Thermal Proteome Profiling Reveals Insight to Antiproliferative and Pro-Apoptotic Effects of Lagunamide A in the Modulation of DNA Damage Repair.

Lagunamide A is a biologically active natural product with a yet unidentified molecular mode of action. Cellular studies revealed that lagunamide A is a potent inhibitor of cancer cell proliferation, promotes apoptosis and causes mitochondrial dysfunction. To decipher the cellular mechanism responsible for these effects, we utilized thermal protein profiling (TPP) and identified EYA3 as a stabilized protein in cells upon lagunamide A treatment. EYA3, involved in the DNA damage repair process, was functionally investigated via siRNA based knockdown studies and corresponding effects of lagunamide A on DNA repair were confirmed. Furthermore, we showed that lagunamide A sensitized tumor cells to treatment with the drug doxorubicin highlighting a putative therapeutic strategy.

Molecular variations to the proteome of zebrafish larvae induced by environmentally relevant copper concentrations.

Contaminants are increasingly accumulating in aquatic environments and biota, with potential adverse effects on individual organisms, communities and ecosystems. However, studies that explore the molecular changes in fish caused by environmentally relevant concentrations of metals, such as copper (Cu), are limited. This study uses embryos of the model organism zebrafish (Danio rerio) to investigate effect of Cu on the proteome and amino acid (AA) composition of fish. Wild-type embryos at 24 h post-fertilisation were exposed to Cu (2 µg L-1 to 120 µg L-1) for 96 h and the number of healthy larvae were determined based on larvae that had hatched and did not display loss of equilibrium (LOE). The effect concentrations where Cu caused a 10 % (EC10) or 50 % (EC50) decrease in the number of healthy larvae were calculated as 3.7 µg L-1 and 10.9 µg L-1, respectively. Proteomics analysis of embryos exposed to the EC10 and EC50 concentrations of Cu revealed the proteome to differ more strongly after 48 h than 96 h, suggesting the acclimatisation of some larvae. Exposure to excess Cu caused differentially expressed proteins (DEPs) involved in oxidative stress, mitochondrial respiration, and neural transduction as well as the modulation of the AAs (Proline, Glycine and Alanine). This is the first study to suggest that LOE displayed by Cu-stressed fish may involve the disruption to GABAergic proteins and the calcium-dependent inhibitory neurotransmitter GABA. Moreover, this study highlights that proteomics and AA analysis can be used to identify potential biomarkers for environmental monitoring.

Zearalenone exposure differentially affects the ovarian proteome in pre-pubertal gilts during thermal neutral and heat stress conditions.

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, causes endocrine disruption and porcine reproductive dysfunction. Heat stress (HS) occurs when exogenous and metabolic heat accumulation exceeds heat dissipation. Independently, HS and ZEN both compromise swine reproduction; thus, the hypothesis investigated was two-pronged: that ZEN exposure would alter the ovarian proteome and that these effects would differ in thermal neutral (TN) and HS pigs. Pre-pubertal gilts (n = 38) were fed ad libitum and assigned to either (TN: 21.0 ±â€…0.1 °C) or HS (12 h cyclic temperatures of 35.0 ±â€…0.2 °C and 32.2 ±â€…0.1 °C). Within the TN group, a subset of pigs were pair-fed (PF) to the amount of feed that the HS gilts consumed to eliminate the confounding effects of dissimilar nutrient intake. All gilts orally received a vehicle control (CT) or ZEN (40 µg/kg/BW) resulting in six treatment groups: thermoneutral (TN) vehicle control (TC; n = 6); TN ZEN (TZ; n = 6); PF vehicle control (PC; n = 6); PF ZEN (PZ; n = 6); HS vehicle control (HC; n = 7); or HS ZEN (HZ; n = 7) for 7 d. When compared to the TC pigs, TZ pigs had 45 increased and 39 decreased proteins (P ≤ 0.05). In the HZ pigs, 47 proteins were increased and 61 were decreased (P ≤ 0.05). Exposure to ZEN during TN conditions altered sec61 translocon complex (40%), rough endoplasmic reticulum membrane (8.2%), and proteasome complex (5.4%), asparagine metabolic process (0.60%), aspartate family amino acid metabolic process (0.14%), and cellular amide metabolic process (0.02%) pathways. During HS, ZEN affected cellular pathways associated with proteasome core complex alpha subunit complex (0.23%), fibrillar collagen trimer (0.14%), proteasome complex (0.05%), and spliceosomal complex (0.03%). Thus, these data identify ovarian pathways altered by ZEN exposure and suggest that the molecular targets of ZEN differ in TN and HS pigs.

Zearalenone (ZEN) is an estrogenic mycotoxin that impairs fertility in swine. This study was designed to identify the ovarian molecular impacts of ZEN exposure in thermal neutral (TN) pre-pubertal pigs. Additionally, whether heat stress (HS) would affect the ovarian ZEN response was also queried. Using a mass spectrometry approach, proteins that are altered in the ovaries of TN and HS pigs were noted to include those involved with chemical detoxification, metabolism, and inflammation. These findings may be of use in developing mitigation strategies to improve fertility in swine exposed to ZEN via contaminated feeds.

Deciphering proteomic mechanisms explaining the role of glutathione as an aid in improving plant fitness and tolerance against cadmium-toxicity in Brassica napus L.

Cadmium (Cd) hazard is a serious limitation to plants, soils and environments. Cd-toxicity causes stunted growth, chlorosis, necrosis, and plant yield loss. Thus, ecofriendly strategies with understanding of molecular mechanisms of Cd-tolerance in plants is highly demandable. The Cd-toxicity caused plant growth retardation, leaf chlorosis and cellular damages, where the glutathione (GSH) enhanced plant fitness and Cd-toxicity in Brassica through Cd accumulation and antioxidant defense. A high-throughput proteome approach screened 4947 proteins, wherein 370 were differently abundant, 164 were upregulated and 206 were downregulated. These proteins involved in energy and carbohydrate metabolism, CO2 assimilation and photosynthesis, signal transduction and protein metabolism, antioxidant defense response, heavy metal detoxification, cytoskeleton and cell wall structure, and plant development in Brassica. Interestingly, several key proteins including glutathione S-transferase F9 (A0A078GBY1), ATP sulfurylase 2 (A0A078GW82), cystine lyase CORI3 (A0A078FC13), ferredoxin-dependent glutamate synthase 1 (A0A078HXC0), glutaredoxin-C5 (A0A078ILU9), glutaredoxin-C2 (A0A078HHH4) actively involved in antioxidant defense and sulfur assimilation-mediated Cd detoxification process confirmed by their interactome analyses. These candidate proteins shared common gene networks associated with plant fitness, Cd-detoxification and tolerance in Brassica. The proteome insights may encourage breeders for enhancing multi-omics assisted Cd-tolerance in Brassica, and GSH-mediated hazard free oil seed crop production for global food security.

Spatial Proteomics Reveals Alcohol-Induced Damages to the Crypts and Villi of the Mouse Small Intestine.

Alcohol consumption perturbs the gut immune barrier and ultimately results in alcoholic liver diseases, but little is known about how immune-related cells in the gut are perturbed in this process. In this study, we employed laser capture microdissection and a label-free proteomics approach to investigate the consequences of alcohol exposure to the proteomes of crypts and villi in the proximal small intestine. Intestinal tissues from alcohol-fed and pair-fed mice were microdissected to selectively capture cells in the crypts and villi regions, followed by one-pot protein digestion and data-independent LC-MS/MS analysis. We successfully identified over 3000 proteins from each of the crypt or villi regions equivalent to ∼3000 cells. Analysis of alcohol-treated tissues indicated an enhanced alcohol metabolism and reduced levels of α-defensins in crypts, alongside increased lipid metabolism and apoptosis in villi. Immunofluorescence imaging further corroborated the proteomic findings. Our work provides a detailed profiling of the proteomic changes in the compartments of the mouse small intestine and aids in molecular-level understanding of alcohol-induced tissue damage.

Transcriptomic and proteomic features of a mouse model of sperm DNA damage induced by benzo(a)pyrene.

This study replicated a mouse model of sperm DNA damage induced by benzo(a)pyrene (BaP), and the transcriptomic and proteomic features of the model were examined to clarify the pathways related to BaP-induced damage to sperm DNA. Male mice in the BaP group were subjected to BaP at a dosage of 100 mg/kg/d or an equivalent quantity of saline solution in the control group for 60 days. Subsequently, the DNA fragmentation index (DFI) in sperm was assessed using a sperm chromatin structure assay (SCSA). RNA-seq and data-independent acquisition (DIA) were used to identify the mRNA and protein expression patterns in the testis. The sperm DFI significantly increased in the BaP group. Compared to the control group, the BaP group exhibited differential expression of 240 genes (referred to as DEGs) and 616 proteins (referred to as DEPs). These molecules included Aldh1a1, Cyb5r3, Fads1, Oxsm, Rcn3, and Prss45. Pathways in cancer, the PI3K-Akt signaling pathway, metabolic pathways, and the MAPK signaling pathway were the primary areas where these genes showed enrichment. BaP can damage the DNA of sperm and affect metabolism, the PI3K-Akt pathway, and pathways associated with cancer signaling.

Insights into the differential proteome landscape of a newly isolated Paramecium multimicronucleatum in response to cadmium stress.

Employing microbial systems for the bioremediation of contaminated waters represent a potential option, however, limited understanding of the underlying mechanisms hampers the implication of microbial-mediated bioremediation. The omics tools offer a promising approach to explore the molecular basis of the bioremediation process. Here, a mass spectrometry-based quantitative proteome profiling approach was conducted to explore the differential protein levels in cadmium-treated Paramecium multimicronucleatum. The Proteome Discoverer software was used to identify and quantify differentially abundant proteins. The proteome profiling generated 7,416 peptide spectral matches, yielding 2824 total peptides, corresponding to 989 proteins. The analysis revealed that 29 proteins exhibited significant (p ≤ 0.05) differential levels, including a higher abundance of 6 proteins and reduced levels of 23 proteins in Cd2+ treated samples. These differentially abundant proteins were associated with stress response, energy metabolism, protein degradation, cell growth, and hormone processing. Briefly, a comprehensive proteome profile in response to cadmium stress of a newly isolated Paramecium has been established that will be useful in future studies identifying critical proteins involved in the bioremediation of metals in ciliates. SIGNIFICANCE: Ciliates are considered a good biological indicator of chemical pollution and relatively sensitive to heavy metal contamination. A prominent ciliate, Paramecium is a promising candidate for the bioremediation of polluted water. The proteins related to metal resistance in Paramecium species are still largely unknown and need further exploration. In order to identify and reveal the proteins related to metal resistance in Paramecia, we have reported differential protein abundance in Paramecium multimicronucleatum in response to cadmium stress. The proteins found in our study play essential roles during stress response, hormone processing, protein degradation, energy metabolism, and cell growth. It seems likely that Paramecia are not a simple sponge for metals but they could also transform them into less toxic derivatives or by detoxification by protein binding. This data will be helpful in future studies to identify critical proteins along with their detailed mechanisms involved in the bioremediation and detoxification of metal ions in Paramecium species.

Thermal Proteome Profiling Strategy Identifies CNPY3 as a Cellular Target of Gambogic Acid for Inducing Prostate Cancer Pyroptosis.

There is an urgent requirement to acquire a comprehensive comprehension of novel therapeutic targets for prostate cancer to facilitate the development of medications with innovative mechanisms. In this study, we identified gambogic acid (GBA) as a specific pyroptosis inducer in prostatic cancer cells. By using a thermal proteome profiling (TPP) strategy, we revealed that GBA induces pyroptosis by directly targeting the canopy FGF signaling regulator (CNPY3), which was previously considered "undruggable". Moreover, through the utilization of the APEX2-based proximity labeling method, we found that GBA recruited delactatease SIRT1, resulting in the elimination of lysine lactylation (Kla) on CNPY3. Of note, SIRT1-mediated delactylation influenced the cellular localization of CNPY3 to promote lysosome rupture for triggering pyroptosis. Taken together, our study identified CNPY3 as a distinctive cellular target for pyroptosis induction and its potential application in prostate cancer therapy.

Phytol and α-Bisabolol Synergy Induces Autophagy and Apoptosis in A549 Cells and Additional Molecular Insights through Comprehensive Proteome Analysis via Nano LC-MS/MS.

BACKGROUND: Non-Small Cell Lung Cancer (NSCLC) is a malignancy with a significant prevalence and aggressive nature, posing a considerable challenge in terms of therapeutic interventions. Autophagy and apoptosis, two intricate cellular processes, are integral to NSCLC pathophysiology, each affecting the other through shared signaling pathways. Phytol (Phy) and α-bisabolol (Bis) have shown promise as potential anticancer agents individually, but their combined effects in NSCLC have not been extensively investigated. OBJECTIVE: The present study was to examine the synergistic impact of Phy and Bis on NSCLC cells, particularly in the context of autophagy modulation, and to elucidate the resulting differential protein expression using LCMS/ MS analysis. METHODS: The A549 cell lines were subjected to the patented effective concentration of Phy and Bis, and subsequently, the viability of the cells was evaluated utilizing the MTT assay. The present study utilized real-time PCR analysis to assess the expression levels of crucial apoptotic genes, specifically Bcl-2, Bax, and Caspase-9, as well as autophagy-related genes, including Beclin-1, SQSTM1, Ulk1, and LC3B. The confirmation of autophagy marker expression (Beclin-1, LC3B) and the autophagy-regulating protein SQSTM1 was achieved through the utilization of Western blot analysis. Differentially expressed proteins were found using LC-MS/MS analysis. RESULTS: The combination of Phy and Bis demonstrated significant inhibition of NSCLC cell growth, indicating their synergistic effect. Real-time PCR analysis revealed a shift towards apoptosis, with downregulation of Bcl-2 and upregulation of Bax and Caspase-9, suggesting a shift towards apoptosis. Genes associated with autophagy regulation, including Beclin-1, SQSTM1 (p62), Ulk1, and LC3B, showed significant upregulation, indicating potential induction of autophagy. Western blot analysis confirmed increased expression of autophagy markers, such as Beclin-1 and LC3B, while the autophagy-regulating protein SQSTM1 exhibited a significant decrease. LC-MS/MS analysis revealed differential expression of 861 proteins, reflecting the modulation of cellular processes. Protein-protein interaction network analysis highlighted key proteins involved in apoptotic and autophagic pathways, including STOML2, YWHAB, POX2, B2M, CDA, CAPN2, TXN, ECHS1, PEBP1, PFN1, CDC42, TUBB1, HSPB1, PXN, FGF2, and BAG3, emphasizing their crucial roles. Additionally, PANTHER pathway analysis uncovered enriched pathways associated with the differentially expressed proteins, revealing their involvement in a diverse range of biological processes, encompassing cell signaling, metabolism, and cellular stress responses. CONCLUSION: The combined treatment of Phy and Bis exerts a synergistic inhibitory effect on NSCLC cell growth, mediated through the interplay of apoptosis and autophagy. The differential protein expression observed, along with the identified proteins and enriched pathways, provides valuable insights into the underlying molecular mechanisms. These findings offer a foundation for further exploration of the therapeutic potential of Phy and Bis in the management of NSCLC.

Screening of polysaccharides from the differently processed products of Angelica sinensis with the best liver protection effect on chicken and the intervention mechanism study based on tandem mass tag proteomics and multiple reaction monitoring approach.

The incidence of colibacillosis in poultry is on the rise, significantly affecting the chicken industry. Ceftiofur sodium (CS) is frequently employed to treat this disease, resulting in lipopolysaccharide (LPS) buildup. Processing plays a vital role in traditional Chinese veterinary medicine. The potential intervention in liver injury by polysaccharides from the differently processed products of Angelica sinensis (PDPPAS) induced by combined CS and LPS remains unclear. This study aims to investigate the protective effect of PDPPAS on chicken liver injury caused by CS combined with LPS buildup and further identify the polysaccharides with the highest hepatoprotective activity in chickens. Furthermore, the study elucidates polysaccharides' intervention mechanism using tandem mass tag (TMT) proteomics and multiple reaction monitoring (MRM) methods. A total of 190 1-day-old layer chickens were randomly assigned into 12 groups, of which 14 chickens were in the control group and 16 in other groups, for a 10-day trial. The screening results showed that charred A. sinensis polysaccharide (CASP) had the most effective and the best hepatoprotective effect at 48 h. TMT proteomics and MRM validation results demonstrated that the intervention mechanism of the CASP high-dose (CASPH) intervention group was closely related to the protein expressions of FCER2, TBXAS1, CD34, AGXT, GCAT, COX7A2L, and CYP2AC1. Conclusively, the intervention mechanism of CASPH had multitarget, multicenter regulatory features.

The effect of mycophenolate mofetil on podocytes in nephrotoxic serum nephritis.

Mycophenolate mofetil (MMF) is applied in proteinuric kidney diseases, but the exact mechanism of its effect on podocytes is still unknown. Our previous in vitro experiments suggested that MMF can ameliorate podocyte damage via restoration of the Ca2+-actin cytoskeleton axis. The goal of this study was to characterize podocyte biology during MMF treatment in nephrotoxic serum (NTS) nephritis (NTN). NTN was induced in three-week old wild-type mice. On day 3, half of the mice were treated with MMF (100 mg/kgBW/d p.o.) for one week. On day 10, we performed proteomic analysis of glomeruli as well as super-resolution imaging of the slit diaphragm. For multiphoton imaging of Ca2+ concentration ([Ca2+]i), the experimental design was repeated in mice expressing podocyte-specific Ca2+ sensor. MMF ameliorated the proteinuria and crescent formation induced by NTS. We identified significant changes in the abundance of proteins involved in Ca2+ signaling and actin cytoskeleton regulation, which was further confirmed by direct [Ca2+]i imaging in podocytes showing decreased Ca2+ levels after MMF treatment. This was associated with a tendency to restoration of podocyte foot process structure. Here, we provide evidence that MPA has a substantial direct effect on podocytes. MMF contributes to improvement of [Ca2+]i and amelioration of the disorganized actin cytoskeleton in podocytes. These data extend the knowledge of direct effects of immunosuppressants on podocytes that may contribute to a more effective treatment of proteinuric glomerulopathies with the least possible side effects.

Lipoylation is dependent on the ferredoxin FDX1 and dispensable under hypoxia in human cells.

Iron-sulfur clusters (ISC) are essential cofactors that participate in electron transfer, environmental sensing, and catalysis. Amongst the most ancient ISC-containing proteins are the ferredoxin (FDX) family of electron carriers. Humans have two FDXs- FDX1 and FDX2, both of which are localized to mitochondria, and the latter of which is itself important for ISC synthesis. We have previously shown that hypoxia can eliminate the requirement for some components of the ISC biosynthetic pathway, but FDXs were not included in that study. Here, we report that FDX1, but not FDX2, is dispensable under 1% O2 in cultured human cells. We find that FDX1 is essential for production of the lipoic acid cofactor, which is synthesized by the ISC-containing enzyme lipoyl synthase. While hypoxia can rescue the growth phenotype of either FDX1 or lipoyl synthase KO cells, lipoylation in these same cells is not rescued, arguing against an alternative biosynthetic route or salvage pathway for lipoate in hypoxia. Our work reveals the divergent roles of FDX1 and FDX2 in mitochondria, identifies a role for FDX1 in lipoate synthesis, and suggests that loss of lipoic acid can be tolerated under low oxygen tensions in cell culture.

Pharos 2023: an integrated resource for the understudied human proteome.

The Illuminating the Druggable Genome (IDG) project aims to improve our understanding of understudied proteins and our ability to study them in the context of disease biology by perturbing them with small molecules, biologics, or other therapeutic modalities. Two main products from the IDG effort are the Target Central Resource Database (TCRD) (http://juniper.health.unm.edu/tcrd/), which curates and aggregates information, and Pharos (https://pharos.nih.gov/), a web interface for fusers to extract and visualize data from TCRD. Since the 2021 release, TCRD/Pharos has focused on developing visualization and analysis tools that help reveal higher-level patterns in the underlying data. The current iterations of TCRD and Pharos enable users to perform enrichment calculations based on subsets of targets, diseases, or ligands and to create interactive heat maps and UpSet charts of many types of annotations. Using several examples, we show how to address disease biology and drug discovery questions through enrichment calculations and UpSet charts.

The importance of transcriptomics and proteomics for studying molecular mechanisms of mycotoxin exposure: A review.

This review aims to highlight recent advances where transcriptomics and proteomics have been used as a key tool to understand molecular toxicity of mycotoxins. The most studied mycotoxin by using transcriptomic approach is deoxynivalenol (DON), followed by aflatoxins (AFs) and zearalenone (ZEA). Instead, proteomics mostly focuses on AFs but also in this case, mildly to ZEA and DON. However, in both omics approaches, fewer studies investigated the toxicological effect of emerging mycotoxins, patulin, ochratoxin A, T-2 toxin, alternariol and amino-14,16-dimethyloctadecan-3-ol. The study of changes in the expression of genes involved in immune system are the most common purposes for transcriptomics whereas cellular processes in proteomics field. Concerning the techniques used to perform the experiments, RT-qPCR is the most employed in gene expression analysis whereas liquid chromatography coupled with mass spectrometry is the master technique for proteomics assays. The gathered data have reported that the interest in using these omic approaches has increased in the last five years. However, in vitro models take precedence over the in vivo and ex vivo ones. Therefore, there is a need to enhance the use of in vivo models and alternative methods to better understand mycotoxins mode of action on animal and human health.

Bisphenol A replacement chemicals, BPF and BPS, induce protumorigenic changes in human mammary gland organoid morphology and proteome.

SignificanceBisphenol A (BPA), found in many plastic products, has weak estrogenic effects that can be harmful to human health. Thus, structurally related replacements-bisphenol S (BPS) and bisphenol F (BPF)-are coming into wider use with very few data about their biological activities. Here, we compared the effects of BPA, BPS, and BPF on human mammary organoids established from normal breast tissue. BPS disrupted organoid architecture and induced supernumerary branching. At a proteomic level, the bisphenols altered the abundance of common targets and those that were unique to each compound. The latter included proteins linked to tumor-promoting processes. These data highlighted the importance of testing the human health effects of replacements that are structurally related to chemicals of concern.

Quantitative Proteomics Analysis Expands the Roles of Lysine ß-Hydroxybutyrylation Pathway in Response to Environmental ß-Hydroxybutyrate.

Lysine ß-hydroxybutyrylation (Kbhb) is a newly identified protein posttranslational modification (PTM) derived from ß-hydroxybutyrate (BHB), a product of ketone body metabolism in liver. BHB could serve as an energy source and play a role in the suppression of oxidative stress. The plasma concentration of BHB could increase up to 20 mM during starvation and in pathological conditions. Despite the progress, how the cells derived from extrahepatic tissues respond to elevated environmental BHB remains largely unknown. Given that BHB can significantly drive Kbhb, we characterized the BHB-induced lysine ß-hydroxybutyrylome and acetylome by quantitative proteomics. A total of 840 unique Kbhb sites on 429 proteins were identified, with 42 sites on 39 proteins increased by more than 50% in response to BHB. The results showed that the upregulated Kbhb induced by BHB was involved in aminoacyl-tRNA biosynthesis, 2-oxocarboxylic acid metabolism, citrate cycle, glycolysis/gluconeogenesis, and pyruvate metabolism pathways. Moreover, some BHB-induced Kbhb substrates were significantly involved in diseases such as cancer. Taken together, we investigate the dynamics of lysine ß-hydroxybutyrylome and acetylome induced by environmental BHB, which reveals the roles of Kbhb in regulating various biological processes and expands the biological functions of BHB.

What Worth the Garlic Peel.

Plants have two types of reproduction: sexual, resulting in embryo production, and asexual, resulting in vegetative bodies commonly derived from stems and roots (e.g., bulb, tuber). Dead organs enclosing embryos (DOEEs, such as seed coat and pericarp) are emerging as central components of the dispersal unit acting to nurture the embryo and ensure its survival in the habitat. Here we wanted to investigate the properties of dead organs enclosing plant asexual reproductive bodies, focusing on the garlic (Allium sativum) bulb. We investigated the biochemical and biological properties of the outer peel enclosing the bulb and the inner peel enclosing the clove using various methodologies, including bioassays, proteomics, and metabolomics. The garlic peels differentially affected germination and post-germination growth, with the outer peel demonstrating a strong negative effect on seed germination of Sinapis alba and on post-germination growth of Brassica juncea. Proteome analysis showed that dead garlic peels possess 67 proteins, including chitinases and proteases, which retained their enzymatic activity. Among primary metabolites identified in garlic peels, the outer peel accumulated multiple sugars, including rhamnose, mannitol, sorbitol, and trehalose, as well as the modified amino acid 5-hydroxylysine, known as a major component of collagen, at a higher level compared to the clove and the inner peel. Growth of Escherichia coli and Staphylococcus aureus was promoted by garlic peel extracts but inhibited by clove extract. All extracts strongly inhibited spore germination of Fusarium oxysporum f.sp. melonis. Thus, the garlic peels not only provide physical protection to vegetative offspring but also appear to function as a refined arsenal of proteins and metabolites for enhancing growth and development, combating potential pathogens, and conferring tolerance to abiotic stresses.

Pterostilbene Promotes Mean Lifespan in Both Male and Female Drosophila Melanogaster Modulating Different Proteins in the Two Sexes.

Aging is a multifactorial phenomenon characterized by degenerative processes closely connected to oxidative damage and chronic inflammation. Recently, many studies have shown that natural bioactive compounds are useful in delaying the aging process. In this work, we studied the effects of an in vivo supplementation of the stilbenoid pterostilbene on lifespan extension in Drosophila melanogaster. We found that the average lifespan of flies of both sexes was increased by pterostilbene supplementation with a higher effect in females. The expression of longevity related genes (Sir2, Foxo, and Notch) was increased in both sexes but with different patterns. Pterostilbene counteracted oxidative stress induced by ethanol and paraquat and up-regulated the antioxidant enzymes Ho e Trxr-1 in male but not in female flies. On the other hand, pterostilbene decreased the inflammatory mediators dome and egr only in female flies. Proteomic analysis revealed that pterostilbene modulates 113 proteins in male flies and only 9 in females. Only one of these proteins was modulated by pterostilbene in both sexes: vacuolar H[+] ATPase 68 kDa subunit 2 (Vha68-2) that was strongly down-regulated. These findings suggest a potential role of pterostilbene in increasing lifespan both in male and female flies by mechanisms that seem to be different in the two sexes, highlighting the need to conduct nutraceutical supplementation studies on males and females separately in order to give more reliable results.

The proteomic landscape of ovarian cancer cells in response to melatonin.

Ovarian cancer (OC) is the most lethal gynecological malignancy with a highly negative prognosis. Melatonin is an indoleamine secreted by the pineal gland during darkness and has shown antitumor activity in both in vitro and in vivo experiments. Herein, we investigated the influence of melatonin on the proteome of human ovarian carcinoma cells (SKOV-3 cell line) using the Ultimate 3000 LC Liquid NanoChromatography equipment coupled to a Q-Exactive mass spectrometry. After 48 h of treatment, melatonin induced a significant cytotoxicity especially with the highest melatonin concentration. The proteomic profile revealed 639 proteins in the control group, and 98, 110, and 128 proteins were altered by melatonin at the doses of 0.8, 1.6, and 2.4 mM, respectively. Proteins associated with the immune system and tricarboxylic acid cycle were increased in the three melatonin-exposed groups of cells. Specifically, the dose of 2.4 mM led to a reduction in molecules associated with protein synthesis, especially those of the ribosomal protein family. We also identified 28 potential genes shared between normal ovarian tissue and OC in all experimental groups, and melatonin was predicted to alter genes encoding ribosomal proteins. Notably, the set of proteins changed by melatonin was linked to a better prognosis for OC patients. We conclude that melatonin significantly alters the proteome of SKOV-3 cells by changing proteins involved with the immune response and mitochondrial metabolism. The concentration of 2.4 mM of melatonin promoted the largest number of protein changes. The evidence suggests that melatonin may be an effective therapeutic strategy against OC.

Novel protein kinase C phosphorylated kinase inhibitor-matrine suppresses replication of hepatitis B virus via modulating the mitogen-activated protein kinase signal.

HBV (hepatitis B virus) infection still threatens human health. Therefore, it is essential to find new effective anti-HBV compounds. Here, we identified matrine as a novel inhibitor of PKC (protein kinase C) phosphorylated kinase by screening a natural compound library. After HepG2.215 cells were treated with matrine, we carried out a phosphorylated proteomics sequence study and analyzed the prediction of related kinase expression level. In the case of HBV infection, it was found that PKC kinase mediates the activation of mitogen-activated protein kinase (MAPK) signaling pathway known as son of sevenless (SOS) activation. It was also found that PKC kinase inhibits the expression of C-X-C Motif Chemokine Ligand 8 (CXCL8) by inhibiting the activity of activating transcription factor 2/ cAMP response element binding protein (ATF2/CREB), and this effect is independent of its activated MAPK signaling pathway. Finally, Western blot was used to detect the expression of MAPK, ATF2, CREB3 phosphorylation and nonphosphorylation in matrine-treated cells and PKC-treated cells. PKC phosphorylated kinase inhibitor-matrine suppresses the replication of HBV via modulating the MAPK/ATF2 signal. Matrine is a good clinical drug to enhance the autoimmunity in the adjuvant treatment of chronic HBV infection.