Angiopoietin-2-induced lymphatic endothelial cell migration drives lymphangiogenesis via the ß1 integrin-RhoA-formin axis.

Lymphangiogenesis is an essential physiological process but also a determining factor in vascular-related pathological conditions. Angiopoietin-2 (Ang2) plays an important role in lymphatic vascular development and function and its upregulation has been reported in several vascular-related diseases, including cancer. Given the established role of the small GTPase RhoA on cytoskeleton-dependent endothelial functions, we investigated the relationship between RhoA and Ang2-induced cellular activities. This study shows that Ang2-driven human dermal lymphatic endothelial cell migration depends on RhoA. We demonstrate that Ang2-induced migration is independent of the Tie receptors, but dependent on ß1 integrin-mediated RhoA activation with knockdown, pharmacological approaches, and protein sequencing experiments. Although the key proteins downstream of RhoA, Rho kinase (ROCK) and myosin light chain, were activated, blockade of ROCK did not abrogate the Ang2-driven migratory effect. However, formins, an alternative target of RhoA, were identified as key players, and especially FHOD1. The Ang2-RhoA relationship was explored in vivo, where lymphatic endothelial RhoA deficiency blocked Ang2-induced lymphangiogenesis, highlighting RhoA as an important target for anti-lymphangiogenic treatments.

Identification of a novel secreted metabolite cyclo(phenylalanyl-prolyl) from Batrachochytrium dendrobatidis and its effect on Galleria mellonella.

BACKGROUND: The fungus, Batrachochytrium dendrobatidis, is the causative agent of chytridiomycosis and a leading cause of global decline in amphibian populations. The first stages of chytridiomycosis include: inflammation, hyperkeratosis, lethargy, loss of righting reflex, and disruption of internal electrolyte levels leading to eventual death of the host. Previous work indicates that B. dendrobatidis can produce immunomodulatory compounds and other secreted molecules that regulate the growth of the fungus. In this study, filtrates of the fungus grown in media and water were subjected to ultra-performance liquid chromatography-mass spectrometry and analyzed using Compound Discoverer 3.0. RESULTS: Identification of cyclo(phenylalanyl-prolyl), chitobiose, and S-adenosylmethionine were verified by their retention times and fragmentation patterns from B. dendrobatidis supernatants. Previous studies have analyzed the effects of B. dendrobatidis on amphibian models, in vitro, or in cell culture. We studied the effects of live B. dendrobatidis cells, spent culture filtrates containing secreted metabolites, and cyclo(pheylalanyl-prolyl) on wax moth larvae (Galleria mellonella). Concentrated filtrates caused melanization within 24 h, while live B. dendrobatidis caused melanization within 48 h. CONCLUSIONS: Here we show B. dendrobatidis produces secreted metabolites previously unreported. The impacts of these chemicals were tested on an alternate non-amphibian model system that has been used for other fungi to study pathogenicity traits in this fungus.

Recent Advances of Functional Proteomics in Gastrointestinal Cancers- a Path towards the Identification of Candidate Diagnostic, Prognostic, and Therapeutic Molecular Biomarkers.

Gastrointestinal (GI) cancer remains one of the common causes of morbidity and mortality. A high number of cases are diagnosed at an advanced stage, leading to a poor survival rate. This is primarily attributed to the lack of reliable diagnostic biomarkers and limited treatment options. Therefore, more sensitive, specific biomarkers and curative treatments are desirable. Functional proteomics as a research area in the proteomic field aims to elucidate the biological function of unknown proteins and unravel the cellular mechanisms at the molecular level. Phosphoproteomic and glycoproteomic studies have emerged as two efficient functional proteomics approaches used to identify diagnostic biomarkers, therapeutic targets, the molecular basis of disease and mechanisms underlying drug resistance in GI cancers. In this review, we present an overview on how functional proteomics may contribute to the understanding of GI cancers, namely colorectal, gastric, hepatocellular carcinoma and pancreatic cancers. Moreover, we have summarized recent methodological developments in phosphoproteomics and glycoproteomics for GI cancer studies.

Recent advances in mass spectrometric analysis of glycoproteins.

Glycosylation is one of the most common posttranslational modifications of proteins that plays essential roles in various biological processes, including protein folding, host-pathogen interaction, immune response, and inflammation and aberrant protein glycosylation is a well-known event in various disease states including cancer. As a result, it is critical to develop rapid and sensitive methods for the analysis of abnormal glycoproteins associated with diseases. Mass spectrometry (MS) in conjunction with different separation methods, such as capillary electrophoresis (CE), ion mobility (IM), and high performance liquid chromatography (HPLC) has become a popular tool for glycoprotein analysis, providing highly informative fragments for structural identification of glycoproteins. This review provides an overview of the developments and accomplishments in the field of glycomics and glycoproteomics reported between 2014 and 2016.

Nanospray liquid chromatography/tandem mass spectrometry analysis of steroids from gray whale blubber.

RATIONALE: Analysis of steroids from precious blubber biopsies obtained from marine mammals, especially endangered species, can provide valuable information on their endocrine status. Challenges with currently used ELISA methodology include lack of absolute quantitation and incompatibility with multiple steroids analysis due to limited biopsy mass. Development of a sensitive, accurate analytical method for this purpose is critical. METHODS: A nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS) method was validated for sensitive, specific and quantitative analysis of three steroid hormones, without derivatization, extracted from 50 mg blubber samples. Data was acquired with an LTQ XL ion trap mass spectrometer in positive ion mode, using single reaction monitoring. All three steroids were analyzed in a single run. Cholic acid was used as a surrogate internal standard for quantitation due to its steroidal structure and lack of measurable endogenous levels in blubber. RESULTS: The lowest limits of quantitation for progesterone, testosterone, and hydrocortisone were significantly improved compared to previous studies using conventional LC/MS/MS. The lowest limit of detection was 7 fg/µL using a 1 µL injection volume. Calibration curves for steroid quantification showed good linearity (r2 >0.99) between 14 and 3620 fg/µL, and accuracy was <20% for interday and <10% for intraday. After validation, the method was successfully applied to quantification of steroids in gray whale blubber samples. CONCLUSIONS: The nanoLC/MS/MS method is more sensitive than traditional LC/MS/MS for steroid analysis. It is also compatible with other important biopsy analyses due to its small blubber mass requirement. This will benefit the reproductive and stress assessments for all marine mammals, particularly endangered populations. Copyright © 2017 John Wiley & Sons, Ltd.

Synthesis of d-labeled and unlabeled ethyl succinic anhydrides and application to quantitative analysis of peptides by isotope differential mass spectrometry.

Ethyl succinic anhydride and its d5-labeled version have been synthesized and applied to quantitative analysis of peptides in combination with MALDI or ESI mass spectrometry. These modifiers react with amino groups in the N-termini and lysine side chains in proteins, and therefore the combination of these modifiers was shown to be a useful tool for quantification of peptides and hence for proteomics research.

A loop unique to ferredoxin-dependent glutamate synthases is not absolutely essential for ferredoxin-dependent catalytic activity.

It had been proposed that a loop, typically containing 26 or 27 amino acids, which is only present in monomeric, ferredoxin-dependent, "plant-type" glutamate synthases and is absent from the catalytic α-subunits of both NADPH-dependent, heterodimeric glutamate synthases found in non-photosynthetic bacteria and NADH-dependent heterodimeric cyanobacterial glutamate synthases, plays a key role in productive binding of ferredoxin to the plant-type enzymes. Site-directed mutagenesis has been used to delete the entire 27 amino acid-long loop in the ferredoxin-dependent glutamate synthase from the cyanobacterium Synechocystis sp. PCC 6803. The specific activity of the resulting loopless variant of this glutamate synthase, when reduced ferredoxin serves as the electron donor, is actually higher than that of the wild-type enzyme, suggesting that this loop is not absolutely essential for efficient electron transfer from reduced ferredoxin to the enzyme. These results are consistent with the results of an in-silico study that suggests that the loop is unlikely to interact directly with ferredoxin in the energetically most favorable model of a 1:1 complex of ferredoxin with the wild-type enzyme.

Isolation and proteomic characterization of the mouse sperm acrosomal matrix.

A critical step during fertilization is the sperm acrosome reaction in which the acrosome releases its contents allowing the spermatozoa to penetrate the egg investments. The sperm acrosomal contents are composed of both soluble material and an insoluble material called the acrosomal matrix (AM). The AM is thought to provide a stable structure from which associated proteins are differentially released during fertilization. Because of its important role during fertilization, efforts have been put toward isolating the AM for biochemical study and to date AM have been isolated from hamster, guinea pig, and bull spermatozoa. However, attempts to isolate AM from mouse spermatozoa, the species in which fertilization is well-studied, have been unsuccessful possibly because of the small size of the mouse sperm acrosome and/or its fusiform shape. Herein we describe a procedure for the isolation of the AM from caput and cauda mouse epididymal spermatozoa. We further carried out a proteomic analysis of the isolated AM from both sperm populations and identified 501 new proteins previously not detected by proteomics in mouse spermatozoa. A comparison of the AM proteome from caput and cauda spermatozoa showed that the AM undergoes maturational changes during epididymal transit similar to other sperm domains. Together, our studies suggest the AM to be a dynamic and functional structure carrying out a variety of biological processes as implied by the presence of a diverse group of proteins including proteases, chaperones, hydrolases, transporters, enzyme modulators, transferases, cytoskeletal proteins, and others.

Roles of four conserved basic amino acids in a ferredoxin-dependent cyanobacterial nitrate reductase.

The roles of four conserved basic amino acids in the reaction catalyzed by the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 have been investigated using site-directed mutagenesis in combination with measurements of steady-state kinetics, substrate-binding affinities, and spectroscopic properties of the enzyme's two prosthetic groups. Replacement of either Lys58 or Arg70 by glutamine leads to a complete loss of activity, both with the physiological electron donor, reduced ferredoxin, and with a nonphysiological electron donor, reduced methyl viologen. More conservative, charge-maintaining K58R and R70K variants were also completely inactive. Replacement of Lys130 by glutamine produced a variant that retained 26% of the wild-type activity with methyl viologen as the electron donor and 22% of the wild-type activity with ferredoxin as the electron donor, while replacement by arginine produces a variant that retains a significantly higher percentage of the wild-type activity with both electron donors. In contrast, replacement of Arg146 by glutamine had minimal effect on the activity of the enzyme. These results, along with substrate-binding and spectroscopic measurements, are discussed in terms of an in silico structural model for the enzyme.

Shotgun label-free quantitative proteomics of water-deficit-stressed midmature peanut (Arachis hypogaea L.) seed.

Legume seeds and peanuts, in particular, are an inexpensive source of plant proteins and edible oil. A comprehensive understanding of seed metabolism and the effects of water-deficit stress on the incorporation of the main storage reserves in seeds, such as proteins, fatty acids, starch, and secondary metabolites, will enhance our ability to improve seed quality and yield through molecular breeding programs. In the present study, we employed a label-free quantitative proteomics approach to study the functional proteins altered in the midmature (65-70 days postanthesis) peanut seed grown under water-deficit stress conditions. We created a pod-specific proteome database and identified 93 nonredundant, statistically significant, and differentially expressed proteins between well-watered and drought-stressed seeds. Mapping of these differential proteins revealed three candidate biological pathways (glycolysis, sucrose and starch metabolism, and fatty acid metabolism) that were significantly altered due to water-deficit stress. Differential accumulation of proteins from these pathways provides insight into the molecular mechanisms underlying the observed physiological changes, which include reductions in pod yield and biomass, reduced germination, reduced vigor, decreased seed membrane integrity, increase in storage proteins, and decreased total fatty acid content. Some of the proteins encoding rate limiting enzymes of biosynthetic pathways could be utilized by breeders to improve peanut seed production during water-deficit conditions in the field. The data have been deposited to the ProteomeXchange with identifier PXD000308.

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803.

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E(m)) value of -165mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E(m) value of -220mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.

Anti-Inflammatory Mechanisms of Curcumin and Its Metabolites in White Adipose Tissue and Cultured Adipocytes.

The plant-derived polyphenol curcumin alleviates the inflammatory and metabolic effects of obesity, in part, by reducing adipose tissue inflammation. We hypothesized that the benefits of curcumin supplementation on diet-induced obesity and systemic inflammation in mice occur through downregulation of white adipose tissue (WAT) inflammation. The hypothesis was tested in adipose tissue from high-fat diet-induced obese mice supplemented with or without curcumin and in 3T3-L1 adipocytes treated with or without curcumin. Male B6 mice were fed a high-fat diet (HFD, 45% kcal fat) with or without 0.4% (w/w) curcumin supplementation (HFC). Metabolic changes in these mice have been previously reported. Here, we determined the serum levels of the curcumin metabolites tetrahydrocurcumin (THC) and curcumin-O-glucuronide (COG) using mass spectrometry. Moreover, we determined interleukin 6 (IL-6) levels and proteomic changes in LPS-stimulated 3T3-L1 adipocytes treated with or without curcumin by using immunoassays and mass spectrometry, respectively, to gain further insight into any altered processes. We detected both curcumin metabolites, THC and COG, in serum samples from the curcumin-fed mice. Both curcumin and its metabolites reduced LPS-induced adipocyte IL-6 secretion and mRNA levels. Proteomic analyses indicated that curcumin upregulated EIF2 and mTOR signaling pathways. Overall, curcumin exerted anti-inflammatory effects in adipocytes, in part by reducing IL-6, and these effects may be linked to the upregulation of the mTOR signaling pathway, warranting additional mechanistic studies on the effects of curcumin and its metabolites on metabolic health.

Anti-inflammatory mechanisms of polyphenols in adipose tissue: role of gut microbiota, intestinal barrier integrity and zinc homeostasis.

Obesity is associated with an imbalance of micro-and macro-nutrients, gut dysbiosis, and a "leaky" gut phenomenon. Polyphenols, such as curcumin, resveratrol, and anthocyanins may alleviate the systemic effects of obesity, potentially by improving gut microbiota, intestinal barrier integrity (IBI), and zinc homeostasis. The essential micronutrient zinc plays a crucial role in the regulation of enzymatic processes, including inflammation, maintenance of the microbial ecology, and intestinal barrier integrity. In this review, we focus on IBI- which prevents intestinal lipopolysaccharide (LPS) leakage - as a critical player in polyphenol-mediated protective effects against obesity-associated white adipose tissue (WAT) inflammation. This occurs through mechanisms that block the movement of the bacterial endotoxin LPS across the gut barrier. Available research suggests that polyphenols reduce WAT and systemic inflammation via crosstalk with inflammatory NF-κB, the mammalian target of rapamycin (mTOR) signaling and zinc homeostasis.

Dietary supplementation of gingerols- and shogaols-enriched ginger root extract attenuate pain-associated behaviors while modulating gut microbiota and metabolites in rats with spinal nerve ligation.

Neuroinflammation is a central factor in neuropathic pain (NP). Ginger is a promising bioactive compound in NP management due to its anti-inflammatory property. Emerging evidence suggests that gut microbiome and gut-derived metabolites play a key role in NP. We evaluated the effects of two ginger root extracts rich in gingerols (GEG) and shogaols (SEG) on pain sensitivity, anxiety-like behaviors, circulating cell-free mitochondrial DNA (ccf-mtDNA), gut microbiome composition, and fecal metabolites in rats with NP. Sixteen male rats were divided into four groups: sham, spinal nerve ligation (SNL), SNL+0.75%GEG in diet, and SNL+0.75%SEG in diet groups for 30 days. Compared to SNL group, both SNL+GEG and SNL+SEG groups showed a significant reduction in pain- and anxiety-like behaviors, and ccf-mtDNA level. Relative to the SNL group, both SNL+GEG and SNL+SEG groups increased the relative abundance of Lactococcus, Sellimonas, Blautia, Erysipelatoclostridiaceae, and Anaerovoracaceae, but decreased that of Prevotellaceae UCG-001, Rikenellaceae RC9 gut group, Mucispirillum and Desulfovibrio, Desulfovibrio, Anaerofilum, Eubacterium siraeum group, RF39, UCG-005, Lachnospiraceae NK4A136 group, Acetatifactor, Eubacterium ruminantium group, Clostridia UCG-014, and an uncultured Anaerovoracaceae. GEG and SEG had differential effects on gut-derived metabolites. Compared to SNL group, SNL+GEG group had higher level of 1'-acetoxychavicol acetate, (4E)-1,7-Bis(4-hydroxyphenyl)-4-hepten-3-one, NP-000629, 7,8-Dimethoxy-3-(2-methyl-3-buten-2-yl)-2H-chromen-2-one, 3-{[4-(2-Pyrimidinyl)piperazino]carbonyl}-2-pyrazinecarboxylic acid, 920863, and (1R,3R,7R,13S)-13-Methyl-6-methylene-4,14,16-trioxatetracyclo[11.2.1.0∼1,10∼.0∼3,7∼]hexadec-9-en-5-one, while SNL+SEG group had higher level for (±)-5-[(tert-Butylamino)-2'-hydroxypropoxy]-1_2_3_4-tetrahydro-1-naphthol and dehydroepiandrosteronesulfate. In conclusion, ginger is a promising functional food in the management of NP, and further investigations are necessary to assess the role of ginger on gut-brain axis in pain management.

Branched-Chain Amino Acids Are Linked with Alzheimer's Disease-Related Pathology and Cognitive Deficits.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder with a complex pathophysiology. Type 2 diabetes (T2D) is a strong risk factor for AD that shares similar abnormal features including metabolic dysregulation and brain pathology such as amyloid and/or Tau deposits. Emerging evidence suggests that circulating branched-chain amino acids (BCAAs) are associated with T2D. While excess BCAAs are shown to be harmful to neurons, its connection to AD is poorly understood. Here we show that individuals with AD have elevated circulating BCAAs and their metabolites compared to healthy individuals, and that a BCAA metabolite is correlated with the severity of dementia. APPSwe mouse model of AD also displayed higher plasma BCAAs compared to controls. In pursuit of understanding a potential causality, BCAA supplementation to HT-22 neurons was found to reduce genes critical for neuronal health while increasing phosphorylated Tau. Moreover, restricting BCAAs from diet delayed cognitive decline and lowered AD-related pathology in the cortex and hippocampus in APP/PS1 mice. BCAA restriction for two months was sufficient to correct glycemic control and increased/restored dopamine that were severely reduced in APP/PS1 controls. Treating 5xFAD mice that show early brain pathology with a BCAA-lowering compound recapitulated the beneficial effects of BCAA restriction on brain pathology and neurotransmitters including norepinephrine and serotonin. Collectively, this study reveals a positive association between circulating BCAAs and AD. Our findings suggest that BCAAs impair neuronal functions whereas BCAA-lowering alleviates AD-related pathology and cognitive decline, thus establishing a potential causal link between BCAAs and AD progression.

Redox properties of a thioredoxin-like Arabidopsis protein, AtTDX.

AtTDX is an enzyme present in Arabidopsis thaliana which is composed of two domains, a thioredoxin (Trx)-motif containing domain and a tetratricopeptide (TPR)-repeat domain. This enzyme has been shown to function as both a thioredoxin and a chaperone. The midpoint potential (E(m)) of AtTDX was determined by redox titrations using the thiol-specific modifiers, monobromobimane (mBBr) and mal-PEG. A NADPH/Trx reductase (NTR) system was used both to validate these E(m) determination methods and to demonstrate that AtTDX is an electron-accepting substrate for NTR. Titrations of full-length AtTDX revealed the presence of a single two-electron couple with an E(m) value of approximately -260 mV at pH 7.0. The two cysteines present in a typical, conserved Trx active site (WCGPC), which are likely to play a role in the electron transfer processes catalyzed by AtTDX, have been replaced by serines by site-directed mutagenesis. These replacements (i.e., C304S, C307S, and C304S/C307S) resulted in a complete loss of the redox process detected using either the mBBr or mal-PEG method to monitor disulfide/dithiol redox couples. This result supports the conclusion that the couple with an E(m) value of -260 mV is a disulfide/dithiol couple involving Cys304 and Cys307. Redox titrations for the separately-expressed Trx-motif containing C-domain also revealed the presence of a single two-electron couple with an E(m) value of approximately -260 mV at 20°C. The fact that these two E(m) values are identical, provides additional support for assignment of the redox couple to a disulfide/dithiol involving C304 and C307. It was found that, while the disulfide/dithiol redox chemistry of AtTDX was not affected by increasing the temperature to 40°C, no redox transitions were observed at 50°C and higher temperatures. In contrast, Escherichia coli thioredoxin was shown to remain redox-active at temperatures as high as 60°C. The temperature-dependence of the AtTDX redox titration is similar to that observed for the redox activity of the protein in enzymatic assays.

Synthesis of d-labeled and unlabeled benzoyloxysuccinimides and application to quantitative analysis of peptides and a protein by isotope differential mass spectrometry.

Benzoyloxysuccinimide and its d(5)-labeled version, which react with amino groups in the N-termini and lysine side chains in proteins, were synthesized and applied to quantitative analysis of peptides and a commercially available protein in combination with a MALDI mass spectrometer.

Analysis of the Neuroproteome Associated With Cell Therapy After Intranigral Grafting in a Mouse Model of Parkinson Disease.

Advances in large-scale proteomics analysis have been very useful in understanding pathogenesis of diseases and elaborating therapeutic strategies. Proteomics has been employed to study Parkinson disease (PD); however, sparse studies reported proteome investigation after cell therapy approaches. In this study, we used liquid chromatography-tandem mass spectrometry and systems biology to identify differentially expressed proteins in a translational mouse model of PD after cell therapy. Proteins were extracted from five nigrostriatal-related brain regions of mice previously lesioned with 6-hydroxydopamine in the substantia nigra. Protein expression was compared in non-grafted brain to 1 and 7 days after intranigral grafting of E12.5 embryonic ventral mesencephalon (VM). We found a total of 277 deregulated proteins after transplantation, which are enriched for lipid metabolism, oxidative phosphorylation and PD, thus confirming that our animal model is similar to human PD and that the presence of grafted cells modulates the expression of these proteins. Notably, seven proteins (Acta1, Atp6v1e1, Eci3, Lypla2, Pip4k2a, Sccpdh, and Sh3gl2) were commonly down-regulated after engraftment in all studied brain regions. These proteins are known to be involved in the formation of lipids and recycling of dopamine (DA) vesicle at the synapse. Moreover, intranigral transplantation of VM cells decreased the expression of proteins related to oxidative stress, especially in the nigrostriatal pathway containing the DA grafted neurons. In the same regions, an up-regulation of several proteins including α-synuclein and tyrosine hydroxylase was observed, whereas expression of tetraspanin 7 was shut down. Overall, these results suggest that intranigral transplantation of VM tissue in an animal model of PD may induce a decrease of oxidative stress in the nigrostriatal pathway and a restoration of the machinery of neurotransmitters, particularly DA release to promote DA transmission through a decrease of D2 DA receptors endocytosis. Identification of new mechanistic elements involved in the nigrostriatal reconstruction process, using translational animal models and systems biology, is a promising approach to enhance the repair of this pathway in PD patients undergoing cell therapy.

Drug-Induced Lipid Remodeling in Leishmania Parasites.

Leishmania parasites efficiently develop resistance against several types of drugs including antimonials, the primary antileishmanial drug historically implemented. The resistance to antimonials is considered to be a major risk factor for effective leishmaniasis treatment. To detect biomarkers/biopatterns for the differentiation of antimony-resistant Leishmania strains, we employed untargeted global mass spectrometry to identify intracellular lipids present in antimony sensitive and resistant parasites before and after antimony exposure. The lipidomic profiles effectively differentiated the sensitive and resistant phenotypes growing with and without antimony pressure. Resistant phenotypes were characterized by significant downregulation of phosphatidylcholines, sphingolipid decrease, and lysophosphatidylcholine increase, while sensitive phenotypes were characterized by the upregulation of triglycerides with long-chain fatty acids and a tendency toward the phosphatidylethanolamine decrease. Our findings suggest that the changes in lipid composition in antimony-resistant parasites contribute to the physiological response conducted to combat the oxidative stress unbalance caused by the drug. We have identified several lipids as potential biomarkers associated with the drug resistance.

Curcumin Reduces Adipose Tissue Inflammation and Alters Gut Microbiota in Diet-Induced Obese Male Mice.

SCOPE: Obesity prevalence continues to increase and contribute to metabolic diseases, potentially by driving systemic inflammation. Curcumin is an anti-inflammatory spice with claimed health benefits. However, mechanisms by which curcumin may reduce obesity-associated inflammation are poorly understood; thus, it is hypothesized that benefits of curcumin consumption may occur through reduced white adipose tissue (WAT) inflammation and/or beneficial changes in gut bacteria. METHODS AND RESULTS: Male B6 mice are fed high-fat diets (HFD, 45% kcal fat) or HFD supplemented with 0.4% (w/w) curcumin (HFC) for 14 weeks. Curcumin supplementation significantly reduces adiposity and total macrophage infiltration in WAT, compared to HFD group, consistent with reduced mRNA levels of M1 (Cd80, Cd38, Cd11c) and M2 (Arginase-1) macrophage markers. Moreover, curcumin supplementation reduces expression of other key pro-inflammatory genes, such as NF-κB p65 subunit (p65), Stat1, Tlr4, and Il6, in WAT (p < 0.05). Using microbial 16S RNA sequencing, it is demonstrated that the relative abundance of the Lactococcus, Parasutterella, and Turicibacter genera are increased in the HFC group versus HFD. CONCLUSIONS: Curcumin exerts protective metabolic effects in dietary obesity, in part through downregulation of adipose tissue inflammation, which may be mediated by alterations in composition of gut microbiota, and metabolism of curcumin into curcumin-O-glucuronide.