Effects of docosahexaenoic acid or arachidonic acid supplementation on the behavior of cardiomyocytes derived from human pluripotent stem cells

Background: Human heart changes its energetic substrates from lactate and glucose to fatty acids during the neonatal period. Noticing the lack of fatty acids in media for the culture of cardiomyocytes derived from human pluripotent stem cells (hiPS-CM), researchers have supplemented mixtures of fatty acids to hiPS-CM and reported the enhancement in the maturation of hiPS-CM. In our previous studies, we separately supplemented two polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) or arachidonic acid (AA), to rat fetal cardiomyocytes and found that the supplementations upregulated the expressions of mRNAs for cardiomyocyte differentiation, fatty acid metabolism, and cellular adhesion. The enhancement in cellular contractility was attributed to the improvement in intercellular connection rather than a direct enhancement of the contractile force. Methods: This study reports the successive results of the effects of DHA or AA supplementation on hiPS-CM. In addition to the contractile force and mRNA measurements used in the previous study, we further investigated the effect of different cellular aggregations on the contractile force output by means of finite element analysis, measured glucose and fatty acids metabolites, and assessed cTNT and MLC2v expressions through immunofluorecsence evaluation. Results: It showed that the sole supplementation of albumin-conjugated DHA or AA can be taken up by hiPS-CM without other uptake-enhancing factors, and the supplementations may activate the CD36_­ERRγ metabolic pathway. DHA or AA supplementation increased the cellular contractile ratio on collagen gels and AA supplementation stimulated hiPS-CM aggregation to form cellular clusters. The enhancement effect on the hiPS-CM contractile force was modest since the increase in contractile force was not significant. AA supplementation was more effective than DHA supplementation because it significantly upregulated mRNA expressions of P300 and CD36. However, finite element analysis showed that the formation of clusters on a collagen gel attenuated the contractile force exerted by the gel on its surroundings. Conclusion: DHA and AA, as having been supplemented in infant formulas, have no direct and significant enhancement effect on the performance of the hiPS-CM when they were supplemented individually, although they were able to enter the cellular metabolic system. The AA supplementation showed some auxiliary effect on the maturation of hiPS-CM, which is worthy of further investigation under the consideration of membrane composition alteration and remodeling of membrane molecules.     

LncRNA <i>ZFAS1</i> regulates cardiomyocyte differentiation of human embryonic stem cells

Background: Cardiomyocytes derived from human embryonic stem cells (hESCs) are regulated by complex and stringent gene networks during differentiation. Long non-coding RNAs (lncRNAs) exert critical epigenetic regulatory functions in multiple differentiation processes. However, the involvement of lncRNAs in the differentiation of hESCs into cardiomyocytes has not yet been fully elucidated. Here, we identified the key roles of ZFAS1 (lncRNA zinc finger antisense 1) in the differentiation of cardiomyocytes from hESCs. Methods: A model of cardiomyocyte differentiation from stem cells was established using the monolayer differentiation method, and the number of beating hESCs-derived cardiomyocytes was calculated. Gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Immunofluorescence assays were performed to assess the expression of cardiac troponin T (cTnT) and α-actinin protein in cardiomyocytes. Results: qRT-PCR showed that ZFAS1 expression in the mesoderm was significantly higher than that in embryonic stem cells, cardiac progenitor cells, and cardiomyocytes. Knockdown of ZFAS1 inhibited cardiomyocyte differentiation from hESCs, which was characterized by reduced expression of the cardiac-specific markers cTnT, α-actinin, myosin heavy chain 6 (MYH6), and myosin heavy chain 7 (MYH7). In contrast, ZFAS1 overexpression remarkably increased the percentage of spontaneously beating cardiomyocytes. In terms of the mechanism, we found that ZFAS1 is an antisense lncRNA at the 5′ end of the protein-coding gene ZNFX1. Knockdown of ZFAS1 could increase the mRNA expression level of ZNFX1. Furthermore, qRT-PCR demonstrated that the silencing of ZNFX1 led to an increase in cardiac-specific markers that predicted the promotion of cardiomyocyte differentiation. Conclusion: Altogether, these data suggest that lncRNA-ZFAS1 is required for cardiac differentiation by functionally inhibiting the expression of ZNFX1, which may provide a reference for the treatment of heart disease to a certain extent.     

Effect of <i>Lycopus lucidus</i> Turcz. supplementation on gut microflora and short chain fatty acid composition in Crj: CD-1 mice

We investigated the diversity and composition of microflora in feces of Lycopus lucidus Turcz.-fed mice. In addition, we evaluated the production of major cytokines (Interleukin-6 and -10) which are related to inflammation and fatty acid composition of several tissues. 16S ribosomal DNA sequencing-based microbiome taxonomic profiling analysis was performed utilizing the EzBioCloud data base. Male mice fed on L. lucidus showed a significantly reduced number of lactic acid bacteria and coliform in the feces compared with the control group (p < 0.05). 16S rDNA sequencing analysis of fecal samples showed that L. lucidus supplementation decreased the community of harmful microflora (Enterobacteriaceae including Escherichia coli and Bacteroides sp.) in feces compared with the control group (p < 0.05). There were no significant differences in mRNA expression of cytokine IL-6 and IL-10 between the control and L. lucidus fed groups. The fecal fatty acid composition in the L. lucidus group had percentages of 4:0, 6:0, 8:0 and 10:0 in the intestine but those short chain fatty acids were not detected in the control group. Our results showed that L. lucidus supplementation influenced gut environment by decreasing harmful microflora and increased the percentages of several short fatty acids.     

LC-MS/MS分析血浆中脂肪酸及代谢产物

建立了高效液相色谱-串联质谱（LC-MS/MS）同时测定血浆中脂肪酸花生四烯酸（AA）及其代谢产物13-羟基十八碳烯酸（13-HODE）和9-羟基十八碳烯酸（9-HODE）的方法。采用Strata-X固相萃取柱净化，BEH C18色谱柱分离，以乙腈 水为流动相，流速0.2 mL/min，等度洗脱；电喷雾离子源负离子模式和多反应监测模式定量。结果表明，3种物质在0.5~50 μg/L范围内线性关系良好，相关系数为0.994 2~0.996 0；低、中、高3个加标水平的平均回收率为97.42%~101.46%；日内相对标准偏差为2.72%~6.11%，日间相对标准偏差为3.87%~6.39%；AA、13-HODE和9-HODE定量限分别为0.5、0.5、1.0 μg/L。该方法简单、灵敏、快速、可靠，可用于血浆中脂肪酸及其代谢产物的检测分析。     

衍生化GC/MS同时测定樟芝菌粉中19种脂肪酸的含量

建立了衍生化气相色谱-质谱联用（GC/MS）同时测定不同批次、不同培植方式樟芝中19种脂肪酸含量的方法。采用HP-VOC毛细管色谱柱(60 m×320 μm×1.8 μm),选择离子扫描方式（SIM）,对不同批次樟芝菌粉超临界CO₂流体萃取物的甲酯化样品进行分析。19种脂肪酸的响应峰面积与其相应浓度的线性关系良好（r＞0.995 6）,加样回收率为93.47%～104.89%。该方法准确、专属、重复性好,能有效测定樟芝菌粉中脂肪酸的含量,可为樟芝菌粉的质量评价奠定基础。     

A 66-kDa protein of bovine hypophyseal <i>Pars tuberalis</i> induces luteinizing hormone release from rat <i>Pars distalis</i>

In this study, evidence for a factor secreted by bovine hypophyseal pars tuberalis that stimulates luteinizing hormone (LH) release from rat pars distalis cells is shown. The secretion products of bovine pars tuberalis cells into the culture medium were assayed on dispersed rat pars distalis cells in 30 min incubations and superfusion experiments. The culture medium from pars tuberalis total cell populations, added at a dose of 6 μg per tube, induced the greater LH release from pars distalis cells, without effect on follicle stimulating hormone (FSH) release. After pars tuberalis cells separation on a discontinuos Percoll gradient, only the culture medium of cells from 50 and 60% strength Percoll were able to release LH from rat pars distalis cells. Therefore, cell fractions from 50 and 60% strenght Percoll were cultured together. To elicit maximal LH release (6 times the basal output), with the addition of 2 μg of pars tuberalis protein was required, suggesting that these cells produce the factor or factors which affect pars distalis gonadotrope cells. After applying the pars tuberalis culture medium on 12% SDS-PAGE, the band with biological activity was that of 66-kDal. Fifty ng protein of its eluate released almost 9 times the basal output of LH from pars distalis cells. Results suggest a modulating effect of a protein from the bovine pars tuberalis on rat cultured gonadotrope cells from the pars distalis.     

荧光显微镜观察银杏内酯B对大鼠心肌细胞凋亡的影响

目的：建立一种简便、快速的细胞凋亡检测方法，探讨银杏内酯B（GB）对心肌细胞凋亡的影响。方法：体外培养大鼠心肌细胞，利用荧光显微镜观察经Hoeehst 33258染色的心肌细胞，记录凋亡细胞核形态学改变并计算凋亡百分率。结果：H2O2可诱导心肌细胞发生凋亡，与H2O2组相比，GB组大鼠心肌细胞凋亡率明显降低，并有显著性差异（P〈0．05）。结论：银杏内酯B对大鼠心肌细胞凋亡具有保护作用。     

深海鱼油,海豹油脂肪酸组份的分析研究

〕首先把深海鱼油、海豹油甲酯化,用石英毛细管色谱／质谱法测定两种油的脂肪酸组份。深海鱼油分离出二十七种脂肪酸,其不饱和脂肪酸含量为７０．１５％;海豹油分离出二十六种脂肪酸,其不饱和脂肪酸的含量为８５．２４％。两种油的主要不饱和脂肪酸均为：十六碳烯酸、亚麻酸、亚油酸、油酸、二十碳五烯酸（ＥＰＡ）、二十碳烯酸、二十二碳六烯酸（ＤＨＡ）、二十二碳五烯酸（ＤＰＡ）、二十二碳烯酸     

Effects of the ascorbic acid supplementation on NADH-diaphorase myenteric neurons in the duodenum of diabetic rats.

We assessed the ascorbic acid (AA) supplementation on the myenteric neurons in the duodenum of rats. Fifteen rats with 90 days of age were divided into three groups: control (C), diabetics (D) and ascorbic acid treated diabetics (DA). After 120 days of daily treatment with AA, the duodenum was submitted to the NADH-diaphorase (NADH-d) histochemical technique, which allowed us to evaluate the neuronal density in an area of 8.96 mm2 for each duodenum, and also to measure the cellular profile area of 500 neurons per group. The supplementation promoted an increase on AA levels. The neuronal density (p < 0.05) was higher in the group DA when compared to group D. There were no significant differences in the neuronal areas, when we compared groups C (204 +/- 16.5) and D (146.3 +/- 35.84) to groups D and DA (184.5 +/- 5.6) (p > 0.05). The AA-supplementation avoided the density reduction of the NADHd myenteric neurons in the duodenum of diabetic rats.     

Organogenesis and plant regeneration of <i>Arachis villosa</i> Benth. (Leguminosae) through leaf culture

With the aim of developing an efficient plant regeneration protocol, leaflet explants of three accessions of Arachis villosa Benth. (S2866, S2867 and L97) were cultured on basic Murashige and Skoog medium supplemented with different combinations of plant growth regulators: α-naphthalenacetic acid, indole-3-butyric acid, 6-benzylaminopurine, kinetin and thidiazuron. The accession L97 was the only one able to differentiate buds through indirect organogenesis. The most suitable combination for bud regeneration was the basic medium added with 13.62 μM thidiazuron and 4.44 μM 6-benzylaminopurine. These results show the important role of the genotype in morphogenetic responses and the organogenetic effect of thidiazuron in Arachis villosa accession L97. A thidiazuron lacking media (only 0.54 μM α-naphthalenacetic acid, 13.95 μM kinetin and 13.32 μM 6-benzylaminopurine were added) promoted the elongation of the regenerated buds. Adventitious rooting was achieved 90 days after the isolated shoots were transferred to a rooting medium containing 0.54 μM α-naphthalenacetic acid.     

Hyaluronic acid inhibited the upregulation of heat shock protein 70 in human chondrocytes from osteoarthritis and Kashin-Beck disease

This study aimed to investigate the effect of hyaluronic acid (HA) on the expression of heat shock protein 70 (HSP70) in chondrocytes isolated from patients with osteoarthritis (OA) and Kashin-Beck disease (KBD). The chondrocytes were collected from OA and KBD patients, and chondrocytes isolated from patients of accident injuries were used as the control. The chondrocytes were treated with HA at different doses. HSP70 expression in chondrocytes at both mRNA and protein levels was tested by PCR and Western blot analysis. Compared with control, both mRNA and protein levels of HSP70 were higher in chondrocytes from KBD and OA. However, HA at the dose of 500 μg/ mL significantly inhibited HSP70 expression levels in both KBD and OA groups (P < 0.05). In conclusion, HSP70 is highly expressed in chondrocytes of patients of OA and KBD. HA intervention inhibits the upregulation of HSP70 in chondrocytes of OA and KBD patients and could be a promising agent for treatment of OA and KBD.     

Inhibitory effect of jasmonic acid and ethylene on epicotyl growth and bud induction in the maritime pine, <i>Pinus pinaster</i> Soland. in Ait

Two independent parameters, epicotyl height (cm) and number of induced buds were studied on Pinus pinaster explants to analyse the effects of three phytohormones (6-benzylaminopurine, jasmonic acid, ethylene) which were combined or not in 11 different treatments. Epicotyle length diminished significantly in relation to the control medium (medium without exogen phytohormones) in presence of jasmonic acid, 6-benzylaminopurine or Ethephon (which is converted to ethylene in plants) in any of treatments. Concentrations of 100 μM of jasmonic acid and Ethephon had a greater inhibitory effect than the treatments with 10 μM. In addition to that, jasmonic acid was a stronger inhibitor than Ethephon in any of the tried combinations. There were no significant differences between the control treatment and the treatments with only 10 μM of jasmonic acid or Ethephon. However, 10 μM 6-benzylaminopurine induced bud formation. The different combinations of 6-benzylaminopurine with jasmonic acid and Ethephon showed that concentrations of 10 to 100 μM did not affect the number of induced buds. Jasmonic acid had an inhibitory effect which Ethephon only showed when combined with 100 μM of jasmonic acid and 10 μM of 6-benzylaminopurine. Three response groups were defined by cluster analysis: group 1 produced the greatest mean number of buds (4 to 5) and a mean epicotyl growth of 1 to 1.5 cm; group 2 produced 2 to 4 buds and a mean growth of 0.5 to 1.2 cm; group 3 produced only one bud and a mean epicotyl length of 1.2 to 2 cm.     

Taxus globosa S. cell lines: Initiation,selection and characterization in terms of growth,and of baccatin III and paclitaxel production

Of the initial six cell lines originating from explants of Taxus globosa, or Mexican yew (stem internode, leaves and meristematic tissue), three were selected for their microbial and oxidation resistance, two from leaves and the other from stem internode. A study of their behavior, both in terms of cell growth, and of baccatin III and paclitaxel production, was developed in suspension cultures with an initially standardized biomass (fresh weight 0.23 g/L) using modified Gamborg’s B5 medium, and an elicitor (methyl jasmonate), on either the first or seventh day of culture, at several levels (0, 0.1, 1, 10, 100 μM). In most of the conditions used, the three cell lines showed growth associated baccatin III production. The cell line from stem internode was the highest producer of baccatin III using 1 μM elicitor, sampling at 10 days (p < 0.01, 6.45 mg/L). This same line also had the highest biomass production (6.85 g/L, p < 0.01) at 10 days of culture but at the higher elicitor concentration of 10 μM. All three cell lines did not produce paclitaxel under experimental conditions used.     

基于椭圆簇轨迹特征的板中超声导波线弧类阵列成像研究

超声导波换能器阵列的布置形式对超声导波成像质量有重要影响,规则的直线阵列、弧形阵列和非规则的贝塞尔阵列等线弧类阵列在板中超声导波成像时有内在联系。讨论线弧类阵列布阵方法,建立线弧类阵列成像系统,分析多通道线弧类阵列信号特点和其图像椭圆簇轨迹特征,基于图像椭圆簇轨迹特征提出线弧类阵列超声导波成像检测的分辨率分析准则,据此研究了线弧类阵列成像检测横向分辨率的影响因素。理论分析和成像结果一致表明,基于图像椭圆簇轨迹特征的板中超声导波合成孔径聚焦可统一运用于以上规则和非规则的线弧类阵列成像,散射点处图像灰度呈现椭圆簇轨迹分布特征,相同基准阵列的超声导波成像检测时弧形阵列有比直线阵列更高的横向分辨率,贝塞尔阵列的横向分辨率与弧形阵列相当,这与分辨率分析准则一致。为进一步开展线弧类阵列统一灵活运用于工业复杂现场的大尺度板材超声导波成像检测研究与应用提供基础。     

Calibrated histochemistry applied to oxygen supply and demand in hypertrophied rat myocardium

Oxygen supply and demand of individual cardiomyocytes during the development of myocardial hypertrophy is studied using calibrated histochemical methods. An oxygen diffusion model is used to calculate the critical extracellular oxygen tension (PO(2,crit)) required by cardiomyocytes to prevent hypoxia during hypertrophic growth, and determinants of PO(2,crit) are estimated using calibrated histochemical methods for succinate dehydrogenase activity, cardiomyocyte cross-sectional area, and myoglobin concentration. The model calculation demonstrates that it is essential to calibrate the histochemical methods, so that absolute values for the relevant parameters are obtained. The succinate dehydrogenase activity, which is proportional to the maximum rate of oxygen consumption, and the myoglobin concentration hardly change while the cardiomyocytes grow. The cross-sectional area of the cardiomyocytes, which increases up to threefold in the right ventricular wall due to pulmonary hypertension in monocrotaline-treated rats, is the most important determinant of PO(2,crit) in this model of myocardial hypertrophy. The relationship between oxygen supply and demand at the level of the cardiomyocyte can be investigated using paired determinations of spatially integrated succinate dehydrogenase activity and capillary density. Hypoxia-inducible factor 1alpha can be demonstrated by immunohistochemistry in cardiomyocytes with high PO(2,crit) and increased spatially integrated succinate dehydrogenase activity, indicating that limited oxygen supply affects gene expression in these cells. We conclude that a mismatch of oxygen supply and demand may develop during hypertrophic growth, which can play a role in the transition from myocardial hypertrophy to heart failure.     

Microscopic,molecular, and biochemical investigations to characterize a benthic cyanoprokaryote Leptolyngbya strainfrom Egypt

Microscopic, molecular, and biochemical investigations were conducted to describe a benthic mat‐forming Leptolyngbya isolate collected from wastewater canal in Helwan area, Egypt. Microscopic examination revealed that the isolate was filamentous, nonheterocystous, with obvious granular surface ornamentation. Electron microscopy was used to reveal the isolate's ultrastructure. Cross walls were thick with uneven deposition. Thylakoids were convoluted and irregularly distributed. Granular content differed from one cell to the other probably due to their physiological stages/position within the filaments and/or their age. Nycridial cells were present. Highly refractile gas vesicle‐like structures were detected and their identity as gas vesicles was confirmed by amplifying the gene coding for the gas vesicle protein GvpA. The presence of gas vesicles in benthic microorganisms is intriguing, and it is possible that those vesicles serve as a floating and dispersal mechanism as they increase in filaments that are about to break and release vacuolated hormogonia. To further confirm the isolate's identity, molecular analysis using 16S rRNA gene was performed. The sequence showed only 94% similarity to Leptolyngbya badia and less than 92% to other leptolyngbya. The phylogenetic analyses showed the coclustering of this strain with other Leptolyngbya strain. The fatty acid composition, used as a chemotaxonomic marker, revealed the presence of a considerable amount of polyunsaturated acids. Nevertheless, saturated fatty acids represented the highest proportion of the total fatty. Surprisingly, fatty acids of relatively limited occurrence within oscillatorian cyanobacteria such as saturated myristic fatty acid and polyunsaturated fatty acid C16:3 were found. Microsc. Res. Tech. 76:249–257, 2013. © 2013 Wiley Periodicals, Inc.     

Molecular Dynamics Simulation on the Aggregation of Lubricant Oxidation Products

The aggregation phenomenon of lubricant oxidation products, fatty acids, fatty alcohols, aliphatic aldehydes and aliphatic ketones in base oil, was probed by molecular dynamics (MD) simulation. The results show that there are aggregates formed in fatty acids or fatty alcohols, and that the aggregation does not occur in aliphatic aldehydes and aliphatic ketones in single component models. Fatty acids or fatty alcohols are connected by hydrogen bonds in the form of dimers or multimers. The hydrogen bond can be formed at a higher temperature in fatty acids, but not in fatty alcohols. In the mixture models, there are dimers and/or multimers constructed by different molecules of lubricant oxidation products, and the multimers have a chain or a ring structure. The number of molecules involved in the formation of aggregates decreases with the increase of temperature. The opportunity to construct hydrogen bonds rises with the increasing concentration of oxidation products. Obvious aggregation occurs at 25 and 100 °C in conditions of high concentration, while it happens only at 25 °C with lower concentration. It is easier for fatty acids and fatty alcohols to form hydrogen bonds and the formed hydrogen bonds are more stable, which result in a higher concentration of fatty acids and fatty alcohols in the aggregates. Fatty acids and fatty alcohols are possibly more significant in the formation of aggregates and it may be beneficial to prevent the aggregation by controlling the concentration of them in lubricating oil.     

Influence of fatty acid composition on the tribological performance of two vegetable‐based lubricants

In light of diminishing natural resources, global climatic change and increased environmental sensitivity, renewable‐based lubricants are being considered potential alternatives to petroleum‐based lubricants. Understanding the tribological performance of vegetable‐based lubricants in relation to their chemical composition is essential for their industrial implementation. This study focuses on the friction and abrasion rate characteristics of soybean and sunflower oils in comparison to a base mineral oil under sliding wear at ambient conditions for various applied loads. It was found that the abrasion rate and friction were the least severe for the soybean, followed by the sunflower oil. The observed trends were attributed to differences in their fatty acid compositions, in particular, a lower percentage of linoleic and oleic acids within the soybean oil. Copyright © 2007 John Wiley & Sons, Ltd.     

牙签电喷雾电离质谱分析脐橙果汁成分

在无需样品预处理的前提下,采用牙签电喷雾电离质谱（wooden-tip ESI-MS）法对脐橙果汁成分进行快速检测。结果表明：在正离子模式下,检测到的主要成分为胆碱、蔗糖、葡萄糖、脯氨酸、正壬醇等,其中胆碱和脯氨酸产生［M+H］⁺信号,蔗糖和葡萄糖产生［M+K］⁺信号;在负离子模式下,检测到的主要成分是脂肪酸、酚酸、羟基酸等有机酸。同时,对抗坏血酸进行了半定量测定,方法检出限为3.7 μg/L,相对标准偏差为7.4%~9.7%,单次检测时间小于0.5 min。该方法无需样品预处理、操作简便、检出限低、分析速度快,有望成为农产品品质分析的一种新方法。