循环水养殖系统中凡纳滨对虾肠道微生物对三种复合益生菌制剂的响应

【目的】在循环养殖系统中应用不同的复合益生菌制剂,探讨凡纳滨对虾肠道菌群结构特征及免疫水平发生的变化。【方法】30 d养殖周期结束后,通过平板计数法分析肠道细菌总数和弧菌总数;基于高通量测序技术分析肠道样品V3+V4区菌群特征;采用实时荧光定量PCR技术分析免疫相关因子TLR1和Dorsal基因表达水平,阐述益生菌制剂应用的意义。【结果】益生菌制剂的应用降低了凡纳滨对虾肠道中细菌总数,抑制弧菌的生长,间接预防疾病的发生。不同益生菌制剂从不同程度上优化了凡纳滨对虾肠道菌群结构,提高高质量序列和有效OTU数量,Chao1、Simpson、Shannon指数显示了丰富度和多样性变化,复合益生菌制剂3效果较好。同时,菌群结构得到优化,其中益生菌制剂1组对拟杆菌门含量百分比产生显著影响。Toll受体TLR1和Toll通路中的Dorsal基因m RNA表达受到益生菌制剂的影响,1、3组促进了TLR 1表达,1、2、3组都促进了Dorsal基因表达。【结论】在循环水养殖系统中添加益生菌制剂可优化凡纳滨对虾肠道菌群特征,提高免疫水平,为病害防控和健康养殖提供理论依据。     

集胞藻PCC 6803中丝氨酸/苏氨酸激酶SpkC对高温胁迫的响应

丝氨酸/苏氨酸激酶是蓝藻感知和转导外界刺激的重要元件,但至今蓝藻中很多丝氨酸/苏氨酸激酶的功能尚属未知。【目的】研究集胞藻PCC6803中的丝氨酸/苏氨酸激酶Spk C是否参与对高温胁迫的响应。【方法】本研究采用同源重组的方法构建spC基因完全敲除突变株,检测突变株与野生株在高温胁迫下的生长状况、色素组成,并对高温胁迫下叶绿素荧光参数差异进行分析,比较光合系统Ⅱ活性差异。此外,通过测定生长速率来判断高温胁迫后藻株的恢复情况。【结果】经过42℃高温胁迫后,与野生株相比,突变株ΔspkC生长减缓,光合色素(叶绿素、类胡萝卜素和藻胆色素)的含量降低;45℃高温胁迫下突变株ΔspkC的光合系统Ⅱ活性下降幅度更大;经过5 d 42℃高温处理后,突变株生长几乎停滞,存活率较野生株明显降低。【结论】集胞藻PCC 6803中spkC基因的缺失导致突变株对高温胁迫响应出现缺陷,提示丝氨酸/苏氨酸激酶SpkC参与响应高温胁迫。     

Analysis of the transformation effect in cytochrome b559 of photosystem II in terms of the model of the heme-quinone redox interaction

Transformation of three-component redox pattern of cytochrome (Cyt) b559 in PS II membrane fragments upon various treatments is manifested in decrease of the relative content (R) of the high potential (HP) redox form of Cyt b559 and concomitant increase in the fractions of the two lower potential forms. Redox titration of Cyt b559 in different types of PS II membrane preparations was performed and revealed that (1) alteration of redox titration curve of Cyt b559 upon treatment of a sample is not specific to the type of treatment; (2) each value of R_HP defines the individual shape of the redox titration curve; (3) population of Cyt b559 may exist in several stable forms with multicomponent redox pattern: three types of three-component redox pattern and one type of two-component redox pattern as well as in the form with a single E_m; (4) transformation of Cyt b559 proceeds as successive conversion between the stable forms with multicomponent redox pattern; (5) upon harsh treatments, Cyt b559 abruptly converts into the state with a single E_m which value is intermediate between the E_m values of the two lower potential forms. Analysis of the data using the model of Cyt b559-quinone redox interaction revealed that diminution of R_HP in a range from 80 to 10% reflects a shift in redox equilibrium between the heme group of Cyt b559 and the interacting quinone, due to a gradual decrease of 90?mV in E_m of the heme group at the virtually unchanged E_m of the quinone component.     

Probing the role of Valine 185 of the D1 protein in the Photosystem II oxygen evolution

In Photosystem II (PSII), the Mn₄CaO₅-cluster of the active site advances through five sequential oxidation states (S₀ to S₄) before water is oxidized and O₂ is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824–6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV–visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking Tyr_Z and the halide. The V185 contributes to the stabilization of S₂ into the low spin (LS), S?=?1/2, configuration. Indeed, in the V185T mutant a high proportion of S₂ exhibits a high spin (HS), S?=?5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S₁Tyr_Z?→?S₂^HSTyr_Z transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S₂^LSTyr_Z?→?S₃Tyr_Z transition was observed and we propose that it occurs in the S₂^LSTyr_Z?→?S₂^HSTyr_Z intermediate step before the S₂^HSTyr_Z?→?S₃Tyr_Z transition occurs. The dramatic slowdown of the S₃Tyr_Z?→?S₀Tyr_Z transition in the V185T mutant does not originate from a structural modification of the Mn₄CaO₅ cluster since the spin S?=?3?S₃ EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein.     

Botulinum hemagglutinin‐mediated in situ break‐up of human induced pluripotent stem cell aggregates for high‐density suspension culture

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high‐density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break‐up using botulinum hemagglutinin (HA), which specifically bound with E‐cadherin and disrupted cell–cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E‐cadherin‐mediated cell–cell connections to facilitate the break‐up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 10⁶ cells ml^?1 was obtained by aggregate break‐up into small ones, which was three times higher than that with the conventional culture without aggregate break‐up. Therefore, the temporary activity of HA for disrupting E‐cadherin‐mediated cell–cell connection was key to establishing a simple in situ method for hiPSC aggregate break‐up in bioreactors, leading to high cell density in suspension culture.