P2Y2 purinergic receptor gene deletion protects mice from bacterial endotoxin and sepsis-associated liver injury and mortality.

The liver plays a significant role in regulating a wide range of metabolic, homeostatic, and host-defense functions. However, the impact of liver injury on the host's ability to control bacteremia and morbidity in sepsis is not well understood. Leukocyte recruitment and activation lead to cytokine and chemokine release, which, in turn, trigger hepatocellular injury and elevate nucleotide levels in the extracellular milieu. P2Y2 purinergic receptors, G protein-coupled and activated by extracellular ATP/UTP, are expressed at the cell surface of hepatocytes and nonparenchymal cells. We sought to determine whether P2Y2 purinergic receptor function is necessary for the maladaptive host response to bacterial infection and endotoxin-mediated inflammatory liver injury and mortality in mice. We report that P2Y2 purinergic receptor knockout mice (P2Y2-/-) had attenuated inflammation and liver injury, with improved survival in response to LPS/galactosamine (LPS/GalN; inflammatory liver injury) and cecal ligation and puncture (CLP; polymicrobial sepsis). P2Y2-/- livers had attenuated c-Jun NH2-terminal kinase activation, matrix metallopeptidase-9 expression, and hepatocyte apoptosis in response to LPS/GalN and attenuated inducible nitric oxide synthase and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 protein expression in response to CLP. Implicating liver injury in the disruption of amino acid homeostasis, CLP led to lower serum arginine and higher bacterial load and morbidity in the WT mice, whereas serum arginine levels were comparable to sham-operated controls in P2Y2-/- mice, which had attenuated bacteremia and improved survival. Collectively, our studies highlight the pathophysiological relevance of P2Y2 purinergic receptor function in inflammatory liver injury and dysregulation of systemic amino acid homeostasis with implications for sepsis-associated immune dysfunction and morbidity in mice.NEW & NOTEWORTHY Our studies provide experimental evidence for P2Y2 purinergic receptor-mediated potentiation of inflammatory liver injury, morbidity, and mortality, in two well-established animal models of inflammatory liver injury. Our findings highlight the potential to target P2Y2 purinergic signaling to attenuate the induction of "cytokine storm" and prevent its deleterious consequences on liver function, systemic amino acid homeostasis, host response to bacterial infection, and sepsis-associated morbidity and mortality.

Protein-Protein Interaction Network Mapping by Affinity Purification Cross-Linking Mass Spectrometry (AP-XL-MS) based Proteomics.

Protein-protein interactions (PPIs) are the physical interactions formed among proteins. These interactions are primarily functional, i.e., they arise from specific biomolecular events, and each interaction interface serves a specific purpose. A significant number of methods have been developed for protein interactions in the field of proteomics in the last decade. Advanced mass spectrometry technology significantly contributed to the development of these methods. The rapid advancement of groundbreaking MS technology has greatly aided the mapping of protein interaction from large-data sets comprehensively. This chapter describes the affinity purification (AP) mass spectrometry (MS)-based methods combined with chemical cross-linking (XL) of protein complexes. This chapter includes sample preparation methods involving cell culture, cell treatments with ligands, drugs, and cross-linkers, protein extractions, affinity purification, sodium dodecyl sulfate (SDS) polyacrylamide gel separation, in-solution or in-gel digestion, liquid-chromatography, and mass spectrometry analysis of samples (LC-MS/MS). Application of a cleavable cross-linker, dual cleavable cross-linking technology (DUCCT) in combination with the affinity purification (AP) method has also been described. Methods for data analysis using unmodified and cross-linked peptide analysis are discussed.

Lipopolysaccharide and statin-mediated immune-responsive protein networks revealed in macrophages through affinity purification spacer-arm controlled cross-linking (AP-SPACC) proteomics.

Toll-like receptor 4 (TLR4), a pattern recognition receptor, is activated by lipopolysaccharides (LPS) and induces the MyD88 pathway, which subsequently produces pro-inflammatory cytokines through activation of transcriptional nuclear factor (NF)-κB. Statins have been widely prescribed to reduce cholesterol synthesis for patients with cardiovascular disease. Statins may have pleiotropic effects, which include anti- and pro-inflammatory effects on cells. The molecular mechanism of the sequential influence of LPS and statin on the innate immune system remains unknown. We employed affinity purification-spacer-arm controlled cross-linking (AP-SPACC) MS-based proteomics analysis to identify the LPS- and statin-LPS-responsive proteins and their networks. LPS-stimulated RAW 264.7 macrophage cells singly and combined with the drug statin used in this study. Two chemical cross-linkers with different spacer chain lengths were utilized to stabilize the weak and transient interactors. Proteomic analysis identified 1631 differentially expressed proteins. We identified 151 immune-response proteins through functional enrichment analysis and visualized their interaction networks. Selected candidate protein-coding genes were validated, specifically squamous cell carcinoma antigens recognized by T cells 3, sphingosine-1-phosphate lyase 1, Ras-related protein Rab-35, and tumor protein D52 protein-coding genes through transcript-level expression analysis. The expressions of those genes were significantly increased upon statin treatment and decreased in LPS-stimulated macrophage cells. Therefore, we presumed that the expression changes of genes occurred due to immune response during activation of inflammation. These results highlight the immune-responsive proteins network, providing a new platform for novel investigations and discovering future therapeutic targets for inflammatory diseases.

Identification of Inflammatory Proteomics Networks of Toll-like Receptor 4 through Immunoprecipitation-Based Chemical Cross-Linking Proteomics.

Toll-like receptor 4 (TLR4) is a receptor on an immune cell that can recognize the invasion of bacteria through their attachment with bacterial lipopolysaccharides (LPS). Hence, LPS is a pro-immune response stimulus. On the other hand, statins are lipid-lowering drugs and can also lower immune cell responses. We used human embryonic kidney (HEK 293) cells engineered to express HA-tagged TLR-4 upon treatment with LPS, statin, and both statin and LPS to understand the effect of pro- and anti-inflammatory responses. We performed a monoclonal antibody (mAb) directed co-immunoprecipitation (CO-IP) of HA-tagged TLR4 and its interacting proteins in the HEK 293 extracted proteins. We utilized an ETD cleavable chemical cross-linker to capture weak and transient interactions with TLR4 protein. We tryptic digested immunoprecipitated and cross-linked proteins on beads, followed by liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the peptides. Thus, we utilized the label-free quantitation technique to measure the relative expression of proteins between treated and untreated samples. We identified 712 proteins across treated and untreated samples and performed protein network analysis using Ingenuity Pathway Analysis (IPA) software to reveal their protein networks. After filtering and evaluating protein expression, we identified macrophage myristoylated alanine-rich C kinase substrate (MARCKSL1) and creatine kinase proteins as a potential part of the inflammatory networks of TLR4. The results assumed that MARCKSL1 and creatine kinase proteins might be associated with a statin-induced anti-inflammatory response due to possible interaction with the TLR4.

IDH1 p.R132H ctDNA and D-2-hydroxyglutarate as CSF biomarkers in patients with IDH-mutant gliomas.

INTRODUCTION: We aimed to evaluate IDH1 p.R132H mutation and 2-hydroxyglutarate (2HG) in cerebrospinal fluid (CSF) as biomarkers for patients with IDH-mutant gliomas. METHODS: CSF was collected from patients with infiltrating glioma, and 2HG levels were measured by liquid chromatography-mass spectrometry. IDH1 p.R132H mutant allele frequency (MAF) in CSF-ctDNA was measured by digital droplet PCR (ddPCR). Tumor volume was measured from standard-of-care magnetic resonance images. RESULTS: The study included 48 patients, 6 with IDH-mutant and 42 with IDH-wildtype gliomas, and 57 samples, 9 from the patients with IDH-mutant and 48 from the patients with IDH-wildtype gliomas. ctDNA was detected in 7 of the 9 samples from patients with IDH-mutant glioma, and IDH1 p.R132H mutation was detected in 5 of the 7 samples. The MAF ranged from 0.3 to 39.95%. Total 2HG level, D-2HG level, and D/L-2HG ratio in CSF were significantly higher in patients with IDH-mutant gliomas than in patients with IDH-wildtype gliomas. D-2HG level and D/L-2HG ratio correlated with total tumor volume in patients with IDH-mutant gliomas but not in patients with IDH-wildtype gliomas. CONCLUSION: Our results suggest that detection of IDH1 p.R132H mutation by ddPCR and increased D-2HG level in CSF may help identify IDH-mutant gliomas. Our results also suggest that D-2HG level and D/L-2HG ratio correlate with tumor volume in patients with IDH-mutant gliomas. Further prospective studies with larger cohorts are needed to validate these findings.

Lipidomic Profiling Identifies a Novel Lipid Signature Associated with Ethnicity-Specific Disparity of Bladder Cancer.

Bladder Cancer (BLCA) is the ninth most frequently diagnosed cancer globally and the sixth most common cancer in the US. African Americans (AA) exhibit half the BLCA incidence compared to European Americans (EA), but they have a 70% higher risk of cancer-related death; unfortunately, this disparity in BLCA mortality remains poorly understood. In this study, we have used an ethnicity-balanced cohort for unbiased lipidomics profiling to study the changes in the lipid fingerprint for AA and EA BLCA tissues collected from similar geographical regions to determine a signature of ethnic-specific alterations. We identified 86 lipids significantly altered between self-reported AA and EA BLCA patients from Augusta University (AU) cohort. The majority of altered lipids belong to phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), ly sophosphatidylcholines (lysoPCs), phosphatidylserines (PSs), and diglycerides (DGs). Interestingly, levels of four lysoPCs (lyso PCs 20:3, lyso PCs 22:1, lyso PCs 22:2, and lyso PCs 26:1) were elevated while, in contrast, the majority of the PCs were reduced in AA BLCA. Significant alterations in long-chain monounsaturated (MonoUN) and polyunsaturated (PolyUN) lipids were also observed between AA and EA BLCA tumor tissues. These first-in-field results implicate ethnic-specific lipid alterations in BLCA.

The COVID-19 vaccination acceptance/hesitancy rate and its determinants among healthcare workers of 91 Countries: A multicenter cross-sectional study.

The aim of this study was to investigate the COVID-19 vaccination acceptance rate and its determinants among healthcare workers in a multicenter study. This was a cross-sectional multi-center survey conducted from February 5 to April 29, 2021. The questionnaire consisted of 26 items in 6 subscales. The English version of the questionnaire was translated into seven languages and distributed through Google Forms using snowball sampling; a colleague in each country was responsible for the forward and backward translation, and also the distribution of the questionnaire. A forward stepwise logistic regression was utilized to explore the variables and questionnaire factors tied to the intention to COVID-19 vaccination. 4630 participants from 91 countries completed the questionnaire. According to the United Nations Development Program 2020, 43.6 % of participants were from low Human Development Index (HDI) regions, 48.3 % high and very high, and 8.1 % from medium. The overall vaccination hesitancy rate was 37 %. Three out of six factors of the questionnaire were significantly related to intention to the vaccination. While 'Perceived benefits of the COVID-19 vaccination' (OR: 3.82, p-value<0.001) and 'Prosocial norms' (OR: 5.18, p-value<0.001) were associated with vaccination acceptance, 'The vaccine safety/cost concerns' with OR: 3.52, p-value<0.001 was tied to vaccination hesitancy. Medical doctors and pharmacists were more willing to take the vaccine in comparison to others. Importantly, HDI with OR: 12.28, 95 % CI: 6.10-24.72 was a strong positive determinant of COVID-19 vaccination acceptance. This study highlighted the vaccination hesitancy rate of 37 % in our sample among HCWs. Increasing awareness regarding vaccination benefits, confronting the misinformation, and strengthening the prosocial norms would be the primary domains for maximizing the vaccination coverage. The study also showed that the HDI is strongly associated with the vaccination acceptance/hesitancy, in a way that those living in low HDI contexts are more hesitant to receive the vaccine.

CHAF1A Blocks Neuronal Differentiation and Promotes Neuroblastoma Oncogenesis via Metabolic Reprogramming.

Neuroblastoma (NB) arises from oncogenic disruption of neural crest (NC) differentiation. Treatment with retinoic acid (RA) to induce differentiation has improved survival in some NB patients, but not all patients respond, and most NBs eventually develop resistance to RA. Loss of the chromatin modifier chromatin assembly factor 1 subunit p150 (CHAF1A) promotes NB cell differentiation; however, the mechanism by which CHAF1A drives NB oncogenesis has remained unexplored. This study shows that CHAF1A gain-of-function supports cell malignancy, blocks neuronal differentiation in three models (zebrafish NC, human NC, and human NB), and promotes NB oncogenesis. Mechanistically, CHAF1A upregulates polyamine metabolism, which blocks neuronal differentiation and promotes cell cycle progression. Targeting polyamine synthesis promotes NB differentiation and enhances the anti-tumor activity of RA. The authors' results provide insight into the mechanisms that drive NB oncogenesis and suggest a rapidly translatable therapeutic approach (DFMO plus RA) to enhance the clinical efficacy of differentiation therapy in NB patients.

Managing adult patients with infectious diseases in emergency departments: international ID-IRI study.

We aimed to explore factors for optimizing antimicrobial treatment in emergency departments. A single-day point prevalence survey was conducted on January 18, 2020, in 53 referral/tertiary hospitals in 22 countries. 1957 (17%) of 11557 patients presenting to EDs had infections. The mean qSOFA score was 0.37 ± 0.74. Sepsis (qSOFA ≥ 2) was recorded in 218 (11.1%) patients. The mean qSOFA score was significantly higher in low-middle (1.48 ± 0.963) compared to upper-middle (0.17 ± 0.482) and high-income (0.36 ± 0.714) countries (P < 0.001). Eight (3.7%) patients with sepsis were treated as outpatients. The most common diagnoses were upper-respiratory (n = 877, 43.3%), lower-respiratory (n = 316, 16.1%), and lower-urinary (n = 201, 10.3%) infections. 1085 (55.4%) patients received antibiotics. The most-commonly used antibiotics were beta-lactam (BL) and BL inhibitors (n = 307, 15.7%), third-generation cephalosporins (n = 251, 12.8%), and quinolones (n = 204, 10.5%). Irrational antibiotic use and inappropriate hospitalization decisions seemed possible. Patients were more septic in countries with limited resources. Hence, a better organizational scheme is required.

Proteomics Network Analysis of Polarized Macrophages.

Macrophages play a critical role in innate immunity through Toll-like receptor (TLR) signaling. Lipopolysaccharides (LPS) are a ligand of microbial origin that can trigger cell signaling in macrophages through TLRs and production of pro-inflammatory cytokines. Statin, a hypercholesterolemia drug, on the contrary, can reduce inflammatory cytokine production, and inflammation at large. Discovery-based quantitative proteomics is a useful method for unraveling complex protein networks and inter-protein interactions. Here, we describe protocols for studying the inflammatory proteomics network in RAW 264.7 cells (a model murine macrophage cell line) with the singular or sequential treatment of LPS and statin. We provide detailed protocols, including a quantitative proteomic analysis by mass spectrometry data, a protein network analysis by bioinformatics, and a validation of target through biochemical methods (e.g., immunocytochemistry, immunoblotting, gene silencing, and real-time PCR).

Improved in-solution trypsin digestion method for methanol-chloroform precipitated cellular proteomics sample.

Methanol-chloroform based protein precipitation is an essential step in many liquid chromatography-tandem mass spectrometry-based cellular proteomics applications. However, re-solubilization of the total protein precipitate is difficult using regular in-solution digestion protocol. Sodium deoxycholate is reported as an efficient surfactant for re-solubilization of membrane fractions. In this study, we demonstrated an application combining methanol-chloroform based protein precipitations and deoxycholic acid assisted re-solubilization of pellets to evaluate the improvement of protein identifications in mass spectrometry-based bottom-up proteomics. We evaluated the modified method using an equal amount of Raw 264.7 mouse macrophage cell lysate. Detailed in-solution trypsin digestion studies were presented on methanol-chloroform precipitated samples with or without deoxycholic acid treatments and compared with popular sample digestion methods. A mass spectrometric analysis confirmed an 82% increase in protein identification in deoxycholic acid-treated samples compared to other established methods. Furthermore, liquid chromatography-tandem mass spectrometry analysis of an equal amount of proteins from methanol-chloroform precipitated, and methanol-chloroform/deoxycholic acid-treated macrophage cell lysate showed a 14% increase and 27% unique protein identifications. We believe this improved digestion method could be a complementary or alternative method for mammalian cell sample preparations where sodium dodecyl sulfate based lysis buffer is frequently used.

Proteomic Investigation of a Diseased Gorgonian Coral Indicates Disruption of Essential Cell Function and Investment in Inflammatory and Other Immune Processes.

As scleractinian coral cover declines in the face of increased frequency in disease outbreaks, future reefs may become dominated by octocorals. Understanding octocoral disease responses and consequences is therefore necessary if we are to gain insight into the future of ecosystem services provided by coral reefs. In Florida, populations of the octocoral Eunicea calyculata infected with Eunicea black disease (EBD) were observed in the field in the fall of 2011. This disease was recognized by a stark, black pigmentation caused by heavy melanization. Histological preparations of E. calyculata infected with EBD demonstrated granular amoebocyte (GA) mobilization, melanin granules in much of the GA population, and the presence of fungal hyphae penetrating coral tissue. Previous transcriptomic analysis also identified immune trade-offs evidenced by increased immune investment at the expense of growth. Our investigation utilized proteogenomic techniques to reveal decreased investment in general cell signaling while increasing energy production for immune responses. Inflammation was also prominent in diseased E. calyculata and sheds light on factors driving the extreme phenotype observed with EBD. With disease outbreaks continuing to increase in frequency, our results highlight new targets within the cnidarian immune system and provide a framework for understanding transcriptomics in the context of an organismal disease phenotype and its protein expression.

Cross-linking Proteomics Indicates Effects of Simvastatin on the TLR2 Interactome and Reveals ACTR1A as a Novel Regulator of the TLR2 Signal Cascade.

Toll-like receptor 2 (TLR2) is a pattern recognition receptor that, upon ligation by microbial molecules, interacts with other proteins to initiate pro-inflammatory responses by the cell. Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors), drugs widely prescribed to reduce hypercholesterolemia, are reported to have both pro- and anti-inflammatory effects upon cells. Some of these responses are presumed to be driven by effects on signaling proteins at the plasma membrane, but the underlying mechanisms remain obscure. We reasoned that profiling the effect of statins on the repertoire of TLR2-interacting proteins might provide novel insights into the mechanisms by which statins impact inflammation. In order to study the TLR2 interactome, we designed a coimmunoprecipitation (IP)-based cross-linking proteomics study. A hemagglutinin (HA)-tagged-TLR2 transfected HEK293 cell line was used to precipitate the TLR2 interactome upon cell exposure to the TLR2 agonist Pam3CSK4 and simvastatin, singly and in combination. To stabilize protein interactors, we used two different chemical cross-linkers with different spacer chain lengths. Proteomic analysis revealed important combinatorial effects of simvastatin and Pam3CSK4 on the TLR2 interactome. After stringent data filtering, we identified alpha-centractin (ACTR1A), an actin-related protein and subunit of the dynactin complex, as a potential interactor of TLR2. The interaction was validated using biochemical methods. RNA interference studies revealed an important role for ACTR1A in induction of pro-inflammatory cytokines. Taken together, we report that statins remodel the TLR2 interactome, and we identify ACTR1A, a part of the dynactin complex, as a novel regulator of TLR2-mediated immune signaling pathways.

Proteome characterization of copper stress responses in the roots of sorghum.

Copper (Cu) is a important micronutrient for plants, but it is extremely toxic to plants at high concentration and can inactivate and disturb protein structures. To explore the Cu stress-induced tolerance mechanism, the present study was conducted on the roots of sorghum seedlings exposed to 50 and 100 µM CuSO4 for 5 days. Accumulation of Cu increased in roots when the seedlings were treated with the highest concentration of Cu2+ ions (100 µM). Elevated Cu concentration provoked notable reduction of Fe, Zn, Ca, and Mn uptake in the roots of sorghum seedlings. In the proteome analysis, high-throughput two-dimensional polyacrylamide gel electrophoresis combined with MALDI-TOF-TOF MS was performed to explore the molecular responses of Cu-induced sorghum seedling roots. In two-dimensional silver-stained gels, 422 protein spots were identified in the 2-D gel whereas twenty-one protein spots (≥1.5-fold) were used to analyze mass spectrometry from Cu-induced sorghum roots. Among the 21 differentially expressed proteins, 10 proteins were increased, while 11 proteins were decreased due to the intake of Cu ions by roots of sorghum. Abundance of most of the identified proteins from the roots that function in stress response and metabolism was remarkably enhanced, while proteins involved in transcription and regulation were severely reduced. Taken together, these results imply insights into a potential molecular mechanism towards Cu stress in C4 plant, sorghum.

Leaf proteome characterization in the context of physiological and morphological changes in response to copper stress in sorghum.

Copper (Cu) is an essential micronutrient required for normal growth and development of plants; however, at elevated concentrations in soil, copper is also generally considered to be one of the most toxic metals to plant cells due to its inhibitory effects against many physiological and biochemical processes. In spite of its potential physiological and economical significance, molecular mechanisms under Cu stress has so far been grossly overlooked in sorghum. To explore the molecular alterations that occur in response to copper stress, the present study was performed in ten-day-old Cu-exposed leaves of sorghum seedlings. The growth characteristics were markedly inhibited, and ionic alterations were prominently observed in the leaves when the seedlings were exposed to different concentrations (0, 100, and 150 µM) of CuSO4. Using two-dimensional gels with silver staining, 643 differentially expressed protein spots (≥1.5-fold) were identified as either significantly increased or reduced in abundance. Of these spots, a total of 24 protein spots (≥1.5-fold) from Cu-exposed sorghum leaves were successfully analyzed by MALDI-TOF-TOF mass spectrometry. Of the 24 differentially expressed proteins from Cu-exposed sorghum leaves, 13 proteins were up-regulated, and 11 proteins were down-regulated. The abundance of most identified protein species, which function in carbohydrate metabolism, stress defense and protein translation, was significantly enhanced, while that of another protein species involved in energy metabolism, photosynthesis and growth and development were severely reduced. The resulting differences in protein expression patterns together with related morpho-physiological processes suggested that these results could help to elucidate plant adaptation to Cu stress and provide insights into the molecular mechanisms of Cu responses in C4 plants.

Morpho-Physiological and Proteome Level Responses to Cadmium Stress in Sorghum.

Cadmium (Cd) stress may cause serious morphological and physiological abnormalities in addition to altering the proteome in plants. The present study was performed to explore Cd-induced morpho-physiological alterations and their potential associated mechanisms in Sorghum bicolor leaves at the protein level. Ten-day-old sorghum seedlings were exposed to different concentrations (0, 100, and 150 µM) of CdCl2, and different morpho-physiological responses were recorded. The effects of Cd exposure on protein expression patterns in S. bicolor were investigated using two-dimensional gel electrophoresis (2-DE) in samples derived from the leaves of both control and Cd-treated seedlings. The observed morphological changes revealed that the plants treated with Cd displayed dramatically altered shoot lengths, fresh weights and relative water content. In addition, the concentration of Cd was markedly increased by treatment with Cd, and the amount of Cd taken up by the shoots was significantly and directly correlated with the applied concentration of Cd. Using the 2-DE method, a total of 33 differentially expressed protein spots were analyzed using MALDI-TOF/TOF MS. Of these, treatment with Cd resulted in significant increases in 15 proteins and decreases in 18 proteins. Major changes were absorbed in the levels of proteins known to be involved in carbohydrate metabolism, transcriptional regulation, translation and stress responses. Proteomic results revealed that Cd stress had an inhibitory effect on carbon fixation, ATP production and the regulation of protein synthesis. Our study provides insights into the integrated molecular mechanisms involved in responses to Cd and the effects of Cd on the growth and physiological characteristics of sorghum seedlings. We have aimed to provide a reference describing the mechanisms involved in heavy metal damage to plants.

Proteins involved in biophoton emission and flooding-stress responses in soybean under light and dark conditions.

To know the molecular systems basically flooding conditions in soybean, biophoton emission measurements and proteomic analyses were carried out for flooding-stressed roots under light and dark conditions. Photon emission was analyzed using a photon counter. Gel-free quantitative proteomics were performed to identify significant changes proteins using the nano LC-MS along with SIEVE software. Biophoton emissions were significantly increased in both light and dark conditions after flooding stress, but gradually decreased with continued flooding exposure compared to the control plants. Among the 120 significantly identified proteins in the roots of soybean plants, 73 and 19 proteins were decreased and increased in the light condition, respectively, and 4 and 24 proteins were increased and decreased, respectively, in the dark condition. The proteins were mainly functionally grouped into cell organization, protein degradation/synthesis, and glycolysis. The highly abundant lactate/malate dehydrogenase proteins were decreased in flooding-stressed roots exposed to light, whereas the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme was increased in both light and dark conditions. Notably, however, specific enzyme assays revealed that the activities of these enzymes and biophoton emission were sharply increased after 3 days of flooding stress. This finding suggests that the source of biophoton emission in roots might involve the chemical excitation of electron or proton through enzymatic or non-enzymatic oxidation and reduction reactions. Moreover, the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme may play important roles in responses in flooding stress of soybean under the light condition and as a contributing factor to biophoton emission.

Jasmonic acid induced protein response to biophoton emissions and flooding stress in soybean.

Biophoton emissions were elevated by the exogenous plant hormone application such as jasmonic (JA) and salicylic acid (SA). To reveal the molecular mechanisms underlying flooding stress responses in soybean treated with JA and SA, biophoton emissions from plants were quantified in combination with proteomic analyses. Furthermore, treatment with exogenous JA inhibited lateral root growth and markedly reduced root weight. Out of 649 proteins identified in the JA- and JA/SA-treated plants, 44 were unique to JA-treated plants, 403 were unique to JA/SA-treated plants, and 202 were shared between the groups. These proteins were involved in stress, signaling, degradation, glycolysis, fermentation, and hormone metabolism. The abundances of glutathione-S-transferase, alanine aminotransferase, and malate dehydrogenase were decreased; however, the activities of these enzymes were increased. In contrast, the abundance and activity of monodehydroascorbate reductase increased in the roots of plants treated with JA and SA under flooding stress. This suggests that the quantity of lateral roots, total root mass, and free radicals generated during oxidation and reduction reactions and reactive oxygen species scavenging largely contribute to biophoton emission. Furthermore, monodehydroascorbate reductase, which is involved in detoxification and controlling hydrogen peroxide levels, may protect plant cells against oxidative damage during flooding. BIOLOGICAL SIGNIFICANCE: To understand the source of biophoton emission and molecular mechanism by the application of jasmonic and salicylic acid under flooding conditions in soybean plants, the label-free quantitative techniques were performed in roots. Root lengths and weights were significantly reduced by the effect of jasmonic and salicylic acid while it inhibited growth of the lateral roots in normal conditions using the jasmonic acid. Finally, identified proteins were functionally annotated by MAPMAN software application; that were assigned to different functional categories, such as stress, signaling, protein, glycolysis, metabolism, cell wall, and cell organization. Consequently, this study offers to learn the photon emission in plants and to know the molecular mechanism under flooding stress in soybean.

Involvement of Reactive Oxygen Species and Mitochondrial Proteins in Biophoton Emission in Roots of Soybean Plants under Flooding Stress.

To understand the mechanism of biophoton emission, ROS and mitochondrial proteins were analyzed in soybean plants under flooding stress. Enzyme activity and biophoton emission were increased in the flooding stress samples when assayed in reaction mixes specific for antioxidant enzymes and reactive oxygen species; although the level of the hydroxyl radicals was increased at day 4 (2 days of flooding) compared to nonflooding at day 4, the emission of biophotons did not change. Mitochondria were isolated and purified from the roots of soybean plants grown under flooding stress by using a Percoll gradient, and proteins were analyzed by a gel-free proteomic technique. Out of the 98 mitochondrial proteins that significantly changed abundance under flooding stress, 47 increased and 51 decreased at day 4. The mitochondrial enzymes fumarase, glutathione-S-transferase, and aldehyde dehydrogenase increased at day 4 in protein abundance and enzyme activity. Enzyme activity and biophoton emission decreased at day 4 by the assay of lipoxygenase under stress. Aconitase, acyl CoA oxidase, succinate dehydrogenase, and NADH ubiquinone dehydrogenase were up-regulated at the transcription level. These results indicate that oxidation and peroxide scavenging might lead to biophoton emission and oxidative damage in the roots of soybean plants under flooding stress.