高粘发酵体系不同搅拌桨的CFD模拟及发酵验证

搅拌桨是高好氧高黏度微生物发酵实现高效反应必不可少的因素之一,不同搅拌桨组合对发酵过程的影响十分重要。威兰胶是由产碱杆菌在高耗氧高粘度发酵体系下合成的胞外微生物多糖,广泛应用于水泥、石油、油墨、食品等行业中。本研究借助于计算流体力学(Computational fluid dynamics,CFD)的方法,以威兰胶发酵液体系为研究体系,研究了6种不同搅拌桨组合在反应器内流体速率分布、剪切速率、和气含率等参数。将模拟效果较好的3种组合用于威兰胶发酵过程。研究表明MB-4-6搅拌桨组合对改善发酵罐内部的溶氧及流场分布效果最明显,威兰胶产量水平提高了13%。同时在该组合下威兰胶的产品粘度得到有效提高。     

Differential responsiveness of vascular endothelial cells to different types of fluid mechanical shear stress

Early atherosclerotic lesions localize preferentially, in arterial regions exposed to low flow, oscillatory flow, or both; however, the cellular basis of this observation remains to be determined. Atherogenesis involves dysfunction of the vascular endothelium, the cellular monolayer lining the inner surfaces of blood vessels. How low flow, oscillatory flow, or both may lead to endothelial dysfunction remains unknown. Over the past two decades, fluid mechanical shear (or frictional) stress has been shown to intricately regulate the structure and function of vascular endothelial cells (ECs). Furthermore, recent data indicate that beyond being merely responsive to shear stress, ECs are able to distinguish among and respond differently to different types of shear stress. This review focuses on EC differential responses to different types of steady and unsteady shear stress and discusses the implications of these responses for the localization of early atherosclerotic lesions. The mechanisms by which endothelial differential responsiveness to different types of flow may occur are also discussed.     

Hemodynamic analysis of a novel stent graft design with slit perforations in thoracic aortic aneurysm

Thoracic endovascular aortic repair (TEVAR) has been introduced as a less invasive approach to the treatment of thoracic aortic aneurysm (TAA). However, the effectiveness of TEVAR in the treatment of TAA is often limited due to the complex anatomy of aortic arch. Flow preservation at the three supra-aortic branches further increases the overall technical difficulty. This study proposes a novel stent graft design with slit perforations that can positively alter the hemodynamics at the aortic arch while maintaining blood flow to supra-aortic branches. We carried out a computational fluid dynamic (CFD) analysis to evaluate flow characteristics near stented aortic arch in simplified TAA models, followed by in-vitro experiments using particle image velocimetry (PIV) in a mock circulatory loop. The hemodynamics result was studied in terms of time-averaged wall shear stress (TAWSS), oscillating shear index (OSI), and endothelial cell action potential (ECAP). The results showed that the stent graft with slit perforations can reduce the disturbed flow region considerably. Furthermore, the effect of the slits on flow preservation to the supra-aortic branches was simulated and compared with experimental results. The effectiveness of the stent graft with slit perforations in preserving flow to the branches was demonstrated by both simulated and experimental results. Low TAWSS and elevated ECAP were observed in the aortic arch aneurysm after the placement of the stent graft with slits, implying the potential of thrombus formation in the aneurysm. On the other hand, the effects of the stent grafts with full-slit design and half-slit design on the shear stress did not differ significantly. The present analysis indicated that not only could the stent graft with slit perforations shield the aneurysm from rupture, but also it resulted in a favorable environment for thrombus that can contribute to the shrinkage of the aneurysm.     

Analysis of the effect of disturbed flow on monocytic adhesion to endothelial cells

The preferential adhesion of monocytes to vascular endothelial cells (ECs) at regions near branches and curvatures of the arterial tree, where flow is disturbed, suggests that hemodynamic conditions play significant roles in monocyte adhesion. The present study aims to elucidate the effects of disturbed flow on monocyte adhesion to ECs and the adhesive properties of ECs. We applied, for the first time, the micron-resolution particle image velocimetry (μPIV) technique to analyze the characteristics of the disturbed flow produced in our vertical-step flow (VSF) chamber. The results demonstrated the existence of a higher near-wall concentration and a longer residence time of the monocytic analog THP-1 cells near the step and the reattachment point. THP-1 cells showed prominent adhesion to ECs pretreated with TNF in the regions near the step and the reattachment point, but they showed virtually no adhesion to un-stimulated ECs. Pre-incubation of the TNF-treated ECs with antibodies against intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin inhibited the THP-1 adhesion; the maximal inhibition was observed with a combination of these antibodies. Pre-exposure of ECs to disturbed flow in VSF for 24 h led to significant increases in their surface expressions of ICAM-1 and E-selectin, but not VCAM-1, and in the adhesion of THP-1 cells. Our findings demonstrate the importance of complex flow environment in modulating the adhesive properties of vascular endothelium and consequently monocyte adhesion in regions of prevalence of atherosclerotic lesions.     

Effects of shear stress on endothelial cells: possible relevance for ultrasound applications

This review forms part of a series of papers resulting from a workshop on safety of ultrasound applications. The physical effects of ultrasound include generation of steady streaming in large fluid volumes, and micro-streaming around contrast bubbles. Such streaming induces shear stress acting on the vascular endothelium. This review provides a discussion on the levels of endothelial shear stress associated with diagnostic ultrasound applications, and on the biological effects of shear stress acting on the endothelial cells. Depending on vessel size and ultrasound characteristics, shear stresses associated with streaming and micro-streaming may exceed the physiological levels associated with the flow of blood by many orders of magnitude. The resulting biological effects could range anywhere from activation of normal shear stress sensors such as ion channels, damage of the endothelial surface layer, reversible perforation of the membrane, to cell detachment and lysis. The possible presence of such biological effects does not necessarily mean that the effects are harmful for the individual. However, considering the ever-increasing use of ultrasound, a further investigation into these shear stress-related effects, using both experiments and modelling, is desired. Apart from safety concerns, such effects may provide a base for strategies aimed at targeted delivery of drugs.     

流体剪切力对内皮细胞miR-21和miR-199a表达的影响

为探讨流体剪切力对内皮细胞micorRNAs表达的影响。采用旋转锥形圆盘剪切力系统对内皮细胞分别加载低(4dyn/cm2)、中(10 dyn/cm2)和高(15 dyn/cm2)3种不同梯度的剪切力作用24h。对照组未加载剪切力。采用高通量筛选芯片检测microRNAs表达变化,qRT-PCR验证,并进行生物信息学分析。与对照组比较,低剪切力组表达差异的microRNAs有33个(FC1.5或0.5倍,P0.05),其中28个上调,5个下调;中剪切力组表达差异的microRNAs有8个(FC1.5或0.5倍,P0.05),其中6个上调,2个下调;高剪切力组表达差异的microRNAs有31个(FC1.5或0.5倍,P0.05),其中25个上调,6个下调。miR-21在高剪切力组中上调最显著(FC=0.026),在低剪切力组中显著下调(FC=3.531)。miR-199a在低剪切力组中上调最显著(FC=0.075),在高剪切力组中显著下调(FC=3.031)。表达差异的microRNA的靶基因主要与内皮细胞的力学信号转导、细胞跨膜迁移、钙离子信号通路、细胞内吞作用等相关。流体剪切力可诱导内皮细胞miR-21和miR-199a表达发生改变。     

低切应力作用下体外培养动脉内皮细胞内皮素的表达

研究了切应力对完整血管的生物学作用以及应力引起血管重建过程中内皮素(ET)的变化．采用血管体外应力培养系统,将一段完整的猪颈总动脉在体外进行培养,设切应力分别为2Pa(％组)和0．5Pa(S5组),设置2、4、6．8．10．12、14．16和18h共9个时相观察点,非平衡法放射免疫检测灌流液中的ET含量．通过Logistic曲线方程拟合,分析切应力作用下完整动脉ET表达变化规律。结果显示：S20组ET总体变化不明显;S5组分泌速率在前12．37h内明显上升,而后又逐渐下降趋于稳定,且始终高于S20组。说明低切应力作用下ET的表达及分泌增高．结果提示,在低切应力引起的血管重建中,ET可能起着重要作用。     

Effects of shear stress on differentiation of stem cells into endothelial cells

Stem cell transplantation is an appealing potential therapy for vascular diseases and an indispensable key step in vascular tissue engineering. Substantial effort has been made to differentiate stem cells toward vascular cell phenotypes, including endothelial cells (ECs) and smooth muscle cells. The microenvironment of vascular cells not only contains biochemical factors that influence differentiation but also exerts hemodynamic forces, such as shear stress and cyclic strain. More recently, studies have shown that shear stress can influence the differentiation of stem cells toward ECs. A deep understanding of the responses and underlying mechanisms involved in this process is essential for clinical translation. This review highlights current data supporting the role of shear stress in stem cell differentiation into ECs. Potential mechanisms and signaling cascades for transducing shear stress into a biological signal are proposed. Further study of stem cell responses to shear stress will be necessary to apply stem cells for pharmacological applications and cardiovascular implants in the realm of regenerative medicine.     

Effects of cold exposure and shear stress on endothelial nitric oxide synthase activation

Endothelial nitric oxide synthase (eNOS) is the primary enzyme that produces nitric oxide (NO), which plays an important role in blood vessel relaxation. eNOS activation is stimulated by various mechanical forces, such as shear stress. Several studies have shown that local cooling of the human finger causes strong vasoconstriction, followed after several minutes by cold-induced vasodilation (CIVD). However, the role played by endothelial cells (ECs) in blood vessel regulation in respond to cold temperatures is not fully understood. In this study, we found that low temperature alone does not significantly increase or decrease eNOS activation in ECs. We further found that the combination of shear stress with temperature change leads to a significant increase in eNOS activation at 37 °C and 28 °C, and a decrease at 4 °C. These results show that ECs play an important role in blood vessel regulation under shear stress and low temperature.     

Effect of spatial gradient in fluid shear stress on morphological changes in endothelial cells in response to flow

Arterial bifurcations are common sites for development of cerebral aneurysms. Although this localization of aneurysms suggests that high shear stress (SS) and high spatial SS gradient (SSG) occurring at the bifurcations may be crucial factors for endothelial dysfunction involved in aneurysm formation, the details of the relationship between the hemodynamic environment and endothelial cell (EC) responses remain unclear. In the present study, we sought morphological responses of ECs under high-SS and high-SSG conditions using a T-shaped flow chamber. Confluent ECs were exposed to SS of 2-10 Pa with SSG of up to 34 Pa/mm for 24 and 72 h. ECs exposed to SS without spatial gradient elongated and oriented to the direction of flow at 72 h through different processes depending on the magnitude of SS. In contrast, cells did not exhibit preferred orientation and elongation under the combination of SS and SSG. Unlike cells aligned to the flow by exposure to only SS, development of actin stress fibers was not observed in ECs exposed to SS with SSG. These results indicate that SSG suppresses morphological changes of ECs in response to flow.     

Pulsatile shear stress increased mitochondrial membrane potential: Implication of Mn-SOD

Mitochondrial dysfunction is intimately involved in cardiovascular diseases. Mitochondrial membrane potential (ΔΨ_m) is coupled with oxidative phosphorylation to drive ATP synthesis. In this study, we examined the effect of physiological pulsatile shear stress (PSS) on ΔΨ_m and the role of Mn-SOD expression on ΔΨ_m. Confluent human aortic endothelial cells (HAEC) were exposed to PSS, and ΔΨ_m was monitored using tetramethylrhodamine methyl ester (TMRM⁺), a mitochondrial membrane potential probe. PSS significantly increased ΔΨ_m and the change in ΔΨ_m was a dynamic process. ΔΨ_m returned to baseline level after PSS for 2 h followed by static state for 4 h. Mitochondrial Mn-SOD expression and activities were also significantly up-regulated in response to PSS. Silencing Mn-SOD attenuated PSS-mediated ΔΨ_m increase while adding Mn-SOD mimetic, MnTMPyP, increased ΔΨ_m to the similar extent as induced by PSS. Our findings suggest that PSS-increased mitochondrial ΔΨ_m, in part, via Mn-SOD up-regulation.     

Direct measurement of shear strain in adherent vascular endothelial cells exposed to fluid shear stress

Functional and morphological responses of endothelial cells (ECs) to fluid shear stress are thought to be mediated by several mechanosensitive molecules. However, how the force due to fluid shear stress applied to the apical surface of ECs is transmitted to the mechanosensors is poorly understood. In the present paper, we performed an analysis of an intracellular mechanical field by observation of the deformation behaviors of living ECs exposed to shear stress with a novel experimental method. Lateral images of human umbilical vein ECs before and after the onset of flow were obtained by confocal microscopy, and image correlation and finite element analysis were performed for quantitative analyses of subcellular strain due to shear stress. The shear strain of the cells changed from 1.06 ± 1.09% (mean ± SD) to 4.67 ± 1.79% as the magnitude of the shear stress increased from 2 to 10 Pa. The nuclei of ECs also exhibited shear deformation, which was similar to that observed in cytoplasm, suggesting that nuclei transmit forces from apical to intracellular components, as well as cytoskeletons. The obtained strain-stress relation resulted in a mean shear modulus of 213 Pa for adherent ECs. These results provide a mechanical perspective on the investigation of flow-sensing mechanisms of ECs.     

3D reconstruction techniques of human coronary bifurcations for shear stress computations

Background

Heterogeneity in plaque composition in human coronary artery bifurcations is associated with blood flow induced shear stress. Shear stress is generally determined by combing 3D lumen data and computational fluid dynamics (CFD). We investigated two new procedures to generate 3D lumen reconstructions of coronary artery bifurcations for shear stress computations.

Methods

We imaged 10 patients with multislice computer tomography (MSCT) and intravascular ultrasound (IVUS). The 3D reconstruction of the main branch was based on the fusion of MSCT and IVUS. The proximal part of side branch was reconstructed using IVUS data or MSCT data, resulting in two different reconstructions of the bifurcation region. The distal part of the side branch was based on MSCT data alone. The reconstructed lumen was combined with CFD to determine the shear stress. Low and high shear stress regions were defined and shear stress patterns in the bifurcation regions were investigated.

Results

The 3D coronary bifurcations were successfully generated with both reconstruction procedures. The geometrical features of the bifurcation region for the two reconstruction procedures did not reveal appreciable differences. The shear stress maps showed a qualitative agreement, and the low and high shear stress regions were similar in size and average shear stress values were identical. The low and high shear stress regions showed an overlap of approximately 75%.

Conclusion

Reconstruction of the side branch with MSCT data alone is an adequate technique to study shear stress and wall thickness in the bifurcation region. The reconstruction procedure can be applied to further investigate the effect of shear stress on atherosclerosis in coronary bifurcations.     

Kinetic analysis of hybridoma cells viability under mechanical shear stress with and without serum protection

The effect of a well-defined mild shear stress on hybridoma cell viability (HB-8852) in a serum-free culture medium has been analysed, and the role as shear protector of different concentrations of fetal bovine serum have been studied. Samples harvested from cultures in their late exponential growth phase, were subjected in a rheometer to a constant shear stress of 0.41 ± 0.02 Pa, and the evolution of viable and total cell concentrations was determined and compared with static controls. A simple segregated kinetic model for the viable and dead cells was used to know the effect of serum concentration on the specific cell growth and death rate of the cells.     

Proteomic analysis of vascular endothelial cells in response to laminar shear stress

Isotope-coded affinity tags (cICAT) coupled with mass spectrometric analysis is one of the leading technologies for quantitative proteomic profiling and protein quantification. We performed proteomic analysis of bovine aortic endothelial cells (BAEC) in response to laminar shear stress using cICAT labeling coupled with LC-MS/MS. Protein expressions in BAEC under 15 dynes/cm2 of shear stress for 10 min, 3 h, and 6 h were compared with matched stationary controls. Analysis of each sample produced 1800-2400 proteins at >or=75% confidence level. We found 142, 213, and 186 candidate proteins that were up- or down-regulated by at least two-fold after 10 min, 3 h, and 6 h of shear stress, respectively. Some of these proteins have known cellular functions and they encompass many signaling pathways. The signaling pathways that respond to shear stress include those of integrins, G-protein-coupled receptors, glutamate receptors, PI3K/AKT, apoptosis, Notch and cAMP-mediated signaling pathways. The validity of the mass spectrometric analysis was also confirmed by Western blot and confocal immunofluorescence microscopy. The present quantitative proteomic analysis suggests novel potential regulatory mechanisms in vascular endothelial cells in response to shear stress. These results provide preliminary footprints for further studies on the signaling mechanisms induced by shear stress.     

Expression of synaptopodin in endothelial cells exposed to laminar shear stress and its role in endothelial wound healing

We examined the hypothesis that certain actin binding proteins might be upregulated by laminar shear stress (LSS) and could contribute to endothelial wound healing. Analysis of mRNA expression profiles of human umbilical vein endothelial cells under static and LSS-exposed conditions provided a list of LSS-induced actin binding proteins including synaptopodin (SYNPO) whose endothelial expression has not been previously reported. Additional studies demonstrated that SYNPO is a key mediator of endothelial wound healing because small interfering RNA-mediated suppression of SYNPO attenuated wound closure under LSS whereas overexpression of exogenous SYNPO enhanced endothelial wound closure in the absence of LSS. This study suggests that LSS-induced actin binding proteins including SYNPO may play a critical role in the endothelial wound healing stimulated by LSS.     

不同肥料组合对冬小麦水分供需状况的研究

采用Ｄ－饱和最优设计对半湿润易旱区冬小麦在各生育阶段土壤水分平衡状况以及不同肥料组合对冬小麦产量和水分利用效率的影响进行了分析和研究。结果表明,根据水量平衡法计算不同施肥条件下土壤供水量明显不同,以Ｎ、Ｐ、有机肥组合土壤供水占的比例最大为２７％,单施氮肥利用降水补给占的比例最高为１００％。整个生育期,单施氮肥冬小麦水分亏缺最严重,而磷肥与有机肥组合水分亏缺最小,Ｎ、Ｐ、有机肥组合能显著提高冬小麦产     

Effects of fluid shear stress on apoptosis of cultured human umbilical vein endothelial cells induced by LPS

The objective of our research was to reveal the effects of shear stress on the apoptosis of cultured human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS). A parallel-plate flow chamber was used to control the strength and duration of shear stress (SS), and apoptosis was measured by immunocytochemistry and radio-immunoassay. Some important conclusions were drawn. In the stationary state, apoptosis of HUVECs could be induced by LPS (50 microg/ml). An SS of 15 dyn/cm(2) could inhibit the apoptosis induced by LPS. However, an SS of 4 dyn/cm(2) had less effect on the same process. At the same time, the experiment demonstrated that the increase in IL-6 secretion by LPS can be inhibited by two different levels of shear stress. Moreover, the inhibition effect was more obvious under high level stress than under low level. We also found that the effect of shear stress on IL-8 was less effective than on IL-6. This research provides data for understanding the mechanism of the contribution of hemodynamic forces to atherosclerosis.     

Cultivation of shear stress sensitive microorganisms in disposable bag reactor systems

Technical scale (≥5 l) cultivations of shear stress sensitive microorganisms are often difficult to perform, as common bioreactors are usually designed to maximize the oxygen input into the culture medium. This is achieved by mechanical stirrers, causing high shear stress. Examples for shear stress sensitive microorganisms, for which no specific cultivation systems exist, are many anaerobic bacteria and fungi, such as basidiomycetes. In this work a disposable bag bioreactor developed for cultivation of mammalian cells was investigated to evaluate its potential to cultivate shear stress sensitive anaerobic Eubacterium ramulus and shear stress sensitive basidiomycetes Flammulina velutipes and Pleurotus sapidus. All cultivations were compared with conventional stainless steel stirred tank reactors (STR) cultivations. Good growth of all investigated microorganisms cultivated in the bag reactor was found. E. ramulus showed growth rates of μ = 0.56 h⁻¹ (bag) and μ = 0.53 h⁻¹ (STR). Differences concerning morphology, enzymatic activities and growth in fungal cultivations were observed. In the bag reactor growth in form of small, independent pellets was observed while STR cultivations showed intense aggregation. F. velutipes reached higher biomass concentrations (21.2 g l⁻¹ DCW vs. 16.8 g l⁻¹ DCW) and up to 2-fold higher peptidolytic activities in comparison to cell cultivation in stirred tank reactors.