Genetically switched d-lactate production in Escherichia coli

During a fermentation process, the formation of the desired product during the cell growth phase competes with the biomass for substrates or inhibits cell growth directly, which results in a decrease in production efficiency. A genetic switch is required to precisely separate growth from production and to simplify the fermentation process. The ldhA promoter, which encodes the fermentative d-lactate dehydrogenase (LDH) in the lactate producer Escherichia coli CICIM B0013-070 (ack-pta pps pflB dld poxB adhE frdA), was replaced with the λ p(R) and p(L) promoters (as a genetic switch) using genomic recombination and the thermo-controllable strain B0013-070B (B0013-070, ldhAp::kan-cI(ts)857-p(R)-p(L)), which could produce two-fold higher LDH activity at 42°C than the B0013-070 strain, was created. When the genetic switch was turned off at 33°C, strain B0013-070B produced 10% more biomass aerobically than strain B0013-070 and produced only trace levels of lactate which could reduce the growth inhibition caused by oxygen insufficiency in large scale fermentation. However, 42°C is the most efficient temperature for switching on lactate production. The volumetric productivity of B0013-070B improved by 9% compared to that of strain B0013-070 when it was grown aerobically at 33°C with a short thermo-induction at 42°C and then switched to the production phase at 42°C. In a bioreactor experiment using scaled-up conditions that were optimized in a shake flask experiment, strain B0013-070B produced 122.8g/l d-lactate with an increased oxygen-limited productivity of 0.89g/g·h. The results revealed the effectiveness of using a genetic switch to regulate cell growth and the production of a metabolic compound.     

Spatial heterogeneity of spring phytoplankton in a large tropical reservoir: could mass effect homogenize the heterogeneity by species sorting?

Dredging Austrian Danube harbours becomes necessary to keep the navigation channel free from bed sediment and to insure safe navigation, especially after major floods. In most cases, the excavated substrate is deposited in the free-flowing river section in the vicinity of the mouth of a harbour. The effects of harbour dredging on the biota need to be studied to minimize possible outcomes that may endanger the ecological status of the river system in order to implement the aims of the European Water Framework Directive. This study presents the first quantitative information on the colonisation of benthic invertebrates with respect to Danube harbours. One sampling at Linz Harbour was conducted before dredging and four samplings were carried out after dredging. Four transects were documented in the affected river section. The study used an air-lift sampler and a grab sampler to investigate if and to what extent the benthic invertebrate community is disturbed. It found that the sediment dredging heavily affected the benthic invertebrates (decline of 82% of biomass), while the dumping of the material had no effects on the benthic Danube biota. As a rough estimate (conferring to a simple trend line analysis) the harbour bottom fauna would recover after 235 days.     

岷江源区两种优势针叶树当年生小枝性状与生物量分配随海拔的分异规律

当年生小枝具有较少的次生组织,同时是植物分支系统中最具活力的部分,木本植物当年生小枝性状与生物量分配的海拔变化是理解物种对不同生境适应策略的重要内容。通过分析青藏高原东缘岷江源区两种优势亚高山针叶乔木(紫果云杉和岷江冷杉)当年生小枝性状(茎长、茎粗、比茎长)与不同部位器官(茎、叶)的生物量随天然生境的海拔(3500-3550 m、3650-3700 m和3800-3850 m)变化,尝试揭示两物种当年生小枝在不同海拔下的生物量权衡及其生长策略。结果表明:(1)不同海拔下茎生物量种间差异大于种内差异,3650-3700 m处的茎生物量变异最大(128.4%)。(2)云杉的茎生物量、总叶生物量与海拔间存在显著的负相关关系(P<0.05),比茎长与海拔呈显著正相关(P<0.05);冷杉的总叶生物量与海拔间为显著负相关关系(P<0.05)。(3)随着海拔的升高,云杉茎生物量分配比逐渐从33.0%降低到27.0%;而冷杉的茎生物量分配比则从23.0%渐增至28.0%。(4)3500-3550 m和3650-3700 m两处的云杉茎生物量与总叶生物量、茎长与茎粗呈异速生长关系;3500-3550 m、3650-3700 m和3800-3850 m三处的冷杉茎生物量与总叶生物量、茎长与茎粗一直呈现异速生长关系。两种针叶树的茎生物量分配比及相关性状随海拔的变化差异表明冷杉更适应高海拔的胁迫环境。     

The Effect of Sign Language Structure on Complex Word Reading in Chinese Deaf Adolescents

The present study was carried out to investigate whether sign language structure plays a role in the processing of complex words (i.e., derivational and compound words), in particular, the delay of complex word reading in deaf adolescents. Chinese deaf adolescents were found to respond faster to derivational words than to compound words for one-sign-structure words, but showed comparable performance for two-sign-structure words. For both derivational and compound words, response latencies to one-sign-structure words were shorter than to two-sign-structure words. These results provide strong evidence that the structure of sign language affects written word processing in Chinese. Additionally, differences between derivational and compound words in the one-sign-structure condition indicate that Chinese deaf adolescents acquire print morphological awareness. The results also showed that delayed word reading was found in derivational words with two signs (DW-2), compound words with one sign (CW-1), and compound words with two signs (CW-2), but not in derivational words with one sign (DW-1), with the delay being maximum in DW-2, medium in CW-2, and minimum in CW-1, suggesting that the structure of sign language has an impact on the delayed processing of Chinese written words in deaf adolescents. These results provide insight into the mechanisms about how sign language structure affects written word processing and its delayed processing relative to their hearing peers of the same age.     

Liver Stiffness by Transient Elastography Predicts Liver-Related Complications and Mortality in Patients with Chronic Liver Disease

Background

Liver stiffness measurement (LSM) by transient elastography (TE, FibroScan) is a validated method for noninvasively staging liver fibrosis. Most hepatic complications occur in patients with advanced fibrosis. Our objective was to determine the ability of LSM by TE to predict hepatic complications and mortality in a large cohort of patients with chronic liver disease.

Methods

In consecutive adults who underwent LSM by TE between July 2008 and June 2011, we used Cox regression to determine the independent association between liver stiffness and death or hepatic complications (decompensation, hepatocellular carcinoma, and liver transplantation). The performance of LSM to predict complications was determined using the c-statistic.

Results

Among 2,052 patients (median age 51 years, 65% with hepatitis B or C), 87 patients (4.2%) died or developed a hepatic complication during a median follow-up period of 15.6 months (interquartile range, 11.0–23.5 months). Patients with complications had higher median liver stiffness than those without complications (13.5 vs. 6.0 kPa; P<0.00005). The 2-year incidence rates of death or hepatic complications were 2.6%, 9%, 19%, and 34% in patients with liver stiffness <10, 10–19.9, 20–39.9, and ≥40 kPa, respectively (P<0.00005). After adjustment for potential confounders, liver stiffness by TE was an independent predictor of complications (hazard ratio [HR] 1.05 per kPa; 95% confidence interval [CI] 1.03–1.06). The c-statistic of liver-stiffness for predicting complications was 0.80 (95% CI 0.75–0.85). A liver stiffness below 20 kPa effectively excluded complications (specificity 93%, negative predictive value 97%); however, the positive predictive value of higher results was sub-optimal (20%).

Conclusions

Liver stiffness by TE accurately predicts the risk of death or hepatic complications in patients with chronic liver disease. TE may facilitate the estimation of prognosis and guide management of these patients.     

阿帕替尼与白蛋白结合型紫杉醇在MDA-MB-231乳腺癌细胞系中的协同抗癌作用

目的阐明阿帕替尼 (apatinib)和白蛋白结合型紫杉醇 (nab-Paclitaxel)诱导MDA-MB-231乳腺癌细胞凋亡的分子机制。方法本研究以MDA-MB-231乳腺癌细胞为研究对象,并以apatinib和nab-Paclitaxel处理细胞后分组:0.1 %DMSO处理为阴性对照组;10 μmol/L apatinib处理组 (APA组);经5、10、15、20 nmol/L nab-Paclitaxel 处理组 (Nab-p 5组、Nab-p 10组、Nab-p 15组和Nab-p 20组);以及10 μmol/L apatinib分别与5、10、15、20 nmol/L nab-Paclitaxel 联合处理组 (APA+Nab-p 5组、APA+Nab-p 10组、APA+Nab-p 15组和APA+Nab-p 20组)。使用乳酸脱氢酶释放测定法测定apatinib和nab-Paclitaxel对MDA-MB-231细胞诱导的细胞毒活性,结合流式细胞术分析不同处理组细胞凋亡情况,通过JC-1染色法测定不同干预方式对MDA-MB-231细胞线粒体膜电位变化 (ΔΨ_m)的影响,借助FAM-FLICA荧光成像检测caspase-8和caspase-9 活性,通过彗星试验评估apatinib和nab-Paclitaxel对非致瘤上皮细胞株MCF-10A细胞DNA损伤的影响。两组之间独立样本t检验或单向ANOVA进行比较,多组之间使用Tukey事后检验。结果细胞毒性检测结果显示,与阴性对照组相比,Nab-p 20组MDA-MB-231细胞杀伤率在24 h时接近90 %;与单药处理组 (Nab-p 5组和Nab-p 10组)相比,APA+Nab-p 5组和APA+Nab-p 10组联合处理24 h和48 h后,分别检测到约85 %和95 %细胞死亡,差异均具有统计学意义 (P 均 < 0.001)。流式细胞术统计结果显示,与Nab-p5组和Nab-p10组相比,APA+Nab-p 5组和APA+Nab-p 10组24 h时MDA-MB-231细胞凋亡率(31.8 %±1.48 %、33.25 %±1.77 %比76.11 %±1.14 %、89.4 %±1.07%)升高 (P 均 < 0.05)。线粒体膜电位检测结果表明,与对照组相比,在单药处理组中,仅APA组和Nab-p 10组24 h时去极化细胞 (12.35 %±1.05%比78.33%±1.11%、46.74%±1.75%)增多;在联用处理组中,APA+Nab-p 5组和APA+Nab-p 10组24 h时去极化细胞 (68.47%±1.94%比90.03%±1.79%)增多,差异均有统计学意义 (P 均 < 0.05)。FAM-FLICA荧光成像结果显示,相较于单一处理组,apatinib和nab-Paclitaxel联用处理组的caspase-8和caspase-9蛋白高度活化。结合彗星试验分析,apatinib和nab-Paclitaxel 干预对MCF-10A非致瘤上皮细胞株DNA完整性没有显著影响。结论 apatinib/nab-Paclitaxel联用通过内源性的线粒体功能扰动和外源性的caspase激活诱导三阴性乳腺癌细胞MDA-MB-231凋亡,发挥协同抗癌作用。     

Effect of Different Culture Systems and 3, 5, 3'-Triiodothyronine/Follicle-Stimulating Hormone on Preantral Follicle Development in Mice

The mechanical method to isolate preantral follicle has been reported for many years. However, the culture systems in vitro are still unstable. The aim of this study was to analyze the effect of the culture system of mice preantral follicles on the follicular development in vitro. The results showed that the 96-well plate system was the most effective method for mice follicle development in vitro (volume change: 51.71%; survival rate: 89%, at day 4). Follicle-stimulating hormone (FSH) and Thyroid hormone (TH) are important for normal follicular development and dysregulation of hormones are related with impaired follicular development. To determine the effect of hormone on preantral follicular development, we cultured follicle with hormones in the 96-well plate culture system and found that FSH significantly increased preantral follicular growth on day 4. The FSH-induced growth action was markedly enhanced by T₃ although T₃ was ineffective alone. We also demonstrated by QRT-PCR that T₃ significantly enhanced FSH-induced up-regulation of Xiap mRNA level. Meanwhile, Bad, cell death inducer, was markedly down-regulated by the combination of hormones. Moreover, QRT-PCR results were also consistent with protein regulation which detected by Western Blotting analysis. Taken together, the findings of the present study demonstrate that 96-well plate system is an effective method for preantral follicle development in vitro. Moreover, these results provide insights on the role of thyroid hormone in increasing FSH-induced preantral follicular development, which mediated by up-regulating Xiap and down-regulating Bad.     

HER2 as a Promising Target for Cytotoxicity T Cells in Human Melanoma Therapy

Anti-HER2/neu antibody therapy has been reported to mediate tumor regression of HER2/ neu⁺ tumors. Here we demonstrated the expression of HER2 in a wide range of human melanoma cells including a primary culture and seven cell lines, and we further investigated whether HER2 could be served as a target for T cell mediated immunotherapy of human melanoma. Specific cytolytic activity of activated T cells (ATC) armed with anti-CD3 x anti-HER2 bispecific antibody (HER2Bi-Ab) against Malme-3M-luc cells was evaluated by bioluminescent signal generated by luciferase reporter which did not alter HER2 expression or proliferation ability of Malme-3M cells. Contrast with unarmed ATC, increased cytotoxic activity of HER2Bi-armed ATC against Malme-3M-luc cells was observed at effector/target (E/T) ratios of 1:1, 5:1, and 20:1. Moreover, HER2Bi-armed ATC expressed higher level of activation marker CD69 and secreted significantly higher level of IFN-γ than unarmed ATC counterpart at the E/T ratio of 20:1. In addition, compared with anti-HER2 mAb (Herceptin®) or unarmed ATC, HER2Bi-armed ATC showed remarkable suppression effect on Malme-3M-luc tumor cells. Furthermore, in melanoma tumor cell xenograft mice, infusion of HER2Bi-armed ATC successfully inhibited the growth of melanoma tumors. The anti-tumor effect of HER2Bi-armed ATC may provide a promising immunotherapy for melanoma in the future.