Proteomic Profiling of Aqueous Humor Exosomes from Age-related Macular Degeneration Patients.

Purpose: The alteration of the exosomal proteins in the aqueous humor (AH) is linked to the development of eye diseases. The goal of this study was to examine the exosomal protein profile of patients with age-related macular degeneration (AMD) to better understand their role in the pathogenesis of AMD. Methods: Exosomes were isolated from the AH of 28 AMD and 25 control eyes. The quality, concentration, and size distribution of exosomes were measured using a nanoparticle tracking analysis system (NTA). Total exosomal proteins from each sample were purified and digested with trypsin for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results: Based on LC-MS/MS analysis, we got 105 exosomal peptides from AMD and control patients. Gene ontology (GO) analysis in the biology process revealed that exosomal proteins of AMD were enriched in the lipoprotein metabolic process. T-test analysis revealed six exosomal proteins in patients with AMD were significantly different from controls. Comparing the exosomal protein profile of AMD patients who were receiving anti-VEGF therapy, we observed the amount of two proteins decreased with the duration of the anti-VEGF treatment time. Conclusions: In this study, we successfully isolated and purified AH exosomes. Our results provide pioneering findings for the exosomal protein profile in AMD development and under therapy. These unique proteins could be the new targets for drug discovery or biological markers for evaluating therapeutic efficacy.

Ovariectomy Interferes with Proteomes of Brown Adipose Tissue in Rats.

Postmenopausal women exhibit a higher prevalence of obesity due to decreased energy expenditure and increased food intake compared to their premenopausal counterparts. Brown adipose tissue (BAT) plays a key role in energy homeostasis, thus providing us with appealing therapeutic targets in obesity. However, how BAT proteomes are altered in response to low levels of estrogen remains unclear. To better understand the underlying mechanisms between the postmenopausal state and BAT proteomic changes, our study aimed to investigate the effect of ovariectomy on the BAT proteome. In this study, eight-week-old female Sprague Dawley rats were randomly allocated into bilateral ovariectomy (Ovx) and sham operation (Sham) groups. Mass spectrometry was used for proteomics assay and Ingenuity Pathway Analysis was applied to examine the differentially regulated proteins. Of the 1,412 identified proteins, 18 proteins were significantly upregulated, whereas 36 proteins were significantly downregulated in the Ovx group as compared to the Sham group. Our findings demonstrate that the proteins involved in BAT morphology, the browning of white adipose tissue, and metabolic substrates for thermogenesis were regulated by ovariectomy. The dysregulation of proteins by ovariectomy might be related to the disruption of BAT function in the postmenopausal status. Understanding how BAT proteomes are altered in response to ovariectomy may illuminate novel therapeutic strategies for the management of postmenopausal weight gain in the future.

Proteomic analysis of Exosomes derived from the Aqueous Humor of Myopia Patients.

Objectives: Myopia is the most common refractive vision disorder. In recent years, several studies have suggested that the alteration of the exosomal protein levels in the aqueous humor (AH) is associated with the development of several eye diseases. Therefore, we aimed to explore the exosomal protein profile of the AH from myopia patients. Methods: Exosomes were isolated from the AH. The quality, concentration, and size distribution of exosomes for each patient were measured using nanoparticle tracking analysis system. Then, the exosomal proteins were purified and digested by trypsin for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results: There was no significant difference observed between the myopia and control when comparing the concentration and size distribution of exosomes in the AH for each sample. Based on LC-MS/MS analysis, myopia patients had higher and more complex exosomal peptide content. We found two proteins that were common in AH exosomes and eight proteins that were highly expressed in the myopia group. Conclusions: Our results provide pioneering findings for the exploration of the exosomal protein profile in myopia development. Further studies may provide significant information for the diagnosis, clinical treatment, and prognosis of myopia.

Feedback Regulation of O-GlcNAc Transferase through Translation Control to Maintain Intracellular O-GlcNAc Homeostasis.

Protein O-GlcNAcylation is a dynamic post-translational modification involving the attachment of N-acetylglucosamine (GlcNAc) to the hydroxyl groups of Ser/Thr residues on numerous nucleocytoplasmic proteins. Two enzymes are responsible for O-GlcNAc cycling on substrate proteins: O-GlcNAc transferase (OGT) catalyzes the addition while O-GlcNAcase (OGA) helps the removal of GlcNAc. O-GlcNAcylation modifies protein functions; therefore, dysregulation of O-GlcNAcylation affects cell physiology and contributes to pathogenesis. To maintain homeostasis of cellular O-GlcNAcylation, there exists feedback regulation of OGT and OGA expression responding to fluctuations of O-GlcNAc levels; yet, little is known about the molecular mechanisms involved. In this study, we investigated the O-GlcNAc-feedback regulation of OGT and OGA expression in lung cancer cells. Results suggest that, upon alterations in O-GlcNAcylation, the regulation of OGA expression occurs at the mRNA level and likely involves epigenetic mechanisms, while modulation of OGT expression is through translation control. Further analyses revealed that the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) contributes to the downregulation of OGT induced by hyper-O-GlcNAcylation; the S5A/S6A O-GlcNAcylation-site mutant of 4E-BP1 cannot support this regulation, suggesting an important role of O-GlcNAcylation. The results provide additional insight into the molecular mechanisms through which cells may fine-tune intracellular O-GlcNAc levels to maintain homeostasis.

Low Levels of IgM and IgA Recognizing Acetylated C1-Inhibitor Peptides Are Associated with Systemic Lupus Erythematosus in Taiwanese Women.

The objective of this study was to identify novel acetylation (Ac) modifications of the C1-inhibitor (C1-INH) and explain the association of the levels of autoantibodies against acetylated C1-INH peptides with the risk of developing systemic lupus erythematosus (SLE). Ac modifications of the C1-INH were identified and validated through in-gel digestion, nano-liquid chromatography-tandem mass spectrometry, immunoprecipitation, and Western blotting by using serum protein samples obtained from patients with SLE and age-matched healthy controls (HCs). In addition, the levels of serum C1-INH, Ac-protein adducts, and autoantibodies against unmodified and acetylated C1-INH peptides were measured. C1-INH levels in patients with SLE were significantly lower than those in HCs by 1.53-fold (p = 0.0008); however, Ac-protein adduct concentrations in patients with SLE were significantly higher than those in HCs by 1.35-fold (p = 0.0009). Moreover, immunoglobulin M (IgM) anti-C1-INH367-385 Ac and IgA anti-C1-INH367-385 Ac levels in patients with SLE were significantly lower than those in HCs. The low levels of IgM anti-C1-INH367-385 (odds ratio [OR] = 4.725, p < 0.001), IgM anti-C1-INH367-385 Ac (OR = 4.089, p = 0.001), and IgA anti-C1-INH367-385 Ac (OR = 5.566, p < 0.001) indicated increased risks for the development of SLE compared with HCs.

Maintenance of neural activities in torpid Rhinolophus ferrumequinum bats revealed by 2D gel-based proteome analysis.

Bats are the only mammals capable of self-powered flying. Many bat species hibernate in winter. A reversible control of cerebral activities is critical for bats to accommodate a repeated torpor-arousal cycle during hibernation. Little is known about the molecular mechanisms that regulate neuronal activities in torpid bats. In this study, Rhinolophus ferrumequinum bat brain proteins were fractionated, and their abundance in active and torpid states was compared. Results of 2D gel-based proteomics showed that 38% of identified proteins with a significant change in abundance are involved in synaptic vesicle recycling and cytoskeletal integrity. Changes in the abundance of proteins related to RNA splicing, proteostasis, redox homeostasis, mitochondrial function, and energy metabolism were also detected. In addition, the levels of GNAO1 (guanine nucleotide-binding protein Gαo subunit), an important modulator of neuronal transmembrane signaling, were significantly increased in the insoluble protein fraction of torpid bats; this may be due to GNAO1 palmitoylation making it insoluble. Our data provide molecular evidence for the maintenance of neuronal activities in torpid bats and suggest that a reversible palmitoylation of the G protein plays a role in the regulation of neuronal activities during bat hibernation.

Proteomics Analysis to Identify and Characterize the Biomarkers and Physical Activities of Non-Frail and Frail Older Adults.

Globally, the proportion of older adults is increasing. Older people face chronic conditions such as sarcopenia and functional decline, which are often associated with disability and frailty. Proteomics assay of potential serum biomarkers of frailty in older adults. Older adults were divided into non-frail and frail groups (n = 6 each; 3 males in each group) in accordance with the Chinese-Canadian Study of Health and Aging Clinical Frailty Scale. Adults were measured for grip power and the 6-min walk test for physical activity, and venous blood was sampled after adults fasted for 8 h. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used for proteomics assay. The groups were compared for levels of biomarkers by t test and Pearson correlation analysis. Non-frail and frail subjects had mean age 77.5±0.4 and 77.7±1.6 years, mean height 160.5±1.3 and 156.6±2.9 cm and mean weight 62.5±1.2 and 62.8±2.9 kg, respectively. Physical activity level was lower for frail than non-frail subjects (grip power: 13.8±0.4 vs 26.1±1.2 kg; 6-min walk test: 215.2±17.2 vs 438.3±17.2 m). Among 226 proteins detected, for 31, serum levels were significantly higher for frail than non-frail subjects; serum levels of Ig kappa chain V-III region WOL, COX7A2, and albumin were lower. The serum levels of ANGT, KG and AT were 2.05-, 1.76- and 2.22-fold lower (all p < 0.05; Figure 1A, 2A and 3A) for non-frail than frail subjects and were highly correlated with grip power (Figure 1B, 2B and 3B). Our study found that ANGT, KG and AT levels are known to increase with aging, so degenerated vascular function might be associated with frailty. In total, 226 proteins were revealed proteomics assay; levels of angiotensinogen (ANGT), kininogen-1 (KG) and antithrombin III (AT) were higher in frail than non-frail subjects (11.26±2.21 vs 5.09±0.74; 18.42±1.36 vs 11.64±1.36; 22.23±1.64 vs 9.52±0.95, respectively, p < 0.05). These 3 factors were highly correlated with grip power (p < 0.05), with higher correlations between grip power and serum levels of ANGT (r = -0.89), KG (r = -0.90), and AT (r = -0.84). In conclusion, this is the first study to demonstrate a serum proteomic profile characteristic of frailty in older adults. Serum ANGT, KG and AT levels could be potential biomarkers for monitoring the development and progression of frailty in older adults.

Characterization of the serum and liver proteomes in gut-microbiota-lacking mice.

Current nutrition research is focusing on health promotion, disease prevention, and performance improvement for individuals and communities around the world. The humans with required nutritional ingredients depend on both how well the individual is provided with balanced foods and what state of gut microbiota the host has. Studying the mutually beneficial relationships between gut microbiome and host is an increasing attention in biomedical science. The purpose of this study is to understand the role of gut microbiota and to study interactions between gut microbiota and host. In this study, we used a shotgun proteomic approach to reveal the serum and liver proteomes in gut-microbiota-lacking mice. For serum, 15 and 8 proteins were uniquely detected in specific-pathogen-free (SPF) and germ-free (GF) mice, respectively, as well as the 3 and 20 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Among the proteins of the serum, major urinary protein 1 (MUP-1) of GF mice was significantly decreased compared to SPF mice. In addition, MUP-1 expression is primarily regulated by testosterone. Lacking in gut flora has been implicated in many adverse effects, and now we have found its pathogenic root maybe gut bacteria can regulate the sex-hormone testosterone levels. In the liver, 8 and 22 proteins were uniquely detected in GF mice and SPF mice, respectively, as well as the 14 and 30 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Furthermore, ingenuity pathway analysis (IPA) indicated that gut microbiota influence the host in cancer, organismal injury and abnormalities, respiratory disease; cell cycle, cellular movement and tissue development; cardiovascular disease, reproductive system disease; and lipid metabolism, molecular transport and small molecule biochemistry. Our findings provide more detailed information of the role of gut microbiota and will be useful to help study gut bacteria and disease prevention.

Exploration of salivary proteins in buffalo: an approach to find marker proteins for estrus.

Saliva is considered as the best source of biological material for biomarker discovery studies since it is noninvasive in comparison to other body sources. Usually buffalo cannot precisely express estrus signals. Hence, there is a need for concise methods to detect the time of estrus to ensure the success of artificial insemination. Therefore, we have established a reference proteome map on the whole saliva of buffalo during their estrous cycle with special reference to estrus. Nearly 12 bands have been observed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole saliva. Collectively, 179 proteins are identified with respect to different phases of the estrous cycle using mass spectrometry. On the whole, 37 proteins are exclusively expressed in the estrus phase, which include ß-enolase, Toll-like receptor (TLR) 4, clusterin, lactoperoxidase, serotransferrin, TGM3, UBA6, and transducin. Among the proteins, ß-enolase and TLR 4 were validated, and their specific expression was found during estrus as compared to other phases using immunoblot. The functional annotation reveals many as binding proteins in the estrus saliva when compared to the other phases. The present findings conclude that the proteomic approach adopted to identify the proteins from buffalo saliva around the estrous cycle may provide a new tool for screening the estrus phase. The results further conclude that the specific expression of ß-enolase and TLR 4 can be taken as the indicator of estrus in buffalo.

Secretion of one adipokine Nampt/Visfatin suppresses the inflammatory stress-induced NF-κB activity and affects Nampt-dependent cell viability in Huh-7 cells.

Nampt/visfatin acts in both intracellular and extracellular compartments to regulate multiple biological roles, including NAD metabolism, cancer, inflammation, and senescence. However, its function in chronic inflammation and carcinogenesis in hepatocellular carcinoma (HCC) has not been well-defined. Here we use Huh-7 hepatoma cells as a model to determine how Nampt/visfatin affects cellular survival under oxidative stress. We found that the transition of Nampt/visfatin from intracellular into extracellular form was induced by H2O2 treatment in 293T cells and confirmed that this phenomenon was not due to cell death but through the secretion of Nampt/visfatin. In addition, Nampt/visfatin suppressed cell viability in oxidative treatment in Huh-7 cells and acted on the inhibition of hepatoma cell growth. Oxidative stress also reduced the Nampt-mediated activation of NF-κB gene expression. In this study, we identify a novel feature of Nampt/visfatin which functions as an adipokine that can be secreted upon cellular stress. Our results provide an example to understand how adipokine interacts with chemotherapeutic treatment by oxidative stress in HCC.

Regulation of mitochondrial F(o)F(1)ATPase activity by Sirt3-catalyzed deacetylation and its deficiency in human cells harboring 4977bp deletion of mitochondrial DNA.

Sirt3, a mitochondrial NAD(+)-dependent deacetylase, is regarded as a potential regulator in cellular metabolism. However, the role of Sirt3 in the regulation of mitochondrial F(o)F(1)ATPase and the linkage to mitochondrial diseases is unclear. In this study, we demonstrated a role of Sirt3 in the regulation of F(o)F(1)ATPase activity in human cells. Knockdown of Sirt3 in 143B cells by shRNA transfection caused increased acetylation levels of the α and OSCP subunits of F(o)F(1)ATPase. We showed that Sirt3 physically interacted with the OSCP and led to its subsequent deacetylation. By incubation of mitochondria with the purified Sirt3 protein, Sirt3 could regulate F(o)F(1)ATPase activity through its deacetylase activity. Moreover, suppression of Sirt3 reduced the F(o)F(1)ATPase activity, consequently decreased the intracellular ATP level, diminished the capacity of mitochondrial respiration, and compromised metabolic adaptability of 143B cells to the use of galactose as the energy source. In human cells harboring ≅85% of mtDNA with 4977bp deletion, we showed that oxidative stress induced a reduction of Sirt3 expression, and an increased acetylation of the OSCP subunit of F(o)F(1)ATPase. Importantly, the expression of Sirt3 was also decreased in the skin fibroblasts from patients with CPEO syndrome. We further demonstrated that oxidative stress induced by 5-10µM of menadione impaired the Sirt3-mediated deacetylation and activation on F(o)F(1)ATPase activity through decreasing the protein level of Sirt3. Our findings suggest that increased intracellular ROS levels might modulate the expression of Sirt3 which deacetylates and activates F(o)F(1)ATPase in human cells with mitochondrial dysfunction caused by a pathogenic mtDNA mutation.

Buffalo cervico-vaginal fluid proteomics with special reference to estrous cycle: heat shock protein (HSP)-70 appears to be an estrus indicator.

Cervico-vaginal fluid (CVF) plays significant roles in coitus, sperm transport, and implantation. It is believed to be a good noninvasive biomarker for various diagnostic purposes. In this study, a comprehensive proteomic analysis of buffalo CVF was performed during the estrous cycle in order to document the protein expressions, utilizing SDS-PAGE, mass spectrometry, and immunoblot. The main objective was to screen the CVF of buffalo for one or more estrus-specific proteins. A total of 416 proteins were identified in the CVF of both estrus and diestrus phases. Out of these proteins, 68 estrus-specific proteins have been extensively reviewed in the protein database. The major physiological functions of estrus CVF proteins appeared to be stress response, immune response, and metabolic. Eventually, the expression level of heat shock protein-70 in the CVF during the estrus phase, as revealed in SDS-PAGE analysis, was higher than during diestrus. The identity of the protein was confirmed by immunoblot analysis as heat shock protein-70. The findings provide a potential lead for the evaluation of these proteins for estrus detection in buffalo because CVF biomarker detection is a noninvasive technique. The mass spectrometric data of identified proteins have been deposited at the ProteomeXchange with the identifier PXD000620.

Purification and characterization of plantaricin Y, a novel bacteriocin produced by Lactobacillus plantarum 510.

Lactobacillus plantarum 510, previously isolated from a koshu vineyard in Japan, was found to produce a bacteriocin-like inhibitory substance which was purified and characterized. Mass spectrometry analysis showed that the mass of this bacteriocin is 4,296.65 Da. A partial sequence, NH2- SSSLLNTAWRKFG, was obtained by N-terminal amino acid sequence analysis. A BLAST search revealed that this is a unique sequence; this peptide is thus a novel bacteriocin produced by Lactobacillus plantarum 510 and was termed plantaricin Y. Plantaricin Y shows strong inhibitory activity against Listeria monocytogenes BCRC 14845, but no activity against other pathogens tested. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was completely inactivated by protease K. Furthermore, trypsin-digested bacteriocin product fragments retained activity against L. monocytogenes BCRC 14845 and exhibited a different inhibitory spectrum.

Bioengineering radioresistance by overproduction of RPA, a mammalian-type single-stranded DNA-binding protein, in a halophilic archaeon.

Halobacterium sp. NRC-1 is a wild-type extremophilic microbe that is naturally tolerant to high levels of ionizing radiation. Mutants of strain NRC-1 with even higher levels of resistance to ionizing radiation, named RAD, were previously isolated after selecting survival to extremely high doses of ionizing radiation. These RAD mutants displayed higher transcription levels for the rfa3 operon, coding two subunits of the RPA-like putative single-stranded binding protein, rfa3 and rfa8, and a third downstream gene, ral. In order to bioengineer cells with increased tolerance to ionizing radiation and further explore the genetic basis of the RAD phenotype, we placed the rfa3 operon under control of the gvpA promoter in a Halobacterium expression plasmid, pDRK1. When pDRK1 was introduced into the wild-type NRC-1 strain, overproduction of the Rfa3 and Rfa8 proteins was observed by Western blotting and proteomic analysis. The Halobacterium strains expressing Rfa3 and Rfa8 also displayed improved survival after exposure to ionizing radiation, similar to the RAD mutants, when compared to wild-type strain NRC-1. The Rfa3 and Rfa8 proteins co-purified by affinity chromatography on single-stranded DNA cellulose columns, confirming the ability of the proteins to bind to single-stranded DNA as well as their relative abundance in the wild-type, RAD mutants, and rfa3 operon overexpression strains. These results clearly establish that overexpression of haloarchaeal RPA promotes ionizing radiation resistance in Halobacterium sp. NRC-1 and that the Rfa3 and Rfa8 subunits bind single-stranded DNA. Bioengineering cells with increased levels of ionizing radiation resistance may have potential value in medical and environmental applications.

Aerobic Exercise Modulates Proteomic Profiles in Gastrocnemius Muscle of db/db Mice, Ameliorating Sarcopenia.

Type-2 diabetes mellitus (T2DM)-induced sarcopenia is intertwined with diminished insulin sensitivity and extracellular matrix (ECM) remodeling in skeletal muscle and other organs. Physical activities such as aerobic exercise play a crucial role in regulating blood glucose levels, insulin sensitivity, metabolic pathways, oxidative stress, fibrosis, ECM remodeling, and muscle regeneration by modulating differentially expressed protein (DEP) levels. The objectives of our research were to investigate the effect of six weeks of aerobic exercise on the gastrocnemius and soleus muscle of db/db mice's DEP levels compared to those of sedentary db/db mice. A total of eight db/db mice were divided into two groups (n = 4 per group), namely sedentary mice (SED) and exercise-trained mice (ET), of which the latter were subjected to six weeks of a moderate-intensity aerobic exercise intervention for five days per week. After the exercise intervention, biochemical tests, including analyses of blood glucose and HbA1c levels, were performed. Histological analysis using H & E staining on tissue was performed to compare morphological characters. Gastrocnemius and soleus muscles were dissected and processed for proteomic analysis. Data were provided and analyzed based on the DEPs using the label-free quantification (LFQ) algorithm. Functional enrichment analysis and Ingenuity Pathway Analysis (IPA) were employed as bioinformatics tools to elucidate the molecular mechanisms involved in the DEPs and disease progression. Significantly reduced blood glucose and HbA1c levels and an increased cross-sectional area (CSA) of gastrocnemius muscle fibers were seen in the ET group after the exercise interventions due to upregulations of metabolic pathways. Using proteomics data analysis, we found a significant decrease in COL1A1, COL4A2, ENG, and LAMA4 protein levels in the ET gastrocnemius, showing a significant improvement in fibrosis recovery, ECM remodeling, and muscle regeneration via the downregulation of the TGF-ß signaling pathway. Upregulated metabolic pathways due to ET-regulated DEPs in the gastrocnemius indicated increased glucose metabolism, lipid metabolism, muscle regeneration, and insulin sensitivity, which play a crucial role in muscle regeneration and maintaining blood glucose and lipid levels. No significant changes were observed in the soleus muscle due to the type of exercise and muscle fiber composition. Our research suggests that engaging in six weeks of aerobic exercise may have a positive impact on the recovery of T2DM-induced sarcopenia, which might be a potential candidate for mitigation, prevention, and therapeutic treatment in the future.

Enterococcus saccharolyticus subsp. taiwanensis subsp. nov., isolated from broccoli.

A coccal strain isolated from fresh broccoli was initially identified as Enterococcus saccharolyticus; however, molecular identification and phenotypic traits did not support this identification. DNA-DNA hybridization with the type strain of E. saccharolyticus (76.4 % relatedness), DNA G+C content (35.7 mol%), phylogenetic analysis based on 16S rRNA, pheS and rpoA gene sequences, rep-PCR fingerprinting and profiles of cellular fatty acids, whole-cell proteins and enzyme activities, together with carbohydrate metabolism characteristics, indicated that this strain is distinct and represents a novel subspecies, for which the name Enterococcus saccharolyticus subsp. taiwanensis subsp. nov. is proposed. The type strain is 812(T) ( = NBRC 109476(T) = BCRC 80575(T)). Furthermore, we present an emended description of Enterococcus saccharolyticus and proposal of Enterococcus saccharolyticus subsp. saccharolyticus subsp. nov. (type strain ATCC 43076(T) = CCUG 27643(T) = CCUG 33311(T) = CIP 103246(T) = DSM 20726(T) = JCM 8734(T) = LMG 11427(T) = NBRC 100493(T) = NCIMB 702594(T)).

Resveratrol protects against physical fatigue and improves exercise performance in mice.

Resveratrol (RES) is a well-known phytocompound and food component which has antioxidative and multifunctional bioactivities. However, there is limited evidence for the effects of RES on physical fatigue and exercise performance. The purpose of this study was to evaluate the potential beneficial effects of trans-RES on fatigue and ergogenic functions following physiological challenge. Male ICR mice from four groups (n = 8 per group) were orally administered RES for 21 days at 0, 25, 50, and 125 mg/kg/day, which were respectively designated the vehicle, RES-25, RES-50, and RES-125 groups. The anti-fatigue activity and exercise performance were evaluated using forelimb grip strength, exhaustive swimming time, and levels of serum lactate, ammonia, glucose, and creatine kinase (CK) after a 15-min swimming exercise. The exhaustive swimming time of the RES-25 group (24.72 ± 7.35 min) was significantly (p = 0.0179) longer than that of vehicle group (10.83 ± 1.15 min). A trend analysis revealed that RES treatments increased the grip strength. RES supplementation also produced dose-dependent decreases in serum lactate and ammonia levels and CK activity and also an increase in glucose levels in dose-dependent manners after the 15-min swimming test. The mechanism was related to the increased energy utilization (as blood glucose), and decreased serum levels of lactate, ammonia, and CK. Therefore, RES could be a potential agent with an anti-fatigue pharmacological effect.

A Proteomics-Based Identification of the Biological Networks Mediating the Impact of Epigallocatechin-3-Gallate on Trophoblast Cell Migration and Invasion, with Potential Implications for Maternal and Fetal Health.

Trophoblast migration and invasion play crucial roles in placental development. However, the effects of (-)-epigallocatechin-3-gallate (EGCG) on trophoblast cell functions remain largely unexplored. In this study, we investigated the impact of EGCG on the survival of trophoblast cells and employed a proteomics analysis to evaluate its influence on trophoblast cell migration and invasion. Be-Wo trophoblast cells were treated with EGCG, and a zone closure assay was conducted to assess the cell migration and invasion. Subsequently, a proteomics analysis was performed on the treated and control groups, followed by a bioinformatics analysis to evaluate the affected biological pathways and protein networks. A quantitative real-time PCR and Western blot analysis were carried out to validate the proteomics findings. Our results showed that EGCG significantly suppressed the trophoblast migration and invasion at a concentration not affecting cell survival. The proteomics analysis revealed notable differences in the protein expression between the EGCG-treated and control groups. Specifically, EGCG downregulated the signaling pathways related to EIF2, mTOR, and estrogen response, as well as the processes associated with the cytoskeleton, extracellular matrix, and protein translation. Conversely, EGCG upregulated the pathways linked to lipid degradation and oxidative metabolism. The quantitative PCR showed that EGCG modulated protein expression by regulating gene transcription, and the Western blot analysis confirmed its impact on cytoskeleton and extracellular matrix reorganization. These findings suggest EGCG may inhibit trophoblast migration and invasion through multiple signaling pathways, highlighting the potential risks associated with consuming EGCG-containing products during pregnancy. Future research should investigate the impact of EGCG intake on maternal and fetal proteoforms.

Comparative secretome analyses using a hollow fiber culture system with label-free quantitative proteomics indicates the influence of PARK7 on cell proliferation and migration/invasion in lung adenocarcinoma.

As the leading cause of cancer death worldwide, lung cancer lacks effective diagnosis tools and treatments to prevent its metastasis. Fortunately, secretome has clinical usages as biomarkers and protein drugs. To discover the secretome that influences lung adenocarcinoma metastasis, the hollow fiber culture (HFC) system was used along with label-free proteomics approach to analyze cell secretomes between CL1-0 and CL1-5 cell lines, which exhibit low and high metastatic potentials. Among the 703 proteins quantified, 50 possessed different levels between CL1-0 and CL1-5. PARK7 was a primary focus because of the lack of research involving lung adenocarcinoma. The cell proliferation, migration, and invasion properties of CL1-0, CL1-5, and A549 cells were significantly diminished when the expression of their PARK7 proteins was reduced. Conversely, these functions were promoted when PARK7 was overexpressed in CL1-0. In clinical expression, PARK7 levels within tissue specimens and plasma samples were significantly higher in the cancer group. This represents the first time the HFC system has been used with label-free quantification to discern the elements of metastasis in lung adenocarcinoma cell secretomes. Likewise, PARK7 has never been researched for its role in promoting lung adenocarcinoma progression.

Qualitative analysis of the fluorophosphonate-based chemical probes using the serine hydrolases from mouse liver and poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis.

The serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in the human genome. Although up to 50 % of this family remains unannotated, there are increasing evidences that activities of certain serine hydrolases are associated with diseases like cancer neoplasia, invasiveness, etc. By now, several activity-based chemical probes have been developed and are applied to profile the global activity of serine hydrolases in diverse proteomes. In this study, two fluorophosphonate (FP)-based chemical probes were synthesized. Further examination of their abilities to label and pull down serine hydrolases was conducted. In addition, the poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis was demonstrated as an appropriate standard serine hydrolase, which can be applied to measure the labeling ability and pull-down efficiency of FP-based probes. Furthermore, mass spectrometry (MS) was used to identify the serine residue that covalently bonded to the active probes. Finally, these FP-based probes were shown capable of establishing the serine hydrolase profiles in diverse mouse tissues; the serine hydrolases pulled down from mouse liver organ were further identified by MS. In summary, our study provides an adequate method to evaluate the reactivity of FP-based probes targeting serine hydrolases.