Correlation of bioactive marker compounds of an orally applied Morus alba root bark extract with toxicity and efficacy in BALB/c mice.

Introduction: In traditional Chinese medicine, the root bark of Morus alba L. is used to treat respiratory infections. Recently, anti-inflammatory and multiple anti-infective activities (against influenza viruses, corona virus 2, S. aureus, and S. pneumoniae) were shown in vitro for a standardized root bark extract from M. alba (MA60). Sanggenons C and D were identified as major active constituents of MA60. The aim of the present preclinical study was to evaluate, whether these findings are transferable to an in vivo setting. Methods: MA60 was orally administered to female BALB/c mice to determine 1) the maximum tolerated dose (MTD) in an acute toxicity study and 2) its anti-influenza virus and anti-inflammatory effects in an efficacy study. A further aim was to evaluate whether there is a correlation between the obtained results and the amount of sanggenons C and D in serum and tissues. For the quantitation of the marker compounds sanggenons C and D in serum and tissue samples an UPLC-ESI-MS method was developed and validated. Results: In our study setting, the MTD was reached at 100 mg/kg. In the efficacy study, the treatment effects were moderate. Dose-dependent quantities of sanggenon C in serum and sanggenon D in liver samples were detected. Only very low concentrations of sanggenons C and D were determined in lung samples and none of these compounds was found in spleen samples. There was no compound accumulation when MA60 was administered repeatedly. Discussion: The herein determined low serum concentration after oral application once daily encourages the use of an alternative application route like intravenous, inhalation or intranasal administration and/or multiple dosing in further trials. The established method for the quantitation of the marker sanggenon compounds in tissue samples serves as a basis to determine pharmacokinetic parameters such as their bioavailability in future studies.

A Versatile Brij-Linker for One-Step Preparation of Targeted Nanoparticles.

Background: Most frequently the functionalization of nanoparticles is hampered by time-consuming, sometimes harsh conjugation and purification procedures causing premature drug release and/or degradation. A strategy to circumvent multi-step protocols is to synthesize building blocks with different functionalities and to use mixtures thereof for nanoparticle preparation in one step. Methods: BrijS20 was converted into an amine derivative via a carbamate linkage. The Brij-amine readily reacts with pre-activated carboxyl-containing ligands such as folic acid. The structures of the building blocks were confirmed by different spectroscopic methods and their utility was assessed by one-step preparation and characterization of nanoparticles applying PLGA as a matrix polymer. Results: Nanoparticles were about 200 nm in diameter independent of the composition. Experiments with human folate expressing single cells and monolayer revealed that the nanoparticle building block Brij mediates a "stealth" effect and the Brij-amine-folate a "targeting" effect. As compared to plain nanoparticles, the stealth effect decreased the cell interaction by 13%, but the targeting effect increased the cell interaction by 45% in the monolayer. Moreover, the targeting ligand density and thus the cell association of the nanoparticles is easily fine-tuned by selection of the initial ratio of the building blocks. Conclusions: This strategy might be a first step towards the one-step preparation of nanoparticles with tailored functionalities. Relying on a non-ionic surfactant is a versatile approach as it might be extended to other hydrophobic matrix polymers and promising targeting ligands from the biotech pipeline.

In Silico and In Vitro Approach to Assess Direct Allosteric AMPK Activators from Nature.

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is an important metabolic regulator. Its allosteric drug and metabolite binding (ADaM) site was identified as an attractive target for direct AMPK activation and holds promise as a novel mechanism for the treatment of metabolic diseases. With the exception of lusianthridin and salicylic acid, no natural product (NP) is reported so far to directly target the ADaM site. For the streamlined assessment of direct AMPK activators from the pool of NPs, an integrated workflow using in silico and in vitro methods was applied. Virtual screening combining a 3D shape-based approach and docking identified 21 NPs and NP-like molecules that could potentially activate AMPK. The compounds were purchased and tested in an in vitro AMPK α 1 ß 1 γ 1 kinase assay. Two NP-like virtual hits were identified, which, at 30 µM concentration, caused a 1.65-fold (± 0.24) and a 1.58-fold (± 0.17) activation of AMPK, respectively. Intriguingly, using two different evaluation methods, we could not confirm the bioactivity of the supposed AMPK activator lusianthridin, which rebuts earlier reports.

Oral intake of xanthohumol attenuates lipoteichoic acid-induced inflammatory response in human PBMCs.

PURPOSE: The aim of the study was to determine if xanthohumol, a prenylated chalcone found in Hop (Humulus lupulus), has anti-inflammatory effects in healthy humans if applied in low doses achievable through dietary intake. METHODS: In a placebo-controlled single-blinded cross-over design study, 14 healthy young men and women either consumed a beverage containing 0.125 mg xanthohumol or a placebo. Peripheral blood mononuclear cells (PBMCs) were isolated before and 1 h after the intake of the beverages. Subsequently, PBMCs were stimulated with or without lipoteichoic acid (LTA) for 24 and 48 h. Concentrations of interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and soluble cluster of differentiation (sCD14) protein were determined in cell culture supernatant. Furthermore, hTLR2 transfected HEK293 cells were stimulated with LTA in the presence or absence of xanthohumol and sCD14. RESULTS: The stimulation of PBMCs with LTA for 24 and 48 h resulted in a significant induction of IL-1ß, IL-6, and sCD14 protein release in PBMCs of both, fasted subjects and subjects after the ingestion of the placebo. In contrast, after ingesting xanthohumol, LTA-dependent induction of IL-1ß, IL-6, and sCD14 protein release from PBMCs was not significantly higher than in unstimulated cells after 48 h. In hTLR2 transfected HEK293 cells xanthohumol significantly suppressed the LTA-dependent activation of cells, an effect attenuated when cells were co-incubated with sCD14. CONCLUSION: The results of our study suggest that an ingestion of low doses of xanthohumol can suppress the LTA-dependent stimulation of PBMCs through mechanisms involving the interaction of CD14 with TLR2. Study registered at ClinicalTrials.gov (NCT04847193, 22.03.2022).

Biochemometry-Based Discovery of Phenylpropanoids from Azadirachta indica Fruits as Inhibitors of In Vitro Osteoclast Formation.

(1) Background: Inhibition of osteoclast differentiation is the key approach in treating osteoporosis. However, using state-of-the-art treatments such as bisphosphonates and estrogen-based therapy is usually accompanied by many side effects. As opposed to this, the use of natural products as an osteoporotic remedy delivers promising outcomes with minimal side effects. (2) Methods: In the present study, we implemented a biochemometric workflow comprising (i) chemometric approaches using NMR and mass spectrometry and (ii) cell biological approaches using an osteoclast cytochemical marker (TRAP). The workflow serves as a screening tool to pursue potential in vitro osteoclast inhibitors. (3) Results: The workflow allowed for the selective isolation of two phenylpropanoids (coniferyl alcohol and sinapyl alcohol) from the fruits of neem tree (Azadirachta indica). These two isolated phenylpropanoids showed a very promising dose-dependent inhibition of osteoclast differentiation with negligible effects in terms of cell viability. (4) Conclusion: The presented workflow is an effective tool in the discovery of potential candidates for osteoclast inhibition from complex extracts. The used biochemometric approach saves time, effort and costs while delivering precise hints to selectively isolate bioactive constituents.

Development and Experimental Validation of Regularized Machine Learning Models Detecting New, Structurally Distinct Activators of PXR.

The pregnane X receptor (PXR) regulates the metabolism of many xenobiotic and endobiotic substances. In consequence, PXR decreases the efficacy of many small-molecule drugs and induces drug-drug interactions. The prediction of PXR activators with theoretical approaches such as machine learning (ML) proves challenging due to the ligand promiscuity of PXR, which is related to its large and flexible binding pocket. In this work we demonstrate, by the example of random forest models and support vector machines, that classifiers generated following classical training procedures often fail to predict PXR activity for compounds that are dissimilar from those in the training set. We present a novel regularization technique that penalizes the gap between a model's training and validation performance. On a challenging test set, this technique led to improvements in Matthew correlation coefficients (MCCs) by up to 0.21. Using these regularized ML models, we selected 31 compounds that are structurally distinct from known PXR ligands for experimental validation. Twelve of them were confirmed as active in the cellular PXR ligand-binding domain assembly assay and more hits were identified during follow-up studies. Comprehensive analysis of key features of PXR biology conducted for three representative hits confirmed their ability to activate the PXR.

Exudates of Picea abies, Pinus nigra, and Larix decidua: Chromatographic Comparison and Pro-Migratory Effects on Keratinocytes In Vitro.

Balms and resins of Picea abies, Larix decidua, and Pinus nigra are traditionally used to treat wounds. Three chromatographic techniques differing in separation capacity and technical demands were employed to distinguish among these plant exudates. A TLC method was established for fingerprint comparison, providing a quick overview of a large number of samples at low cost. HPLC-DAD (RP18) and UHPSFC-DAD (Torus 2-Picolylamin), hyphenated to ESI-MS, represented orthogonal chromatographic systems with high separation performance. The developed methods allow for the separation and detection of major and minor constituents belonging to different compound classes (phenyl carboxylic acids, lignans, diterpene resin acids). The qualitative compositions of the diterpene resin acids, the main compounds in the exudates, were comparable in all three genera. Differences were detected in the distribution of hydroxylated diterpene resin acids, pinoresinol, and hydroxycinnamic acids. The three tested chromatographic systems with varying demands on lab equipment offer appropriate tools for the quality assessment of Picea abies, Larix decidua, and Pinus nigra. The extracts were furthermore tested at three different concentrations (10 µg/mL, 3 µg/mL, and 1 µg/mL) for boosted re-epithelialization, a crucial step in the wound-healing process, in an in vitro HaCaT keratinocyte-based scratch assay. Lysophosphatidic acid (LPA, 10 µM) and extracts of several medicinal plants well known for their wound-healing properties (birch, marigold, St. John's wort, manuka honey) were used as positive controls. Picea abies and Pinus nigra showed concentration dependency; significant activity was measured for Larix decidua at 3 µg/mL.

Insights into the direct anti-influenza virus mode of action of Rhodiola rosea.

BACKGROUND: The anti-influenza A virus activities and contents of previously isolated most active flavonoids (rhodiosin and tricin) from a standardized hydro-ethanolic R. rosea root and rhizome extract (SHR-5®), did not fully explain the efficacy of SHR-5®. Moreover, the mode of antiviral action of SHR-5® is unknown. PURPOSE: To determine the anti-influenza viral principle of SHR-5® we evaluated i) the combined anti-influenza virus effect of rhodiosin and tricin, ii) the impact of its tannin-enriched fraction (TE), iii) its antiviral spectrum and mode of action, and iv) its propensity for resistance development in vitro. METHODS: The combined anti-influenza virus effect of rhodiosin and tricin and the impact of TE were investigated with cytopathic effect (CPE)-inhibition assays in MDCK cells. A tannin-depleted fraction (TD) and TE were prepared by polyamide column chromatography and dereplicated by LC-MS. Plaque-reduction assays provided insights into the anti-influenza virus profile, the mode of action, and the propensity for resistance development of SHR-5®. RESULTS: Our results i) did not reveal synergistic anti-influenza A virus effects of rhodiosin and tricin, but ii) proved a strong impact of TE mainly composed of prodelphinidin gallate oligomers. iii) TE inhibited the plaque-production of influenza virus A(H1N1)pdm09, A(H3N2), and B (Victoria and Yamagata) isolates (including isolates resistant to neuraminidase and/or M2 ion channel inhibitors) with 50% inhibitory concentration values between 0.12 - 0.53 µg/ml similar to SHR-5®. Mechanistic studies proved a virucidal activity, inhibition of viral adsorption, viral neuraminidase activity, and virus spread by SHR-5® and TE. iv) No resistance development was observed in vitro. CONCLUSION: For the first time a comprehensive analysis of the anti-influenza virus profile of a hydro-ethanolic R. rosea extract (SHR-5®) was assessed in vitro. The results demonstrating broad-spectrum multiple direct anti-influenza virus activities, and a lack of resistance development to SHR-5® together with its known augmentation of host defense, support its potential role as an adaptogen against influenza virus infection.

Dermal Delivery of Korean Red Ginseng Extract: Impact on Storage Stability of Different Carrier Systems and Evaluation of Rg1 and Rb1 Skin Permeation Ex Vivo.

The root extract of Panax ginseng C.A. Meyer (Korean red ginseng/KRG extract) is a traditional Asian remedy introduced to dermal products for its antioxidative potential. However, little is known about technological aspects or skin penetration of main ginsenosides. Thus, stable oil-in-water nanoemulsions (NEs) and hydrogels for dermal delivery of KRG extract were developed and characterised using light scattering methods, analysis of flow properties and pH measurements. In addition, Rg1 and Rb1 contents were monitored by UHPLC/MS. Different surfactants (phosphatidylcholine, monoacylphosphatidylcholine and polysorbate 80) and polymers (polyacrylic acid and hydroxyethylcellulose) were tested and compared for their compatibility with KRG extract. The results showed that incorporation of KRG extract led to a significantly reduced formulation pH in hydroxyethylcellulose gels (-22%), NEs (-15%) and carbomer gels (-4-5%). The dynamic viscosity was in the range of 24-28 Pas at 10 s-1 for carbomer gels. The highest storage stability and skin permeation were observed for a hydroalcoholic gel with carbomer 50,000 and TRIS buffer (each of 1% w/w), containing ethanol (20% w/w) and KRG extract (2% w/w). Ex vivo diffusion cell studies confirmed skin permeation of the moderately lipophilic Rg1, but not the more hydrophilic Rb1 with a larger molecular weight.

Impaired Clearance From the Brain Increases the Brain Exposure to Metoclopramide in Elderly Subjects.

The antiemetic and gastroprokinetic drug metoclopramide is a weak substrate of the blood-brain barrier (BBB) efflux transporter P-gp and displays central nervous system (CNS) side effects (i.e., extrapyramidal symptoms and tardive dyskinesia) caused by dopamine D2 receptor blockade in the basal ganglia. These side effects occur with a higher incidence in elderly people. We used positron emission tomography to assess the brain distribution of [11 C]metoclopramide in young (n = 11, 26 ± 3 years) and elderly (n = 7, 68 ± 9 years) healthy men both after administration of a microdose (9 ± 7 µg) and a microdose co-injected with a therapeutic dose of unlabeled metoclopramide (10 mg). For both doses, elderly subjects had a significantly higher total volume of distribution (VT ) of [11 C]metoclopramide in the basal ganglia than young subjects (microdose: +26%, therapeutic dose: +41%). Increases in VT (= K1 /k2 ) were caused by significant decreases in the transfer rate constant of [11 C]metoclopramide from brain into plasma (k2 , microdose: -18%, therapeutic dose: -30%), whereas the distributional clearance from plasma into brain (K1 ) remained unaltered. This reduction in the clearance of [11 C]metoclopramide (k2 ) from the brains of elderly subjects may be caused by an age-related decrease in the activity of P-gp at the BBB and may contribute to the higher incidence of CNS side effects of metoclopramide in the aged population. Our data suggest that an age-associated decrease in the clearance properties of the BBB may modulate the CNS effects or side effects of clinically used P-gp substrates.

Methylated Xanthones from the Rootlets of Metaxya rostrata Display Cytotoxic Activity in Colorectal Cancer Cells.

The tree fern Metaxya rostrata (Kunth) C. Presl is common in the rainforests of Central and South America, where suspensions of the dried rhizome are traditionally used to treat intestinal diseases. Two compounds from this plant, 2-deprenyl-rheediaxanthone B (XB) and 2-deprenyl-7-hydroxy-rheediaxanthone B (OH-XB), have been shown to be biologically highly active against colorectal cancer (CRC) cells in previous studies. The current investigation resulted in the isolation of the previously undescribed methylated xanthones 2-deprenyl-6-O-methyl-7-hydroxy-rheediaxanthone B, 2-deprenyl-5-O-methyl-7-methoxy-rheediaxanthone B, 2-deprenyl-5-O-methyl- 7-hydroxy-rheediaxanthone B and 2-deprenyl-7-methoxy-rheediaxanthone B. All compounds were isolated by column chromatography, structures were elucidated by one- and two-dimensional NMR-experiments and the identities of the compounds were confirmed by LC-HRMS. In logarithmically growing SW480 CRC cell cultures, cytotoxicity by neutral red uptake and MTT assays as well as caspase activation was analyzed. Cellular targets were examined by Western blot, and topoisomerase I (topo I) inhibition potential was tested. Comparing the structure-activity relationship with XB and OH-XB, the monomethylated derivatives showed qualitatively similar effects/mechanisms to their nonmethylated analogues, while dimethylation almost abolished the activity. Inhibition of topo I was dependent on the presence of an unmethylated 7-OH group.

A robust and miniaturized screening platform to study natural products affecting metabolism and survival in Caenorhabditis elegans.

In this study a robust, whole organism screening based on Caenorhabditis elegans is presented for the discovery of natural products (NP) with beneficial effects against obesity and age-related diseases. Several parameters of the elaborated workflow were optimized to be adapted for probing multicomponent mixtures combining knowledge from traditional medicine and NP chemistry by generating optimized small-scale extracts considering scarcity of the natural source, solubility issues, and potential assay interferences. The established miniaturized assay protocol allows for in vivo probing of small amounts of even complex samples (~ 1 mg) to test their ability to increase the nematodes' survival time and the suppression of fat accumulation assessed by Nile red staining as hall marks of "healthy aging". The workflow was applied on 24 herbal and fungal materials traditionally used against symptoms of the metabolic syndrome and revealed promising results for the extracts of Gardenia jasminoides fruits and the sclerotia from Inonotus obliquus. Tested at 100 µg/mL they were able to significantly reduce the Nile red fluorescence and extend the 50% survival rate (DT50) compared to the control groups. This phenotype-directed in vivo approach opens up new horizons for the selection of natural starting materials and the investigation of their active principles as fast drug discovery tool with predictive value for human diseases.

Norway Spruce Balm: Phytochemical Composition and Ability to Enhance Re-epithelialization In Vitro.

The balm of the Norway spruce (Picea abies) is a well-known traditional herbal medicine used to cure wounds. Even though clinical trials have confirmed its empirical use, the active constituents, their mode of action, and the exact composition of this natural product are still unknown. In this study, the balm was subjected to fractionated extraction and further purified employing flash chromatography, HPLC-PDA-ELSD, preparative and analytical TLC. Hydroxycinnamic acids ( 1: - 3: ), the lignan pinoresinol ( 4: ), four hydroxylated derivatives of dehydroabietic acid (DHAA) ( 5:  -  8: ), and dehydroabietic acid ( 9: ) were isolated. Their structures were elucidated by LC-MS, 1D- and 2D-NMR. Four extracts, two commercially available resin acids-pimaric acid ( 10: ) and isopimaric acid ( 11: )-and the isolated compounds were tested for increased re-epithelialization of cell-free areas in a human adult low calcium high temperature keratinocytes monolayer. Lysophosphatidic acid (10 µM) served as positive control and ranged between 100% and 150% rise in cell-covered area related to the vehicle control. Two extracts containing carboxylic acids and non-acidic apolar constituents, respectively, boosted wound closure by 47% and 36% at 10 and 3 µg/mL, respectively. Pinoresinol, DHAA, three of its hydroxylated derivatives, and pimaric and isopimaric acid as well as defined combinations of the hydroxylated DHAA derivatives led to a significantly enhanced wound closure by up to 90% at concentrations between 1 and 10 µM. Overall, lignans and diterpene resin acids, main constituents of Norway spruce balm, are able to increase migration or proliferation of keratinocytes in vitro. The presented data link the phytochemistry of this natural wound healing agent with boosted re-epithelialization.

1H NMR-MS-based heterocovariance as a drug discovery tool for fishing bioactive compounds out of a complex mixture of structural analogues.

Chemometric methods and correlation of spectroscopic or spectrometric data with bioactivity results are known to improve dereplication in classical bio-guided isolation approaches. However, in drug discovery from natural sources the isolation of bioactive constituents from a crude extract containing close structural analogues remains a significant challenge. This study is a 1H NMR-MS workflow named ELINA (Eliciting Nature's Activities) which is based on statistical heterocovariance analysis (HetCA) of 1H NMR spectra detecting chemical features that are positively ("hot") or negatively ("cold") correlated with bioactivity prior to any isolation. ELINA is exemplified in the discovery of steroid sulfatase (STS) inhibiting lanostane triterpenes (LTTs) from a complex extract of the polypore fungus Fomitopsis pinicola.

High-performance thin-layer chromatography/bioautography and liquid chromatography-mass spectrometry hyphenated with chemometrics for the quality assessment of Morus alba samples.

Quality control is a crucial step in the production of effective and safe herbal remedies. The aim of this study was to develop a new, simple, and high throughput procedure for the quality assessment of herbal drugs using a high-performance thin-layer chromatography (HPTLC)/bioautography and UPLC-MS/MS approach combined with chemometrics. This was exemplarily shown for Morus alba L. root bark (sang bái pí; SBP). Bioautography assays were developed for the identification of constituents with radical scavenging (DPPH assay) and antimicrobial activities (Bacillus subtilis, Escherichia coli) of 18 different M. alba samples, which was supported by UPLC-MS/MS analysis. Further, the combination of bioautography and chemometrics identified those samples with the most bioactive constituents. Plant materials collected from Serbia (11 samples) showed higher both radical scavenging and antimicrobial activities compared to samples provided from China (7 samples). Principal component analysis (PCA) confirmed the discrimination of geographically different samples and recognized their main markers responsible for differences between Serbian and Chinese samples. Most importantly for quality assessment, the combined HPTLC/bioautography and UPLC-MS/MS approach not only allowed for a fast chemical profiling of the investigated samples and their unambiguous identification, but also for the disclosure of major and minor bioactive constituents present in SBP.

Time-dependent shotgun proteomics revealed distinct effects of an organoruthenium prodrug and its activation product on colon carcinoma cells.

Activation kinetics of metallo-prodrugs control the types of possible interactions with biomolecules. The intact metallo-prodrug is able to engage with potential targets by purely non-covalent bonding, while the activated metallodrug can form additional coordination bonds. It is hypothesized that the additional coordinative bonding might be favourable with respect to the target selectivity of activated metallodrugs. Thus, a time-dependent shotgun proteomics study was conducted in HCT116 colon carcinoma cells with plecstatins, which are organoruthenium anticancer drug candidates. First, the target selectivity was evaluated in a time-dependent fashion, which accounted for their hydrolysis kinetics. The binding selectivity increased from 50- to 160-fold and the average specificity from 0.72 to 0.86, respectively, from the 2 h to the 4 h target profiling experiment. Target profiling after 19 h did not reveal significant enrichments, possibly due to deactivation of the probe via arene cleavage. Up to 450 interactors were identified in the target profiling experiments. A plecstatin analogue that substituted a hydrogen bond acceptor with a hydrogen bond donor abrogated the target selectivity for plectin in HCT116 whole cell lysates, underlining the necessity of this hydrogen bond acceptor for a strong interaction between plecstatin and plectin. Second, time-dependent response profiling experiments provided evidence that plecstatin-2 induced an integrated stress response (ISR) in HCT116 cell culture. The phosphorylation of eIF2α, a key mediator of the ISR, after 3 h treatment indicated that this perturbation was initiated by the intact plecstatin-2 prodrug, while the effects of plectin-targeting are mediated by activated plecstatin-2.

Collaborative Cardiac Care: A Comprehensive Heart Team Approach to Multiple Severe Vascular Conditions.

Left main artery coronary disease represents the highest risk lesion of ischemic heart disease. Revascularization can be accomplished by surgery or percutaneous interventions. This study highlights the case of a patient with severe multiple peripheral vascular conditions and complex coronary anatomy treated with percutaneous coronary intervention using mechanical circulatory support. (Level of Difficulty: Intermediate.).

Reduction of Sugar-Sweetened Beverage Consumption in Elementary School Students Using an Educational Curriculum of Beverage Sugar Content.

Introduction: Given the known association between sugar-sweetened beverage (SSB) intake and poorer health, we instituted an educational curriculum to reduce student consumption of SSBs. Methods: The program included third- to fifth-grade students. A simple demonstration using teaspoons of sugar or small candies showed students the quantity of added sugar in common beverages. This amount of sugar was compared to the daily limit recommended by the American Heart Association. Key principles were reinforced over a 4-month period. Anonymous beverage recall surveys were distributed to 213 students at baseline and 211 students 6 months after exposure to the curriculum. Primary endpoints included evaluation of SSB, real fruit juice (RFJ), diet soda, and water servings in the last 24 hours. Results: The proportion of children consuming 2 or more beverages daily decreased from 8.9% to 4.3% (P = .0546) for diet soda, from 70.0% to 58.3% (P = .0123) for SSB + RFJ, and from 60.1% to 47.4% (P = .0087) for SSB. At baseline, students reported an average consumption of 3.5 SSB, 4.5 SSB + RFJ, 0.4 diet soda, and 3.3 water servings per day. At 6 months after exposure, the average daily beverage consumption decreased to 2.7 servings per day for SSB (P = .014), 3.8 for SSB + RFJ (P = .039), and 0.2 for diet soda (P = .027). Water consumption increased from 3.3 to 3.6 servings per day (P = .075). Discussion: Our data suggest grade school students are receptive to information about the adverse effects of SSBs on health. Adding similar educational programs to elementary school curriculum may help reduce long-term SSB consumption.

Combined Proteome and Eicosanoid Profiling Approach for Revealing Implications of Human Fibroblasts in Chronic Inflammation.

During inflammation, proteins and lipids act in a concerted fashion, calling for combined analyses. Fibroblasts are powerful mediators of chronic inflammation. However, little is known about eicosanoid formation by human fibroblasts. The aim of this study was to analyze the formation of the most relevant inflammation mediators including proteins and lipids in human fibroblasts upon inflammatory stimulation and subsequent treatment with dexamethasone, a powerful antiphlogistic drug. Label-free quantification was applied for proteome profiling, while an in-house established data-dependent analysis method based on high-resolution mass spectrometry was applied for eicosadomics. Furthermore, a set of 188 metabolites was determined by targeted analysis. The secretion of 40 proteins including cytokines, proteases, and other inflammation agonists as well as 14 proinflammatory and nine anti-inflammatory eicosanoids was found significantly induced, while several acylcarnithins and sphingomyelins were found significantly downregulated upon inflammatory stimulation. Treatment with dexamethasone downregulated most cytokines and proteases, abrogated the formation of pro- but also anti-inflammatory eicosanoids, and restored normal levels of acylcarnithins but not of sphingomyelins. In addition, the chemokines CXCL1, CXCL5, CXCL6, and complement C3, known to contribute to chronic inflammation, were not counter-regulated by dexamethasone. Similar findings were obtained with human mesenchymal stem cells, and results were confirmed by targeted analysis with multiple reaction monitoring. Comparative proteome profiling regarding other cells demonstrated cell-type-specific synthesis of, among others, eicosanoid-forming enzymes as well as relevant transcription factors, allowing us to better understand cell-type-specific regulation of inflammation mediators and shedding new light on the role of fibroblasts in chronic inflammation.

Multi-omics Analysis of Serum Samples Demonstrates Reprogramming of Organ Functions Via Systemic Calcium Mobilization and Platelet Activation in Metastatic Melanoma.

Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia.