media i.e. Murashige and Skoog (MS), Mitra et al. (M) and potato dextrose agar (PDA). Seeds germinated within 15.75+/-0.75 to 35.75+/-0.75 days in the three different media. The protocorms developed therefrom subsequently differentiated into first leaf and root primordia, and complete seedlings were obtained within 111.25+/-1.25 to 141.25+/-1.25 days on MS and M media. The protocorms, though failed to differentiate further on basal PDA medium, despite repeated subculturings, incorporation of peptone (P; 1 gl(-1)), yeast extract (YE; 2 gl(-1)) and coconut water (CW; 20%) in the medium proved beneficial in inducing differentiation, in these germinating entities. Additional use of growth additives (P/YE/CW), in general, favoured better germination, protocorm formation and seedling development. The optimal nutritional combination during seed germination, protocorm growth and multiplication and seedling development was found to be CW (10%) enriched MS medium.     

MALDI-TOF-Based Dermatophyte Identification

MALDI-TOF MS has become increasingly popular for microorganism identification in the routine laboratory. Compared with conventional morphology-based techniques, MALDI-TOF is relatively inexpensive (per-unit identification), involves a rapid result turnaround time and yields more accurate results without the need for highly qualified staff. However, this technology has been technically difficult to implement for filamentous fungi identification. Identification of dermatophytes, a type of filamentous fungi, remains particularly challenging, partly due to the lack of clear species definition for some taxa or within some species complexes. Review of the ten studies published between 2008 and 2015 shows that the accuracy of MALDI-TOF MS-based identification varied between 13.5 and 100 % for dermatophytes. This variability was partly due to inconsistencies concerning critical steps of the routine clinical laboratory process. Use of both a complete formic acid-acetonitrile protein extraction step and a manufacturer library supplemented with homemade reference spectra is essential for an accurate species identification. This technique is conversely unaffected by variations in other routine clinical laboratory conditions such as culture medium type, incubation time and type of mass spectrometry instrument. Provided that a reference spectra library is adequate for dermatophyte identification, MALDI-TOF MS identification is more economical and offers an accuracy comparable to that of DNA sequencing. The technique also represents an advantageous alternative to the protracted and labor-intensive dermatophyte identification via macroscopic and microscopic morphology in the routine clinical laboratory.     

Quantification of sunitinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sunitinib in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.2mL of plasma with 4.0mL tert-butyl-methyl-ether extraction solution containing 25ng/mL of the internal standard clozapine. Separation of compounds was achieved on a C18 (50mmx2.1mm i.d., 3.5microm) analytical column using a mobile phase consisting of acetonitrile/H20 (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.150mL/min for 3min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves in human plasma were generated over the range of 0.2-500ng/mL with values for the coefficient of determination of >0.9950. Within- and between day precision and accuracy were < or =10%. The method was applied to the quantitation of sunitinib in plasma samples from a patient receiving daily oral therapy with sunitinib.     

海水真菌定量分析方法的初步研究

为定量分析海水真菌,分别采用不同的采样方法采样;并采用5种培养基,每种培养基分别用4种不同的处理方式,共20种不同处理组合,筛选出适合定量分析海水中真菌的平板计数方案。结果表明,不同的采样方法下,真菌在同一培养基上的生长无显著性差异。在同一海水水样中,马铃薯培养基和马铃薯葡萄糖琼脂培养基用海水配制时几乎没有真菌生长;麦芽汁培养基在不同的处理条件下真菌略有生长,且各种处理间无明显差别,其种类主要是霉菌;察氏培养基和酵母菌浸出粉胨葡萄糖琼脂培养基在不同处理条件下真菌长势差别较大,种类主要是酵母菌。通过对20种培养组合的比较,选择酵母浸出粉胨葡萄糖琼脂培养基,用海水来配制培养基并用海水稀释样品的处理,可以培养出最大数量的海洋真菌。     

Hydroxamate siderophores of the ectomycorrhizal fungi <Emphasis Type="Italic">Suillus granulatus</Emphasis> and <Emphasis Type="Italic">S. luteus</Emphasis>

Despite indications that S. granulatus and S. luteus release iron-chelating compounds, the exact spectrum of ferric hydroxamates synthesized by these two Suillus species remained unclear. Hence the aim of this study was to identify all of the main siderophores produced by these two ectomycorrhizal fungal species under pure culture conditions. By means of HPLC and LC–MS analyses we show that S. granulatus releases cyclic and linear fusigen, ferrichrome, coprogen and triacetylfusarinine C into the nutrient medium, while S. luteus culture filtrates contain cyclic and linear fusigen, ferricrocin and coprogen. All of the different siderophores were identified on basis of reference compounds and their specific MS spectra which were recorded on a high resolution MS in positive electrospray ionisation mode. Initial HPLC separations were performed on a C-18 stationary phase, using an acidic eluent (0.1% formic acid in water and acetonitrile) in gradient mode. The potential of these two ectomycorrhizal fungal species to produce siderophores representing three different groups of hydroxamates is discussed in relation to its ecological significance.     

Culture of in vitro-produced bovine embryos with vitamin E improves development in vitro and after transfer to recipients

Detrimental effects of oxygen-derived free radicals on embryos during culture have been demonstrated in several species. Vitamin E occurs naturally in cell membranes and protects cells from oxidative stress. Under some conditions, vitamin C acts synergistically to enhance the antioxidant effects of vitamin E, a benefit that may be further enhanced by EDTA. The present experiments concerned culture of bovine embryos derived from in vitro-matured, fertilized oocytes with vitamin E, vitamin C, and EDTA in a chemically defined culture medium + 0.2% BSA at 5% O(2), 5% CO(2), and 90% N(2). In the first experiment, more zygotes developed to expanded blastocysts (17%, n = 224, P < 0.05) when culture medium contained 100 microM vitamin E than in control medium (11%, n = 234). Development to early, expanded, and hatched blastocysts was lower with vitamins E and C combined than with vitamin E alone (15%, 9%, and 2% vs. 24%, 17%, and 5%, respectively; P < 0.05), as was the mean number of cells per blastocyst (56 vs. 84, P < 0.05). Addition of EDTA (3 microM) failed to improve development over that in culture with vitamin E + vitamin C. In experiment 2, in vitro-produced embryos cultured 5.5 days in medium with or without 100 microM vitamin E were transferred nonsurgically to recipient cows and heifers and then collected nonsurgically 7 days later. Embryos cultured with vitamin E (n = 37) were approximately 63% larger in surface area than controls (1.16 mm(2) vs. 0.71 mm(2) surface area; n = 27, P < 0.04).     

Analysis of omeprazole and 5-OH omeprazole in human plasma using hydrophilic interaction chromatography with tandem mass spectrometry (HILIC-MS/MS)--eliminating evaporation and reconstitution steps in 96-well liquid/liquid extraction

Bioanalytical methods using liquid/liquid extraction (LLE) and liquid chromatography with electrospray tandem mass spectrometry (LC-MS/MS) are widely used. The organic extracts need to be evaporated and reconstituted, hampering further improvement of throughput and automation. In this study, we demonstrated a novel approach of eliminating these two steps in 96-well LLE by using hydrophilic interaction chromatography with MS/MS (HILIC-MS/MS) on silica column with high organic/low aqueous mobile phase. Omeprazole, its metabolite 5-OH omeprazole, and internal standard desoxyomeprazole, were extracted from 0.05 ml of human plasma using 0.5 ml of ethyl acetate in a 96-well plate. A portion (0.1 ml) of the ethyl acetate extract was diluted with 0.4 ml of acetonitrile and 10 microl was injected onto a Betasil silica column (50 mm x 3.0 mm, 5 microm) and detected by API 3000 and 4000 with (+) ESI. Mobile phase with linear gradient elution consists of acetonitrile, water, and formic acid (from 95:5:0.1 to 73.5:26.5:0.1 in 2 min). The flow rate was 1.5 ml/min with total run time of 2.75 min. The method was validated for a low limit of quantitation at 2.5 ng/ml for both analytes. The method was also validated for specificity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <4.4% relative standard deviation (R.S.D.) and 4.1% relative error (R.E.) for omeprazole, and 4.5% R.S.D. and 5.6% R.E. for 5-OH omeprazole, respectively.