Analysis of the HNF4A isoform-regulated transcriptome identifies CCL15 as a downstream target in gastric carcinogenesis

Objective:Hepatocyte nuclear factor 4α (HNF4A) has been demonstrated to be an oncogene in gastric cancer (GC). However, the roles of different HNF4A isoforms derived from the 2 different promoters (P1 and P2) and the underlying mechanisms remain obscure.Methods:The expression and prognostic values of P1- and P2-HNF4A were evaluated in The Cancer Genome Atlas (TCGA) databases and GC tissues. Then, functional assays of P1- and P2-HNF4A were conducted both in vivo and in vitro. High-throughput RNA-seq was employed to profile downstream pathways in P1- and P2-HNF4A-overexpressing GC cells. The expression and gene regulation network of the candidate target genes identified by RNA-seq were characterized based on data mining and functional assays.Results:HNF4A amplification was a key characteristic of GC in TCGA databases, especially for the intestinal type and early stage. Moreover, P1-HNF4A expression was significantly higher in tumor tissues than in adjacent non-tumor tissues (P < 0.05), but no significant differences were found in P2-HNF4A expression (P > 0.05). High P1-HNF4A expression indicated poor prognoses in GC patients (P < 0.01). Furthermore, P1-HNF4A overexpression significantly promoted SGC7901 and BGC823 cell proliferation, invasion and migration in vitro (P < 0.01). Murine xenograft experiments showed that P1-HNF4A overexpression promoted tumor growth (P < 0.05). Mechanistically, RNA-seq showed that the cytokine-cytokine receptor interactions pathway was mostly enriched in P1-HNF4A-overexpressing GC cells. Finally, chemokine (C-C motif) ligand 15 was identified as a direct target of P1-HNF4A in GC tissues.Conclusions:P1-HNF4A was the main oncogene during GC progression. The cytokine-cytokine receptor interaction pathway played a pivotal role and may be a promising therapeutic target.     

The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M₁ muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)–linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M₁ and anti–green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M₁-mEGFP was expressed at levels equal to the M₁ receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M₁-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M₁-mEGFP–expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M₁-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

Measuring and understanding the extent and potential significance of quaternary organization of members of the class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs) have both fascinated and frustrated researchers for many years (1, 2). Over time, a wide range of methods have been applied to address this question, and many different GPCRs have been examined. Outcomes have ranged from assertions that such receptors are monomeric and that results consistent with other conclusions reflect either artifacts of the method of measurement or that studies have been performed at nonphysiological levels of expression of the receptor being studied, to those that have suggested rather stable dimeric or tetrameric complexes (1). Only in the case of rhodopsin, the photon receptor expressed at very high levels (in the range of 24,000–30,000 molecules/µm²) in rod outer segments of the eye, have detailed studies been conducted in situ on a class A GPCR. In this example, various studies have shown that rhodopsin is organized as rows of dimers (3, 4). However, to our knowledge, no other GPCR is expressed natively at levels akin to rhodopsin. As such, although a substantial number of studies, generally performed in transfected cell lines or in artificial bilayer systems, have provided evidence that other GPCRs can and do form dimeric and/or higher-order quaternary complexes in a concentration-dependent manner (1, 2), how levels of expression required to observe such complexes relate to expression levels in native cells and tissues has been poorly defined, as is the stability of such complexes and whether they are regulated by ligand binding.Developments in fluorescence fluctuation analysis (FFA) have facilitated efforts to define the oligomeric status of transmembrane receptor proteins (5, 6). Unlike methods based on resonance energy transfer, only a single fluorophore-linked protein is required to be expressed to use FFA. It is, therefore, more practical to use such methods in native cells and tissues if linked to genome-editing approaches and/or the generation of transgenic “knock-in” animal models in which a receptor of interest is replaced with a fluorophore-tagged, modified form of the receptor. Moreover, the recent introduction of fluorescence intensity fluctuation (FIF) spectrometry (7–10) has overcome issues with other methods based on FFA that result in information being compressed due to averaging of oligomer-size data from interrogated regions of interest (RoIs) in which complex mixtures of oligomers of different sizes may be present (7, 8).To define whether the class A M₁ muscarinic acetylcholine receptor is present in hippocampal and cortical neurons as strict monomers or as a range of monomeric, dimeric, and, potentially, oligomeric complexes, we applied FIF spectrometry to images of such neurons isolated from a line of transgenic mice in which we replaced the M₁ receptor with a form of the receptor that includes C-terminally linked monomeric enhanced green fluorescent protein (mEGFP). We first show that both expression levels and function of the introduced M₁-mEGFP construct appear equivalent to the native M₁ receptor in wild-type (WT) mice, using a range of methods and measures ranging from [³H]ligand binding and cell signaling assays to locomotion. We then demonstrate in hippocampal and cortical neurons that in the basal state, the M₁-mEGFP construct is present as a mixture of monomers and dimeric or oligomeric complexes. We also show that the presence of either an agonist or an antagonist ligand promotes monomerization of the receptor. In these studies, we combined analysis of images of a fluorophore-modified receptor in situ with calculation of receptor oligomer complexity. The studies provide a clear and unambiguous answer to a long-standing question that has been the subject of considerable debate (11–13) but that has previously been restricted to studies performed on transfected cell lines. Moreover, these studies are a model for subsequent studies for researchers who plan to explore the topic of dimerization of rhodopsin-family GPCRs.     

抑郁症神经生化机制的研究进展

抑郁症是一种病因相当复杂的疾病,近年来发现抑郁症患者在神经生化方面有许多改变。该文就近年来在抑郁症的神经递质及其受体和转运蛋白、G蛋白和第二信使系统以及神经内分泌系统等方面的研究进展加以综述。     

A case report of IgG4-related respiratory disease with pleural effusion and a literature review

Rationale:IgG4-related respiratory disease (IgG4-RRD) is a chronic autoimmune disease that affects the respiratory system and organs outside the respiratory system. This study explored the diagnosis and treatment of a case of IgG4-RRD with unilateral pleural effusion diagnosed using medical thoracoscopy, and provides an associated literature review. This report summarizes the clinical characteristics of IgG4-RRD involving the pleura to improve the diagnosis of this disease.Patient concerns:A 39-year-old man presented with a 2-week history of cough and chest tightness. Both physical examination and imaging supported the presence of left pleural effusion.Diagnosis:Medical electronic thoracoscopy was performed to obtain a pleural biopsy, which showed lymphoplasmacytic infiltration, 40 IgG4+ plasma cells per High Power Field (HPF) on microscopy, IgG4/IgG ratio >50%, phlebitis obliterans, and storiform fibrosis. The final diagnosis was IgG4-RRD.Interventions and outcomes:The patient was treated with methylprednisolone, after which his symptoms improved, and he was discharged. Oral hormone therapy was continued outside the hospital. After 4 months, the patient returned to the hospital and his condition had improved significantly.Lessons:Pleural involvement in IgG4-RRD is rare, and its diagnosis depends on pleural biopsy. Thoracoscopy usually reveals pleural thickening, pleural nodules, and milky white plaques.     

基于3G的临床实习生教育移动学习资源设计与开发

分析了基于3G移动通讯技术的移动学习特点,探讨了移动学习中学习资源的开发原则,并提出了基于Wap浏览移动学习模式的学习管理系统的设计方案。     

青霉素、硫酸庆大霉素对红剑鱼、孔雀鱼和食蚊鱼的急性毒性实验

目的 :观察青霉素、硫酸庆大霉素对红剑鱼、孔雀鱼和食蚊鱼的急性毒性影响 ,以探讨红剑鱼、孔雀鱼和食蚊鱼作为水生实验动物的可行性及对药物急性毒性的检测效果。方法 :用水毒理学方法测定它们对三种鱼类的 2 4hLC50 。结果 :青霉素对红剑鱼、孔雀鱼和食蚊鱼的急性毒性观察结果 ,2 4hLC50 值分别为 2 83.0 6mg/L、2 79.92mg/L、2 89.4 4mg/L ;硫酸庆大霉素对红剑鱼、孔雀鱼和食蚊鱼的急性毒性观察结果 ,2 4hLC50 值分别为 2 95 .97ug/L、2 73.74ug/L、30 9.0 3ug/L。结论 :红剑鱼、孔雀鱼和食蚊鱼可作为急性毒性的试验材料。     

癌源性免疫球蛋白G全分子相互作用蛋白质的质谱鉴定及其功能分析

目的鉴定与癌源性免疫球蛋白G（IgG）全分子相互作用的蛋白质,为研究其生物功能提供重要的线索。方法分别用兔
抗人IgG全分子抗体和兔IgG在宫颈癌细胞系HeLa中进行免疫共沉淀,将得到的免疫复合物经电泳分离后进行银染。经比较
分析后3条差异条带切下来做质谱鉴定,质谱数据经Swiss-Prot数据库分析。最后对所得的蛋白质进行筛选和功能注释分析。
结果最终得到6个可能与癌源性IgG全分子相互作用的蛋白质。结论通过免疫共沉淀结合质谱分析的方法最终得到了6个
可能与癌源性IgG全分子相互作用的蛋白质,为其功能研究提供了重要线索。
     

Hirschsprung-associated enterocolitis develops independently of NOD2 variants

Backgroud/Purpose

Hirschsprung-associated enterocolitis (HAEC) represents a cause for significant pre- and postoperative morbidity and mortality in Hirschsprung disease (HD). Although multiple studies on HAEC have been performed and several mechanisms have been presumed, the pathogenesis of this condition remains unclear. As changes in colonic mucosal defense are key factors suggested in both Crohn's disease (CD) and HAEC pathogenesis, the aim of the current study was to investigate genetic alterations in the most important susceptibility gene for Crohn's enterocolitis (NOD2) to see whether carriers of polymorphisms within the NOD2 gene are predisposed to the development of HAEC.

Methods

Genotyping for the NOD2 variants in exon 4 (p.Arg702Trp [rs2066844]), exon 8 (p.Gly908Arg [rs2066845]), and exon 11 (p.1007fs [rs2066847]) was performed in 52 white children with HD (41 boys, 11 girls), 152 healthy controls, and 152 children with CD (onset of disease <17 years; mean, 11.8 years). Seventeen patients with HD (32.7%) were carriers of a RET germline mutation, 35 children (67.3%) had short segment disease, and 17 (32.7%) had long segment disease.

Results

Ten children (19.2%) with HD were heterozygous carriers of at least one NOD2 variant vs 17 (11.2%) in the healthy control group and 69 (45.4%) in the CD cohort. Hirschsprung-associated enterocolitis was observed in 7 children (13.5%), with 4 having short segment HD and 3 with long segment HD; but none of them were carriers of NOD2 variants.

Conclusion

Our study shows that NOD2 variants described to be causatively associated with CD do not predispose to the development of HAEC. As data on the molecular basis of HAEC are limited, the distinct mechanisms involved in the pathogenesis of this complication remain unclear.     

Genotoxicity studies of glycidol fatty acid ester (glycidol linoleate) and glycidol

Glycidol fatty acid esters (GEs) are found in refined edible oils. Safety concerns have been alleged due to the possible release of glycidol (G), an animal carcinogen.