Combined Proteomic and Phosphoproteomic Characterization of the Molecular Regulators and Functional Modules During Pancreatic Progenitor Cell Development.

Differentiated multipotent pancreatic progenitors have major advantages for both modeling pancreas development and preventing or treating diabetes. Despite significant advancements in inducing the differentiation of human pluripotent stem cells into insulin-producing cells, the complete mechanism governing proliferation and differentiation remains poorly understood. This study used large-scale mass spectrometry to characterize molecular processes at various stages of human embryonic stem cell (hESC) differentiation toward pancreatic progenitors. hESCs were induced into pancreatic progenitor cells in a five-stage differentiation protocol. A high-performance liquid chromatography-mass spectrometry platform was used to undertake comprehensive proteome and phosphoproteome profiling of cells at different stages. A series of bioinformatic explorations, including coregulated modules, gene regulatory networks, and phosphosite enrichment analysis, were then conducted. A total of 27,077 unique phosphorylated sites and 8122 proteins were detected, including several cyclin-dependent kinases at the initial stage of cell differentiation. Furthermore, we discovered that ERK1, a member of the MAPK cascade, contributed to proliferation at an early stage. Finally, Western blotting confirmed that the phosphosites from SIRT1 and CHEK1 could inhibit the corresponding substrate abundance in the late stage. Thus, this study extends our understanding of the molecular mechanism during pancreatic cell development.

Altered counts and mitochondrial mass of peripheral blood leucocytes in patients with chronic hepatitis B virus infection.

Hepatitis B virus (HBV) damages liver cells through abnormal immune responses. Mitochondrial metabolism is necessary for effector functions of white blood cells (WBCs). The aim was to investigate the altered counts and mitochondrial mass (MM) of WBCs by two novel indicators of mitochondrial mass, MM and percentage of low mitochondrial membrane potential, MMPlow%, due to chronic HBV infection. The counts of lymphocytes, neutrophils and monocytes in the HBV infection group were in decline, especially for lymphocyte (p = 0.034) and monocyte counts (p = 0.003). The degraded MM (p = 0.003) and MMPlow% (p = 0.002) of lymphocytes and MM (p = 0.005) of monocytes suggested mitochondrial dysfunction of WBCs. HBV DNA within WBCs showed an extensive effect on mitochondria metabolic potential of lymphocytes, neutrophils and monocytes indicated by MM; hepatitis B e antigen was associated with instant mitochondrial energy supply indicated by MMPlow% of neutrophils; hepatitis B surface antigen, antiviral therapy by nucleos(t)ide analogues and prolonged infection were also vital factors contributing to WBC alterations. Moreover, degraded neutrophils and monocytes could be used to monitor immune responses reflecting chronic liver fibrosis and inflammatory damage. In conclusion, MM combined with cell counts of WBCs could profoundly reflect WBC alterations for monitoring chronic HBV infection. Moreover, HBV DNA within WBCs may be a vital factor in injuring mitochondria metabolic potential.

Nanoclay Drug-Delivery System Loading Potassium Iodide Promotes Endocytosis and Targeted Therapy in Anaplastic Thyroid Cancer.

The rapid proliferative biological behavior of primary foci of anaplastic thyroid cancer (ATC) makes it a lethal tumor. According to the specific iodine uptake capacity of thyroid cells and enhanced endocytosis of ATC cells, we designed a kind of nanoclay drug-loading system and showed a promising treatment strategy for ATC. Introducing potassium iodide (KI) improves the homoaggregation of clay nanoparticles and then affects the distribution of nanoparticles in vivo, which makes KI@DOX-KaolinMeOH enriched almost exclusively in thyroid tissue. Simultaneously, the improvement of dispersibility of KI@DOX-KaolinMeOH changes the target uptake of ATC cells by improving the endocytosis and nanoparticle-induced autophagy, which regulate the production of autolysosomes and autophagy-enhanced chemotherapy, eventually contributing to a tumor inhibition rate of more than 90% in the primary foci of ATC. Therefore, this facile strategy to improve the homoaggregation of nanoclay by introducing KI has the potential to become an advanced drug delivery vehicle in ATC treatment.

Serum levels of n-3 PUFA and colorectal cancer risk in Chinese population.

Circulating n-3 PUFA, which integrate endogenous and exogenous n-3 PUFA, can be better used to investigate the relationship between n-3 PUFA and disease. However, studies examining the associations between circulating n-3 PUFA and colorectal cancer (CRC) risk were limited, and the results remained inconclusive. This case­control study aimed to examine the association between serum n-3 PUFA and CRC risk in Chinese population. A total of 680 CRC cases and 680 sex- and age-matched (5-year interval) controls were included. Fatty acids were assayed by GC. OR and 95 % CI were calculated using multivariable logistic regression after adjustment for potential confounders. Higher level of serum α-linolenic acid (ALA), docosapentaenoic acid (DPA), DHA, long-chain n-3 PUFA and total n-3 PUFA were associated with lower odds of CRC. The adjusted OR and 95 % CI were 0·34 (0·24, 0·49, Pfor trend < 0·001) for ALA, 0·57 (0·40, 0·80, Pfor trend < 0·001) for DPA, 0·48 (0·34, 0·68, Pfor trend < 0·001) for DHA, 0·39 (0·27, 0·56, Pfor trend < 0·001) for long-chain n-3 PUFA and 0·31 (0·22, 0·45, Pfor trend < 0·001) for total n-3 PUFA comparing the highest with the lowest quartile. However, there was no statistically significant association between EPA and odds of CRC. Analysis stratified by sex showed that ALA, DHA, long-chain n-3 PUFA and total n-3 PUFA were inversely associated with odds of CRC in both sexes. This study indicated that serum ALA, DPA, DHA, long-chain n-3 PUFA and total n-3 PUFA were inversely associated with odds of having CRC in Chinese population.

Development of novel HEPT analogs featuring significantly improved anti-resistance potency against HIV-1 through chemical space exploration of the tolerant region I.

Our recent great interest in developing 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) analogs for HIV therapy identified a potent non-nucleoside reverse transcriptase inhibitor (NNRTI) 3 (EC50 = 0.01681 µM), but its therapeutic efficacy was limited by its poor anti-resistance potency. This prompted us to search for potential HEPT analogs with broad-spectrum activities, leading to the generation of a series of novel HEPT analogs through exploring the chemical space of the solvent - protein interface. Encouraging improvements in anti-resistance efficacy were observed in some of these analogs, with the most promising compound 7 g being 3 to 26 - fold more potent than 3 against five mutant strains (E138K, Y181C, L100I, K103N, and Y188L). This analog surpassed the activity and selectivity of compound 3 by approximately 2-fold (EC50 = 0.007468 µM, SI = 4260). Furthermore, it was found to demonstrate feeble inhibition of CYP and hERG in vitro, and no in vivo acute toxicity. This study will further enrich the structure-activity relationships (SARs) of the HEPT scaffold, providing new guidance for the development of NNRTIs.

Improving the anti-HIV-1 activity and solubility of poorly water-soluble DAPYs by heteroaromatic replacement strategy: From naphthalene-DAPYs to quinoline-DAPYs.

To enhance the anti-HIV-1 efficacy and solubility of our previously documented NNRTI 1, a collection of innovative quinoline-substituted DAPY derivatives were devised using heteroaromatic replacement strategy. The results of biological evaluation revealed that the representative compound 5h possessed the highest inhibitory activity against wild-type HIV-1 and selectivity index (EC50 = 0.0018 µM, SI > 166667), which were obviously better than that of 1 (EC50 = 0.00978 µM, SI > 37764), NVP (EC50 = 0.059 µM, SI > 158), EFV (EC50 = 0.028 µM, SI > 269), and ETR (EC50 = 0.0029 µM, SI > 1519). The water solubility of compound 5h was remarkably improved, surpassing that of 1, ETR and RPV. Additionally, this compound exerted significantly enhanced anti-resistance potency, compared to 1, and displayed comparable activity to ETR against WT RT of HIV-1 (IC50 = 0.011 µM). To elucidate the underlying molecular mechanisms, molecular docking studies were conducted to investigate the crucial interactions between 5h and WT/mutant strains of HIV-1. These findings provide valuable insights and drive further advancements in the development of DAPYs for HIV therapy.

[Research progress in epigenetic pharmacological effects of rhein].

Rhein, which is one of the main active components of Rheum palmatum, has a range of pharmacological activities such as the regulation of the metabolism of glucose and lipids, anti-inflammatory, anti-tumor, anti-fibrosis, etc. Epigenetics refers to the heritable variation of gene expression without altering the DNA sequence. It is involved in the emergence and development of inflammation, renal fibrosis, diabetes, cancer, atherosclerosis, and other diseases, thus becoming a new strategy for the treatment of many di-seases. A series of studies have shown that epigenetic modification may be a common molecular mechanism of various pharmacological effects of rhein. This paper summarized the effects of rhein on the regulation of epigenetic modification and its underlying mechanisms, which involve the regulation of DNA methylation, protein acetylation, and RNA methylation, so as to provide a basis for the development and application of rhein.

Palladium-catalyzed C(sp3)-H nitrooxylation of masked alcohols.

A palladium-catalyzed ß-C(sp3)-H nitrooxylation of aliphatic alcohols with AgNO2 is reported. An 8-formylquinoline-derived oxime is installed as an exo-type directing group for sp3 C-H activation and selectfluor acts as the oxidant. The reaction tolerates a variety of functional groups and shows good selectivity for ß-C-H nitrooxylation of alcohols.

Improvements to the Rice Genome Annotation Through Large-Scale Analysis of RNA-Seq and Proteomics Data Sets.

Rice (Oryza sativa) is one of the most important worldwide crops. The genome has been available for over 10 years and has undergone several rounds of annotation. We created a comprehensive database of transcripts from 29 public RNA sequencing data sets, officially predicted genes from Ensembl plants, and common contaminants in which to search for protein-level evidence. We re-analyzed nine publicly accessible rice proteomics data sets. In total, we identified 420K peptide spectrum matches from 47K peptides and 8,187 protein groups. 4168 peptides were initially classed as putative novel peptides (not matching official genes). Following a strict filtration scheme to rule out other possible explanations, we discovered 1,584 high confidence novel peptides. The novel peptides were clustered into 692 genomic loci where our results suggest annotation improvements. 80% of the novel peptides had an ortholog match in the curated protein sequence set from at least one other plant species. For the peptides clustering in intergenic regions (and thus potentially new genes), 101 loci were identified, for which 43 had a high-confidence hit for a protein domain. Our results can be displayed as tracks on the Ensembl genome or other browsers supporting Track Hubs, to support re-annotation of the rice genome.

Trans triacylglycerols from dairy products and industrial hydrogenated oil exhibit different effects on the function of human umbilical vein endothelial cells via modulating phospholipase A2/arachidonic acid metabolism pathways.

Dairy fat intake has been considered as a risk factor for cardiovascular disease. Rodent models show that trans fatty acids in industrial hydrogenated oil and ruminant milk have different effects on cardiovascular diseases. One of the main reasons is that the distributions of trans fatty acids in triacylglycerols from dairy products and from industrial hydrogenated oil are different, which affects lipid absorption and metabolism. This study investigated the effects of 1,3-olein-2-elaidin (OEO, representing industrial hydrogenated oil triacylglycerols) and 1-vaccenic-2,3-olein (OOV, representing ruminant triacylglycerols in dairy products) on the function of human umbilical vein endothelial cells (HUVEC), including cell viability, lactate dehydrogenase (LDH) exudation rate, and nitric oxide secretory and nitric oxide synthase relative activity. We found that the detrimental effect of OEO on HUVEC was significantly greater than that of OOV. The results also showed that the absorption rate of OEO in HUVEC (78.25%) was significantly greater than that of OOV (63.32%). Mechanistically, based on phospholipidomics analysis, we found that calcium-independent phospholipase A2 (iPLA2) played a key role with regard to the OOV-mediated arachidonic acid (ARA)/COX-2/PG pathway, whereas secretory phospholipase A2 (sPLA2) and cytoplasmic phospholipase A2 (cPLA2) are responsible for the OEO-mediated ARA/COX-2/PG pathway. Moreover, OEO had a greater effect on the protein expression of COX-2 and PG secretion than OOV. In addition, iPLA2, sPLA2, and cPLA2 could mediate the ARA/CYP4A11 pathway in OOV-treated HUVEC, but only iPLA2 could mediate this pathway in HUVEC treated with OEO. We also found that sPLA2 could mediate the ARA/5-LOX pathway in HUVEC treated with OOV, but none of these 3 forms of PLA2 could mediate this pathway in HUVEC treated with OEO. On the other hand, after OOV treatment, trans-11 C18:1 was converted to beneficial forms of fatty acids in HUVEC, including conjugated linoleic acid (CLA) and trans-9 C16:1. In conclusion, we elucidated the potential mechanisms that might account for the diverse effects of triacylglycerols from industrial hydrogenated oil and ruminant milk on the function of HUVEC.

Improving Gene Annotation of the Peanut Genome by Integrated Proteogenomics Workflow.

Peanut (Arachis hypogaea L.) is a staple crop in semiarid tropical and subtropical regions. Although the genome of peanut has been fully sequenced, the current gene annotations are still incomplete. New technologies in genomics and proteomics have resulted in the emergence of proteogenomics, which can integrate genomic, transcriptomic, and proteomic data for improving gene annotation. In the present study, we collected RNA-seq and proteomic data from multiple tissues such as seed, shell, and gynophore of peanut and utilized a proteogenomic approach to improve the gene annotation of peanut based on these data. A total of 1 935 655 904 RNA-seq reads and 7 490 280 MS/MS spectra were collected. Ultimately, 13 767 annotated genes were found with evidence at the protein level, and seven novel protein-coding genes were found with both RNA-seq and proteomics evidence. In addition, 35 gene models were updated based on proteomics data. Proteogenomic approaches improved the gene annotation in certain aspects by integrating both RNA-seq and proteomic data. We expect that these approaches could help improve existing genome annotations of other species.

PPIP: Automated Software for Identification of Bioactive Endogenous Peptides.

Endogenous peptides play an important role in multiple biological processes in many species. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is an important technique for detecting these peptides on a large scale. We present PPIP, which is a dedicated peptidogenomics software for identifying endogenous peptides based on peptidomics and RNA-Seq data. This software automates the de novo transcript assembly based on RNA-Seq data, construction of a protein reference database based on the de novo assembled transcripts, peptide identification, function analysis, and HTML-based report generation. Different function components are integrated using Docker technology. The Docker image of PPIP is available at https://hub.docker.com/r/shawndp/ppip , and the source code under GPL-3 license is available at https://github.com/Shawn-Xu/PPIP . A user manual of PPIP is available at https://shawn-xu.github.io/PPIP .

Comparative Analysis of Proteomics and Transcriptomics during Fertility Transition in a Two-Line Hybrid Rice Line Wuxiang S.

The two-line hybrid rice is an important factor of a global crop, but its fertility transition mechanism is unclear. Here, a comparative proteomics and transcriptomics analysis was completed on the two-line hybrid rice line Wuxiang S (WXS) to explore its molecular mechanism and protein regulation during fertility transition. A total of 340 differentially abundant proteins (DAPs) were identified using iTRAQ between the pollen mother cell formation stage (P2) and the meiosis stage (P3). There were 3541 and 4247 differentially expressed genes (DEGs) in P2 and P3 between WXS (Sterile, S)-WXS(S) and WXS (Fertile, F)-WXS(F), respectively, of which 92 and 71 DEGs had corresponding DAPs. Among the DAPs and DEGs, 65 (SP2 vs. FP2) and 55 (SP3 vs. FP3) corresponding DEGs and DAPs (cor-DEGs-DAPs) showed the same expression trend, indicating the cor-DEGs-DAPs genes might play vital roles in WXS fertility transition. Further analysis indicated that cor-DEGs-DAPs proteins were related to energy metabolism-related proteins in anther development and were accompanied by the activation of the stress response pathway and modifications to the cell wall, which ultimately affected the fertility transition of the PTGMS rice line WXS.

Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings.

In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues; however, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of short-read RNA sequencing, single molecule long-read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data from the two sequencing methods, approximately 83.4% of intron-containing genes were alternatively spliced. Two AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR); however, by contrast to AS events detected under normal conditions, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern, indicated by the increasing number of non-conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding of AS and alternative translation mechanisms under normal conditions, and in response to ABA treatment.

Digging More Missing Proteins Using an Enrichment Approach with ProteoMiner.

Human Proteome Project (HPP) aims at mapping entire human proteins with a systematic effort upon all the emerging techniques, which would enhance understanding of human biology and lay a foundation for development of medical applications. Until now, 2563 missing proteins (MPs, PE2-4) are still undetected even using the most sensitive approach of protein detection. Herein, we propose that enrichment of low-abundance proteins benefits MPs finding. ProteoMiner is an equalizing technique by reducing high-abundance proteins and enriching low-abundance proteins in biological liquids. With triton X-100/TBS buffer extraction, ProteoMiner enrichment, and peptide fractionation, 20 MPs (at least two non-nested unique peptides with more than eight a.a. length) with 60 unique peptides were identified from four human tissues including eight membrane/secreted proteins and five nucleus proteins. Then 15 of them were confirmed with two non-nested unique peptides (≥9 a.a.) identified by matching well with their chemically synthetic peptides in PRM assay. Hence, these results demonstrated ProteoMiner as a powerful means in discovery of MPs.

PGA: an R/Bioconductor package for identification of novel peptides using a customized database derived from RNA-Seq.

BACKGROUND: Peptide identification based upon mass spectrometry (MS) is generally achieved by comparison of the experimental mass spectra with the theoretically digested peptides derived from a reference protein database. Obviously, this strategy could not identify peptide and protein sequences that are absent from a reference database. A customized protein database on the basis of RNA-Seq data is thus proposed to assist with and improve the identification of novel peptides. Correspondingly, development of a comprehensive pipeline, which provides an end-to-end solution for novel peptide detection with the customized protein database, is necessary. RESULTS: A pipeline with an R package, assigned as a PGA utility, was developed that enables automated treatment to the tandem mass spectrometry (MS/MS) data acquired from different MS platforms and construction of customized protein databases based on RNA-Seq data with or without a reference genome guide. Hence, PGA can identify novel peptides and generate an HTML-based report with a visualized interface. On the basis of a published dataset, PGA was employed to identify peptides, resulting in 636 novel peptides, including 510 single amino acid polymorphism (SAP) peptides, 2 INDEL peptides, 49 splice junction peptides, and 75 novel transcript-derived peptides. The software is freely available from http://bioconductor.org/packages/PGA/ , and the example reports are available at http://wenbostar.github.io/PGA/ . CONCLUSIONS: The pipeline of PGA, aimed at being platform-independent and easy-to-use, was successfully developed and shown to be capable of identifying novel peptides by searching the customized protein database derived from RNA-Seq data.

Drug Resistance in Colorectal Cancer Cell Lines is Partially Associated with Aneuploidy Status in Light of Profiling Gene Expression.

A priority in solving the problem of drug resistance is to understand the molecular mechanism of how a drug induces the resistance response within cells. Because many cancer cells exhibit chromosome aneuploidy, we explored whether changes of aneuploidy status result in drug resistance. Two typical colorectal cancer cells, HCT116 and LoVo, were cultured with the chemotherapeutic drugs irinotecan (SN38) or oxaliplatin (QxPt), and the non- and drug-resistant cell lines were selected. Whole exome sequencing (WES) was employed to evaluate the aneuploidy status of these cells, and RNAseq and LC-MS/MS were implemented to examine gene expression at both mRNA and protein level. The data of gene expression was well-matched with the genomic conclusion that HCT116 was a near diploid cell, whereas LoVo was an aneuploid cell with the increased abundance of mRNA and protein for these genes located at chromosomes 5, 7, 12, and 15. By comparing the genomic, transcriptomic, and proteomic data, the LoVo cells with SN38 tolerance showed an increased genome copy in chromosome 14, and the expression levels of the genes on this chromosome were also significantly increased. Thus, we first observed that SN38 could impact the aneuploidy status in cancer cells, which was partially associated with the acquired drug resistance.

IPeak: An open source tool to combine results from multiple MS/MS search engines.

Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) is an important technique for detecting peptides in proteomics studies. Here, we present an open source software tool, termed IPeak, a peptide identification pipeline that is designed to combine the Percolator post-processing algorithm and multi-search strategy to enhance the sensitivity of peptide identifications without compromising accuracy. IPeak provides a graphical user interface (GUI) as well as a command-line interface, which is implemented in JAVA and can work on all three major operating system platforms: Windows, Linux/Unix and OS X. IPeak has been designed to work with the mzIdentML standard from the Proteomics Standards Initiative (PSI) as an input and output, and also been fully integrated into the associated mzidLibrary project, providing access to the overall pipeline, as well as modules for calling Percolator on individual search engine result files. The integration thus enables IPeak (and Percolator) to be used in conjunction with any software packages implementing the mzIdentML data standard. IPeak is freely available and can be downloaded under an Apache 2.0 license at https://code.google.com/p/mzidentml-lib/.

Appraisal of the Missing Proteins Based on the mRNAs Bound to Ribosomes.

Considering the technical limitations of mass spectrometry in protein identification, the mRNAs bound to ribosomes (RNC-mRNA) are assumed to reflect the mRNAs participating in the translational process. The RNC-mRNA data are reasoned to be useful for appraising the missing proteins. A set of the multiomics data including free-mRNAs, RNC-mRNAs, and proteomes was acquired from three liver cancer cell lines. On the basis of the missing proteins in neXtProt (release 2014-09-19), the bioinformatics analysis was carried out in three phases: (1) finding how many neXtProt missing proteins have or do not have RNA-seq and/or MS/MS evidence, (2) analyzing specific physicochemical and biological properties of the missing proteins that lack both RNA-seq and MS/MS evidence, and (3) analyzing the combined properties of these missing proteins. Total of 1501 missing proteins were found by neither RNC-mRNA nor MS/MS in the three liver cancer cell lines. For these missing proteins, some are expected higher hydrophobicity, unsuitable detection, or sensory functions as properties at the protein level, while some are predicted to have nonexpressing chromatin structures on the corresponding gene level. With further integrated analysis, we could attribute 93% of them (1391/1501) to these causal factors, which result in the expression products scarcely detected by RNA-seq or MS/MS.

Tissue-Based Proteogenomics Reveals that Human Testis Endows Plentiful Missing Proteins.

Investigations of missing proteins (MPs) are being endorsed by many bioanalytical strategies. We proposed that proteogenomics of testis tissue was a feasible approach to identify more MPs because testis tissues have higher gene expression levels. Here we combined proteomics and transcriptomics to survey gene expression in human testis tissues from three post-mortem individuals. Proteins were extracted and separated with glycine- and tricine-SDS-PAGE. A total of 9597 protein groups were identified; of these, 166 protein groups were listed as MPs, including 138 groups (83.1%) with transcriptional evidence. A total of 2948 proteins are designated as MPs, and 5.6% of these were identified in this study. The high incidence of MPs in testis tissue indicates that this is a rich resource for MPs. Functional category analysis revealed that the biological processes that testis MPs are mainly involved in are sexual reproduction and spermatogenesis. Some of the MPs are potentially involved in tumorgenesis in other tissues. Therefore, this proteogenomics analysis of individual testis tissues provides convincing evidence of the discovery of MPs. All mass spectrometry data from this study have been deposited in the ProteomeXchange (data set identifier PXD002179).