Protective effect of sucrose esters from cape gooseberry (Physalis peruviana L.) in TNBS-induced colitis.

Phytotherapy is an attractive strategy to treat inflammatory bowel disease (IBD) that could be especially useful in developing countries. We previously demonstrated the intestinal anti-inflammatory effect of the total ethereal extract from the Physalis peruviana (Cape gooseberry) calyces in TNBS-induced colitis. This work investigates the therapeutic potential of Peruviose A and B, two sucrose esters that constitute the major metabolites of its calyces. The effect of the Peruvioses A and B mixture on TNBS-induced colitis was studied after 3 (preventive) and 15-days (therapy set-up) of colitis induction in rats. Colonic inflammation was assessed by measuring macroscopic/histologic damage, MPO activity, and biochemical changes. Additionally, LPS-stimulated RAW 264.7 macrophages were treated with test compounds to determine the effect on cytokine imbalance in these cells. Peruvioses mixture ameliorated TNBS-induced colitis in acute (preventive) or established (therapeutic) settings. Although 3-day treatment with compounds did not produce a potent effect, it was sufficient to significantly reduce the extent/severity of tissue damage and the microscopic disturbances. Beneficial effects in the therapy set-up were substantially higher and involved the inhibition of pro-inflammatory enzymes (iNOS, COX-2), cytokines (TNF-α, IL-1ß, and IL-6), as well as epithelial regeneration with restoration of goblet cells numbers and expression of MUC-2 and TFF-3. Consistently, LPS-induced RAW 264.7 cells produced less NO, PGE2, TNF-α, IL-6, and MCP-1. These effects might be related to the inhibition of the NF-κB signaling pathway. Our results suggest that sucrose esters from P. peruviana calyces, non-edible waste from fruit production, might be useful as an alternative IBD treatment.

Impact of Saccharomyces cerevisiae yeast inoculation mode on wine composition.

Inoculation modes are known to affect yeast behavior. Here, we characterized the impact of ADY and pre-culturing on the composition of the resulting wine, fermented by four commercial strains of Saccharomyces cerevisiae. Classical oenological parameters were not affected by the yeast inoculation mode. Using an untargeted metabolomic approach, a significant distinction in wine composition was noted regardless of the strain between the two inoculation modes, each associated with a specific metabolomic signature. 218 and 895 biomarkers were annotated, respectively, for ADYs associated with the preservation of wine polyphenols, and for pre-cultures related to the modulation of yeast nitrogen metabolism. Volatilome analysis revealed that the ester family was that most impacted by the inoculation mode whatever the strain. Ester production was enhanced in ADY condition. For the first time, the complete reprogramming of the yeast metabolism was revealed as a function of yeast preparation, which significantly impacts its volatilome and exometabolome.

Reverse metabolomics for the discovery of chemical structures from humans.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.

A simple method to isolate fatty acids and fatty alcohols from wax esters in a wax-ester rich marine oil.

Calanus finmarchicus is one of the most important zooplankton species in the North Atlantic. The zooplankton is currently being harvested and industrially processed to a marine oil product for human consumption as a marine nutraceutical containing long-chain omega-3 polyunsaturated fatty acids. This oil is very rich in wax esters, a lipid class where fatty acids are esterified to long chain fatty alcohols. In this paper we describe a simple method to 1) isolate the wax esters from the other lipid classes present in the oil, 2) hydrolyze the wax esters, and 3) separate the fatty acids from the fatty alcohol, all by means of solid phase extraction. Starting with an average of 322 mg Calanus oil, we obtained 75 mg fatty alcohols and 63 mg fatty acids. Contrary to previously described techniques, our method neither oxidize the fatty alcohols to fatty acids, nor are the fatty acids methylated, allowing the native, unesterified fatty acids and fatty alcohols to be used for further studies, such as in cell culture experiments to study the metabolic effects of these specific lipid fractions rather than the intact oil or wax esters.

Environmental health risks induced by interaction between phthalic acid esters (PAEs) and biological macromolecules: A review.

As a kind of compounds abused in industry productions, phthalic acid esters (PAEs) cause serious problems in natural environment. PAEs pollution has penetrated into environmental media and human food chain. This review consolidates the updated information to assess the occurrence and distribution of PAEs in each transmission section. It is found that micrograms per kilogram of PAEs are exposed to humans through daily diets. After entering the human body, PAEs often undergo the metabolic process of hydrolysis to monoesters phthalates and conjugation process. Unfortunately, in the process of systemic circulation, PAEs will interact with biological macromolecules in vivo under the action of non-covalent binding, which is also the essence of biological toxicity. The interactions usually operate in the following pathways: (a) competitive binding; (b) functional interference; and (c) abnormal signal transduction. While the non-covalent binding forces mainly contain hydrophobic interaction, hydrogen bond, electrostatic interaction, and π interaction. As a typical endocrine disruptor, the health risks of PAEs often start with endocrine disorder, further leading to metabolic disruption, reproductive disorders, and nerve injury. Besides, genotoxicity and carcinogenicity are also attributed to the interaction between PAEs and genetic materials. This review also pointed out that the molecular mechanism study on biological toxicity of PAEs are deficient. Future toxicological research should pay more attention to the intermolecular interactions. This will be beneficial for evaluating and predicting the biological toxicity of pollutants at molecular scale.

Sinapic and ferulic acid phenethyl esters increase the expression of steroidogenic genes in MA-10 tumor Leydig cells.

During aging, the production of androgens by the testis Leydig cells gradually decreases. Phenolic compounds can improve testosterone biosynthesis and delay the onset of hypogonadal symptoms in males. In this study, sinapic acid phenethyl ester was evaluated for its ability to regulate gene expression and steroid production in Leydig cells. Specifically, the effects of this ester on the transcriptome of MA-10 Leydig cells were investigated by RNA-Seq. To better establish a structure-function relationship of the hydroxy-methoxyphenyl moiety of sinapic and phenethyl ester, its influences on gene expression were compared to those of its ferulic acid phenethyl ester analogue. According to the transcriptomic analysis, most genes encoding enzymes related to cholesterol biosynthesis are increased in response to sinapic acid phenethyl ester treatment of MA-10 Leydig cells. Interestingly, treatments with 10 µM of ferulic acid phenethyl ester increased cAMP-dependent Star promoter activation, gene expression and protein levels. In addition, treatments of MA-10 Leydig cells with 10 µM of sinapic or ferulic acid phenethyl ester resulted in increased progesterone production. Thus, our results indicate that sinapic and ferulic acid phenethyl esters can improve cholesterol and steroid biosynthesis in testicular Leydig cells.

Structural Analysis and Anti-Inflammatory Effect of a Digalactosyldiacylglycerol-Monoestolide, a Characteristic Glycolipid in Oats.

Digalactosyldiacylglycerol- (DGDG-) monoestolide is a characteristic glycolipid in oats. DGDG-monoestolides possess a unique structure whereby a fatty acid of DGDG is replaced by a fatty acid ester of hydroxy fatty acid (FAHFA). While the physiological effects of DGDG and FAHFA have been reported previously, the effects of DGDG-monoestolides are unknown. Hence, we isolated a major DGDG-monoestolide molecular species from oats, analyzed its structure, and evaluated its anti-inflammatory effect. Based on GC-MS, MS/MS, and NMR analyses, the isolated compound was identified as a DGDG-monoestolide that contains the linoleic acid ester of 15-hydroxy linoleic acid (LAHLA) and linoleic acid (i.e., DGDG-LAHLA). The isolated DGDG-LAHLA was evaluated for its anti-inflammatory effect on LPS-stimulated RAW264 cells. The production of nitric oxide and cytokines (IL-6, TNF-α, and IL-10) were significantly decreased by DGDG-LAHLA, suggesting the anti-inflammatory effect of DGDG-LAHLA for the first time. In addition, our data showed a pronounced uptake of DGDG-LAHLA by cells. Some compounds corresponding to the predicted DGDG-LAHLA metabolites were also detected, suggesting that both intact DGDG-LAHLA and its metabolites may contribute to the above anti-inflammatory activities. These results are expected to expand the availability of oats as a functional food.

Notch Signaling Mediates Radiation-Induced Smooth Muscle Cell Hypermuscularization and Cerebral Vasculopathy.

BACKGROUND: Emerging evidence highlighted vascular injury in aggravating radiation-induced brain injury (RIBI), a common complication of radiotherapy. This study aimed to delineate the pathological feature of cerebral small vessel and investigate the functional roles of Notch signaling in RIBI. METHODS: Brain tissue and functional MRI from RIBI patients were collected and analyzed for radiation-induced vasculopathy. A RIBI mouse model was induced by a single dose of 30-Gy cranial irradiation. Vascular morphology, pulsatility, and reactivity to pharmacological interventions, such as nimodipine and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, were monitored by 2-photon imaging in mice at 6 weeks postirradiation. Western blot, real-time quantitative PCR, immunofluorescence staining, and behavioral tests were performed. The effect of N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester, a Notch inhibitor, was used to investigate the vascular pathogenesis of RIBI mouse model. RESULTS: Morphologically, radiation resulted in vascular malformation featured by focal contractile rings together with general stenosis. Functionally, radiation also led to hypoperfusion, attenuated vascular pulsatility, and decreased dilation to nimodipine and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid. Mechanically, Notch activation and increased expression of α-SMA protein were found in both surgical specimens of RIBI patients and the irradiated mice. Importantly, Notch inhibition by N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester significantly alleviated cerebral hypoperfusion, vasculopathy, and cognitive deficits in the RIBI mouse model. CONCLUSIONS: Radiation-induced cerebral vasculopathy showed bead-like shape and increased contractile state. Inhibition of Notch signaling by N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester effectively attenuated vasculopathy and relieved cognitive impairment, suggesting Notch signaling as a therapeutic target for the treatment of RIBI.

Rate-limiting hydrolysis in ribosomal release reactions revealed by ester activation.

Translation terminates by releasing the polypeptide chain in one of two chemical reactions catalyzed by the ribosome. Release is also a target for engineering, as readthrough of a stop codon enables incorporation of unnatural amino acids and treatment of genetic diseases. Hydrolysis of the ester bond of peptidyl-tRNA requires conformational changes of both a class I release factor (RF) protein and the peptidyl transferase center of a large subunit rRNA. The rate-limiting step was proposed to be hydrolysis at physiological pH and an RF conformational change at higher pH, but evidence was indirect. Here, we tested this by activating the ester electrophile at the Escherichia coli ribosomal P site using a trifluorine-substituted amino acid. Quench-flow kinetics revealed that RF1-catalyzed release could be accelerated, but only at pH 6.2-7.7 and not higher pH. This provided direct evidence for rate-limiting hydrolysis at physiological or lower pH and a different rate limitation at higher pH. Additionally, we optimized RF-free release catalyzed by unacylated tRNA or the CCA trinucleotide (in 30% acetone). We determined that these two model release reactions, although very slow, were surprisingly accelerated by the trifluorine analog but to a different extent from each other and from RF-catalyzed release. Hence, hydrolysis was rate limiting in all three reactions. Furthermore, in 20% ethanol, we found that there was significant competition between fMet-ethyl ester formation and release in all three release reactions. We thus favor proposed mechanisms for translation termination that do not require a fully-negatively-charged OH- nucleophile.

Antiskin Aging Effects of Indole Alkaloid N-Glycoside from Ginkgo Fruit (Ginkgo biloba fruit) on TNF-α-Exposed Human Dermal Fibroblasts.

Human skin aging has internal and external factors, both of which are characterized by TNF-α overproduction. Therefore, we aimed to identify a natural product that suppresses the damage that occurs in cutaneous dermal fibroblasts exposed to TNF-α. The protective effects of the indole alkaloid N-glycoside, ginkgoside B dimethyl ester (GBDE), isolated from ginkgo fruit (Ginkgo biloba fruit) were evaluated in TNF-α stimulated human dermal fibroblasts (HDFs). GBDE inhibited TNF-α-induced MMP-1 expression to 2.2 ± 0.1-fold (p < 0.01) and reversed the decrease in collagen levels to 0.4 ± 0.00-fold (p < 0.01) at 50 µM. The effect of GBDE was due to the suppression of the phospolylaton of MAPKs (ERK, 0.47 ± 0.05; JNK, 1.21 ± 0.07; p38, 0.77 ± 0.07-folds, p < 0.001) and Akt (0.14 ± 0.03-fold, p < 0.001) compared to the TNF-α group. GBDE also reduced the expression of COX-2 to 2.06 ± 0.12-fold (p < 0.001) and increased the expression of HO-1 to 10.64 ± 0.2-fold (p < 0.001). In addition, GBDE inhibited the expression of the pro-inflammatory cytokines (IL-8, 2.2 ± 0.0; IL-1ß, 1.6 ± 0.0; IL-6, 2.0 ± 0.10-folds, p < 0.05). These results provide experimental evidence that GBDE can protect against skin damage, including aging.

TGF-ß3 induces lactate production in Sertoli cell through inhibiting the Notch pathway.

BACKGROUNDS: In the testis, spermatocytes and spermatids rely on lactate produced by Sertoli cells (SCs) as energy source. Transforming growth factor-beta 3 (TGF-ß3) is one of the generally accepted paracrine regulatory factors of SC-created blood-testis barrier (BTB), yet its role in SC glycolysis and lactate production still remains unclear. OBJECTIVES: To investigate the effect of TGF-ß3 on glycolysis and lactate production in SCs and determine the role of lethal giant larvae 2 (Lgl2) and Notch signaling activity during this process. MATERIALS AND METHODS: Primary cultured rat SCs and TM4 cells were treated with different concentrations of TGF-ß3. In some experiments, cells were transfected with siRNA specifically targeting Lgl2 and then treated with TGF-ß3 or N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Lactate concentration, glucose and glutamine (Gln) consumption in the culture medium, activity of phosphofructokinase (PFK), lactate dehydrogenase (LDH), and glutaminase (Gls), ATP level, oxygen consumption, extracellular acidification, and mitochondrial respiration complex activity were detected using commercial kits. The protein level of Lgl2, LDH, monocarboxylate transporter 4 (MCT4), and activity of Akt, ERK, p38 MAPK, and Notch pathway were detected by Western blot. The stage-specific expression of Jagged1 was examined by immunohistochemistry (IHC) and qPCR after laser capture microdissection. Spermatogenesis in rat testis injected with recombinant Jagged1 (re-Jagged1) was observed by HE staining, and lactate concentration in testis lysate was measured at a different day point after re-Jagged1 treatment. RESULTS: Significant enhancement of lactate concentration was detected in a culture medium of both primary SCs and TM4 cells treated with TGF-ß3 at 3 or 5 ng/ml. Besides, other parameters of glycolysis, that is, glucose and Gln consumption, enzyme activity of PFK, LDH, and Gls displayed different levels of increment in primary SCs and TM4 cells after TGF-ß3 treatment. Mitochondria respiration of SCs was shown to decrease in response to TGF-ß3. Lgl2, MCT4, activity of ERK, and p38 MAPK were up-regulated, whereas Akt and Notch pathway activity were inhibited by TGF-ß3. Silencing of Lgl2 in SCs affected lactate production and attenuated the previous effects of TGF-ß3 on SC glycolysis except for Gln consumption, Gls activity, and activity of Akt, ERK, and p38. N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment in SCs antagonized glycolysis suppression caused by Lgl2-silencing. In vivo analysis revealed a stage-specific expression of Jagged1 in contrary with TGF-ß3. Activating Notch signaling by re-Jagged1 resulted in restorable hypospermatogenesis and lowered lactate level in rat testis. CONCLUSION: TGF-ß3 induces lactate production in SC through up-regulating Lgl2, which weakened the Notch signaling activity and intensified glycolysis in SCs. Thus, besides the known function of TGF-ß3 as the BTB regulator, TGF-ß3-Lgl2-Notch may be considered an important pathway controlling SC glycolysis and spermatogenesis.

In Vitro Reconstitution of Fimsbactin Biosynthesis from Acinetobacter baumannii.

Siderophores produced via nonribosomal peptide synthetase (NRPS) pathways serve as critical virulence factors for many pathogenic bacteria. Improved knowledge of siderophore biosynthesis guides the development of inhibitors, vaccines, and other therapeutic strategies. Fimsbactin A is a mixed ligand siderophore derived from human pathogenic Acinetobacter baumannii that contains phenolate-oxazoline, catechol, and hydroxamate metal chelating groups branching from a central l-Ser tetrahedral unit via amide and ester linkages. Fimsbactin A is derived from two molecules of l-Ser, two molecules of 2,3-dihydroxybenzoic acid (DHB), and one molecule of l-Orn and is a product of the fbs biosynthetic operon. Here, we report the complete in vitro reconstitution of fimsbactin A biosynthesis in a cell-free system using purified enzymes. We demonstrate the conversion of l-Orn to N1-acetyl-N1-hydroxy-putrescine (ahPutr) via ordered action of FbsJ (decarboxylase), FbsI (flavin N-monooxygenase), and FbsK (N-acetyltransferase). We achieve conversion of l-Ser, DHB, and l-Orn to fimsbactin A using FbsIJK in combination with the NRPS modules FbsEFGH. We also demonstrate chemoenzymatic conversion of synthetic ahPutr to fimsbactin A using FbsEFGH and establish the substrate selectivity for the NRPS adenylation domains in FbsH (DHB) and FbsF (l-Ser). We assign a role for the type II thioesterase FbsM in producing the shunt metabolite 2-(2,3-dihydroxyphenyl)-4,5-dihydrooxazole-4-carboxylic acid (DHB-oxa) via cleavage of the corresponding thioester intermediate that is tethered to NRPS peptidyl carrier domains during biosynthetic assembly. We propose a mechanism for branching NRPS-derived peptides via amide and ester linkages via the dynamic equilibration of N-DHB-Ser and O-DHB-Ser thioester intermediates via hydrolysis of DHB-oxa thioester intermediates. We also propose a genetic signature for NRPS "branching" in the presence of a terminating C-T-C motif (FbsG).

SIRT1 Protects Against Particulate Matter-Induced Oxidative Stress in Human Corneal and Conjunctival Epithelial Cells.

Purpose: Sirtuin1 (SIRT1) as a hot therapeutic target for oxidative stress-associated diseases that has been extensively studied. This study aimed to determine the changes in SIRT1 expression in particulate matter (PM)-induced corneal and conjunctival epithelial cell damage and explore potential drugs to reduce PM-associated ocular surface injury. Methods: Immortalized human corneal epithelial cells (HCECs) and human conjunctival epithelial cells (HCjECs) were exposed to an ambient PM sample. Cytotoxicity was evaluated by water-soluble tetrazolium salt-8 assay. SIRT1 expression was measured by Western blot analysis. Reactive oxygen species (ROS) production, cell apoptosis, mitochondrial function, and cell senescence were assessed by using 2',7'-dichlorofluorescein diacetate assay, annexin V apoptosis assay, tetramethylrhodamine ethyl ester assay, and senescence ß-galactosidase staining, respectively. Results: PM-induced cytotoxicity of HCECs and HCjECs occurred in a dose-dependent manner. Increased ROS production, as well as decreased SIRT1 expression, were observed in HCECs and HCjECs after 200 µg/mL PM exposure. In addition, PM induced oxidative stress-mediated cellular damage, including cell apoptosis, mitochondrial damage, and cell senescence. Interestingly, SRT1720, a SIRT1 activator, increased SIRT1 expression and decreased ROS production and attenuated PM-induced cell damage in HCECs and HCjECs. Conclusions: This study determined that SIRT1 was involved in PM-induced oxidative stress in HCECs and HCjECs and found that ROS overproduction may a key factor in PM-induced SIRT1 downregulation. The SIRT1 activator, SRT1720, can effectively upregulate SIRT1 expression and inhibit ROS production, thereby reversing PM-induced cell damage. This study provides a new potential target for clinical treatment of PM-associated ocular surface diseases.

Extracellular Metabolites of Heterotrophic Auxenochlorella protothecoides: A New Source of Bio-Stimulants for Higher Plants.

The biodiversity of microalgal species is enormous, and their versatile metabolism produces a wide diversity of compounds that can be used in food, healthcare, and other applications. Microalgae are also a potential source of bio-stimulants that enhance nutrition efficiency, abiotic stress tolerance, and/or crop quality traits. In this study, the extracellular metabolites of Auxenochlorella protothecoides (EAp) were prepared using three different culture strategies, and their effects on plant growth were examined. Furthermore, the composition of EAp was analyzed by GC-MS. The elongation of lateral roots and the cold-tolerance of Arabidopsis thaliana and Nicotiana benthamiana were promoted by EAp. Moreover, EAp from high-cell-density fermentation stimulated the growth of the leafy vegetables Brassica rapa and Lactuca sativa at dilutions as high as 500- and 1000-fold. Three major groups of compounds were identified by GC-MS, including organic acids or organic acid esters, phenols, and saccharides. Some of these compounds have known plant-stimulating effects, while the rest requires further investigation in the future. Our study demonstrates that EAp is a potential bio-stimulant, while also providing an environmentally friendly and economical microalgae fermentation process.

Fluroxypyr-1-methylheptyl ester induced ROS production and mitochondrial apoptosis through the MAPK signaling cascade in porcine trophectoderm and uterine luminal epithelial cells.

FPMH (Fluroxypyr-1-methylheptyl ester) is a synthetic auxin herbicide used in agriculture. The mechanism by which FPMH induces adverse effects in porcine trophectoderm (pTr) and porcine uterine luminal epithelial (pLE) cells, which are involved in porcine implantation, have not been studied yet. Therefore, the present study investigates the toxicological effects of FPMH on pTr and pLE cells. We confirmed that FPMH induced cytotoxic effects on the cells, including apoptosis induction, mitochondrial membrane potential (MMP) depolarization, and ROS production. The phosphorylation of the MAPK pathway (ERK1/2, JNK, and p38) was dysregulated by FPMH administration. In addition, FPMH could suppress cell-cell adhesion and migration abilities of pTr and pLE, which are crucial for implantation. Therefore, exposure to FPMH induced adverse effects in pTr and pLE cells and could result in implantation failure.

Induction of Systemic Resistance to Tobacco mosaic virus in Tomato through Foliar Application of Bacillus amyloliquefaciens Strain TBorg1 Culture Filtrate.

The application of microbe-derived products as natural biocontrol agents to boost systemic disease resistance to virus infections in plants is a prospective strategy to make agriculture more sustainable and environmentally friendly. In the current study, the rhizobacterium Bacillus amyloliquefaciens strain TBorg1 was identified based on 16S rRNA, rpoB, and gyrA gene sequences, and evaluated for its efficiency in conferring protection of tomato from infection by Tobacco mosaic virus (TMV). Under greenhouse circumstances, foliar sprays of TBorg1 culture filtrate (TBorg1-CF) promoted tomato growth, lowered disease severity, and significantly decreased TMV accumulation in systemically infected leaves of treated plants relative to untreated controls. TMV accumulation was reduced by 90% following the dual treatment, applied 24 h before and after TMV infection. Significant increases in levels of total soluble carbohydrates, proteins, and ascorbic acid were also found. In addition, a significant rise in activities of enzymes capable of scavenging reactive oxygen species (PPO and POX), as well as decreased levels of non-enzymatic oxidative stress markers (H2O2 and MDA) were observed, compared to untreated plants. Enhanced systemic resistance to TMV was indicated by significantly increased transcript accumulation of polyphenolic pathway (C4H, HCT, and CHI) and pathogenesis-related (PR-1 and PR-5) genes. Out of the 15 compounds identified in the GC-MS analysis, 1,2-benzenedicarboxylic acid mono(2-ethylhexyl) ester and phenol, 2,4-bis(1,1-dimethylethyl), as well as L-proline, N-valeryl-, and heptadecyl ester were present in the highest concentrations in the ethyl acetate extract of TBorg1-CF. In addition, significant amounts of n-hexadecanoic acid, pyrrolo [1,2-a] pyrazine-1,4-dione hexahydro-3-(2-methylpropyl)-, nonane, 5-butyl-, and eicosane were also detected. These compounds may act as inducers of systemic resistance to viral infection. Our findings indicate that the newly isolated B. amyloliquefaciens strain TBorg1 could be a potentially useful rhizobacterium for promoting plant growth and a possible source of biocontrol agents for combating plant virus infections.

The human fecal microbiome contributes to the biotransformation of the PFAS surfactant 8:2 monosubstituted polyfluoroalkyl phosphate ester.

Polyfluoroalkyl phosphate esters (PAPs) can be found throughout society due to their numerous commercial applications. However, they also pose an environmental and health concern given their ability to undergo hydrolysis and oxidation to several bioactive and persistent products, including the perfluorocarboxylic acids (PFCAs). The metabolism of PAPs has been shown to occur in mammalian liver and intestine, however metabolism by the gut microbiome has not yet been investigated. In this study, human fecal samples were used to model the microbial population of the colon, to test whether these anaerobic microbes could facilitate 8:2 monosubstituted PAP (monoPAP) transformation. In vitro testing was completed by incubating the fecal samples with 8:2 monoPAP (400-10,000 nM) up to 120 minutes in an anaerobic chamber. Reactions were then terminated and the samples prepared for GC- and LC-MS/MS analysis. Metabolites of interest were the immediate hydrolysis product, the 8:2 fluorotelomer alcohol (FTOH), and 11 additional metabolites previously shown to form from 8:2 FTOH in both oxic and anoxic environments. The kinetics of 8:2 monoPAP transformation by gut microbiota were compared to those in human S9 liver and intestine fractions, both of which have active levels of hydrolyzing and oxidative enzymes that transform 8:2 monoPAP. Transformation rates from 8:2 monoPAP to 8:2 FTOH were highest in liver S9 > intestine S9 > fecal suspensions. The gut microbiome also produced a unique composition of oxidative metabolites, where the following intermediate metabolites were more abundant than terminal PFCAs: 8:2 fluorotelomer unsaturated carboxylic acid (FTUCA) > 8:2 fluorotelomer carboxylic acid (FTCA) > 7:2 Ketone ≈ perfluorohexanoic acid (PFHxA). Hydrolytic and oxidative metabolites contributed up to 30% of the molar balance after microbial 8:2 monoPAP transformation. Together, the results suggest that the gut microbiome can play a notable role in PAP biotransformation.

Impaired ketogenesis ties metabolism to T cell dysfunction in COVID-19.

Anorexia and fasting are host adaptations to acute infection, and induce a metabolic switch towards ketogenesis and the production of ketone bodies, including ß-hydroxybutyrate (BHB)1-6. However, whether ketogenesis metabolically influences the immune response in pulmonary infections remains unclear. Here we show that the production of BHB is impaired in individuals with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) but not in those with  influenza-induced ARDS. We found that BHB promotes both the survival of and the production of interferon-γ by CD4+ T cells. Applying a metabolic-tracing analysis, we established that BHB provides an alternative carbon source to fuel oxidative phosphorylation (OXPHOS) and the production of bioenergetic amino acids and glutathione, which is important for maintaining the redox balance. T cells from patients with SARS-CoV-2-induced ARDS were exhausted and skewed towards glycolysis, but could be metabolically reprogrammed by BHB to perform OXPHOS, thereby increasing their functionality. Finally, we show in mice that a ketogenic diet and the delivery of BHB as a ketone ester drink restores CD4+ T cell metabolism and function in severe respiratory infections, ultimately reducing the mortality of mice infected with SARS-CoV-2. Altogether, our data reveal that BHB is an alternative source of carbon that promotes T cell responses in pulmonary viral infections, and highlight impaired ketogenesis as a potential confounding factor in severe COVID-19.

Comparing the mitochondrial signatures in ESCs and iPSCs and their neural derivations.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have distinct origins: ESCs are derived from pre-implanted embryos while iPSCs are reprogrammed somatic cells. Both have their own characteristics and lineage specificity, and both are valuable tools for studying human neurological development and disease. Thus far, few studies have analyzed how differences between stem cell types influence mitochondrial function and mitochondrial DNA (mtDNA) homeostasis during differentiation into neural and glial lineages. In this study, we compared mitochondrial function and mtDNA replication in human ESCs and iPSCs at three different stages - pluripotent, neural progenitor and astrocyte. We found that while ESCs and iPSCs have a similar mitochondrial signature, neural and astrocyte derivations manifested differences. At the neural stem cell (NSC) stage, iPSC-NSCs displayed decreased ATP production and a reduction in mitochondrial respiratory chain (MRC) complex IV expression compared to ESC-NSCs. IPSC-astrocytes showed increased mitochondrial activity including elevated ATP production, MRC complex IV expression, mtDNA copy number and mitochondrial biogenesis relative to those derived from ESCs. These findings show that while ESCs and iPSCs are similar at the pluripotent stage, differences in mitochondrial function may develop during differentiation and must be taken into account when extrapolating results from different cell types.Abbreviation: BSA: Bovine serum albumin; DCFDA: 2',7'-dichlorodihydrofluorescein diacetate; DCX: Doublecortin; EAAT-1: Excitatory amino acid transporter 1; ESCs: Embryonic stem cells; GFAP: Glial fibrillary acidic protein; GS: Glutamine synthetase; iPSCs: Induced pluripotent stem cells; LC3B: Microtubule-associated protein 1 light chain 3ß; LC-MS: Liquid chromatography-mass spectrometry; mito-ROS: Mitochondrial ROS; MMP: Mitochondrial membrane potential; MRC: Mitochondrial respiratory chain; mtDNA: Mitochondrial DNA; MTDR: MitoTracker Deep Red; MTG: MitoTracker Green; NSCs: Neural stem cells; PDL: Poly-D-lysine; PFA: Paraformaldehyde; PGC-1α: PPAR-γ coactivator-1 alpha; PPAR-γ: Peroxisome proliferator-activated receptor-gamma; p-SIRT1: Phosphorylated sirtuin 1; p-ULK1: Phosphorylated unc-51 like autophagy activating kinase 1; qPCR: Quantitative PCR; RT: Room temperature; RT-qPCR: Quantitative reverse transcription PCR; SEM: Standard error of the mean; TFAM: Mitochondrial transcription factor A; TMRE: Tetramethylrhodamine ethyl ester; TOMM20: Translocase of outer mitochondrial membrane 20.

Metabolism of the active carfentanil metabolite, 4-Piperidinecarboxylic acid, 1-(2-hydroxy-2-phenylethyl)-4-[(1-oxopropyl)phenylamino]-, methyl ester in vitro.

Carfentanil, a µ-opioid receptor (MOR) agonist with an analgesic potency 10,000 times that of morphine, is extensively metabolized to norcarfentanil (M1), 4-Piperidinecarboxylic acid, 1-(2-hydroxy-2-phenylethyl)-4-[(1-oxopropyl)phenylamino]-, methyl ester (M0 in this article), and other low abundant metabolites in human hepatocytes and liver/lung microsomes. M0 possessed comparable MOR activity to carfentanil, and accounted for approximately 12 % of the total carfentanil metabolite formation in human liver microsomes (HLMs). Little is known about the subsequent elimination of M0. This study investigated its metabolic pathway in HLMs, separation and preliminary identification of metabolites by liquid chromatography-tandem mass spectrometry, and possible involvement of cytochrome P450 enzymes in M0 metabolism with kinetic analysis. M0 produced 9 metabolites via N-dealkylation (M1), oxidation (M3, M6-9), N-dealkylation followed by oxidation (M2 and M4), and glucuronidation (M5). Formation of the major metabolite M1 fitted typical Michaelis-Menten kinetics. Recombinant human CYP3A5 showed the highest activity toward M1 formation followed by CYP3A4 and CYP2C8, while M8 was primarily formed by CYP3A4 followed by CYP2C19 and CYP2C8. These findings reveal the main involvement of CYP3A5 and 3A4 in human hepatic elimination of M0 with a kinetic profile similar to carfentanil which may inform development of treatment protocols for carfentanil exposure.