Optimizing MS-Based Multi-Omics: Comparative Analysis of Protein, Metabolite, and Lipid Extraction Techniques.

Multi-omics integrates diverse types of biological information from genomic, proteomic, and metabolomics experiments to achieve a comprehensive understanding of complex cellular mechanisms. However, this approach is also challenging due to technical issues such as limited sample quantities, the complexity of data pre-processing, and reproducibility concerns. Furthermore, existing studies have primarily focused on technical performance assessment and the presentation of modified protocols through quantitative comparisons of the identified protein counts. Nevertheless, the specific differences in these comparisons have been minimally investigated. Here, findings obtained from various omics approaches were profiled using various extraction methods (methanol extraction, the Folch method, and Matyash methods for metabolites and lipids) and two digestion methods (filter-aided sample preparation (FASP) and suspension traps (S-Trap)) for resuspended proteins. FASP was found to be more effective for the identification of membrane-related proteins, whereas S-Trap excelled in isolating nuclear-related and RNA-processing proteins. Thus, FASP may be suitable for investigating the immune response and bacterial infection pathways, whereas S-Trap may be more effective for studies focused on the mechanisms of neurodegenerative diseases. Moreover, regarding the choice of extraction method, the single-phase method identified organic compounds and compounds related to fatty acids, whereas the two-phase extraction method identified more hydrophilic compounds such as nucleotides. Lipids with strong hydrophobicity, such as ChE and TG, were identified in the two-phase extraction results. These findings highlight that significant differences among small molecules are primarily identified due to the varying polarities of extraction solvents. These results, obtained by considering variables such as human error and batch effects in the sample preparation step, offer comprehensive and detailed results not previously provided by existing studies, thereby aiding in the selection of the most suitable pre-processing approach.

Unveiling Neuroprotection and Regeneration Mechanisms in Optic Nerve Injury: Insight from Neural Progenitor Cell Therapy with Focus on Vps35 and Syntaxin12.

Axonal degeneration resulting from optic nerve damage can lead to the progressive death of retinal ganglion cells (RGCs), culminating in irreversible vision loss. We contrasted two methods for inducing optic nerve damage: optic nerve compression (ONCo) and optic nerve crush (ONCr). These were assessed for their respective merits in simulating traumatic optic neuropathies and neurodegeneration. We also administered neural progenitor cells (NPCs) into the subtenon space to validate their potential in mitigating optic nerve damage. Our findings indicate that both ONCo and ONCr successfully induced optic nerve damage, as shown by increases in ischemia and expression of genes linked to neuronal regeneration. Post NPC injection, recovery in the expression of neuronal regeneration-related genes was more pronounced in the ONCo model than in the ONCr model, while inflammation-related gene expression saw a better recovery in ONCr. In addition, the proteomic analysis of R28 cells in hypoxic conditions identified Vps35 and Syntaxin12 genes. Vps35 preserved the mitochondrial function in ONCo, while Syntaxin12 appeared to restrain inflammation via the Wnt/ß-catenin signaling pathway in ONCr. NPCs managed to restore damaged RGCs by elevating neuroprotection factors and controlling inflammation through mitochondrial homeostasis and Wnt/ß-catenin signaling in hypoxia-injured R28 cells and in both animal models. Our results suggest that ischemic injury and crush injury cause optic nerve damage via different mechanisms, which can be effectively simulated using ONCo and ONCr, respectively. Moreover, cell-based therapies such as NPCs may offer promising avenues for treating various optic neuropathies, including ischemic and crush injuries.

Bottom-Up Proteomics: Advancements in Sample Preparation.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics is a powerful technique for profiling proteomes of cells, tissues, and body fluids. Typical bottom-up proteomic workflows consist of the following three major steps: sample preparation, LC-MS/MS analysis, and data analysis. LC-MS/MS and data analysis techniques have been intensively developed, whereas sample preparation, a laborious process, remains a difficult task and the main challenge in different applications. Sample preparation is a crucial stage that affects the overall efficiency of a proteomic study; however, it is prone to errors and has low reproducibility and throughput. In-solution digestion and filter-aided sample preparation are the typical and widely used methods. In the past decade, novel methods to improve and facilitate the entire sample preparation process or integrate sample preparation and fractionation have been reported to reduce time, increase throughput, and improve reproducibility. In this review, we have outlined the current methods used for sample preparation in proteomics, including on-membrane digestion, bead-based digestion, immobilized enzymatic digestion, and suspension trapping. Additionally, we have summarized and discussed current devices and methods for integrating different steps of sample preparation and peptide fractionation.

The Role of Extracellular Vesicles in Optic Nerve Injury: Neuroprotection and Mitochondrial Homeostasis.

Stem cell therapies hold great promise as alternative treatments for incurable optic nerve disorders. Although mesenchymal stem cells exhibit various tissue regeneration and recovery capabilities that may serve as valuable therapies, the clinical applications remain limited. Thus, we investigated the utility of extracellular vesicles (EVs) from human placenta-derived mesenchymal stem cells (hPSCs) in this context. Hypoxically preconditioned hPSCs (HPPSCs) were prepared via short-term incubation under 2.2% O2 and 5.5% CO2. The EVs were then isolated. R28 cells (retinal precursor cells) were exposed to CoCl2 and treated with EVs for 24 h. Cell proliferation and regeneration were measured using a BrdU assay and immunoblotting; ATP quantification revealed the extent of the mitochondrial function. The proteome was determined via liquid chromatography-tandem mass spectroscopy. Differentially expressed proteins (DEPs) were detected and their interactions identified. HPPSC_EVs functions were explored using animal models of optic nerve compression. HPPSC_EVs restored cell proliferation and mitochondrial quality control in R28 cells damaged by CoCl2. We identified DEPs (p < 0.05) that aided recovery. The mitochondrial DEPs included LONP1; PARK7; VDAC1, 2, and 3; HSPD1; and HSPA9. EVs regulated the levels of mitophagic proteins in R28 cells injured by hypoxia; the protein levels did not increase in LONP1 knockdown cells. LONP1 is a key mediator of the mitophagy that restores mitochondrial function after hypoxia-induced optic nerve injury.

Lipid Profiling of Pacific Abalone (Haliotis discus hannai) at Different Developmental Stages Using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry.

Pacific abalone (Haliotis discus hannai) is a commercially important mollusk; therefore, improvement of its growth performance and quality has been emphasized. During embryonic development, abalones undergo a series of distinct larval stages, including swimming veliger larvae, juveniles, and mature individuals, and their biomolecular composition varies depending on the developmental stage. Therefore, in the present study, we performed untargeted lipid profiling of abalone tissues at different developmental stages as well as the hemolymph of mature female and male abalones using ultrahigh-performance liquid chromatography-tandem mass spectrometry. These profiles can provide meaningful information to understand compositional changes in lipids through abalone metamorphosis and development. A total of 132 lipids belonging to 15 classes were identified from abalone tissues at different developmental stages. Moreover, 21 lipids belonging to 8 classes were identified from the hemolymph of mature abalones. All data were processed following strict criteria to provide accurate information. Triglycerides and phosphatidylcholines were the major lipid components identified in both tissues and hemolymph, accounting for, respectively, 27% and 15% of all lipids in tissues and, respectively, 24% and 38% of all lipids in the hemolymph. Of note, lysophosphatidylcholine was only detected in the tissues of mature abalones, paving the way for further analyses of abalone lipids based on developmental stages. The present findings offer novel insights into the lipidome of abalone tissues and hemolymph at different developmental stages, building a foundation for improving the efficiency and quality of abalone aquaculture.

A review of suspension trapping digestion method in bottom-up proteomics.

The standard bottom-up proteomic workflow is comprised of sample preparation, data acquisition, and data analysis. While the latter two parts have made considerable advances in the last decade, sample preparation has remained an important challenge within the workflow due to the multi-step nature of complex biological samples, and still requires much development. Several sample preparation methods have been developed and used in the last two decades, including in-gel, in-solution, on-bead, filter-aided sample preparation, and suspension trapping, to improve reproducibility, efficiency, scalability, and reduce the handling time of this process. One of the most recent methods developed and applied in proteomics studies in recent years is suspension trapping, which combines rapid detergent removal, reactor-type protein digestion, and peptide clean-up in a tip or spin column. Suspension trapping is a simple, rapid, and reproducible digestion method that can effectively handle proteins in low microgram or sub-microgram amounts. This review discusses the benefits of the suspension trapping digestion method in relation to its development and application in bottom-up proteomics studies. We also discuss recent applications of suspension trapping digestion to different sample types and the features of the suspension trapping digestion method compared with other sample preparation methods.

Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14.

Apoptotic cells are rapidly engulfed and removed by phagocytes after displaying cell surface eat-me signals. Among many phospholipids, only phosphatidylserine (PS) is known to act as an eat-me signal on apoptotic cells. Using unbiased proteomics, we identified externalized phosphatidylinositides (PIPs) as apoptotic eat-me signals recognized by CD14+ phagocytes. Exofacial PIPs on the surfaces of early and late-apoptotic cells were observed in patches and blebs using anti-PI(3,4,5)P3 antibody, AKT- and PLCδ PH-domains, and CD14 protein. Phagocytosis of apoptotic cells was blocked either by masking exofacial PIPs or by CD14 knockout in phagocytes. We further confirmed that exofacial PIP+ thymocytes increased dramatically after in vivo irradiation and that exofacial PIP+ cells represented more significant populations in tissues of Cd14-/- than WT mice, especially after induction of apoptosis. Our findings reveal exofacial PIPs to be previously unknown cell death signals recognized by CD14+ phagocytes.

Different Regulatory Modes of Synechocystis sp. PCC 6803 in Response to Photosynthesis Inhibitory Conditions.

Cyanobacteria are promising industrial platforms owing to their ability to produce diverse natural secondary metabolites and nonnative value-added biochemicals from CO2 and light. To fully utilize their industrial potency, it is critical to understand their photosynthetic efficiency under various environmental conditions. In this study, we elucidated the inhibitory mechanisms of photosynthesis under high-light and low-temperature stress conditions in the model cyanobacterium Synechocystis sp. PCC 6803. Under each stress condition, the transcript abundance and translation efficiency were measured using transcriptome sequencing (RNA-seq) and ribosome profiling, and the genome-wide transcription unit architecture was constructed by data integration of transcription start sites and transcript 3'-end positions obtained from differential RNA-seq and sequencing of 3'-ends (Term-seq), respectively. Our results suggested that the mode of photosynthesis inhibition differed between the two stress conditions; high light stress induced photodamage responses, while low temperature stress impaired the translation efficiency of photosynthesis-associated genes. In particular, poor translation of photosystem I resulted from ribosome stalling at the untranslated regions, affecting the overall photosynthetic yield under low temperature stress. Our comprehensive multiomics analysis with transcription unit architecture provides foundational information on photosynthesis for future industrial strain development. IMPORTANCE Cyanobacteria are a compelling biochemical production platform for their ability to propagate using light and atmospheric CO2 via photosynthesis. However, the engineering of strains is hampered by limited understanding of photosynthesis under diverse environmental conditions such as high-light and low-temperature stresses. Herein, we decipher the transcriptomic and translatomic responses of the photosynthetic efficiency to stress conditions using the integrative analysis of multiomic data generated by RNA-seq and ribosome profiling, respectively. Through the generated massive data, along with the guide of the genome-wide transcription unit architecture constructed by transcription start sites and transcript 3'-end positions, we identified the factors affecting photosynthesis at transcription, posttranscription, and translation levels. Importantly, the high-light stress induces photodamage responses, and the low-temperature stress cripples the translation efficiency of photosynthesis-associated genes. The resulting insights provide pivotal information for future cyanobacterial cell factories powered by the engineering toward robust photosynthesis ability.

Multi-Omic Analyses Reveal Habitat Adaptation of Marine Cyanobacterium Synechocystis sp. PCC 7338.

Cyanobacteria are considered as promising microbial cell factories producing a wide array of bio-products. Among them, Synechocystis sp. PCC 7338 has the advantage of growing in seawater, rather than requiring arable land or freshwater. Nonetheless, how this marine cyanobacterium grows under the high salt stress condition remains unknown. Here, we determined its complete genome sequence with the embedded regulatory elements and analyzed the transcriptional changes in response to a high-salt environment. Complete genome sequencing revealed a 3.70 mega base pair genome and three plasmids with a total of 3,589 genes annotated. Differential RNA-seq and Term-seq data aligned to the complete genome provided genome-wide information on genetic regulatory elements, including promoters, ribosome-binding sites, 5'- and 3'-untranslated regions, and terminators. Comparison with freshwater Synechocystis species revealed Synechocystis sp. PCC 7338 genome encodes additional genes, whose functions are related to ion channels to facilitate the adaptation to high salt and high osmotic pressure. Furthermore, a ferric uptake regulator binding motif was found in regulatory regions of various genes including SigF and the genes involved in energy metabolism, suggesting the iron-regulatory network is connected to not only the iron acquisition, but also response to high salt stress and photosynthesis. In addition, the transcriptomics analysis demonstrated a cyclic electron transport through photosystem I was actively used by the strain to satisfy the demand for ATP under high-salt environment. Our comprehensive analyses provide pivotal information to elucidate the genomic functions and regulations in Synechocystis sp. PCC 7338.

Discovery of Post-Translational Modifications in Emiliania huxleyi.

Emiliania huxleyi is a cosmopolitan coccolithophore that plays an essential role in global carbon and sulfur cycling, and contributes to marine cloud formation and climate regulation. Previously, the proteomic profile of Emiliania huxleyi was investigated using a three-dimensional separation strategy combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current study reuses the MS/MS spectra obtained, for the global discovery of post-translational modifications (PTMs) in this species without specific enrichment methods. Twenty-five different PTM types were examined using Trans-Proteomic Pipeline (Comet and PeptideProphet). Overall, 13,483 PTMs were identified in 7421 proteins. Methylation was the most frequent PTM with more than 2800 modified sites, and lysine was the most frequently modified amino acid with more than 4000 PTMs. The number of proteins identified increased by 22.5% to 18,780 after performing the PTM search. Compared to intact peptides, the intensities of some modified peptides were superior or equivalent. The intensities of some proteins increased dramatically after the PTM search. Gene ontology analysis revealed that protein persulfidation was related to photosynthesis in Emiliania huxleyi. Additionally, various membrane proteins were found to be phosphorylated. Thus, our global PTM discovery platform provides an overview of PTMs in the species and prompts further studies to uncover their biological functions. The combination of a three-dimensional separation method with global PTM search is a promising approach for the identification and discovery of PTMs in other species.

Establishment of functional epithelial organoids from human lacrimal glands.

BACKGROUND: Tear deficiency due to lacrimal gland (LG) dysfunction is one of the major causes of dry eye disease (DED). Therefore, LG stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue; however, the number of stem cells in the LG tissue is low, and 2D long-term cultivation reduces the differentiation capacity of stem cells. Nevertheless, 3D LG organoids could be an alternative for a DED therapy because it is capable of prolonged growth while maintaining the characteristics of the LG tissue. Here, we report the development of LG organoids and their application as cell therapeutics. METHODS: Digested cells from human LG tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, specific marker expression, and histological characteristics were analyzed to authenticate the formation of LG organoids. The secretory function of LG organoids was confirmed  through calcium influx or proteomics analysis after pilocarpine treatment. To explore the curability of the developed organoids, mouse-derived LG organoids were fabricated and transplanted into the lacrimal tissue of a mouse model of DED. RESULTS: The histological features and specific marker expression of LG organoids were similar to those of normal LG tissue. In the pilocarpine-treated LG organoid, levels of internal Ca2+ ions and ß-hexosaminidase, a lysosomal protein in tear fluid, were increased. In addition, the secreted proteins from pilocarpine-treated lacrimal organoids were identified through proteomics. More than 70% of the identified proteins were proven to exosome through gene ontology analysis. These results indicate that our developed organoid was pilocarpine reactive, demonstrating the function of LG. Additionally, we developed LG organoids from patients with Sjogren's syndrome patients (SS) and confirmed that their histological features were similar to those of SS-derived LG tissue. Finally, we confirmed that the mouse LG organoids were well engrafted in the lacrimal tissue two weeks after transplantation. CONCLUSION: This study demonstrates that the established LG organoids resemble the characteristics of normal LG tissue and may be used as a therapy for patients with DED.

Photosynthetic production of biodiesel in Synechocystis sp. PCC6803 transformed with insect or plant fatty acid methyltransferase.

Biodiesel contains methyl or ethyl esters of long-chain fatty acids and has recently attracted increasing attention. Microalgae have emerged as a sustainable biodiesel production system owing to their photosynthetic potential. However, the conversion of microalgal biomass to biodiesel requires high energy and is costly. This study aimed to overcome the high cost of the pretreatment process by generating cyanobacteria converting fatty acids to fatty acids methyl ester (FAME) in vivo by introducing the fatty acid methyl ester transferase (FAMT) gene. Two FAMT genes from Drosophila melanogaster and Arabidopsis thaliana were selected and their codons were optimized for insertion in the Synechocystis sp. PCC6803 genome through homologous recombination, respectively. FAMT mRNA and protein expression levels were confirmed through reverse-transcription PCR and western blot analysis, respectively. Furthermore, heterologous expression of the FAMT genes yielded FAME, which was analyzed by gas chromatography. We found that FAMT transformants can be further metabolically optimized and applied for commercial production of biodiesel.

Enhanced Production of Photosynthetic Pigments and Various Metabolites and Lipids in the Cyanobacteria Synechocystis sp. PCC 7338 Culture in the Presence of Exogenous Glucose.

Synechocystis strains are cyanobacteria that can produce useful biomaterials for biofuel and pharmaceutical resources. In this study, the effects of exogenous glucose (5-mM) on cell growth, photosynthetic pigments, metabolites, and lipids in Synechocystis sp. PCC 7338 (referred to as Synechocystis 7338) were investigated. Exogenous glucose increased cell growth on days 9 and 18. The highest production (mg/L) of chlorophyll a (34.66), phycocyanin (84.94), allophycocyanin (34.28), and phycoerythrin (6.90) was observed on day 18 in Synechocystis 7338 culture under 5-mM glucose. Alterations in metabolic and lipidomic profiles under 5-mM glucose were investigated using gas chromatography-mass spectrometry (MS) and nanoelectrospray ionization-MS. The highest production (relative intensity/L) of aspartic acid, glutamic acid, glycerol-3-phosphate, linolenic acid, monogalactosyldiacylglycerol (MGDG) 16:0/18:1, MGDG 16:0/20:2, MGDG 18:1/18:2, neophytadiene, oleic acid, phosphatidylglycerol (PG) 16:0/16:0, and PG 16:0/17:2 was achieved on day 9. The highest production of pyroglutamic acid and sucrose was observed on day 18. We suggest that the addition of exogenous glucose to Synechocystis 7338 culture could be an efficient strategy for improving growth of cells and production of photosynthetic pigments, metabolites, and intact lipid species for industrial applications.

Current Status and Future Strategies to Increase Secondary Metabolite Production from Cyanobacteria.

Cyanobacteria, given their ability to produce various secondary metabolites utilizing solar energy and carbon dioxide, are a potential platform for sustainable production of biochemicals. Until now, conventional metabolic engineering approaches have been applied to various cyanobacterial species for enhanced production of industrially valued compounds, including secondary metabolites and non-natural biochemicals. However, the shortage of understanding of cyanobacterial metabolic and regulatory networks for atmospheric carbon fixation to biochemical production and the lack of available engineering tools limit the potential of cyanobacteria for industrial applications. Recently, to overcome the limitations, synthetic biology tools and systems biology approaches such as genome-scale modeling based on diverse omics data have been applied to cyanobacteria. This review covers the synthetic and systems biology approaches for advanced metabolic engineering of cyanobacteria.

UBA2 activates Wnt/ß-catenin signaling pathway during protection of R28 retinal precursor cells from hypoxia by extracellular vesicles derived from placental mesenchymal stem cells.

BACKGROUND: Stem cell transplantation has been proposed as an alternative treatment for intractable optic nerve disorders characterized by irrecoverable loss of cells. Mesenchymal stem cells, with varying tissue regeneration and recovery capabilities, are being considered for potential cell therapies. To overcome the limitations of cell therapy, we isolated exosomes from human placenta-derived mesenchymal stem cells (hPMSCs) and investigated their therapeutic effects in R28 cells (retinal precursor cells) exposed to CoCl2. METHOD: After 9 h of exposure to CoCl2, the hypoxic damaged R28 cells were divided into the non-treatment group (CoCl2 + R28 cells) and treatment group (CoCl2 + R28 cells treated with exosome). Immunoblot analysis was performed for Pcna, Hif-1α, Vegf, Vimentin, Thy-1, Gap43, Ermn, Neuroflament, Wnt3a, ß-catenin, phospo-GSK3ß, Lef-1, UBA2, Skp1, ßTrcp, and ubiquitin. The proteomes of each group were analyzed by liquid chromatography/tandem mass (LC-MS/MS) spectrometry. Differentially expressed proteins (DEPs) were detected by label-free quantification, and the interactions of the proteins were examined through signal transduction pathway and gene ontology analysis. RESULT: We observed that exosome could significantly recover proliferation damaged by CoCl2 treatment. In addition, the treatment group presented the decreased expression of Hif-1α protein (P < 0.05) and increased expression of proliferation marker, Pcna, and nerve regeneration-related factors such as Vimentin, Thy-1, and Neuroflament (P < 0.05) compared with the non-treatment group. In total, 200 DEPs were identified in the non-treatment group and treatment group (fold change ≥ 2, p < 0.05). Catenin and ubiquitin systems (UBA2, UBE2E3, UBE2I) were found in both the DEP lists of downregulated proteins from the non-treatment group and upregulated proteins from the treatment group. The mRNA expressions of ubiquitin systems were significantly decreased under hypoxic conditions. Moreover, UBA2 and Wnt/ß-catenin protein were associated with the rescue of the hypoxic damaged R28 cells. Using a siRNA system, we could find it out that hPMSC exosomes could not repair altered expressions of target proteins by CoCl2 in lacking UBA2 R28 cells. CONCLUSION: This study reported that hypoxic damaged expression of regeneration markers in R28 cells was significantly recovered by hPMSC exosomes. We could also demonstrate that UBA2 played a key role in activating the Wnt/ß-catenin signaling pathway during protection of hypoxic damaged R28 cells, induced by hPMSC exosomes.

Proteomic Profiling of Emiliania huxleyi Using a Three-Dimensional Separation Method Combined with Tandem Mass Spectrometry.

Emiliania huxleyi is one of the most abundant marine planktons, and it has a crucial feature in the carbon cycle. However, proteomic analyses of Emiliania huxleyi have not been done extensively. In this study, a three-dimensional liquid chromatography (3D-LC) system consisting of strong cation exchange, high- and low-pH reversed-phase liquid chromatography was established for in-depth proteomic profiling of Emiliania huxleyi. From tryptic proteome digest, 70 fractions were generated and analyzed using liquid chromatography-tandem mass spectrometry. In total, more than 84,000 unique peptides and 10,000 proteins groups were identified with a false discovery rate of ≤0.01. The physicochemical properties of the identified peptides were evaluated. Using ClueGO, approximately 700 gene ontology terms and 15 pathways were defined from the identified protein groups with p-value ≤0.05, covering a wide range of biological processes, cellular components, and molecular functions. Many biological processes associated with CO2 fixation, photosynthesis, biosynthesis, and metabolic process were identified. Various molecular functions relating to protein binding and enzyme activities were also found. The 3D-LC strategy is a powerful approach for comparative proteomic studies on Emiliania huxleyi to reveal changes in its protein level and related mechanism.

Dietary walnut as food factor to rescue from NSAID-induced gastrointestinal mucosal damages.

Nuclear factor erythroid-derived 2-like 2 (Nrf-2) is transcription factor implicated in the antioxidant response element-mediated induction of endogenous antioxidant enzyme such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase, and NAD(P)H quinone dehydrogenase 1, among which HO-1 is an enzyme catalyzing the degradation of heme.producing biliverdin, ferrous iron, and carbon monoxide. In the stomach, as much as regulating gastric acid secretions, well-coordinated establishment of defense system stands for maintaining gastric integrity. In previous study, author et al. for the first time discovered HO-1 induction was critical in affording faithful gastric defense against various irritants including Helicobacter pylori infection, stress, alcohol, non-steroidal anti-inflammatory drugs (NSAIDs), aspirin, and toxic bile acids. In this review article, we can add the novel evidence that dietary walnut intake can be reliable way to rescue from NSAIDs-induced gastrointestinal damages via the induction of HO-1 transcribed with Nrf-2 through specific inactivation of Keap-1. From molecular exploration to translational animal model of indomethacin-induced gastrointestinal damages, significant induction of HO-1 contributed to rescuing from damages. In addition to HO-1 induction action relevant to walnut, we added the description the general actions of walnut extracts or dietary intake of walnut regarding cytoprotection and why we have focused on to NSAID damages.

Discovery of plasma biomarkers for predicting the severity of coronary artery atherosclerosis by quantitative proteomics.

INTRODUCTION: Cardiovascular disease (CVD) in patients with diabetes is the leading cause of death. Finding early biomarkers for detecting asymptomatic patients with CVD can improve survival. Recently, plasma proteomics-targeted selected reaction monitoring/multiple reaction monitoring analyses (MRM)-has emerged as highly specific and sensitive tools compared with classic ELISA methods. The objective was to identify differentially regulated proteins according to the severity of the coronary artery atherosclerosis. RESEARCH DESIGN AND METHODS: A discovery cohort, a verification cohort and a validation cohort consisted of 18, 53, and 228 subjects, respectively. The grade of coronary artery stenosis was defined as a percentage of luminal stenosis of the major coronary arteries. Participants were divided into six groups, depending on the presence of diabetes and the grade of coronary artery stenosis. Two mass spectrometric approaches were employed: (1) conventional shotgun liquid chromatography tandem mass spectrometry for a discovery and (2) quantitative MRM for verification and validation. An analysis of the covariance was used to examine the biomarkers' predictivity beyond conventional cardiovascular risks. RESULTS: A total of 1349 different proteins were identified from a discovery cohort. We selected 52 proteins based on the tandem mass tag quantitative analysis then summarized as follows: chemokine (C-X-C motif) ligand 7 (CXCL7), apolipoprotein C-II (APOC2), human lipopolysaccharide-binding protein (LBP) and dedicator of cytokinesis 2 (DOCK2) in diabetes; CXCL7, APOC2, LBP, complement 4A (C4A), vitamin D-binding protein (VTDB) and laminin ß1 subunit in non-diabetes. Analysis of covariance showed that APOC2, DOCK2, CXCL7 and VTDB were upregulated and C4A was downregulated in patients with diabetes showing severe coronary artery stenosis. LBP and VTDB were downregulated in patients without diabetes, showing severe coronary artery stenosis. CONCLUSION: We identified significant associations between circulating APOC2, C4A, CXCL7, DOCK2, LBP and VTDB levels and the degree of coronary artery stenosis using the MRM technique.

Review of Three-Dimensional Liquid Chromatography Platforms for Bottom-Up Proteomics.

Proteomics is a large-scale study of proteins, aiming at the description and characterization of all expressed proteins in biological systems. The expressed proteins are typically highly complex and large in abundance range. To fulfill high accuracy and sensitivity of proteome analysis, the hybrid platforms of multidimensional (MD) separations and mass spectrometry have provided the most powerful solution. Multidimensional separations provide enhanced peak capacity and reduce sample complexity, which enables mass spectrometry to analyze more proteins with high sensitivity. Although two-dimensional (2D) separations have been widely used since the early period of proteomics, three-dimensional (3D) separation was barely used by low reproducibility of separation, increased analysis time in mass spectrometry. With developments of novel microscale techniques such as nano-UPLC and improvements of mass spectrometry, the 3D separation becomes a reliable and practical selection. This review summarizes existing offline and online 3D-LC platforms developed for proteomics and their applications. In detail, setups and implementation of those systems as well as their advances are outlined. The performance of those platforms is also discussed and compared with the state-of-the-art 2D-LC. In addition, we provide some perspectives on the future developments and applications of 3D-LC in proteomics.

De novo transcriptome profile of coccolithophorid alga Emiliania huxleyi CCMP371 at different calcium concentrations with proteome analysis.

The haptophyte alga Emiliania huxleyi is the most abundant coccolithophore in the modern ocean and produces elaborate calcite crystals, called coccolith, in a separate intracellular compartment known as the coccolith vesicle. Despite the importance of biomineralization in coccolithophores, the molecular mechanism underlying it remains unclear. Understanding this precise machinery at the molecular level will provide the knowledge needed to enable further manipulation of biomineralization. In our previous study, altering the calcium concentration modified the calcifying ability of E. huxleyi CCMP371. Therefore in this study, we tested E. huxleyi cells acclimated to three different calcium concentrations (0, 0.1, and 10 mM). To understand the whole transcript profile at different calcium concentrations, RNA-sequencing was performed and used for de novo assembly and annotation. The differentially expressed genes (DEGs) among the three different calcium concentrations were analyzed. The functional classification by gene ontology (GO) revealed that 'intrinsic component of membrane' was the most enriched of the GO terms at the ambient calcium concentration (10 mM) compared with the limited calcium concentrations (0 and 0.1 mM). Moreover, the DEGs in those comparisons were enriched mainly in 'secondary metabolites biosynthesis, transport and catabolism' and 'signal transduction mechanisms' in the KOG clusters and 'processing in endoplasmic reticulum', and 'ABC transporters' in the KEGG pathways. Furthermore, metabolic pathways involved in protein synthesis were enriched among the differentially expressed proteins. The results of this study provide a molecular profile for understanding the expression of transcripts and proteins in E. huxleyi at different calcium concentrations, which will help to identify the detailed mechanism of its calcification.