Generation of APOE knock-down SK-N-SH human neuroblastoma cells using CRISPR/Cas9: a novel cellular model relevant to Alzheimer’s disease research

APOE ε4 is the major genetic risk factor for Alzheimer’s disease (AD). A precise role for apolipoprotein E (apoE) in the pathogenesis of the disease remains unclear in part due to its expression in multiple cell types of the brain. APOE is highly expressed in astrocytes and microglia, however its expression can also be induced in neurons under various conditions. The neuron-like cell line SK-N-SH is a useful model in the study of the cellular and molecular effects of apoE as it can be differentiated with retinoic acid to express and secrete high levels of apoE and it also shows the same apoE fragmentation patterns observed in the human brain. We previously found that apoE is cleaved into a 25-kDa fragment by high temperature-requirement serine protease A1 (HtrA1) in SK-N-SH cells. To further understand the endogenous functions of apoE, we used CRISPR/Cas9 to generate SK-N-SH cell lines with APOE expression knocked-down (KD). APOE KD cells showed lower APOE and HTRA1 expression than parental SK-N-SH cells but no overt differences in neuritogenesis or cell proliferation compared with the CRISPR/Cas9 control cells. This research shows that the loss of apoE and HtrA1 has a negligible effect on neuritogenesis and cell survival in SK-N-SH neuron-like cells.     

Using life cycle assessment to achieve a circular economy

The International Journal of Life Cycle Assessment - The current global interest in circular economy (CE) opens an opportunity to make society’s consumption and production patterns more...     

Plant ontogenetic changes in vein and stomatal traits and their relationship with economic traits in leaves of three Mediterranean oaks

3种地中海橡树在叶脉和气孔性状上的个体发育性变化及其与叶片经济性状的关系 我们比较了3个地中海栎属(Quercus)树种幼苗和成龄树的叶脉和气孔性状。之前的研究表明,幼苗的气体交换率往往比成龄树的要高。我们的研究目标是确认叶脉和气孔的性状在整个植物个体发育过程中是否会随着气体交换率的变化而同步改变。我们提出了以下两个备选假设：幼苗比成龄树有着更高的叶脉和气孔密度(假设1);幼苗在维管组织上的投资更少,从而降低了建设成本(假设2)。本研究是在西班牙中西部进行的,我们对每个树种不同生长阶段的植株随机采集了10个样本。我们测量了气孔和叶脉的平均性状(单位叶面积里的气孔大小和数量、叶脉密度、叶脉体积、叶脉到表皮的距离),比叶质量和叶片厚度。研究结果表明,细脉密度和单位面积叶脉体积随着树龄的增加而增加,这似乎与气体交换率方面的个体发育趋势不一致。这种差异支持了我们的假设2,说明植株在幼苗阶段减少对维管组织的投资以最大限度地提高生长速度是其当务之急。幼苗叶片中较大的叶脉间距可以由叶脉到表皮的较短距离所补偿。因此,薄叶片可能是幼苗的一个必要性状,它可以让水分沿较短路径输送到蒸发点,而无需对高成本的维管组织进行大量投资。     

Eutrophic status influences the impact of pesticide mixtures and predation on Daphnia pulex populations

Pesticides, nutrients, and ecological stressors such as competition or predation co‐occur in freshwater ecosystems impacted by agriculture. The extent to which combinations of these stressors affect aquatic populations and the role of nutrients availability in modulating these responses requires further understanding. In this study, we assessed how pesticides affecting different taxonomic groups and predation influence the response of Daphnia pulex populations under different trophic conditions. An outdoor experiment was designed following a factorial design, with the insecticide chlorpyrifos, the herbicide diuron, and the predation by Notonecta sp. individuals as key stressors. The single impact of each of these stressors, and their binary and tertiary combinations, was evaluated on D. pulex abundance and population structure under mesotrophic and eutrophic conditions for 21 days. Data were analyzed using generalized linear mixed models estimated by means of a novel Bayesian shrinkage technique. Our study shows a significant influence of each of the evaluated stressors on D. pulex abundance; however, the impacts of the herbicide and predation were lower under eutrophic conditions as compared to the mesotrophic ones. We found that binary stressor interactions were generally additive in the mesotrophic scenario, except for the herbicide–predation combination, which resulted in synergistic effects. The impacts of the binary stressor combinations in the eutrophic scenario were classified as antagonistic, except for the insecticide–herbicide combination, which was additive. The tertiary interaction resulted in significant effects on some sampling dates; however, these were rather antagonistic and resembled the most important binary stressor combination in each trophic scenario. Our study shows that the impact of pesticides on freshwater populations depends on the predation pressure, and demonstrates that the combined effect of pesticides and ecological stressors is influenced by the food availability and organism fitness related to the trophic status of freshwater ecosystems.     

The Phosphorylated Pathway of Serine Biosynthesis Is Essential Both for Male Gametophyte and Embryo Development and for Root Growth in Arabidopsis

This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular development. The expression of PSP1 in the tapetum at critical stages of microspore development suggests that PSP1 activity in this cell layer is essential in pollen development. In addition to embryo death and male sterility, conditional psp1 mutants displayed a short-root phenotype, which was reverted in the presence of Ser. A metabolomic study demonstrated that the PPSB plays a crucial role in plant metabolism by affecting glycolysis, the tricarboxylic acid cycle, and the biosynthesis of amino acids. We provide evidence of the crucial role of the PPSB in embryo, pollen, and root development and suggest that this pathway is an important link connecting primary metabolism with development.     

Inflammation-induced reactive nitrogen species cause proteasomal degradation of dimeric peroxiredoxin-1 in a mouse macrophage cell line

Peroxiredoxin 1 (PRDX1) is an antioxidant enzyme that, when secreted, can act as a proinflammatory signal. Here we studied the regulation of intracellular PRDX1 by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) in the RAW 264.7 mouse macrophage cell line. While LPS or IFN-γ alone did not affect PRDX1 protein levels, their combination led to an almost complete loss of the PRDX1 dimer. This was likely mediated by the increased production of nitric oxide (NO) as it was reversed by the NO synthase inhibitor L-N-methylarginine (L-NMMA), while a NO-releasing agent decreased PRDX1 levels. Inhibition of the proteasome with MG132 also prevented the loss of the PRDX1 dimer, suggesting that the decrease is due to a NO-activated proteasomal degradation pathway. By contrast with the decrease in protein levels, LPS increased PRDX1 mRNA and this effect was amplified by IFN-γ. Two other Nrf2 target genes, thioredoxin reductase (TXNRD1) and haem oxygenase (HMOX1), were also induced by LPS but IFN-γ did not increase their expression further. This study shows that inflammation differentially regulates PRDX1 at the levels of protein stability and gene expression, and that NO plays a key role in this mechanism.     

Placental tissue metabolome analysis by GC-MS: Oven-drying is a viable sample preparation method

Abstract

Analysis of the human placenta metabolome has great potential to advance the understanding of complicated pregnancies and deleterious fetal outcomes in remote populations, but samples preparation can present unique challenges. Herein, we introduce oven-drying as a simple and widely available method of sample preparation that will facilitate investigations of the placental metabolome from remote and under-studied populations. Placentae from complicated and uncomplicated pregnancies were prepared in three ways (oven-dried at 60?°C, fresh, lyophilized) for metabolome analysis via gas chromatography-mass spectrometry (GC-MS). Multiple computer models (e.g. PLS-DA, ANN) were employed to classify and determine if there was a difference in placentae metabolome and a group of metabolites with high variable importance in projection scores across the three preparations and by complicated vs. control groups. The analyses used herein were shown to be thorough and sensitive. Indeed, significant differences were detected in metabolomes of complicated vs. uncomplicated pregnancies; however, there were no statistical differences in the metabolome of placentae prepared by oven-drying vs. lyophilization vs. fresh placentae. Oven-drying is a viable sample preparation method for placentae intended for use in metabolite analysis via GC-MS. These results open many possibilities for researching metabolome patterns associated with fetal outcomes in remote and resource-poor communities worldwide.