PubMed

Related Articles Emerging Molecular Technologies in Renal Cell Carcinoma: Liquid Biopsy. Cancers (Basel). 2019 Feb 07;11(2): Authors: Cimadamore A, Gasparrini S, Massari F, Santoni M, Cheng L, Lopez-Beltran A, Scarpelli M, Montironi R Abstract Liquid biopsy, based on the circulating tumor cells (CTCs) and cell-free nucleic acids has potential applications at multiple points throughout the natural course of cancer, from diagnosis to follow-up. The advantages of doing ctDNA assessment vs. tissue-based genomic profile are the minimal procedural risk, the possibility to serial testing in order to monitor disease-relapse and response to therapy over time and to reduce hospitalization costs during the entire process. However, some critical issues related to ctDNA assays should be taken into consideration. The sensitivity of ctDNA assays depends on the assessment technique and genetic platforms used, on tumor-organ, stage, tumor heterogeneity, tumor clonality. The specificity is usually very high, whereas the concordance with tumor-based biopsy is generally low. In patients with renal cell carcinoma (RCC), qualitative analyses of ctDNA have been performed with interesting results regarding selective pressure from therapy, therapeutic resistance, exceptional treatment response to everolimus and mutations associated with aggressive behavior. Quantitative analyses showed variations of ccfDNA levels at different tumor stage. Compared to CTC assay, ctDNA is more stable than cells and easier to isolate. Splice variants, information at single-cell level and functional assays along with proteomics, transcriptomics and metabolomics studies can be performed only in CTCs. PMID: 30736478 [PubMed]

Related Articles Current Developments in µMAS NMR Analysis for Metabolomics. Metabolites. 2019 Feb 06;9(2): Authors: Lucas-Torres C, Wong A Abstract Analysis of microscopic specimens has emerged as a useful analytical application in metabolomics because of its capacity for characterizing a highly homogenous sample with a specific interest. The undeviating analysis helps to unfold the hidden activities in a bulk specimen and contributes to the understanding of the fundamental metabolisms in life. In NMR spectroscopy, micro(µ)-probe technology is well-established and -adopted to the microscopic level of biofluids. However, this is quite the contrary with specimens such as tissue, cell and organism. This is due to the substantial difficulty of developing a sufficient µ-size magic-angle spinning (MAS) probe for sub-milligram specimens with the capability of high-quality data acquisition. It was not until 2012; a µMAS probe had emerged and shown promises to µg analysis; since, a continuous advancement has been made striving for the possibility of µMAS to be an effective NMR spectroscopic analysis. Herein, the mini-review highlights the progress of µMAS development-from an impossible scenario to an attainable solution-and describes a few demonstrative metabolic profiling studies. The review will also discuss the current challenges in µMAS NMR analysis and its potential to metabolomics. PMID: 30736341 [PubMed]

27 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2019/02/09PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

Related Articles Serum-based metabolomics characterization of patients with reticular oral lichen planus. Arch Oral Biol. 2019 Jan 30;99:183-189 Authors: Li XZ, Zhang SN, Yang XY Abstract OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory mucosal lesion and systemic disease. In OLP, reticular type is the most common presentation of the disease. However, little is known about it. The aim of this study was to analyze the pathogenesis of reticular OLP and its possible associations with the pathological changes in other organ systems through serum-based metabolomics. METHODS: Blood samples were obtained from 16 reticular OLP patients and 24 control subjects. Liquid chromatography (LC)-mass spectrometry (MS) system was used to identify differentially expressed metabolites. The pathways analysis was performed by MetaboAnalyst. Pathological network was constructed by Cytoscape software. RESULTS: Totally, 31 modulated metabolites were identified, whose dysregulations affected 25 metabolic pathways and 7 pathological processes in the disease. Through an impact-value screen (impact-value＞0.1), 6 pathways were selected as the significantly dysregulated pathways. Pathological network showed that these metabolites participated in 7 pathological processes, that is, apoptosis process, DNA damage and repair disorder, oxidative stress injury, carbohydrate metabolism disorder, mood dysfunction, inflammatory lesion, and other pathological process. CONCLUSION: The study demonstrated that reticular OLP could cause the dysregulations of the metabolites in serum, which might be also further linked to other organ and systemic diseases through the blood system, such as diabetes, sleep disorders, and depression, etc. PMID: 30731368 [PubMed - as supplied by publisher]

Related Articles Comparative proteomics and metabolomics of JAZ7-mediated drought tolerance in Arabidopsis. J Proteomics. 2019 Feb 04;: Authors: Meng L, Zhang T, Geng S, Scott PB, Li H, Chen S Abstract Jasmonates (JAs) are important phytohormones that regulate a wide range of plant processes, including growth, development and stress responses. Jasmonate ZIM-domain (JAZ) proteins are transcriptional repressors in JA signaling. Overexpression of JAZ7 was found to confer drought tolerance in Arabidopsis thaliana (A. thaliana), but the molecular mechanisms are not known. Using Tandem Mass Tag (TMT) quantitative proteomics and targeted metabolomics approaches, we found that 394 unique proteins and 96 metabolites were differentially expressed under drought and/or among the three genotypes (wild type (WT), JAZ7 knock out (KO) and JAZ7 overexpression (OE)). Unique and differential proteins/metabolites after each comparison were analyzed to gauge their potential functions in drought tolerance. The proteins and metabolites are enriched in JA and abscisic acid (ABA) signaling pathways, response to stress, photosynthesis, redox and metabolic process. BIOLOGICAL SIGNIFICANCE: Drought stress is a global challenge that affects agricultural production. JAZ7 overexpression led to drought tolerance in A. thaliana through modulating photosynthesis, redox, and amino acids, phytohormones and defense metabolites. The results have provided important insights into the JAZ7 regulated molecular networks of drought tolerance. The knowledge may facilitate effort to enhance crop drought tolerance in the era of climate change. PMID: 30731210 [PubMed - as supplied by publisher]

Related Articles Metabolomic analysis of serum and myocardium in compensated heart failure after myocardial infarction. Life Sci. 2019 Feb 04;: Authors: McKirnan MD, Ichikawa Y, Zhang Z, Zemljic-Harpf AE, Fan S, Barupal DK, Patel HH, Hammond HK, Roth DM Abstract AIMS: To determine the metabolic adaptations to compensated heart failure using a reproducible model of myocardial infarction and an unbiased metabolic screen. To address the limitations in sample availability and model variability observed in preclinical and clinical metabolic investigations of heart failure. MAIN METHODS: Metabolomic analysis was performed on serum and myocardial tissue from rabbits after myocardial infarction (MI) was induced by cryo-injury of the left ventricular free wall. Rabbits followed for 12 weeks after MI exhibited left ventricular dilation and depressed systolic function as determined by echocardiography. Serum and tissue from the viable left ventricular free wall, interventricular septum and right ventricle were analyzed using a gas chromatography time of flight mass spectrometry-based untargeted metabolomics assay for primary metabolites. KEY FINDINGS: Unique results included: a two- three-fold increase in taurine levels in all three ventricular regions of MI rabbits and similarly, the three regions had increased inosine levels compared to sham controls. Reduced myocardial levels of myo-inositol in the myocardium of MI animals point to altered phospholipid metabolism and membrane receptor function in heart failure. Metabolite profiles also provide evidence for responses to oxidative stress and an impairment in TCA cycle energy production in the failing heart. SIGNIFICANCE: Our results revealed metabolic changes during compensated cardiac dysfunction and suggest potential targets for altering the progression of heart failure. PMID: 30731143 [PubMed - as supplied by publisher]

Related Articles Discovery of novel glycerolated quinazolinones from Streptomyces sp. MBT27. J Ind Microbiol Biotechnol. 2019 Feb 07;: Authors: Machushynets NV, Wu C, Elsayed SS, Hankemeier T, van Wezel GP Abstract Actinobacteria are a major source of novel bioactive natural products. A challenge in the screening of these microorganisms lies in finding the favorable growth conditions for secondary metabolite production and dereplication of known molecules. Here, we report that Streptomyces sp. MBT27 produces 4-quinazolinone alkaloids in response to elevated levels of glycerol, whereby quinazolinones A (1) and B (2) form a new sub-class of this interesting family of natural products. Global Natural Product Social molecular networking (GNPS) resulted in a quinazolinone-related network that included anthranilic acid (3), anthranilamide (4), 4(3H)-quinazolinone (5), and 2,2-dimethyl-1,2-dihydroquinazolin-4(3H)-one (6). Actinomycins D (7) and X2 (8) were also identified in the extracts of Streptomyces sp. MBT27. The induction of quinazolinone production by glycerol combined with biosynthetic insights provide evidence that glycerol is integrated into the chemical scaffold. The unprecedented 1,4-dioxepane ring, that is spiro-fused into the quinazolinone backbone, is most likely formed by intermolecular etherification of two units of glycerol. Our work underlines the importance of varying the growth conditions for the discovery of novel natural products and for understanding their biosynthesis. PMID: 30729343 [PubMed - as supplied by publisher]

Related Articles Imbalanced sphingolipid signaling is maintained as a core proponent of a cancerous phenotype in spite of metabolic pressure and epigenetic drift. Oncotarget. 2019 Jan 11;10(4):449-479 Authors: Speirs MMP, Swensen AC, Chan TY, Jones PM, Holman JC, Harris MB, Maschek JA, Cox JE, Carson RH, Hill JT, Andersen JL, Prince JT, Price JC Abstract Tumor heterogeneity may arise through genetic drift and environmentally driven clonal selection for metabolic fitness. This would promote subpopulations derived from single cancer cells that exhibit distinct phenotypes while conserving vital pro-survival pathways. We aimed to identify significant drivers of cell fitness in pancreatic adenocarcinoma (PDAC) creating subclones in different nutrient formulations to encourage differential metabolic reprogramming. The genetic and phenotypic expression profiles of each subclone were analyzed relative to a healthy control cell line (hTert-HPNE). The subclones exhibited distinct variations in protein expression and lipid metabolism. Relative to hTert-HPNE, PSN-1 subclones uniformly maintained modified sphingolipid signaling and specifically retained elevated sphingosine-1-phosphate (S1P) relative to C16 ceramide (C16 Cer) ratios. Each clone utilized a different perturbation to this pathway, but maintained this modified signaling to preserve cancerous phenotypes, such as rapid proliferation and defense against mitochondria-mediated apoptosis. Although the subclones were unique in their sensitivity, inhibition of S1P synthesis significantly reduced the ratio of S1P/C16 Cer, slowed cell proliferation, and enhanced sensitivity to apoptotic signals. This reliance on S1P signaling identifies this pathway as a promising drug-sensitizing target that may be used to eliminate cancerous cells consistently across uniquely reprogrammed PDAC clones. PMID: 30728898 [PubMed]

Related Articles Modulation of Metabolome and Bacterial Community in Whole Crop Corn Silage by Inoculating Homofermentative Lactobacillus plantarum and Heterofermentative Lactobacillus buchneri. Front Microbiol. 2018;9:3299 Authors: Xu D, Ding W, Ke W, Li F, Zhang P, Guo X Abstract The present study investigated the species level based microbial community and metabolome in corn silage inoculated with or without homofermentative Lactobacillus plantarum and heterofermentative Lactobacillus buchneri using the PacBio SMRT Sequencing and time-of-flight mass spectrometry (GC-TOF/MS). Chopped whole crop corn was treated with (1) deionized water (control), (2) Lactobacillus plantarum, or (3) Lactobacillus buchneri. The chopped whole crop corn was ensiled in vacuum-sealed polyethylene bags containing 300 g of fresh forge for 90 days, with three replicates for each treatment. The results showed that a total of 979 substances were detected, and 316 different metabolites were identified. Some metabolites with antimicrobial activity were detected in whole crop corn silage, such as catechol, 3-phenyllactic acid, 4-hydroxybenzoic acid, azelaic acid, 3,4-dihydroxybenzoic acid and 4-hydroxycinnamic acid. Catechol, pyrogallol and ferulic acid with antioxidant property, 4-hydroxybutyrate with nervine activity, and linoleic acid with cholesterol lowering effects, were detected in present study. In addition, a flavoring agent of myristic acid and a depression mitigation substance of phenylethylamine were also found in this study. Samples treated with inoculants presented more biofunctional metabolites of organic acids, amino acids and phenolic acids than untreated samples. The Lactobacillus species covered over 98% after ensiling, and were mainly comprised by the L. acetotolerans, L. silagei, L. parafarraginis, L. buchneri and L. odoratitofui. As compared to the control silage, inoculation of L. plantarum increased the relative abundances of L. acetotolerans, L. buchneri and L. parafarraginis, and a considerable decline in the proportion of L. silagei was observed; whereas an obvious decrease in L. acetotolerans and increases in L. odoratitofui and L. farciminis were observed in the L. buchneri inoculated silage. Therefore, inoculation of L. plantarum and L. buchneri regulated the microbial composition and metabolome of the corn silage with different behaviors. The present results indicated that profiling of silage microbiome and metabolome might improve our current understanding of the biological process underlying silage formation. PMID: 30728817 [PubMed]

Related Articles Single Cell Omics of Breast Cancer: An Update on Characterization and Diagnosis. Indian J Clin Biochem. 2019 Jan;34(1):3-18 Authors: Dwivedi S, Purohit P, Misra R, Lingeswaran M, Vishnoi JR, Pareek P, Misra S, Sharma P Abstract Breast cancer is recognized for its different clinical behaviors and patient outcomes, regardless of common histopathological features at diagnosis. The heterogeneity and dynamics of breast cancer undergoing clonal evolution produces cells with distinct degrees of drug resistance and metastatic potential. Presently, single cell analysis have made outstanding advancements, overshadowing the hurdles of heterogeneity linked with vast populations. The speedy progression in sequencing analysis now allow unbiased, high-output and high-resolution elucidation of the heterogeneity from individual cell within a population. Classical therapeutics strategies for individual patients are governed by the presence and absence of expression pattern of the estrogen and progesterone receptors and human epidermal growth factor receptor 2. However, such tactics for clinical classification have fruitfulness in selection of targeted therapies, short-term patient responses but unable to predict the long-term survival. In any phenotypic alterations, like breast cancer disease, molecular signature have proven its implication, as we aware that individual cell's state is regulated at diverse levels, such as DNA, RNA and protein, by multifaceted interplay of intrinsic biomolecules pathways existing in the organism and extrinsic stimuli such as ambient environment. Thus for complete understanding, complete profiling of single cell requires a synchronous investigations from different levels (multi-omics) to avoid incomplete information produced from single cell. In this article, initially we briefed on novel updates of various methods available to explore omics and then we finally pinpointed on various omics (i.e. genomics, transcriptomics, epigenomics, proteomics and metabolomics) data and few special aspects of circulating tumor cells, disseminated tumor cells and cancer stem cells, so far available from various studies that can be used for better management of breast cancer patients. PMID: 30728668 [PubMed]

Related Articles Therapeutic modulation of autophagy: which disease comes first? Cell Death Differ. 2019 Feb 06;: Authors: Maiuri MC, Kroemer G Abstract The relentless efforts of thousands of researchers have allowed deciphering the molecular machinery that regulates and executes autophagy, thus identifying multiple molecular targets to enhance or block the process, rendering autophagy "druggable". Autophagy inhibition may be useful for preserving the life of cells that otherwise would succumb to excessive self-digestion. Moreover, autophagy blockade may reduce the fitness of cancer cells or interrupt metabolic circuitries required for their growth. Autophagy stimulation is probably useful for the prevention or treatment of aging, cancer (when stimulation of immunosurveillance is the therapeutic goal), cardiovascular disease, cystic fibrosis, infection by intracellular pathogens, obesity, and intoxication by heavy metals, just to mention a few examples. Epidemiological evidence suggests broad health-improving effects for lifestyles, micronutrients, and drugs that favor autophagy. In this review, we discuss the role of autophagy in disease pathogenesis while focusing on the question, which disease will become the first clinically approved indication for therapeutic autophagy modulation. PMID: 30728461 [PubMed - as supplied by publisher]

Related Articles Short-Term and Long-Term Outcomes of Infliximab and Tacrolimus Treatment for Moderate to Severe Ulcerative Colitis: Retrospective Observational Study. Kobe J Med Sci. 2018 Dec 04;64(4):E140-E148 Authors: Otsuka T, Ooi M, Tobimatsu K, Wakahara C, Watanabe D, Adachi S, Yasutomi E, Yamairi H, Ku Y, Yoshida M, Hoshi N, Kodama Y Abstract BACKGROUND/AIMS: While some studies have shown that IFX and TAC exhibit similar efficacy against UC in the short-term, it is unclear which drug produces better long-term outcomes. In this study, we compared the long-term efficacy of IFX and TAC in patients with moderate to severe UC. METHODS: This retrospective study was conducted from 2009 to 2017. It included patients with no history of IFX or TAC treatment. We analyzed the clinical response and remission rates at 12 and 52 weeks, and colectomy-free and relapse-free survival were evaluated until the end of the study. RESULTS: At 12 weeks, 94.4% and 77.8% of the patients in the IFX group (n = 18) had demonstrated clinical responses and clinical remission, respectively, whereas 72.7% of the patients in the TAC group (n = 11) exhibited clinical responses and clinical remission. The clinical response, clinical remission, and colectomy-free rates did not differ significantly between the groups. At 52 weeks, clinical responses and clinical remission had been achieved in 76.5% and 70.6% of the patients both in the IFX group, respectively. In the TAC group, clinical responses and clinical remission were achieved in 50.0% of patients. Relapse-free and colectomy-free survival were estimated significantly better in IFX group evaluated by Kaplan-Meier curves. CONCLUSION: This study indicates that IFX and TAC produce similar short-term outcomes in UC patients, but IFX produces better long-term outcomes than TAC especially with avoidance of colectomy. Our data suggest that IFX therapy may be prioritized over TAC for the treatment of moderate to severe UC. PMID: 30728340 [PubMed - in process]

Related Articles Branched-Chain Amino Acid Catabolism Impacts Triacylglycerol Homeostasis in Chlamydomonas reinhardtii. Plant Physiol. 2019 Feb 06;: Authors: Liang Y, Kong F, Torres Romero I, Burlacot A, Cuiné S, Legeret B, Billon E, Brotman Y, Alseekh S, Fernie AR, Beisson F, Peltier G, Li-Beisson Y Abstract Nitrogen (N) starvation-induced triacylglycerol (TAG) synthesis and its complex relationship with starch metabolism in algal cells has been intensively studied; however, few studies have examined the interaction between amino acid metabolism and TAG biosynthesis. Here, via a forward genetic screen for TAG homeostasis, we isolated a Chlamydomonas mutant (bkdE1α) that is deficient in the E1α subunit of the branched-chain ketoacid dehydrogenase (BCKDH) complex. Metabolomics analysis revealed a defect in the catabolism of branched-chain amino acids (BCAAs) in bkdE1α. Furthermore, this mutant accumulated 30% less TAG than the parental strain during N starvation, and was compromised in TAG remobilization upon N resupply. Intriguingly, the rate of mitochondrial respiration was 20-35% lower in bkdE1α compared to the parental strains. Three additional knock-out mutants of the other components of the BCKDH complex exhibited phenotypes similar to that of bkdE1α. Transcriptional responses of BCKDH to different N statuses were consistent with its role in TAG homeostasis. Collectively, these results indicate that BCAA catabolism contributes to TAG metabolism by providing carbon precursors and ATP, thus highlighting the complex interplay between distinct subcellular metabolisms for oil storage in green microalgae. PMID: 30728273 [PubMed - as supplied by publisher]

Related Articles A reverse metabolic approach to weaning: in silico identification of immune-beneficial infant gut bacteria, mining their metabolism for prebiotic feeds and sourcing these feeds in the natural product space. Microbiome. 2018 09 21;6(1):171 Authors: Michelini S, Balakrishnan B, Parolo S, Matone A, Mullaney JA, Young W, Gasser O, Wall C, Priami C, Lombardo R, Kussmann M Abstract BACKGROUND: Weaning is a period of marked physiological change. The introduction of solid foods and the changes in milk consumption are accompanied by significant gastrointestinal, immune, developmental, and microbial adaptations. Defining a reduced number of infections as the desired health benefit for infants around weaning, we identified in silico (i.e., by advanced public domain mining) infant gut microbes as potential deliverers of this benefit. We then investigated the requirements of these bacteria for exogenous metabolites as potential prebiotic feeds that were subsequently searched for in the natural product space. RESULTS: Using public domain literature mining and an in silico reverse metabolic approach, we constructed probiotic-prebiotic-food associations, which can guide targeted feeding of immune health-beneficial microbes by weaning food; analyzed competition and synergy for (prebiotic) nutrients between selected microbes; and translated this information into designing an experimental complementary feed for infants enrolled in a pilot clinical trial ( http://www.nourishtoflourish.auckland.ac.nz/ ). CONCLUSIONS: In this study, we applied a benefit-oriented microbiome research strategy for enhanced early-life immune health. We extended from "classical" to molecular nutrition aiming to identify nutrients, bacteria, and mechanisms that point towards targeted feeding to improve immune health in infants around weaning. Here, we present the systems biology-based approach we used to inform us on the most promising prebiotic combinations known to support growth of beneficial gut bacteria ("probiotics") in the infant gut, thereby favorably promoting development of the immune system. PMID: 30241567 [PubMed - indexed for MEDLINE]

Related Articles Comprehensive Profiling of Fecal Metabolome of Mice by Integrated Chemical Isotope Labeling-Mass Spectrometry Analysis. Anal Chem. 2018 03 06;90(5):3512-3520 Authors: Yuan BF, Zhu QF, Guo N, Zheng SJ, Wang YL, Wang J, Xu J, Liu SJ, He K, Hu T, Zheng YW, Xu FQ, Feng YQ Abstract Gut microbiota plays important roles in the host health. The host and symbiotic gut microbiota coproduce a large number of metabolites during the metabolism of food and xenobiotics. The analysis of fecal metabolites can provide a noninvasive manner to study the outcome of the host-gut microbiota interaction. Herein, we reported the comprehensive profiling of fecal metabolome of mice by an integrated chemical isotope labeling combined with liquid chromatography-mass spectrometry (CIL-LC-MS) analysis. The metabolites are categorized into several submetabolomes based on the functional moieties (i.e., carboxyl, carbonyl, amine, and thiol) and then analysis of the individual submetabolome was performed. The combined data from the submetabolome form the metabolome with relatively high coverage. To this end, we synthesized stable isotope labeling reagents to label metabolites with different groups, including carboxyl, carbonyl, amine, and thiol groups. We detected 2302 potential metabolites, among which, 1388 could be positively or putatively identified in feces of mice. We then further confirmed 308 metabolites based on our established library of chemically labeled standards and tandem mass spectrometry analysis. With the identified metabolites in feces of mice, we established mice fecal metabolome database, which can be used to readily identify metabolites from feces of mice. Furthermore, we discovered 211 fecal metabolites exhibited significant difference between Alzheimer's disease (AD) model mice and wild type (WT) mice, which suggests the close correlation between the fecal metabolites and AD pathology and provides new potential biomarkers for the diagnosis of AD. PMID: 29406693 [PubMed - indexed for MEDLINE]

28 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2019/02/07PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

28 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2019/02/07PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

31 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2019/02/06PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

31 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2019/02/06PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

18 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2019/02/05PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.