基于加权基因共表达网络识别糖尿病视网膜病变与免疫相关的关键基因

目的:通过生物信息学的方法探究糖尿病视网膜病变(DR)中免疫相关的关键基因以及免疫细胞的浸润情况。方法:2022-09/10从GEO数据库获取基因芯片数据集,采用“limma”R包获得差异表达基因(DEGs),并进行GO功能注释和KEGG通路富集分析,基于CIBERSORT算法分析免疫细胞浸润情况。通过加权基因共表达网络分析(WGCNA)筛选与免疫相关基因模块中的DEGs,利用STRING在线数据库及Cytoscape软件构建蛋白质互作网络,利用MCODE以及cytoHubba插件进一步并筛选出关键基因。结果:共筛选出上调差异基因1 426个,下调差异基因206个。原始B细胞、血浆细胞、记忆型CD4⁺T细胞、调节性T细胞(Tregs)、M0型巨噬细胞、M1型巨噬细胞以及中性粒细胞7种免疫细胞显著高表达(P<0.05);NK cells activated这种免疫细胞低表达(P<0.05)。WGCNA分析后,与免疫最相关模块中差异基因820个,构建PPI网络后利用插件筛选出10个关键基因,利用各差异基因在PPI中的相关性程度进一步筛选出2个关键基因为DL...     

Prognostic value of sorting nexin 10 weak expression in stomach adenocarcinoma revealed by weighted gene coexpression network analysis

AIM To detect significant clusters of co-expressed genes associated with tumorigenesis that might help to predict stomach adenocarcinoma(SA) prognosis.METHODS The Cancer Genome Atlas database was used to obtain RNA sequences as well as complete clinical data of SA and adjacent normal tissues from patients. Weighted gene co-expression network analysis(WGCNA) was used to investigate the meaningful module along with hub genes. Expression of hub genes was analyzed in 362 paraffin-embedded SA biopsy tissues by immunohistochemical staining. Patients were classified into two groups(according to expression of hub genes): Weak expression and over-expression groups. Correlation of biomarkers with clinicopathological factors indicated patient survival.RESULTS Whole genome expression level screening identified 6,231 differentially expressed genes. Twenty-four coexpressed gene modules were identified using WGCNA. Pearson's correlation analysis showed that the tan module was the most relevant to tumor stage(r = 0.24, P = 7 × 10-6). In addition, we detected sorting nexin(SNX)10 as the hub gene of the tan module. SNX10 expression was linked to T category(P = 0.042, χ~2 = 8.708), N category(P = 0.000, χ~2 = 18.778), TNM stage(P = 0.001, χ~2 = 16.744) as well as tumor differentiation(P = 0.000, χ~2 = 251.930). Patients with high SNX10 expression tended to have longer diseasefree survival(DFS; 44.97 mo vs 33.85 mo, P = 0.000) as well as overall survival(OS; 49.95 vs 40.84 mo, P = 0.000) in univariate analysis. Multivariate analysis showed that dismal prognosis could be precisely predicted clinicopathologically using SNX10 [DFS: P = 0.014, hazard ratio(HR) = 0.698, 95% confidence interval(CI): 0.524-0.930, OS: P = 0.017, HR = 0.704, 95%CI: 0.528-0.940].CONCLUSION This study provides a new technique for screening prognostic biomarkers of SA. Weak expression of SNX10 is linked to poor prognosis, and is a suitable prognostic biomarker of SA.     

Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system

Objective

CYP1A2 and NADPH-CYP450 oxidoreductase (POR) were expressed in the baculovirus/Spodoptera frugiperda (sf9) system. The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR.

Methods

The heme precursors [δ-Aminolaevulinic Acid (5-ALA), Fe³⁺ and hemin] were introduced into the system to evaluate their effects on the expression of CYP1A2, POR and their co-expression. All the proteins were identified using immunoblotting, CO-difference spectroscopy, or cytochrome c assay.

Results

In the present study, functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system, and both of them showed high activities. Co-addition of 5-ALA and Fe³⁺ significantly improved expression of CYP1A2 by about 50% compared with the addition of 5-ALA, Fe³⁺ or hemin alone. Either co-addition of 5-ALA and Fe³⁺ or addition of 5-ALA or Fe³⁺ alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control (no addition). However, unlike CYP1A2, there was no difference between the co-addition and addition of these heme precursors alone. Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR, with a 3:1 ratio of BvCYP1A2 / BvPOR significantly increasing their co-expression. Surprisingly, the addition of 0.1 mM 5-ALA or Fe³⁺ alone, but not their co-addition, could significantly improve the CYP1A2 and POR co-expression (P < 0.05).

Conclusion

5-ALA and Fe³⁺ increased the expression of CYP1A2 and POR in a baculovirus/sf9 system, but the pattern of their expression was different between their expression alone and co-expression.     

加权重基因共表达网络分析识别慢性肾脏病 足细胞损伤关键节点基因MAGI 2

目的 通过加权基因共表达网络分析（WGCNA）识别慢性肾脏病（CKD）足细胞损伤相关的基 因模块与关键基因。方法 从基因表达综合数据库（GEO）中下载包含足细胞损伤体外模型及CKD 患者肾小 球组份的表达谱GSE66107、GSE93798、GSE30528、GSE32591 4 个数据集。利用R 软件包做数据预处理并选 取错误发现率（FDR）<0.05 及差异倍数≥ 1.5 作为差异表达基因（DEG）;结合数据集GSE993395（133 例 CKD 患者肾小球样本）用WGCNA 进行模块化分析并对模块内基因进行功能注释（GO）,根据GO 结果和 含有文献报道的足细胞标准基因（PSG ）情况确定足细胞损伤相关模块,根据模块内基因连接度（Kwithin） 挑选枢纽（hub ）基因;利用Cytoscape 软件及插件Cluego 构建足细胞损伤相关模块基因的加权共表达网络。 结果 4 个数据集共得到 趋势一致的DEG 7 957 个,其中15 个DEG 在4 个数据集中均有差异（coDEG）。 GSE993395 中包含的4 031 个DEG 被用于WGCNA 分析;最终得到12 个基因模块,其中green 模块包含最多的 coDEG 和PSG,进一步通过Kwithin 发现其中同时作为coDEG 和PSG 的MAGI 2 基因是其hub 基因之一。结论 WGCNA 表达网络分析方法是一个高效的系统生物学方法,应用该方法发现CKD 时关键节点基因MAGI 2 在 足细胞损伤中可能起到重要作用。     

CD4+ CD8+ (thymocyte-like) T lymphocytes present in blood and skin from patients with atopic dermatitis suggest immune dysregulation

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease expressed early in life. Disease development is primarily determined by as yet unknown genetic factors, leading to the accumulation of activated T lymphocytes in the skin. OBJECTIVES: To investigate the nature of these T cells. METHODS: T-cell lines could be established from AD skin biopsies, but not from normal skin or AD peripheral blood, when placed in RPMI 1640 medium with 10% human AB serum, antibiotics, and the T-lymphocyte growth factors interleukins 2 and 4. The cell lines were subjected to phenotypic analysis using a fluorescence-activated cell sorter and compared with lymphocytes from AD and normal control peripheral blood. RESULTS: T-cell lines from 22 of 24 consecutive skin biopsies taken from 24 adult patients with AD were established. All cells were T lymphocytes expressing several activation markers. A significant proportion of the lymphocytes had stable expression of a CD4+ CD8+ phenotype (26% +/- 6%; mean +/- SEM). Such double-positive T lymphocytes are normally only seen in the thymus and not in the peripheral immune system. CD4+ CD8+ cells in peripheral blood of the patients (12.5% +/- 3.3%) were also detected. CONCLUSIONS: We suggest that a basic pathophysiological change in AD may be a faulty maturation of the T-lymphocyte system, leading to skin inflammation with CD4+ CD8+ T lymphocytes resembling immature T cells. This is likely to lead to skewing of many immune reactions in the patients.     

Chinese infant recovers from bird flu infection

It has been well documented that bone morphogenetic .proteins （BMPs）, a group of proteins belonging to the transforming growth factor-β （TGFβ） superfamily, can induce bone formation, both in vivo and in vitro. Bone morphogenetic protein-2 （BMP2） is a potent osteoinductive factor and is being evaluated as a bone growth inducer for orthopedic applications.1 Vascular endothelial growth factor （VEGF）, the best-characterized angiogenic factor,     

前列腺癌中致病基因与核心转录因子的识别与功能分析

目的：通过分析识别前列腺癌功能基因模块并挖掘影响这些模块的核心转录因子与关键基因,探讨前列腺癌的发病 机制。方法：通过加权基因共表达网络分析（weighted gene co-expressed network analysis, WGCNA）识别与前列腺癌病理分期、 Gleason分级等重要临床特征相关的枢纽基因模块,利用OPOSSUM在线工具分析富集调控这些基因的转录因子,应用通路富集 分析和蛋白质相互作用网络分析识别前列腺癌的关键基因及其对前列腺癌患者重要临床特征和无病生存率（disease free survival, DFS）的影响。结果：识别出3个枢纽模块,分别与前列腺癌病理T分期、病理N分期、Gleason分级高度相关。进一步筛选得到 13个前列腺癌核心转录因子参与调控这3个枢纽模块。这些转录因子调控的差异表达基因显著富集于Calcium、cGMP-PKG、 cAMP 前列腺癌相关信号通路, 其组成的基因网络中 14 个关键基因（PRKG1、PRKG2、CYSLTR2、GRPR、CHRM3、ADCY5、 ADRA1D、EDNRA、EDNRB、CYSLTR2、AGTR1、GRPR、GRIA1和OXT）处于重要节点位置,其中ADRA1A、PRKG2、CHRM3、 ADRA1D和EDN3高表达显著延长了前列腺癌患者的DFS（均P<0.01）。结论：ADRA1A、PRKG2、CHRM3、ADRA1D和EDN3 受前列腺癌核心转录因子调控,与前列腺癌重要临床特征高度相关,且高表达会显著增加前列腺癌的DFS,对前列腺癌机制的后 续研究具有重要的参考价值。     

细粒棘球蚴AgB亚单位基因的联合表达和血清学评价

目的为进一步提高AgB抗原在诊断中的敏感性和特异性,将细粒棘球蚴AgB1和AgB2两个亚单位基因在同一载体中进行联合表达,并对联合表达的重组抗原和单基因表达的AgB1、AgB2在血清抗体检测中的差异进行比较研究。方法在表达载体PET32a中构建AgB1＋AgB2（AgBs）联合基因的重组质粒,用ELISA检测重组抗原与病人血清的反应性。结果构建了AgBs重组质粒,经测序证实插入的联合基因片段序列正确。AgBs重组质粒表达的融合蛋白分子量为38kDa,为不溶性蛋白。联合表达抗原AgBs对细粒棘球蚴病（CE）血清的敏感性为84．4％,特异性为80．5％;单基因表达的AgB1和AgB2的敏感性分别为75．6％和57．8％,特异性分别为72．6％和91．2％。结论成功构建了AgB亚单位联合表达基因,联合表达的AgBs抗原对CE血清的诊断价值优于单基因表达的AgB1或AgB2抗原。