Persister cells in a biofilm treated with a biocide

This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC = 0.55 ± 0.20 mM for planktonic cells; MBC = 1.7 ± 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA, and the number of viable and culturable cells increased over time. The MBC of the recovered biofilm cells after a second exposure to OPA was 0.58 ± 0.40 mM, a concentration similar to the MBC of planktonic cells. This study demonstrates that persister cells may survive in biocide-treated biofilms, even in the absence of EPS.     

Bovine Serum Albumin protofibril-like aggregates formation: solo but not simple mechanism

We report an experimental study on the model protein Bovine Serum Albumin (BSA), with the aim of elucidating the mechanisms by which a fully folded globular protein undergoes different aggregation pathways leading to the formation of amyloid fibrils or amorphous aggregates. We observe thermally induced formation of fibrillar structures at pH far from the protein isoelectric point. The increase of electrostatic repulsion results in protein destabilization and in modifications of inter and intra-molecular interactions leading to the growth of fibril-like aggregates stabilized by inter-molecular-β sheets. The aggregation kinetics is studied by means of fluorescence techniques, light scattering, Circular Dichroism (CD), infrared spectroscopy (FTIR) and Atomic Force Microscopy (AFM). Changes in protein secondary structures turn out to be the driving mechanism of the observed aggregation and they progress in parallel with the growth of Thioflavin T emission intensity and scattering signal. This concurrent behavior suggests a mutual stabilization of elongated protofibril-like structures and of protein conformational and structural changes, which lead to a more rigid and ordered structures. Our results give new insights on BSA self-assembly process in alkaline conditions clearly providing new pieces of evidences of the interplay of several and interconnected mechanisms occurring on different time and length scales.     

Optimization of 3D hydrogel microenvironment for enhanced hepatic functionality of primary human hepatocytes

Although primary human hepatocytes (PHHs) are the gold standard in drug efficacy and metabolism studies, long-term survival of PHHs and maintenance of their hepatic function are still challenging. In this study, we focused on the effect of the initial microenvironment on upregulation and long-term preservation of hepatic function of PHHs encapsulated within biodegradable hydrogel systems. PHHs were encapsulated in RGD-functionalized hybrid hydrogels with various degrees of degradability, and their hepatic functionality was analyzed. Regardless of the hydrogel elastic modulus, the combination with nondegradable hydrogels had a predominantly negative effect on the prompt engraftment of PHHs, whereas a degradable hydrogel with intermediate initial degradability was most effective in maintaining hepatic function. Efficient network formation by PHHs and cocultured cells, along with the control of hydrogel degradation, governed the hepatic functionality at an early stage and upon long-term cultivation. Under optimized conditions, expression of genes involved in biological processes such as focal adhesions, cell survival, cytoskeleton formation, and extracellular matrix interactions was significantly higher than that in a control with relatively delayed initial degradation. Thus, we suggest that the orchestrated control of initial cellular remodeling may play an important role in the maintenance of hepatic function in a three-dimensional PHH culture.     

长期施肥对土壤氨氧化微生物的影响

长期施肥可改变土壤碳氮等养分供应,进而影响微生物数量与群落组成。本研究基于棕壤长期定位实验站,分析不同施肥方式下(不施肥,CK;低量无机氮肥,N₂;高量无机氮肥,N₄;有机无机氮肥配施,M₂N₂)土壤氨氧化古菌(AOA)和细菌(AOB)的变化,为土壤氮素转化的微生物学机制和培肥土壤提供依据。结果表明:不同施肥方式下,土壤AOA与AOB的数量比值为2.28～61.95。与CK相比,施肥后土壤AOA数量降低了1.6%～13.6%。N₄处理AOB数量随土壤深度增加呈先降低后升高的趋势,其他处理则相反。土壤AOB群落Shannon多样性指数、均匀度指数和Simpson指数均高于AOA。M₂N₂处理0～20 cm土层土壤AOB多样性增加,但AOA多样性降低。土壤AOB主要因土壤深度发生聚类,AOB和AOA均未因施肥方式发生聚类。综上,长期施肥改变了土壤AOA和AOB的构成状况,AOA对环境变化较为敏感,AOB较为丰富和稳定。     

氮添加量和施氮频率对温带半干旱草原土壤呼吸及组分的影响

日益加剧的氮沉降已经对陆地生态系统生产力和碳循环过程产生了显著影响。草原生态系统近90%的碳储存在土壤中, 明确土壤呼吸及其组分对氮添加的响应对评估大气氮沉降背景下草原生态系统碳平衡和土壤碳库稳定性是非常重要的。以往关于草原土壤呼吸对氮沉降响应的理解多是基于短期(<5年)和低频(每年1-2次)氮添加实验研究, 而关于长期氮添加和不同施氮频率对土壤呼吸及其组分的影响尚缺乏实验证据。该研究基于2008年建立在内蒙古半干旱草原的长期氮添加实验平台, 包括6个氮添加水平和2个施氮频率处理, 通过连续两年(2018-2019年)土壤呼吸及其组分的测定, 发现: 1)氮添加显著降低了土壤总呼吸速率(R_s), 且R_s下降程度随着氮添加量的增加而增强。土壤异养呼吸速率(R_h)的显著下降是R_s下降的主要原因。2)不同氮添加频率并未显著影响土壤呼吸及其组分对氮添加处理的响应。3)长期氮添加造成的土壤酸化降低了土壤微生物活性并改变了微生物群落结构(真菌/细菌比), 进而导致土壤呼吸及其异养组分呈现显著的负响应。以上结果表明, 长期(>10年)氮添加对土壤地下碳循环过程的抑制作用非常明显, 特别是异养呼吸组分的下降会降低土壤有机碳分解速率, 有助于土壤碳库稳定性的维持。同时, 随着氮添加处理时间的延长, 不同施氮频率影响效应的差异减弱, 表明目前长期的低频氮添加实验监测数据可以为评估自然生态系统对大气氮沉降的响应提供较为可靠的参考。     

海南岛干旱地区昌化江径流变化及影响因素

识别降水量和径流量变化过程及其影响因素对流域生态安全有重要意义。以海南岛干旱区昌化江流域主要控制水文站宝桥站58年(1956-2013年)水文资料为基础, 采用肯德尔秩次相关等统计方法和GIS技术研究流域内降水径流的变化特点及变化原因。结果表明: (1)降水和径流序列均呈缓慢上升趋势, 但2003年后径流序列有明显下降。(2)应用有序聚类分析径流系数跳跃成分, 结果显示1989年和2003年前后发生了较大改变。(3)将1956-1988年作为无人类活动影响的基准期, 建立降水-径流关系, 分析人类活动对径流变化过程的影响, 表明人类活动的间接影响使昌化江径流量减小, 流域植被覆盖率减少使蒸发作用加大, 导致径流量降低。(4)2003-2013年人类的活动影响使得昌化江径流量大幅减少, 其中直接影响减少量63.5 mm, 间接影响减少量为53.7 mm, 总减少量为117.1 mm, 人类活动综合影响对径流量下降的贡献率为13.9%。禁止砍伐热带雨林, 减少种植经济作物对维护流域生态安全尤为关键。     

Differences in circadian time course and level for the plasma concentrations of glucose,free fatty acids and amino acids between ad lib‐eating and fasting rabbits

Abstract

The entrained rhythm in food approaches, representative of the circadian fluctuation in locomotor and food intake activity was recorded from 12 rabbits eating ad libitum during exposure to an LD 12:12 h‐regimen. They were also subjected to 53 h‐sessions with the same LD alternation, in which blood samples were taken at 2 h‐intervals. During these sessions 6 of them were permitted to eat freely whereas the others were food‐deprived. It appeared that in eating rabbits plasma levels are higher for glucose and a‐amino nitrogen but lower for free fatty acids and, further, that eating specimens exhibit marked circadian fluctuations in the levels of glucose and FFA which are closely correlated with, and probably a consequence of, the circadian rhythm in the amounts of food actually taken up in the course of the sessions. In fasting rabbits, however, the same plasma parameters show rhythms with quite another time course, that can be attributed to the circadian fluctuations in (locomotor) activity and endocrine balance.