Reasonable management of perennial planting grassland contributes to positive succession of soil microbial community in Sanjiangyuan of Qinghai-Tibetan Plateau,China

合理管理多年生人工建植草地有助于中国青藏高原三江源土壤微生物群落的正向演替 摘要：草地重建是缓解青藏高原三江源“黑土滩”的一种主要方法,同时了解如何管理建植草地也至关重要。而哪种人工管理模式更能有效地恢复“黑土滩”退化草地？为恢复“黑土滩”提供科学依据,我们研究了不同管理模式下人工草地植被特性、土壤理化性质和土壤微生物群落结构的变化,并探讨了不同管理模式对人工草地群落的影响。在本研究中,植被特性和土壤理化性质分别通过实地调查和实验室分析等方法得出,并且运用高通量测序技术测定了土壤微生物群落组成。研究结果表明,在不同管理模式下的人工建植草地植被特性、土壤理化性质和土壤微生物群落结构存在明显差异,而且植被植物多样性、地上生物量、土壤有机碳显著控制着放线菌门和担子菌门。当建植一次时Shannon-Wiener指数、地上生物量和土壤有机碳达到峰值,此时放线菌门和担子菌门所被注释的ASVs的相对丰度显著富集。此外,该管理模式下土壤的细菌多样性最高,真菌多样性最低,土壤逐渐成为“细菌型”土壤。由此得出,建植一次的人工草地植被特性和土壤环境更有利于整体群落的正向演替,是恢复“黑土滩”最合理的管理模式。     

多能干细胞体外分化为类神经管模型的研究进展

近年来多能干细胞(PSCs)的体外培养与分化技术发展迅速,并广泛应用于再生医学和发育生物学等领域。PSCs能够在体外神经诱导的条件下分化为类神经管模型,这为探索体内早期神经发育与中枢神经系统发育疾病的形成机制提供了全新的实验平台。本文总结了近年来应用小鼠和人PSCs建立体外类神经管模型的研究进展,其中体外模型主要包括在不同培养体系下诱导获得的二维(2D)与三维(3D)类神经管模型,并针对早期类神经管模型在神经系统发育性疾病机制研究中的前景和挑战作进一步探讨,同时为疾病预防和治疗提供新的思路。     

Using Serum α-Fetoprotein for Prognostic Prediction in Patients with Hepatocellular Carcinoma: What is the Most Optimal Cutoff?

Background and Aims

The prognostic ability of α-fetoprotein (AFP) for patients with hepatocellular carcinoma (HCC) was examined by using different cutoff values. The optimal AFP cutoff level is still unclear.

Methods

A total of 2579 HCC patients were consecutively enrolled in Taiwan, where hepatitis B is the major etiology of chronic liver disease. Four frequently used AFP cutoff levels, 20, 200, 400, 1000 ng/mL, were investigated. One-to-one matched pairs between patients having AFP higher and lower than the cutoffs were selected by using the propensity model. The adjusted hazard ratios of survival difference were calculated with Cox proportional hazards model.

Results

Patients with a higher AFP level were associated with more severe cirrhosis, more frequent vascular invasion, higher tumor burden and poorer performance status (all p<0.0001). In the propensity model, 4 groups of paired patients were selected, and there was no difference found in the comparison of baseline characteristics (all p>0.05). Patients with AFP <20 ng/mL had significantly better long-term survival than patients with AFP ≧20 ng/mL (p<0.0001), and patients with AFP <400 ng/mL had significantly better overall outcome than patients with AFP ≧400 ng/mL (p = 0.0186). There was no difference of long-term survival between patients divided by AFP levels of 200 and 1000 ng/mL. The adjusted hazard ratios of AFP ≧20 ng/mL and AFP ≧400 ng/mL were 1.545 and 1.471 (95% confidence interval: 1.3–1.838 and 1.178–1.837), respectively.

Conclusions

This study shows the independently predictive ability of baseline serum AFP level in HCC patients. AFP levels of 20 and 400 ng/mL are considered feasible cutoffs to predict long-term outcome in unselected HCC patients.     

Primary Pulmonary Mucoepidermoid Carcinoma: Histopathological and Moleculargenetic Studies of 26 Cases

Introduction

Pulmonary mucoepidermoid carcinoma (PMEC) is an uncommon neoplasm of the lung and the main salivary gland-type lung carcinoma. The aims of this study were to review the clinicopathological and immunohistochemical features of PMEC and characterize the genetic events in PMEC.

Methods

We reviewed the pathology cases in our hospital and found 34 initially diagnosed PMEC cases, 26 of which were confirmed as PMEC after excluding 8 cases of MEC-like pulmonary carcinoma. The clinicopathological characteristics of the 26 PMEC cases and the 8 cases of MEC-like pulmonary carcinoma were retrospectively reviewed. MAML2 rearrangement was detected by fluorescence In Situ Hybridization (FISH). Immunostains of ALK, calponin, collagen IV, CK7, EGFR, HER2, Ki-67, Muc5Ac, p63, p40, and TTF-1 were performed. DNA was extracted from 23 cases of PMEC. Mutation profiling of the EGFR, KRAS, BRAF, ALK, PIK3CA, PDGFRA, and DDR2 genes were carried out using next-generation sequencing (NGS), Sanger sequencing, and quantitative polymerase chain reaction (QPCR) in 9 successfully amplified cases.

Results

Twenty-six cases of PMEC (18 low-grade, 8 high-grade) included 13 men and 13 women aged 12–79 years. Twenty-two cases had a central/endobronchial growth pattern, and 4 cases had a peribronchial growth pattern. Immunohistochemically, CK7, Muc5Ac, p40, and p63 were positive in all cases (26/26);EGFR was positive in 11 cases (11/26); TTF-1, Calponin, HER2 and ALK were negative in all cases (0/26). MAML2 rearrangement was identified in 12 of 18 PMEC cases. No mutations were detected in any of the 7 genes in the 9 cases that qualified for mutation analysis. Twenty-three PMEC patients had follow-up information with a median interval of 32.6 months. Both the 5- and 10-year overall survival rates (OS) were 72.1%, and a high-grade tumor was an adverse prognostic factor in PMEC. There were 8 cases of MEC-like pulmonary carcinoma aged 36–78 years: 2 cases were located in the bronchus, and 6 cases were located in the lung. p63 and TTF-1 were positive in all cases (8/8), p40 was positive in 5 cases (5/8), and ALK was positive in 5 cases (5/8). No cases of MAML2 rearrangement were detected, but there were 5 cases of ALK rearrangement.

Conclusions

PMEC is a primary malignant pulmonary tumor with a relatively good prognosis that is historically characterized by the presence of mucous cells and a lack of keratinization. There are distinct differences between PMEC and MEC-like pulmonary carcinoma in tumor location preference, immunophenotype, and molecular genetics, and the differential diagnosis is critical due to the therapeutic and prognostic considerations.     

Selection of Genes Associated with Variations in the Circle of Willis in Gerbils Using Suppression Subtractive Hybridization

Deformities in the Circle of Willis (CoW) can significantly increase the risk of cerebrovascular disease in humans. However, the molecular mechanisms underlying these deformities have not been understood. Based on our previous studies, variations in the CoW of gerbils are hereditary. A normal CoW is observed in approximately 60% of gerbils, a percentage that also applies to humans. Thus, gerbil is an ideal experimental model for studying variations in the CoW. To study the mechanisms underlying these variations, we selected genes associated with different types of the CoW using suppression subtractive hybridization (SSH). After evaluating the efficiency of SSH using quantitative real-time polymerase chain reaction (qPCR) on subtracted and unsubtracted cDNA and Southern blotting on SSH PCR products, 12 SSH libraries were established. We identified 4 genes (CST3, GNAS, GPx4 and PFN2) associated with variations in the CoW. These genes were identified with qPCR and Western blotting using 70 expressed sequence tags from the SSH libraries. Cloning and sequencing allowed us to demonstrate that the 4 genes were closely related to mouse genes. We may assume that these 4 genes play an important role in the development of variations in the CoW. This study provides a foundation for further research of genes related to development of variations in the CoW and the mechanisms of dysmorphosis of cerebral vessels.     

Discovery of triterpenoids as potent dual inhibitors of pancreatic lipase and human carboxylesterase 1

Pancreatic lipase (PL) is a well-known key target for the prevention and treatment of obesity. Human carboxylesterase 1A (hCES1A) has become an important target for the treatment of hyperlipidaemia. Thus, the discovery of potent dual-target inhibitors based on PL and hCES1A hold great potential for the development of remedies for treating related metabolic diseases. In this study, a series of natural triterpenoids were collected and the inhibitory effects of these triterpenoids on PL and hCES1A were determined using fluorescence-based biochemical assays. It was found that oleanolic acid (OA) and ursolic acid (UA) have the excellent inhibitory effects against PL and hCES1A, and highly selectivity over hCES2A. Subsequently, a number of compounds based on the OA and UA skeletons were synthesised and evaluated. Structure–activity relationship (SAR) analysis of these compounds revealed that the acetyl group at the C-3 site of UA (compound 41) was very essential for both PL and hCES1A inhibition, with IC₅₀ of 0.75 µM and 0.014 µM, respectively. In addition, compound 39 with 2-enol and 3-ketal moiety of OA also has strong inhibitory effects against both PL and hCES1A, with IC₅₀ of 2.13 µM and 0.055 µM, respectively. Furthermore, compound 39 and 41 exhibited good selectivity over other human serine hydrolases including hCES2A, butyrylcholinesterase (BChE) and dipeptidyl peptidase IV (DPP-IV). Inhibitory kinetics and molecular docking studies demonstrated that both compounds 39 and 41 were effective mixed inhibitors of PL, while competitive inhibitors of hCES1A. Further investigations demonstrated that both compounds 39 and 41 could inhibit adipocyte adipogenesis induced by mouse preadipocytes. Collectively, we found two triterpenoid derivatives with strong inhibitory ability on both PL and hCES1A, which can be served as promising lead compounds for the development of more potent dual-target inhibitors targeting on PL and hCES1A.     

A组轮状病毒我国地方株VP6基因的序列测定及其在杆状病毒系统中的表达

通过反转录－ 聚合酶链反应（ Ｒ Ｔ－ Ｐ Ｃ Ｒ） 获得了轮状病毒地方株 Ｔ１１４ Ｖ Ｐ６ 全基因的ｃ Ｄ Ｎ Ａ 片段,将其克隆入转移载体质粒ｐ Ｖ Ｌ１３９３ 中,构建成重组质粒ｐ Ｖ Ｌ１３９３ － Ｖ Ｐ６ 。对克隆的 Ｖ Ｐ６ 基因进行序列测定,并用它和杆状病毒（ Ａｃ Ｍ Ｎ Ｐ Ｖ） 野毒株 Ｄ Ｎ Ａ 共转染 Ｓｆ９ 细胞,筛选纯化得到含 Ｖ Ｐ６ 基因插入片段的重组杆状病毒,并进行了表达重组蛋白 Ｖ Ｐ６ 的检测。测序结果显示 Ｖ Ｐ６ 基因全长１ ３５６ｂｐ ,序列分析显示与 Ｗａ 株非常接近,提示 Ｔ１１４ 为亚组Ⅱ病毒株。用高价免疫血清经 Ｗｅｓｔｅｒｎ ｂｌｏｔ 检测表达产物,结果显示,重组病毒感染细胞裂解液样品中可见大小约４５ｋ Ｄ 的特异条带;亚组Ⅱ特异性单抗检测到大小约１２０ｋ Ｄ 的条带,提示重组蛋白 Ｖ Ｐ６ 获得了表达,具有正常的抗原反应性和天然 Ｖ Ｐ６ 的三体结构。     

共聚焦镜观察凋亡巨噬细胞内pH的变化

用透射电镜观察巨噬细胞的形态学改变,结果显示,地塞米松处理８小时后,大部分巨噬细胞发生凋亡特征变化：胞突缩短、减少,胞膜完整。胞体皱缩,胞质密度增加,其中出现大量空泡。胞核染色质边聚、浓缩。另外用激光扫描共聚焦显微镜（ＡＣＡＳ５７０）和ｐＨ荧光探针ＳＮＡＲＦ┐１／ＡＭ实时检测地塞米松处理巨噬细胞胞浆ｐＨ的动态变化。加入地塞米松,多数巨噬细胞胞浆马上发生快速和短期的碱化。随后,胞浆ｐＨ缓慢降低,胞浆酸化。结果表明,胞浆酸化是细胞凋亡发展的必然过程,胞浆碱化则很可能与细胞凋亡的发生相关,也可能与细胞种类、细胞功能状态相关