Disrupting the methionine biosynthetic pathway in Candida guilliermondii: characterization of the MET2 gene as counter‐selectable marker

Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the fungal CTG clade. This yeast remains actively studied as a result of its moderate clinical importance and most of all for its potential uses in biotechnology. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both a methionine auxotroph recipient strain and a functional MET gene as selection marker. We first disrupted the MET2 and MET15 genes encoding homoserine‐O‐acetyltransferase and O‐acetylserine O‐acetylhomoserine sulphydrylase, respectively. The met2 mutant was shown to be a methionine auxotroph in contrast to met15 which was not. Interestingly, met2 and met15 mutants formed brown colonies when cultured on lead‐containing medium, contrary to the wild‐type strain, which develop as white colonies on this medium. The MET2 wild‐type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene‐expressing vector into the met2 recipient strain. In addition, we showed that the loss of the MET2‐containing YFP‐expressing plasmid can be easily observed on lead‐containing medium. The MET2 wild‐type allele, flanked by two short repeated sequences, was then used to disrupt the LYS2 gene (encoding the α‐aminoadipate reductase) in the C. guilliermondii met2 recipient strain. The resulting lys2 mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop‐out the MET2 marker (following the recombination of the bordering repeat sequences) from a target lys2 locus were unsuccessful using white/brown colony colour screening. Nevertheless, this MET2 transformation/disruption system represents a new versatile genetic tool for C. guilliermondii. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of starvation on the performance and re-acclimation of biotrickling filters for air pollution control

Biotrickling filters for air pollution control are expected to encounter fluctuating conditions or periods without pollutant supply. In the present study, we investigated the effect of pollutant starvation in bench-scale biotrickling filters treating toluene. The experimental protocol consisted of starving biotrickling filters under various conditions: with or without airflow, with or without liquid recycle, and with or without an alternate carbon source (glucose) supply. The duration of the period without toluene was varied from 2 to 9 days, during time which the biotrickling filters were monitored for biomass content, endogenous and toluene-induced oxygen uptake rates during starvation, and toluene overall elimination capacity after restart. During starvation, all reactors lost their ability to degrade toluene within 5 days, regardless of the mode of starvation. The biomass content significantly decreased during starvation, in particular in those reactors where the recycle liquid was maintained, but this decrease was not critical for future re-acclimation. Glucose addition to starved biotrickling filters had several detrimental effects. It resulted in a faster decrease of the biomass content and slowed the reacclimation phase. Overall, the results show that the reacclimation of toluene-degrading biotrickling filters after periods of nonuse is short (10-24 h to re-establish full performance), and they suggest that, in the case of toluene-degrading biotrickling filters, re-acclimation time is largely governed by the induction of key pollutant-degrading enzymes.     

糖尿病大鼠骨髓来源内皮祖细胞体外生物学活性研究

【摘要】 目的 探讨糖尿病大鼠骨髓来源内皮祖细胞与正常大鼠在数量及生物学活性方面的差异。方法 雄性SD大鼠按60 mg/kg腹腔快速注射1％ STZ的枸椽酸缓冲液建立糖尿病动物模型。密度梯度法分离糖尿病大鼠和正常大鼠骨髓单个核细胞,用EGMTM- 2MV BulletKitTM培养基进行诱导培养,观察细胞形态学特征,流式细胞仪检测细胞表面抗原,摄取Dil- Ac- LDL 与结合 FITC- UEA- 1 双荧光鉴定细胞及检测细胞比例,CCK- 8、Transwell小盒等检测细胞增殖、黏附及迁移能力。结果 内皮祖细胞在体外培养时呈梭形,贴壁生长,均可表达CD34、CD133和VEGFR- 2,且能摄取Dil- Ac- LDL并结合FITC- UEA- 1,证明所培养细胞为内皮祖细胞。糖尿病大鼠骨髓来源的内皮祖细胞数目与正常大鼠相比无明显差异,但其黏附能力明显低于正常大鼠,增殖能力、黏附及迁移能力均降低。结论 与正常大鼠相比,糖尿病大鼠骨髓来源内皮祖细胞数量无明显差异,但生物学活性降低,主要表现在增殖能力、黏附及迁移能力均减弱。     

经皮椎体成形术临床应用—— — 附２９５例５５３节椎体报告

目的评价经皮椎体成形术（PVP）临床疗效、适应证及相关技术。方法对２９５例患者５５３个椎体在X线C臂血管机导引下行PVP,包括骨质疏松症１７１例,转移性肿瘤１０３例,骨髓瘤１２例,血管瘤２例,外伤７例。一般采用单侧椎弓根穿刺,骨水泥量为２～７ml 。结果骨质疏松症患者总有效率为９４．１％（１６１／１７１）,转移性肿瘤总有效率为８１．５％,骨髓瘤患者有效为８例（８／９）,外伤患者有效为７例（７／７）。本组患者无严重并发症;１８例转移瘤PVP后３～４d行放射治疗;１２例采用大平板血管机旋转X线采集和椎体三维重建成像指导手术操作。结论PVP已成为疼痛性压缩性骨折标准治疗方法之一,伴随手术方法和器材不断改进其适应证必将不断扩大。     

碘油平阳霉素乳剂联合利多卡因浸泡明胶海绵栓塞治疗子宫肌瘤的临床观察

目的探讨平阳霉素、利多卡因、碘化油乳剂及经利多卡因浸泡的明胶海绵颗粒栓塞子宫动脉治疗子宫肌瘤的可行性和疗效。方法对６８例子宫肌瘤患者应用平阳霉素、利多卡因、碘油乳剂及经利多卡因浸泡的明胶海绵颗粒栓塞治疗,观察术中、术后的疼痛反应、临床症状和肌瘤大小的变化、子宫体积变化及标本病理学表现。结果术中、术后患者疼痛反应较轻,需肌注镇痛剂控制难以忍受疼痛者１３例,占１９．１％;症状缓解率为９２．３％,其中８９．５％月经异常得以恢复,盆腔疼痛与腹胀及压迫症状消失达９０．８％。３～６个月随防肌瘤平均缩小７８．９％,子宫体积平均缩小４７．５％。结论平阳霉素、利多卡因、碘油乳剂及经利多卡因浸泡的明胶海绵颗粒是治疗子宫肌瘤较为理想的栓塞材料     

Emulsifying properties of β-lactoglobulin and Quillaja bark saponin mixtures: Effects of number of homogenization passes,pH, and NaCl concentration

The effects of number of homogenization passes, pH, and NaCl concentration on the formation and stability of oil-in-water emulsions comprising a mixture of a biosurfactant (Quillaja bark saponin) and a globular protein (β-lactoglobulin) were investigated. The emulsions were characterized as to visual appearance, droplet size, droplet surface charge, and rheology. The emulsions obtained by different conditions (4, 6, or 8 passes; pH 7, 8, or 9; and 0, 100, or 200 mmol L^?1 of NaCl) were polydisperse, presented relatively small average droplet sizes (z-average < 323 nm) as well as negative droplet charge (between –20 and –79.6 mV) in all evaluated conditions. Regardless of the number of homogenization passes, the emulsions exhibited low apparent viscosity and pseudoplastic behavior with small yield stress. Viscoelastic behavior was also observed, thus the emulsions were characterized as weak gels. Four homogenization passes were enough to obtain small droplets in the evaluated conditions. Droplet size was not significantly affected by NaCl concentration and pH (p > 0.05). On the other hand, the absolute ζ-potential values significantly decreased and increased upon increased NaCl content and pH, respectively. Regardless of the tested conditions, all emulsions had good stability against phase separation and droplet aggregation, since no significant changes in average droplet size were observed throughout storage (p > 0.05). In the presence of NaCl, in which droplet charge significantly decreased, emulsion was also stable. Thus, we can conclude that electrostatic repulsion as well as steric repulsion was responsible for stabilization.     

Antioxidant activity, browning and volatile Maillard compounds in Pedro Ximénez sweet wines under accelerated oxidative aging

Pedro Ximénez wine, aged by thermal treatment during 10, 20 and 30 days in the presence or absence of oak chips, was studied in terms of antioxidant activity, browning and volatile Maillard compounds. The phenolic fractions obtained by the SepPack tC-18 column revealed that the antioxidant activity after 10 days of thermal treatment was due to the presence of phenols and polar compounds possibly formed by the Maillard reaction. After 30 days, however, the antioxidant activity was also due to high-molecular weight procyanidins. Volatile Maillard compounds increased throughout the thermal treatment. It should be noted that there was an increase in the 5-ethoxymethylfurfural content and the presence of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-2-one, which was recently shown to inhibit growth of colon tumor cells. The presence of oak chips had a substantial effect on the contents in volatile Maillard compounds after the thermal treatment with the exception of corylon. The wine obtained after 10 days of thermal treatment was judged to be the best in terms of sensory properties. In fact, it exhibited the optimum balance between varietal and aging aromas due to Maillard compounds.     

Sucrose hydrolysis by gelatin-immobilized inulinase from Kluyveromyces marxianus var. bulgaricus

The crude cell-free medium from a culture of Kluyveromyces marxianus var. bulgaricus was immobilized in a gelatin-water support, with an immobilization yield of 82.60% for inulinase activity. The optimum pH for both free and immobilized inulinase was the same (3.5) and the optimum temperatures were 55 °C for the free and 60 °C for the immobilized enzyme. The Arrhenius plots were linear and activation energies were 56.20 (free enzyme) and 20.27 kJ/mol K (immobilized enzyme). The kinetic parameters were calculated by Lineweaver–Burk plots and the V_max and K_m were 37.60 IU/mg protein and 61.83 mM for the free inulinase and 31.45 IU/mg protein and 149.28 mM for the immobilized enzyme, respectively. The operational stability of the immobilized inulinase was studied in a continuous fixed-bed column reactor for 33 days, at the end of which the sucrose conversion was 58.12%.     

TBP,PPIA, YWHAZ and EF1A1 Are the Most Stably Expressed Genes during Osteogenic Differentiation

RT-qPCR is the gold standard and the most commonly used method for measuring gene expression. Selection of appropriate reference gene(s) for normalization is a crucial part of RT-qPCR experimental design, which allows accurate quantification and reliability of the results. Because there is no universal reference gene and even commonly used housekeeping genes’ expression can vary under certain conditions, careful selection of an appropriate internal control must be performed for each cell type or tissue and experimental design. The aim of this study was to identify the most stable reference genes during osteogenic differentiation of the human osteosarcoma cell lines MG-63, HOS, and SaOS-2 using the geNorm, NormFinder, and BestKeeper statistical algorithms. Our results show that TBP, PPIA, YWHAZ, and EF1A1 are the most stably expressed genes, while ACTB, and 18S rRNA expressions are most variable. These data provide a basis for future RT-qPCR normalizations when studying gene expression during osteogenic differentiation, for example, in studies of osteoporosis and other bone diseases.