Stimulation of retrotrapezoid nucleus Phox2b‐expressing neurons rescues breathing dysfunction in an experimental Parkinson’s disease rat model

Emerging evidence from multiple studies indicates that Parkinson’s disease (PD) patients suffer from a spectrum of autonomic and respiratory motor deficiencies in addition to the classical motor symptoms attributed to substantia nigra degeneration of dopaminergic neurons. Animal models of PD show a decrease in the resting respiratory rate as well as a decrease in the number of Phox2b‐expressing retrotrapezoid nucleus (RTN) neurons. The aim of this study was to determine the extent to which substantia nigra pars compact (SNc) degeneration induced RTN biomolecular changes and to identify the extent to which RTN pharmacological or optogenetic stimulations rescue respiratory function following PD‐induction. SNc degeneration was achieved in adult male Wistar rats by bilateral striatal 6‐hydroxydopamine injection. For proteomic analysis, laser capture microdissection and pressure catapulting were used to isolate the RTN for subsequent comparative proteomic analysis and Ingenuity Pathway Analysis (IPA). The respiratory parameters were evaluated by whole‐body plethysmography and electromyographic analysis of respiratory muscles. The results confirmed reduction in the number of dopaminergic neurons of SNc and respiratory rate in the PD‐animals. Our proteomic data suggested extensive RTN remodeling, and that pharmacological or optogenetic stimulations of the diseased RTN neurons promoted rescued the respiratory deficiency. Our data indicate that despite neuroanatomical and biomolecular RTN pathologies, that RTN‐directed interventions can rescue respiratory control dysfunction.     

The Staphylococcus aureus proteome

Staphylococcus aureus is a Gram-positive commensal bacterium that is regarded as a major threat for modern health care systems. This relates both to the ability of S. aureus to overcome antibiotic therapy by developing high-level resistance against multiple antibiotics and this bacterium's extensive arsenal of virulence factors. Understanding the mechanisms of resistance and functional studies on stress and starvation responses are the main goals of proteomics in staphylococcal research. This review high-lights recent advances in gel-based and gel-free proteomics analyses of S. aureus and pinpoints the importance of location-specific proteomics studies targeting the cytosol, the membrane, the cell surface and the extracellular milieu in combination with integrated global proteome studies. Emerging hot topics in staphylococcal proteomics are discussed with special focus on in vivo proteomics, membrane vesicles, biofilm formation and the acquisition of absolute proteome data for systems biological modeling approaches.     

大鼠髁突软骨细胞发育中相关信号通路的初步筛查

目的:通过建立的大鼠髁突软骨细胞发育过程中蛋白表达差异谱,分析可能参与的信号通路,为进一步认识髁突软骨生理性及病理性生长改建的信号调控机制提供相关信息。方法:收获出生后1、7、14、28 d共4组SD大鼠髁突软骨细胞,甲苯胺蓝染色、Ⅱ型胶原免疫组化染色鉴定软骨细胞。提取各组软骨细胞总蛋白,采用i TRAQ标记定量蛋白,2D nano-HPLC和基质辅助激光解吸/电离串联飞行时间质谱(MALDI-TOF-TOF),获取出生后大鼠髁突软骨细胞发育过程中差异蛋白的表达谱,所得数据用MASCOT软件处理,筛选样本之间有意义的差异蛋白,运用GO法及David软件进行KEGG信号通路分析。结果 :共鉴定137种具有可信度表达的蛋白,有44种蛋白参与至少27条KEGG信号通路,其中ECM-受体相互作用、焦点粘连、actin骨架调节、Ca2+信号通路、血管平滑肌收缩、Gn RH信号通路、肌醇三磷酸代谢、磷脂酰肌醇信号系统以及核糖体等信号通路具有统计学意义。结论:在大鼠髁突软骨发育中,各信号通路蛋白在时间、空间上差异性表达,共同形成复杂的信号传递网络。     

鼻息肉的2-DE图谱建立和蛋白质组学分析

目的:建立鼻息肉及鼻黏膜双向凝胶电泳(two-dimensional polyacrylamide gel electrophoresis,2-DE)图谱,鉴定差异表达蛋白质。方法:收集鼻息肉及鼻黏膜样本各7例,采用固相pH梯度2-DE,凝胶银染,扫描图像,ImageMaster2-DE软件比较分析等方法,识别差异表达蛋白质,通过质谱分析得到相应肽质指纹图谱(peptide mass fingerprint,PMF),采用PeptIdent软件查询Swiss-Protand TreMBL数据库,鉴定差异表达蛋白质。结果:建立了鼻息肉和鼻黏膜蛋白质的2-DE图谱。鼻息肉和鼻黏膜3块凝胶的平均蛋白质点数分别为825±78和936±62;平均匹配点数为682±96和821±78,匹配百分率为82.7%和87.7%;同一鼻息肉的3块胶在蛋白质点位置上有较好的重复性,不同胶间蛋白质点在等电聚焦(IEF)方向偏差为(1.06±0.14)mm,在SDS-PAGE方向偏差为(1.45±0.21)mm。比较分析两种组织各7例样本的2-DE图谱,鼻息肉和鼻黏膜蛋白质点数为1458个和1617个,平均匹配点数为1026个。质谱分析差异蛋白质点40个,获取34张PMF,查询数据库鉴定出蛋白质24个。结论:建立了分辨率高和重复性好的鼻息肉及鼻黏膜2-DE图谱,识别鉴定出一些与鼻息肉病变相关的蛋白质。     

Data-independent acquisition-based proteomics analysis correlating type 2 diabetes mellitus with osteoarthritis in total knee arthroplasty patients

Background:To explore the effects of type 2 diabetes mellitus (T2DM) on osteoarthritis (OA), 12 bone tissue samples were obtained surgically from the human total knee arthroplasty patients and analyzed by quantitative proteomics.Methods:Based on patient clinical histories, patient samples were assigned to diabetes mellitus osteoarthritis (DMOA) and OA groups. A data-independent acquisition method for data collection was used with proteomic data analysis to assess intergroup proteomic differences. Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genome pathway enrichment analysis were used to further find the correlation between T2DM and OA.Results:GO functional analysis found 153 differentially expressed proteins between DMOA and OA groups, of which 92 differentially expressed proteins were significantly up-regulated and 61 were significantly down-regulated. Kyoto Encyclopedia of Genes and Genome pathway analysis found 180 pathways, including 9 pathways significantly enriched. Further data analysis revealed that 6 signaling pathways were closely associated with T2DM and OA.Conclusion:OA and DMOA onset and progression were closely related to synthesis and metabolism of extracellular matrix components (e.g., fibronectin, decorin, etc.). The effects of T2DM on OA occur though 2 major ways of oxidative stress and low-grade chronic inflammation, involving in 2 inhibited signaling pathways and 4 activated signaling pathways.     

Ⅲ~Ⅳ期糖尿病肾病不同中医证型的血清蛋白组学研究

目的:探索Ⅲ~Ⅳ期糖尿病肾病(DN)患者血清差异蛋白,同时筛选不同中医证型的血清差异蛋白。方法:收集Ⅲ~Ⅳ期DN患者70例(气阴两虚证19例,脾肾气虚证18例,血瘀证16例,湿热证17例),同时选择健康受试者35例(健康对照组),应用表面加强激光解吸电离-飞行时间-质谱(SELDI-TOF-MS)技术检测各组血清蛋白指纹图谱,并对差异蛋白峰进行对比分析。结果:1) DN组与健康对照组之间共有9个蛋白峰存在显著差异,质荷比(M/Z)分别为2042.57、3291.28、4986.15、5312.69、5564.09、9861.47、10786.53、13392.89、17395.27;2)气阴两虚证与脾肾气虚证之间共有4个蛋白峰存在显著差异,M/Z分别为2994.77、4986.15、7937.25、2758.91;3)气阴两虚证与血瘀证之间共有4个蛋白峰存在显著差异,M/Z分别为4986.15、16982.62、6819.69、9947.36;4)气阴两虚证与湿热证之间共有3个蛋白峰存在显著差异,M/Z分别为4986.15、11741.33、7001.54;5)脾肾气虚证与血瘀证之间共有3个蛋白峰存在显著差异,M/Z分别为3448.22、8063.43、9787.21;6)脾肾气虚证与湿热证之间共有5个蛋白峰存在显著差异,M/Z分别为2144.34、3992.01、8871.35、10568.32、14643.47;7)血瘀证与湿热证之间共有6个蛋白峰存在显著差异,M/Z分别为4233.15、5771.32、5987.18、8496.76、6651.25、13551.94。结论:Ⅲ~Ⅳ期DN组与健康对照组血清蛋白表达存在差异,该差异或可作为相关临床诊断的标志物,对探索DN的发病机制具有重要的参考价值;在不同中医证型之间能够找到差异血清蛋白,可能反映"证"的实质内涵,对今后建立诊断决策模型具有积极意义。     

基于蛋白质组学大黄异病同治急性中风大鼠脑物质的基础及相关机制

目的:基于定量蛋白质组学和生物信息学分析,探索大黄异病同治急性中风的物质基础及机制。方法:采用线栓法制备缺血再灌注的缺血性中风(IS)大鼠模型和胶原酶来诱导的出血性中风(ICH)模型。将60只SD大鼠随机分为缺血性中风假手术组(Sham1),缺血性中风组(IS),出血性中风+大黄治疗组(DH1),出血性中风假手术组(Sham2),出血性中风(ICH)组和出血性中风+大黄治疗组(DH2),每组10只。IS,Sham1和DH1组在24 h后,ICH,Sham2和DH2组在48 h后,生理盐水灌注后取脑组织行定量蛋白质组学分析,鉴定差异表达蛋白(DEPs)。对共性DEPs进行生物信息学分析,并对相关的DEPs进行蛋白免疫印迹验证。结果:大黄调节急性中风疾病相关共性DEPs 21个(上调12个,下调9个)。京都基因和基因组百科全书(KEGG)分析显示,富集了肌萎缩侧索硬化症(ALS)通路,通路中包含神经丝蛋白轻链多肽(Nefl),神经丝蛋白中链多肽(Nefm),神经丝蛋白重链多肽(Nefh)。大黄异病同治急性中风共性机制主要包括能量代谢、离子稳态、突触相关蛋白的调节、细胞周期及神经形成。共性DEPs验证,大黄治疗后,GTP结合蛋白REM2(Rem2),酪氨酸3-单加氧酶(Th),Nefl和神经调制蛋白(Gap43)表达量与相应模型组比较差异均有统计学意义(P0.05)。其中,治疗后Nefl表达为下调,而Rem2,Th和Gap43表达为上调,此结果与蛋白质组学检测结果一致。结论:该研究建立了大黄-异病同治-差异蛋白质表达谱,能量代谢、离子稳态、突触相关蛋白调节、细胞周期及神经形成是其共性机制。     

Searching for prognostic biomarkers for small renal masses in the urinary proteome

Renal cell carcinoma (RCC) is frequently diagnosed incidentally as an early-stage small renal mass (SRM; pT1a, ≤4 cm). Overtreatment of patients with benign or clinically indolent SRMs is increasingly common and has resulted in a recent shift in treatment recommendations. There are currently no available biomarkers that can accurately predict clinical behavior. Therefore, we set out to identify early biomarkers of RCC progression. We employed a quantitative label-free liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomics approach and targeted parallel-reaction monitoring to identify and validate early, noninvasive urinary biomarkers for RCC-SRMs. In total, we evaluated 115 urine samples, including 33 renal oncocytoma (≤4 cm) cases, 30 progressive and 26 nonprogressive clear cell RCC (ccRCC)-SRM cases, in addition to 26 healthy controls. We identified six proteins, which displayed significantly elevated expression in clear cell RCC-SRMs (ccRCC-SRMs) relative to healthy controls. Proteins C12ORF49 and EHD4 showed significantly elevated expression in ccRCC-SRMs compared to renal oncocytoma (≤4 cm). Additionally, proteins EPS8L2, CHMP2A, PDCD6IP, CNDP2 and CEACAM1 displayed significantly elevated expression in progressive relative to nonprogressive ccRCC-SRMs. A two-protein signature (EPS8L2 and CCT6A) showed significant discriminatory ability (areas under the curve: 0.81, 95% CI: 0.70–0.93) in distinguishing progressive from nonprogressive ccRCC-SRMs. Patients (Stage I–IV) with EPS8L2 and CCT6A mRNA alterations showed significantly shorter overall survival (p = 1.407 × 10⁻⁶) compared to patients with no alterations. Our in-depth proteomic analysis identified novel biomarkers for early-stage RCC-SRMs. Pretreatment characterization of urinary proteins may provide insight into early RCC progression and could potentially help assign patients to appropriate management strategies.