microRNA-145-5p attenuates acute lung injury via targeting ETS2.

The protective effect of microRNA (miR)-145-5p in acute lung injury (ALI) has been discovered previously. Thus, in this study, we attempted to further investigate the mechanism of miR-145-5p in ALI through the downstream E26 transformation-specific proto-oncogene 2 (ETS2)/transforming growth factor ß1 (TGF-ß1)/Smad pathway. A lipopolysaccharide (LPS)-induced ALI rat model was established. The expression of miR-145-5p in ALI rat lung tissues was up-regulated. Afterward, pathological damage in the lung tissue, the wet/dry (W/D) ratio, apoptosis, and serum inflammatory factor contents were observed. miR-145-5p, ETS2, TGF-ß1, Smad2/3, and phosphorylated Smad2/3 levels were measured in rats. miR-145-5p expression was down-regulated, ETS2 expression was up-regulated, and the TGF-ß1/Smad pathway was activated in LPS-exposed rats. Overexpression of miR-145-5p inactivated the TGF-ß1/Smad pathway and attenuated ALI, as reflected by relieved pathological damage, a decreased W/D ratio, reduced apoptosis, and suppressed inflammatory response. In contrast, loss of miR-145-5p or elevated ETS2 levels worsened ALI and activated the TGF-ß1/Smad pathway. Moreover, elevation of ETS2 diminished miR-145-5p-mediated protection against ALI. Evidently, miR-145-5p negatively regulates ETS2 expression and inactivates the TGF-ß1/Smad pathway to ameliorate ALI in rats.

Systems Signatures Reveal Unique Remission-path of Type 2 Diabetes Following Roux-en-Y Gastric Bypass Surgery.

Roux-en-Y Gastric bypass surgery (RYGB) is emerging as a powerful tool for treatment of obesity and may also cause remission of type 2 diabetes. However, the molecular mechanism of RYGB leading to diabetes remission independent of weight loss remains elusive. In this study, we profiled plasma metabolites and proteins of 10 normal glucose-tolerant obese (NO) and 9 diabetic obese (DO) patients before and 1-week, 3-months, 1-year after RYGB. 146 proteins and 128 metabolites from both NO and DO groups at all four stages were selected for further analysis. By analyzing a set of bi-molecular associations among the corresponding network of the subjects with our newly developed computational method, we defined the represented physiological states (called the edge-states that reflect the interactions among the bio-molecules), and the related molecular networks of NO and DO patients, respectively. The principal component analyses (PCA) revealed that the edge states of the post-RYGB NO subjects were significantly different from those of the post-RYGB DO patients. Particularly, the time-dependent changes of the molecular hub-networks differed between DO and NO groups after RYGB. In conclusion, by developing molecular network-based systems signatures, we for the first time reveal that RYGB generates a unique path for diabetes remission independent of weight loss.

Personalized evaluation based on quantitative proteomics for drug-treated patients with chronic kidney disease.

The patient's response to drug treatment is usually systems-wide based on multi-spots through either direct or indirect targets. Thus, the evaluation of the treatment cannot rely on single targeted biomarker, especially for complex diseases such as chronic kidney disease. In the present study, we performed a systems-wide analysis using proteomic approach to quantify changes in the proteomic profiles of the plasma from IgA nephropathy (IgAN) patients before and after treatment. In particular, the patient-to-health distances based on global proteome quantification before and after treatment were calculated and considered as quantitative readouts to measure patient divergences from the healthy condition. We found that the patient-to-health distance nicely correlated with the patient's response to drug treatment and long-term prognosis, which created a self-tracking platform for personalized evaluation. In addition, the steroid treatment plays a role in immunosuppression, while the Chinese Traditional Medicine (TCM) can modulate whole-body systems. Our results indicated that STC therapy normalized the proteomic profile more significantly than SA therapy. This work provides an omics-based and systematic platform for personalized evaluation of disease treatment. This strategy could help us to evaluate treatment outcomes and predict prognosis in patients with IgAN and other complex diseases.

[Plasma D-dimer changes and prognostic implication in severe acute pancreatitis].

OBJECTIVE: To study the role of plasma D-dimer in human severe acute pancreatitis (SAP) and its relationship with disease severity. METHODS: A prospective study was performed. From February 2010 to June 2011, plasma concentration of D-dimer and the results of other routine laboratory tests were measured in 32 SAP inpatients at admission immediately and 24, 48, 72 hours after admission. Ranson scores were also recorded. The relationship between plasma concentration of D-dimer and the results of other routine laboratory tests, Ranson scores were analyzed, and so did the relationship between plasma concentration of D-dimer and severity of patient's condition. RESULTS: (1) There were no obvious changes for plasma concentration of D-dimer and the results of other routine laboratory index in 32 SAP inpatients at each time points after admission. (2) Plasma concentration of D-dimer of SAP was positively related to white blood count (WBC), creatinine (Cr), prothrombin time (PT) and activated partial thromboplastin time (APTT) and negatively related to hematocrit (Hct), albumin and calcium (P<0.05 or P<0.01). Plasma concentration of D-dimer of SAP at 48 hours after admission had positive relationship with Ranson scores (r=0.729, P=0.001). (3) Plasma concentration of D-dimer of SAP inpatients in CT grade D, E stage were significantly higher than that in CT grade B, C stage (1.18±0.02 mg/L, 1.23±0.01 mg/L vs. 0.90±0.02 mg/L, 0.93±0.01 mg/L, all P<0.05). (4) Plasma concentration of D-dimer of SAP in inpatients with multiple organ failure (MOF) at 24 hours and 48 hours were significantly higher than that in inpatients without MOF (24 hours: 1.26±0.02 mg/L vs. 0.93±0.02 mg/L, 48 hours:1.25±0.02 mg/L vs. 0.93±0.02 mg/L, both P<0.05). CONCLUSION: Plasma concentration of the D-dimer is significantly increased in SAP patients, and is related to the severity. The first plasma concentration of D-dimer of SAP after admission can well reflect severity of patient's condition.

Secretome-derived isotope tags (SDIT) reveal adipocyte-derived apolipoprotein C-I as a predictive marker for cardiovascular disease.

We developed a quantitative strategy, named secretome-derived isotopic tag (SDIT), to concurrently identify and quantify the adipocyte-secreted plasma proteins from normal and high-fat-diet (HFD) induced obese mice, based on the application of isotope-labeled secreted proteins from cultured mouse adipocytes as internal standards. We detected 197 proteins with significant changes between normal and obese mice plasma. Importantly, a novel adipocyte-secreted plasma protein, apolipoprotein C-I (apoC-I), significantly increased in the obese mice plasma. The expression and secretion of adipocyte apoC-I was detected in differentiated 3T3-L1 and primary rat adipocytes. Our in vitro experiments proved that functional Golgi apparatus was required for apoC-I secretion. Additionally, obese mice had increased apoC-I production in adipose tissue. Population survey of 367 participants showed that the plasma level of apoC-I was significantly increased in obese individuals compared with healthy individuals. After multiple adjustments for age and sex, the odds ratios for risk factors of cardiovascular disease including high LDL cholesterol, hypercholesterolemia, and hypertriglyceridemia, respectively, were used to compare the highest with the lowest apoC-I quartile. Taken together, our studies provide a novel strategy to concurrently identify and quantify tissue-specific secreted proteins. This strategy can be used to identify the largest global characterization of adipocyte-derived plasma proteome and provides a potential disease-related biomarker for clinical diagnoses. By selectively analyzing adipocyte-secreted proteins in plasma from obese vs lean murine and/or human subjects, we discovered that apoC-I is an adipocyte-secreted plasma protein and a predictive marker for cardiovascular disease.

Quantitative detection of single amino acid polymorphisms by targeted proteomics.

Single-nucleotide polymorphisms (SNPs) are recognized as one kind of major genetic variants in population scale. However, polymorphisms at the proteome level in population scale remain elusive. In the present study, we named amino acid variances derived from SNPs within coding regions as single amino acid polymorphisms (SAPs) at the proteome level, and developed a pipeline of non-targeted and targeted proteomics to identify and quantify SAP peptides in human plasma. The absolute concentrations of three selected SAP-peptide pairs among 290 Asian individuals were measured by selected reaction monitoring (SRM) approach, and their associations with both obesity and diabetes were further analyzed. This work revealed that heterozygotes and homozygotes with various SAPs in a population could have different associations with particular traits. In addition, the SRM approach allows us for the first time to separately measure the absolute concentration of each SAP peptide in the heterozygotes, which also shows different associations with particular traits.

Large scale phosphoproteome profiles comprehensive features of mouse embryonic stem cells.

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Analyses of copy number variation of GK rat reveal new putative type 2 diabetes susceptibility loci.

Large efforts have been taken to search for genes responsible for type 2 diabetes (T2D), but have resulted in only about 20 in humans due to its complexity and heterogeneity. The GK rat, a spontanous T2D model, offers us a superior opportunity to search for more diabetic genes. Utilizing array comparative genome hybridization (aCGH) technology, we identifed 137 non-redundant copy number variation (CNV) regions from the GK rats when using normal Wistar rats as control. These CNV regions (CNVRs) covered approximately 36 Mb nucleotides, accounting for about 1% of the whole genome. By integrating information from gene annotations and disease knowledge, we investigated the CNVRs comprehensively for mining new T2D genes. As a result, we prioritized 16 putative protein-coding genes and two microRNA genes (rno-mir-30b and rno-mir-30d) as good candidates. The catalogue of CNVRs between GK and Wistar rats identified in this work served as a repository for mining genes that might play roles in the pathogenesis of T2D. Moreover, our efforts in utilizing bioinformatics methods to prioritize good candidate genes provided a more specific set of putative candidates. These findings would contribute to the research into the genetic basis of T2D, and thus shed light on its pathogenesis.

A comprehensive and non-prefractionation on the protein level approach for the human urinary proteome: touching phosphorylation in urine.

Increasing attention has been paid to the urinary proteome because it holds the promise of discovering various disease biomarkers. However, most of the urine proteomics studies routinely relied on protein pre-fractionation and so far did not present characterization on phosphorylation status. Two robust approaches, integrated multidimensional liquid chromatography (IMDL) and Yin-yang multidimensional liquid chromatography (MDLC) tandem mass spectrometry, were recently developed in our laboratory, with high-coverage identification of peptide mixtures. In this study, we adopted a strategy without pre-fractionation on the protein level for urinary proteome identification, using both the IMDL and the Yin-yang MDLC methods for peptide fractionation followed by identification using a linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometer with high resolution and mass accuracy. A total of 1310 non-redundant proteins were highly confidently identified from two experiments, significantly including 59 phosphorylation sites. More than half the annotated identifications were membrane-related proteins. In addition, the lysosomal as well as kidney-associated proteins were detected. Compared with the six largest datasets of urinary proteins published previously, we found our data included most of the reported proteins. Our study developed a robust approach for exploring the human urinary proteome, which would provide a catalogue of urine proteins on a global scale. It is the first report, to our best knowledge, to profile the urinary phosphoproteome. This work significantly extends current comprehension of urinary protein modification and its potential biological significance. Moreover, the strategy could further serve as a reference for biomarker discovery.

Dynamic proteomics for investigating the response of individual cancer cells under drug action.

Evaluation of: Cohen AA, Geva-Zatorsky N, Eden E et al. Dynamic proteomics of individual cancer cells in response to a drug. Science 322(5907), 1511-1516 (2008). One of the greatest challenges in cancer chemotherapy is that seemingly identical cancer cells can respond differently to drug treatment. The pioneering work reported by Cohen and colleagues moves one step closer to solving this challenge. They develop a dynamic proteomics approach that utilizes fluorescent markers and a time-lapse microscope to detect the fluctuating locations and levels of approximately 1000 proteins in individual cancer cells at high temporal resolution. After adminstration of the cancer drug camptothecin, certain proteins display similar spatiotemporal distribution patterns in individual cells; for example, the drug target topoisomerase-1 shows a rapid decrease in protein level and in nuclear location. However, two particular proteins demonstrate cell-cell variability in their behavior corresponding to cell fate, which may help to explain drug resistance. This method offers an effective way to investigate drug mechanisms in individual cells.

Fractionation of complex protein mixture by virtual three-dimensional liquid chromatography based on combined pH and salt steps.

The complexity and diversity of biological samples in proteomics require intensive fractionation ahead of mass spectrometry identification. This work developed a chromatographic method called virtual three-dimensional chromatography to fractionate complex protein mixtures. By alternate elution with different pHs and salt concentrations, we implemented pH and salt steps by turns on a single strong cation exchange column to fully exploit its chromatographic ability. Given standard proteins that were not resolved solely by pH or salt gradient elution could be successfully separated using this combined mode. With a reversed phase column tandem connected behind, we further fractionated as well as desalted proteins as the third dimension. This present strategy could readily be adapted with respect to special complexity of biological samples. Crude plasma without depleting high abundance proteins were fractionated by this three-dimensional mode and then analyzed by reversed phase liquid chromatography coupled with LTQ mass spectrometry. In total, 1933 protein groups with wide dynamic ranges were identified from a single experiment. Some characteristics that correlated to the behavior of proteins on strong cation exchange columns are also discussed.

Localized-statistical quantification of human serum proteome associated with type 2 diabetes.

BACKGROUND: Recent advances in proteomics have shed light to discover serum proteins or peptides as biomarkers for tracking the progression of diabetes as well as understanding molecular mechanisms of the disease. RESULTS: In this work, human serum of non-diabetic and diabetic cohorts was analyzed by proteomic approach. To analyze total 1377 high-confident serum-proteins, we developed a computing strategy called localized statistics of protein abundance distribution (LSPAD) to calculate a significant bias of a particular protein-abundance between these two cohorts. As a result, 68 proteins were found significantly over-represented in the diabetic serum (p<0.01). In addition, a pathway-associated analysis was developed to obtain the overall pathway bias associated with type 2 diabetes, from which the significant over-representation of complement system associated with type 2 diabetes was uncovered. Moreover, an up-stream activator of complement pathway, ficolin-3, was observed over-represented in the serum of type 2 diabetic patients, which was further validated with statistic significance (p = 0.012) with more clinical samples. CONCLUSIONS: The developed LSPAD approach is well fit for analyzing proteomic data derived from biological complex systems such as plasma proteome. With LSPAD, we disclosed the comprehensive distribution of the proteins associated with diabetes in different abundance levels and the involvement of ficolin-related complement activation in diabetes.

A fully automated 2-D LC-MS method utilizing online continuous pH and RP gradients for global proteome analysis.

The conventional 2-D LC-MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong-cation-exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2-D LC-MS/MS system is introduced that provided excellent separation and identification power. The pH gradient-based elution provided more basic peptides than that of salt-based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution-based 2-D LC-MS/MS approaches were compared by analyzing the mouse liver proteome.

Large-scale identification of human biliary proteins from a cholesterol stone patient using a proteomic approach.

Gallbladder bile, one of the most important body fluids, is composed of water, inorganic ions, conjugated bile salts, phospholipids, cholesterol, bilirubin, mucin and proteins. The separation and identification of bile proteins remain difficult due to the complexity of this matrix. In the present study, human gallbladder bile was obtained from a cholesterol stone patient, and the proteins were isolated and purified by dialysis, precipitation and delipidation procedures. The resulting proteins were divided into several aliquots. One aliquot was subjected to two-dimensional gel electrophoresis (2DE). The protein spots were then in-gel digested and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). Another aliquot was directly digested and analyzed by a combination of strong cation-exchange (SCX) and reversed-phase (RP) chromatography prior to tandem mass spectrometry (2D-LC/MS/MS). Eventually, 48 and 218 unique proteins were identified from 2DE/MS and 2D-LC/MS/MS, respectively, resulting in a total of 222 unique identified proteins. Of the 218 proteins identified by 2D-LC/MS/MS, 92 were identified based on more than one unique tryptic peptide, and, of the total 222 proteins, 98 were identified based on more than one unique tryptic peptide.

Prefractionation of proteome by liquid isoelectric focusing prior to two-dimensional liquid chromatography mass spectrometric identification.

Due to the complexity of proteomes, developing methods of sample fractionation, separation, concentration, and detection have become urgent to the identification of large numbers of proteins, as well as the acquisition of those proteins in low abundance. In this work, liquid isoelectric focusing (LIEF) combined with 2D-LC-MS/MS was applied to the proteome of Saccharomyces cerevisiae. This yielded a total of 1795 proteins that were detected and identified by 30 fractions of protein prefractionation. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis without protein fractionation. LIEF-2D-LC-MS/MS also produced improved resolution of low-abundance proteins. Furthermore, we analyzed the characteristics of proteins obtained by LIEF-2D-LC-MS/MS. 1103 proteins with CAI under 0.2 were identified, allowing us to specifically obtain detailed biochemical information on these kind proteins. It was observed that LIEF-2D-LC-MS/MS is useful for large-scale proteome analysis and may be specifically applied to systems with wide dynamic ranges.

Def1p is involved in telomere maintenance in budding yeast.

Saccharomyces Rrm3p, a member of Pif1 5'-3' DNA helicase subfamily, helps replication forks traverse protein-DNA complexes, including the telomere. Here we have identified an Rrm3p interaction protein known to be Def1p. In def1 mutants, telomeres were approximately 200-bp shorter than that in wild-type cells. DEF1 is also required for the stable maintenance of mitochondrial DNA, and the telomere shortening phenotype seen in def1 cells is not a secondary consequence of the mitochondrion defect. A combination of DEF1 null mutation with deletion of EST2 or EST3 resulted in an accelerated senescence phenotype, suggesting that Def1p is not involved in the telomerase recruitment pathway. In the absence of telomerase, cells escape senescence by either amplifying Y' regions or TG-telomeric repeats to generate type I or type II survivors, respectively. Only type I survivors were recovered from both def1Delta est2Delta and def1Delta est3Delta double mutant cells, further suggesting that the function of Def1p in telomere maintenance is specific. Our novel findings of the functions of Def1p in telomere and mitochondria suggested that Def1p plays multiple roles in yeast.