Identification of novel biomarkers in the early diagnosis of malignant melanoma by untargeted liquid chromatography coupled to high-resolution mass spectrometry-based metabolomics: a pilot study.

BACKGROUND: Malignant melanoma (MM) is a highly aggressive form of skin cancer whose incidence continues to rise worldwide. If diagnosed at an early stage, it has an excellent prognosis, but mortality increases significantly at advanced stages after distant spread. Unfortunately, early detection of aggressive melanoma remains a challenge. OBJECTIVES: To identify novel blood-circulating biomarkers that may be useful in the diagnosis of MM to guide patient counselling and appropriate disease management. METHODS: In this study, 105 serum samples from 26 healthy patients and 79 with MM were analysed using an untargeted approach by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to compare the metabolomic profiles of both conditions. Resulting data were subjected to both univariate and multivariate statistical analysis to select robust biomarkers. The classification model obtained from this analysis was further validated with an independent cohort of 12 patients with stage I MM. RESULTS: We successfully identified several lipidic metabolites differentially expressed in patients with stage I MM vs. healthy controls. Three of these metabolites were used to develop a classification model, which exhibited exceptional precision (0.92) and accuracy (0.94) when validated on an independent sample. CONCLUSIONS: These results demonstrate that metabolomics using LC-HRMS is a powerful tool to identify and quantify metabolites in bodily fluids that could serve as potential early diagnostic markers for MM.

Melanoma is a type of skin cancer that can be deadly if it is not detected at an early stage. Unfortunately, the early detection of melanoma is challenging. Our team has developed a model that could be used to predict whether a person has stage I malignant melanoma based on blood serum analysis. The model was trained on data from a group of people with melanoma and it was found to be accurate in predicting melanoma at an early stage. This means that the model could be used to identify people who have skin cancer before it progresses and becomes more complicated to treat. Although the researchers recommend that further studies are conducted to validate the model in a larger population of people, this research could help with the early diagnosis of melanoma and work toward improving survival rates.

Evaluation of pro-regenerative and anti-inflammatory effects of isolecanoric acid in the muscle: Potential treatment of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating degenerative disease of skeletal muscles caused by loss of dystrophin, a key protein that maintains muscle integrity, which leads to progressive muscle degeneration aggravated by chronic inflammation, muscle stem cells' (MuSCs) reduced regenerative capacity and replacement of muscle with fibroadipose tissue. Previous research has shown that pharmacological GSK-3ß inhibition favors myogenic differentiation and plays an important role in modulating inflammatory processes. Isolecanoric acid (ILA) is a natural product isolated from a fungal culture displaying GSK-3ß inhibitory properties. The present study aimed to investigate the proregenerative and anti-inflammatory properties of this natural compound in the DMD context. Our results showed that ILA markedly promotes myogenic differentiation of myoblasts by increasing ß-Catenin signaling and boosting the myogenic potential of mouse and human stem cells. One important finding was that the GSK-3ß/ß-Catenin pathway is altered in dystrophic mice muscle and ILA enhances the myofiber formation of dystrophic MuSCs. Treatment with this natural compound improves muscle regeneration of dystrophic mice by, in turn, improving functional performance. Moreover, ILA ameliorates the inflammatory response in both muscle explants and the macrophages isolated from dystrophic mice to, thus, mitigate fibrosis after muscle damage. Overall, we show that ILA modulates both inflammation and muscle regeneration to, thus, contribute to improve the dystrophic phenotype.

Preclinical Evaluation of an Imidazole-Linked Heterocycle for Alzheimer's Disease.

Humanity is facing a vast prevalence of neurodegenerative diseases, with Alzheimer's disease (AD) being the most dominant, without efficacious drugs, and with only a few therapeutic targets identified. In this scenario, we aim to find molecular entities that modulate imidazoline I2 receptors (I2-IRs) that have been pointed out as relevant targets in AD. In this work, we explored structural modifications of well-established I2-IR ligands, giving access to derivatives with an imidazole-linked heterocycle as a common key feature. We report the synthesis, the affinity in human I2-IRs, the brain penetration capabilities, the in silico ADMET studies, and the three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of this new bunch of I2-IR ligands. Selected compounds showed neuroprotective properties and beneficial effects in an in vitro model of Parkinson's disease, rescued the human dopaminergic cell line SH-SY5Y from death after treatment with 6-hydroxydopamine, and showed crucial anti-inflammatory effects in a cellular model of neuroinflammation. After a preliminary pharmacokinetic study, we explored the action of our representative 2-(benzo[b]thiophen-2-yl)-1H-imidazole LSL33 in a mouse model of AD (5xFAD). Oral administration of LSL33 at 2 mg/Kg for 4 weeks ameliorated 5XFAD cognitive impairment and synaptic plasticity, as well as reduced neuroinflammation markers. In summary, this new I2-IR ligand that promoted beneficial effects in a well-established AD mouse model should be considered a promising therapeutic strategy for neurodegeneration.

Crossiellidines A-F, Unprecedented Pyrazine-Alkylguanidine Metabolites with Broad-Spectrum Antibacterial Activity from Crossiella sp.

Crosiellidines are intriguing pyrazine-alkylguanidine metabolites isolated from the minor actinomycete genus Crossiella. Their structures present an unprecedented 2-methoxy-3,5,6-trialkyl pyrazine scaffold and uncommon guanidine prenylations, including an exotic O-prenylated N-hydroxyguanidine moiety. The novel substitution pattern of the 2-methoxypyrazine core inaugurates a new class of naturally occurring pyrazine compounds, the biosynthetic implications of which are discussed herein. Isotopic feeding and genome analysis allowed us to propose a biosynthetic pathway from arginine. The crossiellidines exhibited remarkable, broad-spectrum antibacterial activity.

Untargeted Metabolomics by Liquid Chromatography-Mass Spectrometry in Biomedical Research.

Metabolomics, alone or in combination with other omics sciences, has shown great relevance in a large number of investigations in different branches of biomedicine, often providing novel discoveries and helping to expand the knowledge. Metabolomics analyses are carried out using different techniques, but in this chapter, we focus on liquid chromatography coupled to high-resolution mass spectrometry. The designated methodology consists of an untargeted approach for the analysis of plasma samples. The use of this method, with a reverse-phase column and electrospray ionization in positive mode, covers the detection of a broad range of metabolites, mainly of nonpolar and of intermediate polarity. This chapter also reviews the mass fragmentation spectra for the identification of bile acids, acylcarnitines, and glycerophospholipids.

Pharmacokinetics and Endocrine Effects of an Oral Dose of D-Pinitol in Human Fasting Healthy Volunteers.

The present study characterizes the oral pharmacokinetics of D-Pinitol, a natural insulin mimetic inositol, in human healthy volunteers (14 males and 11 females). D-Pinitol absorption was studied in (a) subjects receiving a single oral dose of 15 mg/kg (n = 10), or (b) 5 mg/kg pure D-Pinitol (n = 6), and (c) subjects receiving D-Pinitol as part of carbohydrate-containing carob pods-derived syrup with a 3.2% D-Pinitol (Dose of 1600 mg/subject, n = 9). The volunteers received a randomly assigned single dose of either D-Pinitol or carob pod-derived syrup. Blood samples were collected at 0, 15, 30, 45, 60, 90, 120, 180, 240, 360 and 1440 min after intake. Plasma concentration of D-Pinitol was measured and pharmacokinetic parameters obtained. The data indicate that when given alone, the oral absorption of D-Pinitol is dose-dependent and of extended duration, with a Tmax reached after almost 4 h, and a half-life greater than 5 h. When the source of D-Pinitol was a carob pods-derived syrup, Cmax was reduced to 40% of the expected based on the data of D-Pinitol alone, suggesting a reduced absorption probably because of competition with monosaccharide transport. In this group, Tmax was reached before that of D-Pinitol alone, but the estimated half-life remained the same. In the D-Pinitol groups, plasma concentrations of glucose, insulin, glucagon, ghrelin, free fatty acids, and pituitary hormones were additionally measured. A dose of 15 mg/kg of D-Pinitol did not affect glucose levels in healthy volunteers, but reduced insulin and increased glucagon and ghrelin concentrations. D-Pinitol did not increase other hormones known to enhance plasma glucose, such as cortisol or GH, which were surprisingly reduced after the ingestion of this inositol. Other pituitary hormones (gonadotropins, prolactin, and thyroid-stimulating hormone) were not affected after D-Pinitol ingestion. In a conclusion, D-Pinitol is absorbed through the oral route, having an extended half-life and displaying the pharmacological profile of an endocrine pancreas protector, a pharmacological activity of potential interest for the treatment or prevention of insulin resistance-associated conditions.

Kribbellichelins A and B, Two New Antibiotics from Kribbella sp. CA-293567 with Activity against Several Human Pathogens.

Current needs in finding new antibiotics against emerging multidrug-resistant superbugs are pushing the scientific community into coming back to Nature for the discovery of novel active structures. Recently, a survey of halophilic actinomyectes from saline substrates of El Saladar del Margen, in the Cúllar-Baza depression (Granada, Spain), led us to the isolation and identification of 108 strains from the rhizosphere of the endemic plant Limonium majus. Evaluation of the potential of these strains to produce new anti-infective agents against superbug pathogens was performed through fermentation in 10 different culture media using an OSMAC approach and assessment of the antibacterial and antifungal properties of their acetone extracts. The study allowed the isolation of two novel antibiotic compounds, kribbellichelin A (1) and B (2), along with the known metabolites sandramycin (3), coproporphyrin III (4), and kribelloside C (5) from a bioassay-guided fractionation of scaled-up active extracts of the Kribbella sp. CA-293567 strain. The structures of the new molecules were elucidated by ESI-qTOF-MS/MS, 1D and 2D NMR, and Marfey's analysis for the determination of the absolute configuration of their amino acid residues. Compounds 1-3 and 5 were assayed against a panel of relevant antibiotic-resistant pathogenic strains and evaluated for cytotoxicity versus the human hepatoma cell line HepG2 (ATCC HB-8065). Kribbellichelins A (1) and B (2) showed antimicrobial activity versus Candida albicans ATCC-64124, weak potency against Acinetobacter baumannii MB-5973 and Pseudomonas aeruginosa MB-5919, and an atypical dose-dependent concentration profile against Aspergillus fumigatus ATCC-46645. Sandramycin (3) confirmed previously reported excellent growth inhibition activity against MRSA MB-5393 but also presented clear antifungal activity against C. albicans ATCC-64124 and A. fumigatus ATCC-46645 associated with lower cytotoxicity observed in HepG2, whereas Kribelloside C (5) displayed high antifungal activity only against A. fumigatus ATCC-46645. Herein, we describe the processes followed for the isolation, structure elucidation, and potency evaluation of these two new active compounds against a panel of human pathogens as well as, for the first time, the characterization of the antifungal activities of sandramycin (3).

Insights into the Pharmacokinetics and In Vitro Cell-Based Studies of the Imidazoline I2 Receptor Ligand B06.

The impact of neurodegenerative diseases (ND) is becoming unbearable for humankind due to their vast prevalence and the lack of efficacious treatments. In this scenario, we focused on imidazoline I2 receptors (I2-IR) that are widely distributed in the brain and are altered in patients with brain disorders. We took the challenge of modulating I2-IR by developing structurally new molecules, in particular, a family of bicyclic α-iminophosphonates, endowed with high affinity and selectivity to these receptors. Treatment of two murine models, one for age-related cognitive decline and the other for Alzheimer's disease (AD), with representative compound B06 ameliorated their cognitive impairment and improved their behavioural condition. Furthermore, B06 revealed beneficial in vitro ADME-Tox properties. The pharmacokinetics (PK) and metabolic profile are reported to de-risk B06 for progressing in the preclinical development. To further characterize the pharmacological properties of B06, we assessed its neuroprotective properties and beneficial effect in an in vitro model of Parkinson's disease (PD). B06 rescued the human dopaminergic cell line SH-SY5Y from death after treatment with 6-hydroxydopamine (6-OHDA) and showed a crucial anti-inflammatory effect in a cellular model of neuroinflammation. This research reveals B06 as a putative candidate for advancing in the difficult path of drug discovery and supports the modulation of I2-IR as a fresh approach for the therapy of ND.

The Mycobacterium tuberculosis PhoPR virulence system regulates expression of the universal second messenger c-di-AMP and impacts vaccine safety and efficacy.

Cyclic (di)nucleotides act as universal second messengers endogenously produced by several pathogens. Specifically, the roles of c-di-AMP in Mycobacterium tuberculosis immunity and virulence have been largely explored, although its contribution to the safety and efficacy of live tuberculosis vaccines is less understood. In this study, we demonstrate that the synthesis of c-di-AMP is negatively regulated by the M. tuberculosis PhoPR virulence system. Accordingly, the live attenuated tuberculosis vaccine candidate M. tuberculosis vaccine (MTBVAC), based on double phoP and fadD26 deletions, produces more than 25- and 45-fold c-di-AMP levels relative to wild-type M. tuberculosis or the current vaccine bacille Calmette-Guérin (BCG), respectively. Secretion of this second messenger was exclusively detected in MTBVAC but not in M. tuberculosis or in BCG. We also demonstrate that c-di-AMP synthesis during in vitro cultivation of M. tuberculosis is a growth-phase- and medium-dependent phenotype. To uncover the role of this metabolite in the vaccine properties of MTBVAC, we constructed and validated knockout and overproducing/oversecreting derivatives by inactivating the disA or cnpB gene, respectively. All MTBVAC derivatives elicited superior interleukin-1ß (IL-1ß) responses compared with BCG during an in vitro infection of human macrophages. However, both vaccines failed to elicit interferon ß (IFNß) activation in this cellular model. We found that increasing c-di-AMP levels remarkably correlated with a safer profile of tuberculosis vaccines in the immunodeficient mouse model. Finally, we demonstrate that overproduction of c-di-AMP due to cnpB inactivation resulted in lower protection of MTBVAC, while the absence of c-di-AMP in the MTBVAC disA derivative maintains the protective efficacy of this vaccine in mice.

Predicting dynamic response to neoadjuvant chemotherapy in breast cancer: a novel metabolomics approach.

Neoadjuvant chemotherapy (NACT) outcomes vary according to breast cancer (BC) subtype. Since pathologic complete response is one of the most important target endpoints of NACT, further investigation of NACT outcomes in BC is crucial. Thus, identifying sensitive and specific predictors of treatment response for each phenotype would enable early detection of chemoresistance and residual disease, decreasing exposures to ineffective therapies and enhancing overall survival rates. We used liquid chromatography-high-resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics to detect molecular changes in plasma of three different BC subtypes following the same NACT regimen, with the aim of searching for potential predictors of response. The metabolomics data set was analyzed by combining univariate and multivariate statistical strategies. By using ANOVA-simultaneous component analysis (ASCA), we were able to determine the prognostic value of potential biomarker candidates of response to NACT in the triple-negative (TN) subtype. Higher concentrations of docosahexaenoic acid and secondary bile acids were found at basal and presurgery samples, respectively, in the responders group. In addition, the glycohyocholic and glycodeoxycholic acids were able to classify TN patients according to response to treatment and overall survival with an area under the curve model > 0.77. In relation to luminal B (LB) and HER2+ subjects, it should be noted that significant differences were related to time and individual factors. Specifically, tryptophan was identified to be decreased over time in HER2+ patients, whereas LysoPE (22:6) appeared to be increased, but could not be associated with response to NACT. Therefore, the combination of untargeted-based metabolomics along with longitudinal statistical approaches may represent a very useful tool for the improvement of treatment and in administering a more personalized BC follow-up in the clinical practice.

Impact of the immune response modification by lysophosphatidylcholine in the efficacy of antibiotic therapy of experimental models of peritoneal sepsis and pneumonia by Pseudomonas aeruginosa: LPC therapeutic effect in combined therapy.

INTRODUCTION: Immune response stimulation may be an adjuvant to antimicrobial treatment. Here, we evaluated the impact of immune response modification by lysophosphatidylcholine (LPC), combined with imipenem or ceftazidime, in murine models of peritoneal sepsis (PS) and pneumonia induced by Pseudomonas aeruginosa. METHODS: The imipenem and ceftazidime-susceptible strain (Pa39) and imipenem and ceftazidime-resistant strain (Pa238) were used. Ceftazidime pharmacokinetic and pharmacodynamic parameters were determined. The therapeutic efficacy and TNF-α and IL-10 levels were determined in murine models of PS and pneumonia induced by Pa39 and Pa238 and treated with LPC, imipenem or ceftazidime, alone or in combination. RESULTS: In the PS model, LPC+ceftazidime reduced spleen and lung Pa238 concentrations (-3.45 and -3.56log10CFU/g; P<0.05) to a greater extent than ceftazidime monotherapy, while LPC+imipenem maintained the imipenem efficacy (-1.66 and -1.45log10CFU/g; P>0.05). In the pneumonia model, LPC+ceftazidime or LPC+imipenem reduced the lung Pa238 concentrations (-2.37log10CFU/g, P=0.1, or -1.35log10CFU/g, P=0.75). For Pa39, no statistically significant difference was observed in the PS and pneumonia models between combined therapy and monotherapy. Moreover, LPC+imipenem and LPC+ceftazidime significantly decreased and increased the TNF-α and IL-10 levels, respectively, in comparison with the untreated controls and monotherapies. CONCLUSIONS: These results demonstrate the impact of immune response modification by LPC plus antibiotics on the prognosis of infections induced by ceftazidime-resistant P. aeruginosa.

Impact of the immune response modification by lysophosphatidylcholine in the efficacy of antibiotic therapy of experimental models of peritoneal sepsis and pneumonia by Pseudomonas aeruginosa: LPC therapeutic effect in combined therapy / Impacto de la modificación de la respuesta inmunitaria por la lisofosfatidilcolina en la eficacia de la terapia antibiótica en un modelo experimental de sepsis peritoneal y de neumonía por Pseudomonas aeruginosa

IntroductionImmune response stimulation may be an adjuvant to antimicrobial treatment. Here, we evaluated the impact of immune response modification by lysophosphatidylcholine (LPC), combined with imipenem or ceftazidime, in murine models of peritoneal sepsis (PS) and pneumonia induced by Pseudomonas aeruginosa.MethodsThe imipenem and ceftazidime-susceptible strain (Pa39) and imipenem and ceftazidime-resistant strain (Pa238) were used. Ceftazidime pharmacokinetic and pharmacodynamic parameters were determined. The therapeutic efficacy and TNF-α and IL-10 levels were determined in murine models of PS and pneumonia induced by Pa39 and Pa238 and treated with LPC, imipenem or ceftazidime, alone or in combination.ResultsIn the PS model, LPC+ceftazidime reduced spleen and lung Pa238 concentrations (−3.45 and −3.56log10CFU/g; P<0.05) to a greater extent than ceftazidime monotherapy, while LPC+imipenem maintained the imipenem efficacy (−1.66 and −1.45log10CFU/g; P>0.05). In the pneumonia model, LPC+ceftazidime or LPC+imipenem reduced the lung Pa238 concentrations (−2.37log10CFU/g, P=0.1, or −1.35log10CFU/g, P=0.75). For Pa39, no statistically significant difference was observed in the PS and pneumonia models between combined therapy and monotherapy. Moreover, LPC+imipenem and LPC+ceftazidime significantly decreased and increased the TNF-α and IL-10 levels, respectively, in comparison with the untreated controls and monotherapies.ConclusionsThese results demonstrate the impact of immune response modification by LPC plus antibiotics on the prognosis of infections induced by ceftazidime-resistant P. aeruginosa.

IntroducciónLa estimulación de la respuesta inmunitaria podría ser adyuvante al tratamiento antimicrobiano. En este estudio, hemos evaluado el impacto de la modificación de la respuesta inmunitaria por la lisofosfatidilcolina (LPC), combinada con imipenem ó ceftazidima, en modelos murinos de sepsis peritoneal (SP) y de neumonía por Pseudomonas aeruginosa (P. aeruginosa).MétodosLa cepa sensible a imipenem y ceftazidima (Pa39) y la cepa resistente a ambos antibióticos (Pa238) fueron usadas. Los parámetros farmacocinéticos/farmacodinámicos de ceftazidima fueron determinados. La eficacia terapéutica y los niveles de TNF-α and IL-10 fueron determinados en los modelos murinos de SP y de neumonía por Pa39 y Pa238 y tratados con LPC, imipenem o ceftazidima, en monoterapia ó en combinación.ResultadosEn el modelo de SP, LPC + ceftazidima redujo la concentración de Pa238 en el bazo y el pulmón (–3,45 y –3,56 log10 UFC/g; p < 0,05) en comparación con ceftazidima, mientras LPC + impenem mantuvo la eficacia de imipenem (–1,66 y –1,45 log10 UFC/g; p > 0,05). En el modelo de neumonía, LPC + ceftazidima o LPC + imipenem redujo la concentración de Pa238 en pulmón (–2,37 log10 UFC/g, p = 0,1 o –1,35 log10 UFC/g, p = 0,75). Para Pa39, no se observó diferencia estadística significativa entre la terapia combinada y la monoterapia en los modelos de SP y de neumonía. Además, LPC + imipenem y LPC + ceftazidime redujeron y aumentaron los niveles de TNF-α y IL-10, respectivamente, en comparación con los controles no tratados y las monoterapias.ConclusionesEstos resultados demuestran el impacto de la modificación de la respuesta inmunitaria por LPC en combinación con antibióticos en el pronóstico de las infecciones por P. aeruginosa ceftazidima-resistente.

Preclinical evaluation of strasseriolides A-D, potent antiplasmodial macrolides isolated from Strasseria geniculata CF-247,251.

BACKGROUND: Malaria is a global health problem for which novel therapeutic compounds are needed. To this end, a recently published novel family of antiplasmodial macrolides, strasseriolides A-D, was herein subjected to in vivo efficacy studies and preclinical evaluation in order to identify the most promising candidate(s) for further development. METHODS: Preclinical evaluation of strasseriolides A-D was performed by MTT-based cytotoxicity assay in THLE-2 (CRL-2706) liver cells, cardiotoxicity screening using the FluxOR™ potassium assay in hERG expressed HEK cells, LC-MS-based analysis of drug-drug interaction involving CYP3A4, CYP2D6 and CYP2C9 isoforms inhibition and metabolic stability assays in human liver microsomes. Mice in vivo toxicity studies were also accomplished by i.v. administration of the compounds (vehicle: 0.5% HPMC, 0.5% Tween 80, 0.5% Benzyl alcohol) in mice at 25 mg/kg dosage. Plasma were prepared from mice blood samples obtained at different time points (over a 24-h period), and analysed by LC-MS to quantify compounds. The most promising compounds, strasseriolides C and D, were subjected to a preliminary in vivo efficacy study in which transgenic GFP-luciferase expressing Plasmodium berghei strain ANKA-infected Swiss Webster female mice (n = 4-5) were treated 48 h post-infection with an i.p. dosage of strasseriolide C at 50 mg/kg and strasseriolide D at 22 mg/kg for four days after which luciferase activity was quantified on day 5 in an IVIS® Lumina II imager. RESULTS: Strasseriolides A-D showed no cytotoxicity, no carditoxicity and no drug-drug interaction problems in vitro with varying intrinsic clearance (CLint). Only strasseriolide B was highly toxic to mice in vivo (even at 1 mg/kg i.v. dosage) and, therefore, discontinued in further in vivo studies. Strasseriolide D showed statistically significant activity in vivo giving rise to lower parasitaemia levels (70% lower) compared to the controls treated with vehicle. CONCLUSIONS: Animal efficacy and preclinical evaluation of the recently discovered potent antiplasmodial macrolides, strasseriolides A-D, led to the identification of strasseriolide D as the most promising compound for further development. Future studies dealing on structure optimization, formulation and establishment of optimal in vivo dosage explorations of this novel compound class could enhance their clinical potency and allow for progress to later stages of the developmental pipeline.

Untargeted Metabolomics for the Diagnosis of Exocrine Pancreatic Insufficiency in Chronic Pancreatitis.

Background and Objectives: The clinical manifestations and course of chronic pancreatitis (CP) are often nonspecific and variable, hampering diagnosis of the risk of exocrine pancreatic insufficiency (EPI). Development of new, reproducible, and non-invasive methods to diagnose EPI is therefore a major priority. The objective of this metabolomic study was to identify novel biomarkers associated with EPI. Materials and Methods: We analyzed 53 samples from patients with CP, 32 with and 21 without EPI, using an untargeted metabolomics workflow based on hydrophilic interaction chromatography coupled to high-resolution mass spectrometry. Principal component and partial least squares-discriminant analyses showed significant between-group differentiation, and univariate and multivariate analyses identified potential candidate metabolites that significantly differed between samples from CP patients with EPI and those without EPI. Results: Excellent results were obtained using a six-metabolic panel to diagnose the presence of EPI in CP patients (area under the ROC curve = 0.785). Conclusions: This study confirms the usefulness of metabolomics in this disease setting, allowing the identification of novel biomarkers to differentiate between the presence and absence of EPI in CP patients.

Discovery and Characterization of Epemicins A and B, New 30-Membered Macrolides from Kutzneria sp. CA-103260.

Actinobacteria have been a rich source of novel, structurally complex natural products for many decades. Although the largest genus is Streptomyces, from which the majority of antibiotics in current and past clinical use were originally isolated, other less common genera also have the potential to produce a wealth of novel secondary metabolites. One example is the Kutzneria genus, which currently contains only five reported species. One of these species is Kutzneria albida DSM 43870T, which has 46 predicted biosynthetic gene clusters and is known to produce the macrolide antibiotic aculeximycin. Here, we report the isolation and structural characterization of two novel 30-membered glycosylated macrolides, epemicins A and B, that are structurally related to aculeximycin, from a rare Kutzneria sp. The absolute configuration for all chiral centers in the two compounds is proposed based on extensive 1D and 2D NMR studies and bioinformatics analysis of the gene cluster. Through heterologous expression and genetic inactivation, we have confirmed the link between the biosynthetic gene cluster and the new molecules. These findings show the potential of rare Actinobacteria to produce new, structurally diverse metabolites. Furthermore, the gene inactivation represents the first published report to genetically manipulate a representative of the Kutzneria genus.

Efficacy of Fosfomycin and Its Combination With Aminoglycosides in an Experimental Sepsis Model by Carbapenemase-Producing Klebsiella pneumoniae Clinical Strains.

Carbapenemase-producing Klebsiella pneumoniae infections are an increasing global threat with scarce and uncertain treatment options. In this context, combination therapies are often used for these infections. The bactericidal and synergistic activity of fosfomycin plus amikacin and gentamicin was studied trough time-kill assays against four clonally unrelated clinical isolates of carbapenemase-producing K. pneumoniae, VIM-1, VIM-1 plus DHA-1, OXA-48 plus CTXM-15, and KPC-3, respectively. The efficacy of antimicrobials that showed synergistic activity in vitro against all the carbapenemase-producing K. pneumoniae were tested in monotherapy and in combination, in a murine peritoneal sepsis model. In vitro, fosfomycin plus amikacin showed synergistic and bactericidal effect against strains producing VIM-1, VIM-1 plus DHA-1, and OXA-48 plus CTX-M-15. Fosfomycin plus gentamicin had in vitro synergistic activity against the strain producing KPC-3. In vivo, fosfomycin and amikacin and its combination reduced the spleen bacterial concentration compared with controls groups in animals infected by K. pneumoniae producing VIM-1 and OXA-48 plus CTX-M-15. Moreover, amikacin alone and its combination with fosfomycin reduced the bacteremia rate against the VIM-1 producer strain. Contrary to the in vitro results, no in vivo efficacy was found with fosfomycin plus amikacin against the VIM-1 plus DHA-1 producer strain. Finally, fosfomycin plus gentamicin reduced the bacterial concentration in spleen against the KPC-3 producer strain. In conclusion, our results suggest that fosfomycin plus aminoglycosides has a dissimilar efficacy in the treatment of this severe experimental infection, when caused by different carbapenemase-producing K. pneumoniae strains. Fosfomycin plus amikacin or plus gentamycin may be useful to treat infections by OXA-48 plus CTX-M-15 or KPC-3 producer strains, respectively.

Identification of Compounds with Potential Therapeutic Uses from Sweet Pepper (Capsicum annuum L.) Fruits and Their Modulation by Nitric Oxide (NO).

Plant species are precursors of a wide variety of secondary metabolites that, besides being useful for themselves, can also be used by humans for their consumption and economic benefit. Pepper (Capsicum annuum L.) fruit is not only a common food and spice source, it also stands out for containing high amounts of antioxidants (such as vitamins C and A), polyphenols and capsaicinoids. Particular attention has been paid to capsaicin, whose anti-inflammatory, antiproliferative and analgesic activities have been reported in the literature. Due to the potential interest in pepper metabolites for human use, in this project, we carried out an investigation to identify new bioactive compounds of this crop. To achieve this, we applied a metabolomic approach, using an HPLC (high-performance liquid chromatography) separative technique coupled to metabolite identification by high resolution mass spectrometry (HRMS). After chromatographic analysis and data processing against metabolic databases, 12 differential bioactive compounds were identified in sweet pepper fruits, including quercetin and its derivatives, L-tryptophan, phytosphingosin, FAD, gingerglycolipid A, tetrahydropentoxylin, blumenol C glucoside, colnelenic acid and capsoside A. The abundance of these metabolites varied depending on the ripening stage of the fruits, either immature green or ripe red. We also studied the variation of these 12 metabolites upon treatment with exogenous nitric oxide (NO), a free radical gas involved in a good number of physiological processes in higher plants such as germination, growth, flowering, senescence, and fruit ripening, among others. Overall, it was found that the content of the analyzed metabolites depended on the ripening stage and on the presence of NO. The metabolic pattern followed by quercetin and its derivatives, as a consequence of the ripening stage and NO treatment, was also corroborated by transcriptomic analysis of genes involved in the synthesis of these compounds. This opens new research perspectives on the pepper fruit's bioactive compounds with nutraceutical potentiality, where biotechnological strategies can be applied for optimizing the level of these beneficial compounds.

Pentaminomycins F and G, Nonribosomal Peptides Containing 2-Pyridylalanine.

Pentaminomycins F-H (1-3), a group of three new hydroxyarginine-containing cyclic pentapeptides, were isolated from cultures of a Streptomyces cacaoi subsp. cacaoi strain along with the known pentaminomycins A-E. The structures of the new peptides were determined by a combination of mass spectrometry, NMR, and Marfey's analyses. Pentaminomycins F (1) and G (2) were shown to contain the rare amino acid 3-(2-pyridyl)-alanine. This finding represents the first reported examples of nonribosomal peptides containing this residue. The ldlld chiral sequence found for the three compounds was in agreement with that reported for previously isolated pentaminomycins and consistent with the epimerization domains present in the putative nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster.

Human Plasma Metabolomics for Biomarker Discovery: Targeting the Molecular Subtypes in Breast Cancer.

PURPOSE: The aim of this study is to identify differential metabolomic signatures in plasma samples of distinct subtypes of breast cancer patients that could be used in clinical practice as diagnostic biomarkers for these molecular phenotypes and to provide a more individualized and accurate therapeutic procedure. METHODS: Untargeted LC-HRMS metabolomics approach in positive and negative electrospray ionization mode was used to analyze plasma samples from LA, LB, HER2+ and TN breast cancer patients and healthy controls in order to determine specific metabolomic profiles through univariate and multivariate statistical data analysis. RESULTS: We tentatively identified altered metabolites displaying concentration variations among the four breast cancer molecular subtypes. We found a biomarker panel of 5 candidates in LA, 7 in LB, 5 in HER2 and 3 in TN that were able to discriminate each breast cancer subtype with a false discovery range corrected p-value < 0.05 and a fold-change cutoff value > 1.3. The model clinical value was evaluated with the AUROC, providing diagnostic capacities above 0.85. CONCLUSION: Our study identifies metabolic profiling differences in molecular phenotypes of breast cancer. This may represent a key step towards therapy improvement in personalized medicine and prioritization of tailored therapeutic intervention strategies.