Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction

Assembly of cell wall polysaccharides into specific patterns is required for plant growth. A complex of RAPID ALKALINIZATION FACTOR 4 (RALF4) and its cell wall-anchored LEUCINE-RICH REPEAT EXTENSIN 8 (LRX8)-interacting protein is crucial for cell wall integrity during pollen tube growth, but its molecular connection with the cell wall is unknown. Here, we show that LRX8-RALF4 complexes adopt a heterotetrametric configuration in vivo, displaying a dendritic distribution. The LRX8-RALF4 complex specifically interacts with demethylesterified pectins in a charge-dependent manner through RALF4's polycationic surface. The LRX8-RALF4-pectin interaction exerts a condensing effect, patterning the cell wall's polymers into a reticulated network essential for wall integrity and expansion. Our work uncovers a dual structural and signaling role for RALF4 in pollen tube growth and in the assembly of complex extracellular polymers

16p11.2 600 kb Duplications confer risk for typical and atypical Rolandic epilepsy

Rolandic epilepsy (RE) is the most common idiopathic focal childhood epilepsy. Its molecular basis is largely unknown and a complex genetic etiology is assumed in the majority of affected individuals. The present study tested whether six large recurrent copy number variants at 1q21, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 previously associated with neurodevelopmental disorders also increase risk of RE. Our association analyses revealed a significant excess of the 600 kb genomic duplication at the 16p11.2 locus (chr16: 29.5-30.1 Mb) in 393 unrelated patients with typical (n = 339) and atypical (ARE; n = 54) RE compared with the prevalence in 65 046 European population controls (5/393 cases versus 32/65 046 controls; Fisher's exact test P = 2.83 × 10−6, odds ratio = 26.2, 95% confidence interval: 7.9-68.2). In contrast, the 16p11.2 duplication was not detected in 1738 European epilepsy patients with either temporal lobe epilepsy (n = 330) and genetic generalized epilepsies (n = 1408), suggesting a selective enrichment of the 16p11.2 duplication in idiopathic focal childhood epilepsies (Fisher's exact test P = 2.1 × 10−4). In a subsequent screen among children carrying the 16p11.2 600 kb rearrangement we identified three patients with RE-spectrum epilepsies in 117 duplication carriers (2.6%) but none in 202 carriers of the reciprocal deletion. Our results suggest that the 16p11.2 duplication represents a significant genetic risk factor for typical and atypical R

Evaluation of presumably disease causing SCN1A variants in a cohort of common epilepsy syndromes

Objective: The SCN1A gene, coding for the voltage-gated Na+ channel alpha subunit NaV1.1, is the clinically most relevant epilepsy gene. With the advent of high-throughput next-generation sequencing, clinical laboratories are generating an ever-increasing catalogue of SCN1A variants. Variants are more likely to be classified as pathogenic if they have already been identified previously in a patient with epilepsy. Here, we critically re-evaluate the pathogenicity of this class of variants in a cohort of patients with common epilepsy syndromes and subsequently ask whether a significant fraction of benign variants have been misclassified as pathogenic. Methods: We screened a discovery cohort of 448 patients with a broad range of common genetic epilepsies and 734 controls for previously reported SCN1A mutations that were assumed to be disease causing. We re-evaluated the evidence for pathogenicity of the identified variants using in silico predictions, segregation, original reports, available functional data and assessment of allele frequencies in healthy individuals as well as in a follow up cohort of 777 patients. Results and Interpretation: We identified 8 known missense mutations, previously reported as path

Endocytic and Secretory Traffic in Arabidopsis Merge in the Trans-Golgi Network/Early Endosome, an Independent and Highly Dynamic Organelle[W]

This study examines secretory and endocytotic trafficking in Arabidopsis by tracking the movement of a brassinosteroid receptor and a boron exporter through the endomembrane system. Both endocytotic and secretory cargo travel through the trans-Golgi network/early endosome (TGN/EE), and the TGN/EE is shown to be an independent organelle that only transiently associates with the Golgi

A suberized exodermis is required for tomato drought tolerance.

Plant roots integrate environmental signals with development using exquisite spatiotemporal control. This is apparent in the deposition of suberin, an apoplastic diffusion barrier, which regulates flow of water, solutes and gases, and is environmentally plastic. Suberin is considered a hallmark of endodermal differentiation but is absent in the tomato endodermis. Instead, suberin is present in the exodermis, a cell type that is absent in the model organism Arabidopsis thaliana. Here we demonstrate that the suberin regulatory network has the same parts driving suberin production in the tomato exodermis and the Arabidopsis endodermis. Despite this co-option of network components, the network has undergone rewiring to drive distinct spatial expression and with distinct contributions of specific genes. Functional genetic analyses of the tomato MYB92 transcription factor and ASFT enzyme demonstrate the importance of exodermal suberin for a plant water-deficit response and that the exodermal barrier serves an equivalent function to that of the endodermis and can act in its place

利用即時聚合酶連鎖反應及酵素免疫連結分析法評估黃連素對BALB/c小鼠脾臟細胞分泌細胞激素之影響

In Chinese medicine, bitter foods are described as “heat-removing” agents for reducing fever and have been widely used in therapeutic application. The major bitter compounds in bitter foods are alkaloids, such as aloperine, amygdalin, berberine, crotaline and naringenin. The bitter compounds are suggested to have multiple physiological functions, including anti-pyretic, anti-nociceptive and anti-tumor activities. However, there are few discussions on immunomodulatory effects of bitter compounds. Therefore, the objective of this study was to investigate the anti-inflammatory effects of five selected bitter compounds including aloperine, amygdalin, berberine, crotaline and naringenin administrations on cytokine secretions by BALB/c mouse splenocytes under four different in vitro experimental models. The potent bitter compound, berberine, was further selected to evaluate its immunomodulatory mechanism using real time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) methods. In this study, there were four different in vitro experimental models used to detect the immunomodulatory effects of bitter compound administrations on BALB/c primary splenocytes. In the spontaneous splenocyte culture model, the primary splenocytes were just co-cultured with samples. In the inflammatory model, the lipopolysaccharide (LPS)-stimulated splenocytes were co-cultured with samples. In the preventive model, splenocyte cultures were first incubated with samples, then washed out samples, and finally stimulated with LPS. In the repair model, splenocyte cultures were first stimulated with LPS, then washed out LPS, and finally treated with samples. In the first part, the results showed that five selected bitter compounds including aloperine, amygdalin, berberine, crotaline and naringenin, significantly (P<0.05) decreased the productions of pro-inflammatory cytokines by spontaneous splenocytes, such as interleukin (IL)-6 or tumor necrosis factor alpha (TNF-α). The levels of IL-6 and TNF-α significantly decreased in the inflammatory and preventive models at moderate concentrations of the selected bitter compounds expect crotaline. In addition, the ratios of pro-/anti- inflammatory cytokine, displayed as either IL-6/IL-10 or TNF-α/IL-10, also significantly diminished in both inflammatory and preventive models by the selected bitter compounds expect crotaline. It is suggested that the selected bitter compounds expect crotaline might have anti-inflammatory effects in vitro. Among the five selected bitter compounds, berberine has a significant effect on inhibiting the inflammation induced by LPS. Therefore, berberine was further administered to splenocyte cultures to detect its regulation on gene expression. In the second part, an investigation was further done to clarify the effect of berberine on cytokine mRNA expressions of primary splenocyte. Results showed that berberine had no significant effect on the mRNA expressions of IL-6, IL-10 and TNF-α, suggesting that berberine administration might affect cytokine expression via post-transcriptional regulation. However, berberine administration could down-regulate the gene expression ratio of Th2 (IL-5)/Th1 (IL-2) cytokines in the inflammatory model. The results suggested that berberine administration might have potential effects on modulating the Th2/Th1 immune balance toward the Th1 pole in vitro. In conclusion, berberine administration at moderate concentrations may significantly decrease the inflammation induced by LPS in vitro, via decreasing the ratio of pro-/anti-inflammatory cytokine (IL-6/IL-10 or TNF-α/IL-10). Besides, it is suggested that berberine could modulate immune responses through down-regulating the gene expression ratio of Th2/Th1 cytokines.傳統中醫普遍認為苦味食物具有良好的”退火”功效，已經被廣泛運用在食療保健上。苦豆鹼(aloperine)、苦杏仁苷(amygdalin)、黃連素(berberine)、野百合鹼(crotaline)以及柚苦苷(naringenin)等，為苦味食物中主要的苦味生物鹼，研究證實其具有許多生理活性，包含退燒、鎮痛以及抗癌等活性，但對苦味物質的免疫調節功能，至今仍無完整研究，因此本篇主要研究目的為，選取五種苦味物質：苦豆鹼、苦杏仁苷、黃連素、野百合鹼以及柚苦苷，在動物體外四種不同實驗模式下，探討其抗發炎活性，並從其中選出黃連素，利用即時聚合酶連鎖反應及酵素免疫連結分析法評估苦味物質黃連素對BALB/c小鼠脾臟細胞分泌細胞激素之影響，以探討其免疫調節功能。本研究使用四種不同體外實驗來評估苦味物質對BALB/c小鼠初代脾臟細胞之免疫調節作用，實驗設計如下：單獨添加樣品與初代脾臟細胞共同培養(單獨添加樣品模式)、以內毒素脂多醣(lipopolysaccharide, LPS)刺激脾臟細胞同時添加樣品與細胞共同培養(發炎模式)、樣品先與脾臟細胞培養，洗去樣品，再加入LPS刺激脾臟細胞(預防模式)以及先以LPS刺激脾臟細胞，洗去LPS，再加入樣品與脾臟細胞培養(恢復模式)。 第一部分研究結果顯示，單獨添加五種苦味物質苦豆鹼、苦杏仁苷、黃連素、野百合鹼以及柚苦苷與脾臟細胞共同培養，可顯著抑制促發炎細胞激素介白質(interleukin, IL)-6或腫瘤壞死因子-α (tumor necrosis factor alpha, TNF-α)分泌量。脾臟細胞在發炎及預防的模式下，添加苦豆鹼、苦杏仁苷、黃連素以及柚苦苷可顯著抑制促發炎細胞激素分泌量，並調節促發炎IL-6 (TNF-α)/抗發炎細胞激素IL-10分泌量的比值，顯示苦味物質具有抗發炎的潛力，而五種苦味物質中又以黃連素抗發炎的能力最顯著，故以黃連素探討其對初代脾臟細胞細胞激素基因表現量之影響。 第二部分研究黃連素在不同細胞培養模式下對脾臟細胞細胞激素mRNA表現量的影響，發現黃連素對IL-6、IL-10及TNF-α mRNA的表現無顯著調控作用，推測黃連素對脾臟細胞細胞激素分泌之調控，可能屬於轉錄後調節作用(post-transcriptional regulation)。另外，在發炎模式下，黃連素可以下調Th2 (IL-5)/Th1 (IL-2)細胞激素mRNA表現量的比值，顯示黃連素具有調節免疫的功能，可使免疫平衡傾向Th1反應。 綜合本實驗結果發現，黃連素可以藉由降低促發炎/抗發炎細胞激素(IL-6/IL-10或TNF-α/IL-10)分泌量的比值，而減緩LPS所誘導的發炎反應。除此之外，黃連素可以下調Th2/Th1細胞激素mRNA表現量的比值，進而調節小鼠脾臟細胞的免疫反應變化。總目錄 .......................................................... i 表目錄 .......................................................... vi 圖目錄 .......................................................... ix 縮寫對照表 ...................................................... x 中文摘要 ........................................................ xi 英文摘要 ........................................................ xiii 第一章 緒言 ................................................ 1 第二章 文獻回顧 ............................................ 2 第一節 免疫介紹 ............................................ 2 一、 免疫系統 ........................................ 2 (一) 先天性免疫反應 ................................. 3 (二) 適應性免疫反應 ................................. 5 二、 發炎反應 ........................................ 7 三、 輔助型T細胞第一型及第二型之平衡 ................ 8 四、 細胞激素簡介 .................................... 8 五、 細胞激素基因表現與蛋白質生成量的關係 ............ 10 第二節 苦味物質介紹 ........................................ 11 一、 五種苦味物質之相關研究與介紹 .................... 11 (一) 苦豆鹼(aloperine) ................................ 11 (二) 苦杏仁苷(amygdalin) ............................. 11 (三) 黃連素(berberine) ................................ 12 (四) 野百合鹼(crotaline) ............................... 13 (五) 柚苦苷(naringenin) ............................... 14 第三節 研究動機及目的 ...................................... 14 第四節 實驗設計 ............................................ 16 第三章 材料與方法 ......................................... 18 第一節 五種苦味物質樣品之製備與其對初代脾臟細胞體外免疫調節 功能之研究 .......................................... 18 一、 五種苦味物質樣品之製備 .......................... 18 二、 免疫初代細胞之取得與培養 ........................ 20 (一) 免疫初代細胞之來源 ............................ 20 (二) 脾臟細胞之取得與培養 .......................... 21 三、 五種苦味物質對小鼠初代脾臟細胞存活率之評估 ...... 22 四、 不同實驗模式下五種苦味物質對小鼠脾臟細胞分泌細胞 激素之影響 ...................................... 24 (一) 模式A：單獨添加樣品對細胞的影響 .............. 24 (二) 模式B：模擬在發炎模式下內毒素脂多醣與樣品同時 存在對細胞的影響 ............................. 24 (三) 模式C：模擬在預防模式下樣品對細胞的影響 ..... 24 (四) 模式D：模擬在恢復模式下樣品對細胞的影響 ..... 25 (五) 脾臟細胞細胞激素之測定 ....................... 25 五、 統計分析 ........................................ 27 第二節 小鼠脾臟細胞培養不同時間其TNF-α, IL-6, IL-10, IL-4, IL-5 與IL-2 mRNA表現的變化 ............................. 27 一、 初代脾臟細胞自發性及脂多醣刺激下之培養 .......... 27 (一) 初代脾臟細胞之自發性培養 ..................... 27 (二) 初代脾臟細胞以脂多醣誘導發炎狀態之培養 ....... 28 二、 以real-time PCR (RT-PCR)方法分析初代脾臟RNA表現量 28 (一) 總RNA之抽取 ................................ 28 (二) RNA電泳 .................................... 29 (三) RNA轉成cDNA (Reverse transcription reaction) ..... 31 (四) Real time polymerase chain reaction (RT-PCR) ....... 33 三、 統計分析 ........................................ 38 第三節 黃連素對小鼠脾臟細胞細胞激素mRNA表現量之影響 ..... 38 一、 不同實驗模式下黃連素對初代脾臟細胞細胞激素mRNA 表現量之影響 .................................... 38 (一) 模式a：單獨添加黃連素對脾臟細胞的影響 ........ 38 (二) 模式b：模擬在發炎模式下脂多醣與黃連素同時存在對 細胞的影響 .................................... 38 (三) 模式c：模擬在預防模式下添加黃連素對細胞的影響.. 39 (四) 模式d：模擬在恢復模式下添加黃連素對細胞的影響.. 39 二、 統計分析 ......................................... 39 第四章 結果 ................................................. 40 第一節 五種苦味物質對初代脾臟細胞體外免疫調節功能之研究 .... 40 一、 五種苦味物質對BALB/c小鼠初代脾臟細胞存活率之影響 40 二、 不同實驗模式下五種苦味物質對小鼠脾臟細胞分泌細胞激 素之影響 ......................................... 47 (一) 模式A：單獨添加樣品對脾臟細胞的影響 .......... 47 (二) 模式B：模擬在發炎狀態下脂多醣與樣品同時存在對 脾臟細的影響 ................................. 53 (三) 模式C：模擬在預防模式下樣品對脾臟細胞的影響... 60 (四) 模式D：模擬在恢復模式下樣品對脾臟細胞的影響... 66 第二節 小鼠脾臟細胞培養不同時間其TNF-α, IL-6, IL-10, IL-4, IL-5 與IL-2 mRNA表現的變化 ............................. 73 第三節 黃連素對小鼠脾臟細胞細胞激素mRNA表現量之影響 ..... 75 (一) 添加黃連素在實驗模式a下對BALB/c小鼠脾臟細胞 細胞激素mRNA表現量之影響 .................. 75 (二) 添加黃連素在實驗模式b下對BALB/c小鼠脾臟細胞 細胞激素mRNA表現量之影響 .................. 77 (三) 添加黃連素在實驗模式c下對BALB/c小鼠脾臟細胞 細胞激素mRNA表現量之影響 .................. 79 (四) 添加黃連素在實驗模式d下對BALB/c小鼠脾臟細胞 細胞激素mRNA表現量之影響 .................. 81 第五章 討論 ................................................. 83 一、 五種苦味物質對初代脾臟細胞體外免疫調節功能之研究 ..... 83 二、 黃連素對小鼠脾臟細胞細胞激素mRNA表現量之影響 ...... 87 第六章 結論 ................................................. 92 第七章 參考文獻 ............................................. 9

Las cartas de patrocinio

Las cartas de patrocinio son un instrumento jurídico con muy poca tradición en la jurisprudencia española que se ha hecho bastante popular recientemente en el ámbito financiero de los grupos de sociedades debido tanto a la facilidad o simpleza con la que se emiten, como a las ventajas fiscales que ostentan frente a otras garantías clásicas (fianza, aval…). El Tribunal Supremo se ha visto obligado desde 1985, a pronunciarse sobre sus efectos e interpretación de un instrumento que no estaba regulado ni siquiera en el Código Civil. No sin problemas en su interpretación, se ha diferenciado entre cartas débiles y fuertes, pasando en estas últimas de ser equiparadas a una fianza a considerarse que generan una obligación contractual para la emisora de la carta.Comfort letters are a legal instrument with not much tradition in Spanish Law which have recently become very popular in financial aspects of business holdings. Their rising popularity has been possible due to the facility and simplicity being issued and also because of the fiscal benefits they have against other security interests (bond, endorsement…). The High Court has been forced since 1985 to make statement about the Comfort Letters effects and their interpretation because they were not even regulated in the Spanish Civil Code. Counting also with some interpretation troubles, it has been also possible to discern between weak and strong comfort letters. The last ones, were compared to a bond and now they are considered a contractual obligation for their issuer.Graduado o Graduada en Administración y Dirección de Empresas por la Universidad Pública de NavarraEnpresen Administrazio eta Zuzendaritzan Graduatua Nafarroako Unibertsitate Publikoa

Exome-wide analysis of mutational burden in patients with typical and atypical Rolandic epilepsy

Rolandic epilepsy (RE) is the most common focal epilepsy in childhood. To date no hypothesis-free exome-wide mutational screen has been conducted for RE and atypical RE (ARE). Here we report on whole-exome sequencing of 194 unrelated patients with RE/ARE and 567 ethnically matched population controls. We identified an exome-wide significantly enriched burden for deleterious and loss-of-function variants only for the established RE/ARE gene GRIN2A. The statistical significance of the enrichment disappeared after removing ARE patients. For several disease-related gene-sets, an odds ratio >1 was detected for loss-of-function variants

Investigation of GRIN2A in common epilepsy phenotypes

Recently, mutations and deletions in the GRIN2A gene have been identified to predispose to benign and severe idiopathic focal epilepsies (IFE), revealing a higher incidence of GRIN2A alterations among the more severe phenotypes. This study aimed to explore the phenotypic boundaries of GRIN2A mutations by investigating patients with the two most common epilepsy syndromes: (i) idiopathic generalized epilepsy (IGE) and (ii) temporal lobe epilepsy (TLE). Whole exome sequencing data of 238 patients with IGE as well as Sanger sequencing of 84 patients with TLE were evaluated for GRIN2A sequence alterations. Two additional independent cohorts comprising 1469 IGE and 330 TLE patients were screened for structural deletions (>40. kb) involving GRIN2A. Apart from a presumably benign, non-segregating variant in a patient with juvenile absence epilepsy, neither mutations nor deletions were detected in either cohort. These findings suggest that mutations in GRIN2A preferentially are involved in genetic variance of pediatric IFE and do not contribute significantly to either adult focal epilepsies as TLE or generalized epilepsies