RESUMEN
Tectona grandis Linn, commonly known as teak, is traditionally used to treat a range of diseases, including the common cold, headaches, bronchitis, scabies, diabetes, inflammation, and others. The present study was conducted with the purpose of isolating and identifying the active compounds in T. grandis leaf against a panel of Vibrio spp., which may induce vibriosis in shrimp, using bioassay-guided purification. The antimicrobial activity was assessed using the microdilution method, followed by the brine shrimp lethality assay to determine toxicity. Following an initial screening with a number of different solvents, it was established that the acetone extract was the most effective. The acetone extract was then exposed to silica gel chromatography followed by reversed-phase HPLC and further UHPLC-orbitrap-ion trap mass spectrometry to identify the active compounds. Three compounds called 1-hydroxy-2,6,8-trimethoxy-9,10-anthraquinone, deoxyanserinone B, and khatmiamycin were identified with substantial anti-microbial action against V. parahaemolyticus, V. alginolyticus, V. harveyi, V. anguillarum, and V. vulnificus. The IC50 values of the three compounds viz. 1-hydroxy-2,6,8-trimethoxy-9,10-anthraquinone, deoxyanserinone B, and khatmiamycin varied between 2 and 28, 7 and 38, and 7 and 56 µg/mL, respectively, which are as good as the standard antibiotics such as amoxicillin and others. The in vivo toxicity test revealed that the compounds were non-toxic to shrimp. The results of the study suggest that T. grandis leaf can be used as a source of bioactive compounds to treat Vibrio species in shrimp farming.
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Antibacterianos , Artemia , Bioensayo , Pruebas de Sensibilidad Microbiana , Extractos Vegetales , Hojas de la Planta , Vibrio , Animales , Vibrio/efectos de los fármacos , Extractos Vegetales/farmacología , Extractos Vegetales/química , Hojas de la Planta/química , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Artemia/efectos de los fármacos , Combretaceae/química , Fraccionamiento QuímicoRESUMEN
To find drivers of healthy ageing, a genome-wide association study (GWAS) was performed in healthy and unhealthy older individuals. Healthy individuals were defined as free from cardiovascular disease, stroke, heart failure, major adverse cardiovascular event, diabetes, dementia, cancer, chronic obstructive pulmonary disease (COPD), asthma, rheumatism, Crohn's disease, malabsorption or kidney disease. Six single nucleotide polymorphisms (SNPs) with unknown function associated with ten human genes were identified as candidate healthspan markers. Thirteen homologous or closely related genes were selected in the model organism C. elegans for evaluating healthspan after targeted RNAi-mediated knockdown using pathogen resistance, muscle integrity, chemotaxis index and the activity of known longevity and stress response pathways as healthspan reporters. In addition, lifespan was monitored in the RNAi-treated nematodes. RNAi knockdown of yap-1, wwp-1, paxt-1 and several acdh genes resulted in heterogeneous phenotypes regarding muscle integrity, pathogen resistance, chemotactic behaviour, and lifespan. Based on these observations, we hypothesize that their human homologues WWC2, CDKN2AIP and ACADS may play a role in health maintenance in the elderly.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anciano , Animales , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras , Estudio de Asociación del Genoma Completo , Humanos , Longevidad/genética , Fenotipo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Parasitic helminths continue to pose problems in human and veterinary medicine, as well as in agriculture. Semen pharbitidis, the seeds of Pharbitis nil (Linn.) Choisy (Convolvulaceae), is a well-known traditional Chinese medicinal botanical preparation widely used for treating intestinal parasites in China owing to its desirable efficacy. However, the anthelmintic compounds in Semen pharbitidis and their mechanism of action have not been investigated yet. This study aimed to identify the compounds active against helminths from Semen pharbitidis, and to establish the mechanism of action of these active compounds. Bioassay-guided fractionation was used to identify the anthelmintic compounds from Semen pharbitidis. The anthelmintic assay was performed by monitoring Caenorhabditis elegans (C. elegans) motility with a WMicrotracker instrument. Active compounds were identified by high-resolution mass spectrometry. Several (analogues of) fragments of the anthelmintic compounds were purchased and tested to explore the structure-activity relationship, and to find more potent compounds. A panel of C. elegans mutant strains resistant to major currently used anthelmintic drugs was used to explore the mechanism of action of the active compounds. The bioassay-guided isolation from an ethanol extract of Semen pharbitidis led to a group of glycosides, namely pharbitin (IC50: 41.0 ± 9.4 µg/mL). Hit expansion for pharbitin fragments yielded two potent analogues: 2-bromohexadecanoic acid (IC50: 1.6 ± 0.7 µM) and myristoleic acid (IC50: 35.2 ± 7.6 µM). One drug-resistant mutant ZZ37 unc-63 (x37) demonstrated a ~17-fold increased resistance to pharbitin compared with wild-type worms. Collectively, we provide further experimental scientific evidence to support the traditional use of Semen pharbitidis for the treatment of intestinal parasites. The anthelmintic activity of Semen pharbitidis is due to pharbitin, whose target could be UNC-63 in C. elegans.
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Antihelmínticos , Extractos Vegetales , Animales , Humanos , Extractos Vegetales/química , Caenorhabditis elegans , Semillas , Antihelmínticos/farmacología , Antihelmínticos/química , Glicósidos/farmacología , Bioensayo/métodosRESUMEN
Helminths, with an estimated 1.5 billion annual global infections, are one of the major health challenges worldwide. The current strategy of the World Health Organization to prevent helminth infection includes increasing hygienic awareness, providing better sanitation and preventative anthelmintic drug therapy in vulnerable populations. Nowadays, anthelmintic drugs are used heavily in livestock, both in case of infection and as a preventative measure. However, this has led to the development of resistance against several of the most common drugs, such as levamisole, ivermectin and thiabendazole. As many as 70% of the livestock in developed countries now has helminths that are drug resistant, and multiple resistance is common. Because of this, novel anthelmintics are urgently needed to help combat large-scale production losses. Prior to this review, no comprehensive review of the anthelmintic effects of essential oils and their components existed. Multiple review articles have been published on the uses of a single plant and its extracts that only briefly touch upon their anthelmintic activity. This review aims to provide a detailed overview of essential oils and their components as anthelmintic treatment against a wider variety of helminths.
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Antihelmínticos , Helmintiasis , Helmintos , Aceites Volátiles , Animales , Humanos , Aceites Volátiles/farmacología , Aceites Volátiles/uso terapéutico , Antihelmínticos/farmacología , Antihelmínticos/uso terapéutico , Helmintiasis/tratamiento farmacológico , Levamisol/farmacología , Resistencia a MedicamentosRESUMEN
Several biogerontology databases exist that focus on genetic or gene expression data linked to health as well as survival, subsequent to compound treatments or genetic manipulations in animal models. However, none of these has yet collected experimental results of compound-related health changes. Since quality of life is often regarded as more valuable than length of life, we aim to fill this gap with the "Healthy Worm Database" ( http://healthy-worm-database.eu ). Literature describing health-related compound studies in the aging model Caenorhabditis elegans was screened, and data for 440 compounds collected. The database considers 189 publications describing 89 different phenotypes measured in 2995 different conditions. Besides enabling a targeted search for promising compounds for further investigations, this database also offers insights into the research field of studies on healthy aging based on a frequently used model organism. Some weaknesses of C. elegans-based aging studies, like underrepresented phenotypes, especially concerning cognitive functions, as well as the convenience-based use of young worms as the starting point for compound treatment or phenotype measurement are discussed. In conclusion, the database provides an anchor for the search for compounds affecting health, with a link to public databases, and it further highlights some potential shortcomings in current aging research.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Envejecimiento , Animales , Longevidad , Calidad de VidaRESUMEN
Recently, nine Caenorhabditis elegans genes, grouped into two pathways/clusters, were found to be implicated in healthspan in C. elegans and their homologues in humans, based on literature curation, WormBase data mining and bioinformatics analyses. Here, we further validated these genes experimentally in C. elegans. We downregulated the nine genes via RNA interference (RNAi), and their effects on physical function (locomotion in a swim assay) and on physiological function (survival after heat stress) were analysed in aged nematodes. Swim performance was negatively affected by the downregulation of acox-1.1, pept-1, pak-2, gsk-3 and C25G6.3 in worms with advanced age (twelfth day of adulthood) and heat stress resistance was decreased by RNAi targeting of acox-1.1, daf-22, cat-4, pig-1, pak-2, gsk-3 and C25G6.3 in moderately (seventh day of adulthood) or advanced aged nematodes. Only one gene, sad-1, could not be linked to a health-related function in C. elegans with the bioassays we selected. Thus, most of the healthspan genes could be re-confirmed by health measurements in old worms.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Estrés Fisiológico , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Humanos , Longevidad/genéticaRESUMEN
Disease epidemics in shrimp aquaculture increase apace with the development of aquaculture systems throughout the world. The disease caused by Vibrio spp. (vibriosis) is considered the most devastating, which has made it the most feared bacterial disease in the shrimp sector. In aquaculture, several strategies have already been applied to control Vibrio strains, including chemicals, probiotics, antibiotics, natural products from plants, including plant oils; hence, there has been considerable attention for using plants in shrimp aquaculture to provide sustainable, eco-friendly and safe compounds, such as alkaloids, saponins, terpenoids and flavonoids for replacing chemical compounds and antibiotics in current aquaculture. Medicinal plants may also have immunostimulating activity, increase growth and resistance in shrimps. The present paper aims to review the inhibition of Vibrio spp. in shrimp by medicinal plants, using both in vitro or/and in vivo techniques. Several medicinal plants appear capable of inhibiting growth of Vibrio pathogens outside living shrimp or in the body of shrimp, through enhancing growth and immune capacity when shrimps are fed or injected with them. In the current review Gracilaria spp. (Gracilariaceae family) and Sargassum spp. (family Sargassaceae) have been used most for in vitro and in vivo experiments. Among the terrestrial plants, Eucalyptus camaldulensis, Psidium guajava, Rhodomyrtus tomentosa, and Syzygium cumini (Myrtaceae family) had significant activity against Vibrio.
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Adyuvantes Inmunológicos/farmacología , Antibacterianos/farmacología , Penaeidae/inmunología , Extractos Vegetales/farmacología , Plantas Medicinales , Vibrio/efectos de los fármacos , Animales , Acuicultura/métodos , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/prevención & control , Infecciones por Bacterias Gramnegativas/veterinariaRESUMEN
Polygodial is a "hot" peppery-tasting sesquiterpenoid that was first described for its anti-feedant activity against African armyworms. Using the haploid deletion mutant library of Saccharomyces cerevisiae, a genome-wide mutant screen was performed to shed more light on polygodial's antifungal mechanism of action. We identified 66 deletion strains that were hypersensitive and 47 that were highly resistant to polygodial treatment. Among the hypersensitive strains, an enrichment was found for genes required for vacuolar acidification, amino acid biosynthesis, nucleosome mobilization, the transcription mediator complex, autophagy and vesicular trafficking, while the resistant strains were enriched for genes encoding cytoskeleton-binding proteins, ribosomal proteins, mitochondrial matrix proteins, components of the heme activator protein (HAP) complex, and known regulators of the target of rapamycin complex 1 (TORC1) signaling. WE confirm that polygodial triggers a dose-dependent vacuolar alkalinization and that it increases Ca2+ influx and inhibits glucose-induced Ca2+ signaling. Moreover, we provide evidence suggesting that TORC1 signaling and its protective agent ubiquitin play a central role in polygodial resistance, suggesting that they can be targeted by polygodial either directly or via altered Ca2+ homeostasis.
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Antifúngicos/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Antifúngicos/química , Calcio , Farmacorresistencia Fúngica/genética , Homeostasis/efectos de los fármacos , Concentración de Iones de Hidrógeno , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Pruebas de Sensibilidad Microbiana , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Nucleosomas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/química , Sesquiterpenos/farmacología , Transducción de Señal , ATPasas de Translocación de Protón VacuolaresRESUMEN
Bio-active peptides are involved in the regulation of most physiological processes in the body. Classical bio-active peptides (CBAPs) are cleaved from a larger precursor protein and stored in secretion vesicles from which they are released in the extracellular space. Recently, another non-classical type of bio-active peptides (NCBAPs) has gained interest. These typically are not secreted but instead appear to be translated from short open reading frames (sORF) and released directly into the cytoplasm. In contrast to CBAPs, these peptides are involved in the regulation of intra-cellular processes such as transcriptional control, calcium handling and DNA repair. However, bio-chemical evidence for the translation of sORFs remains elusive. Comprehensive analysis of sORF-encoded polypeptides (SEPs) is hampered by a number of methodological and biological challenges: the low molecular mass (many 4-10 kDa), the low abundance, transient expression and complications in data analysis. We developed a strategy to address a number of these issues. Our strategy is to exclude false positive identifications. In total sample, we identified 926 peptides originated from 37 known (neuro)peptide precursors in mouse striatum. In addition, four SEPs were identified including NoBody, a SEP that was previously discovered in humans and three novel SEPS from 5' untranslated transcript regions (UTRs).
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High attrition of new oncology drug candidates in clinical trials is partially caused by the poor predictive capacity of artificial monolayer cell culture assays early in drug discovery. Monolayer assays do not take the natural three-dimensional (3D) microenvironment of cells into account. As a result, false positive compounds often enter clinical trials, leading to high dropout rates and a waste of time and money. Over the past 2 decades, tissue engineers and cell biologists have developed a broad range of 3D in vitro culturing tools that better represent in vivo cell biology. These tools preserve the 3D architecture of cells and can be used to predict toxicity of and resistance against antitumor agents. Recent progress in tissue engineering further improves 3D models by taking into account the tumor microenvironment, which is important for metastatic progression and vascularization. However, the widespread implementation of 3D cell cultures into cell-based research programs has been limited by various factors, including their cost and reproducibility. In addition, different 3D cell culture techniques often produce spheroids of different size and shape, which can strongly influence drug efficacy and toxicity. Hence, it is imperative to morphometrically characterize multicellular spheroids to avoid generalizations among different spheroid types. Standardized 3D culturing procedures could further reduce data variability and enhance biological relevance. Here, we critically evaluate the benefits and challenges inherent to growing cells in 3D, along with an overview of the techniques used to form spheroids. This is done with a specific focus on antitumor drug screening.
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Técnicas de Cultivo de Célula/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Animales , Humanos , Esferoides Celulares/patologíaRESUMEN
Human longevity continues to increase world-wide, often accompanied by decreasing birth rates. As a larger fraction of the population thus gets older, the number of people suffering from disease or disability increases dramatically, presenting a major societal challenge. Healthy ageing has therefore been selected by EU policy makers as an important priority ( http://www.healthyageing.eu/european-policies-and-initiatives ); it benefits not only the elderly but also their direct environment and broader society, as well as the economy. The theme of healthy ageing figures prominently in the Horizon 2020 programme ( https://ec.europa.eu/programmes/horizon2020/en/h2020-section/health-demographic-change-and-wellbeing ), which has launched several research and innovation actions (RIA), like "Understanding health, ageing and disease: determinants, risk factors and pathways" in the work programme on "Personalising healthcare" ( https://ec.europa.eu/research/participants/portal/desktop/en/opportunities/h2020/topics/693-phc-01-2014.html ). Here we present our research proposal entitled "ageing with elegans" (AwE) ( http://www.h2020awe.eu/ ), funded by this RIA, which aims for better understanding of the factors causing health and disease in ageing, and to develop evidence-based prevention, diagnostic, therapeutic and other strategies. The aim of this article, authored by the principal investigators of the 17 collaborating teams, is to describe briefly the rationale, aims, strategies and work packages of AwE for the purposes of sharing our ideas and plans with the biogerontological community in order to invite scientific feedback, suggestions, and criticism.
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Envejecimiento/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Estilo de Vida Saludable/fisiología , Longevidad/fisiología , Modelos Animales , AnimalesRESUMEN
We report the design, synthesis and antibacterial activity analysis of conjugates of vancomycin and cathelicidin-related antimicrobial peptides (CRAMP). Vancomycin inhibits the nascent peptidoglycan synthesis and is highly active against Gram-positive bacteria, whereas Gram-negative bacteria are generally insensitive due to a protective outer membrane. CRAMP is known to translocate across the Gram-negative outer membrane by a self-promoted uptake mechanism. Vancomycin-CRAMP conjugates were synthesized using click chemistry with diverse hydrophilic and hydrophobic linkers, with CRAMP functioning as a carrier peptide for the transfer of vancomycin through the outer membrane. Small hydrophobic linkers with an aromatic group result in the most active conjugates against planktonic Gram-negative bacteria, while maintaining the high activity of vancomycin against Gram-positive bacteria. These conjugates thus show a broad-spectrum activity, which is absent in CRAMP or vancomycin alone, and which is strongly improved compared to an equimolar mixture of CRAMP and vancomycin. In addition, these conjugates also show a strong inhibitory activity against S. Typhimurium biofilm formation.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Catelicidinas/farmacología , Vancomicina/farmacología , Secuencia de Aminoácidos , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Biopelículas/crecimiento & desarrollo , Catelicidinas/química , Cromatografía Liquida , Interacciones Hidrofóbicas e Hidrofílicas , Isomerismo , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Vancomicina/químicaRESUMEN
We identified a 26-amino-acid truncated form of the 34-amino-acid cathelicidin-related antimicrobial peptide (CRAMP) in the islets of Langerhans of the murine pancreas. This peptide, P318, shares 67% identity with the LL-37 human antimicrobial peptide. As LL-37 displays antimicrobial and antibiofilm activity, we tested antifungal and antibiofilm activity of P318 against the fungal pathogen Candida albicans. P318 shows biofilm-specific activity as it inhibits C. albicans biofilm formation at 0.15 µM without affecting planktonic survival at that concentration. Next, we tested the C. albicans biofilm-inhibitory activity of a series of truncated and alanine-substituted derivatives of P318. Based on the biofilm-inhibitory activity of these derivatives and the length of the peptides, we decided to synthesize the shortened alanine-substituted peptide at position 10 (AS10; KLKKIAQKIKNFFQKLVP). AS10 inhibited C. albicans biofilm formation at 0.22 µM and acted synergistically with amphotericin B and caspofungin against mature biofilms. AS10 also inhibited biofilm formation of different bacteria as well as of fungi and bacteria in a mixed biofilm. In addition, AS10 does not affect the viability or functionality of different cell types involved in osseointegration of an implant, pointing to the potential of AS10 for further development as a lead peptide to coat implants.
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Antibacterianos/farmacología , Antifúngicos/farmacología , Bacterias/efectos de los fármacos , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Catelicidinas/farmacología , Anfotericina B/farmacología , Animales , Péptidos Catiónicos Antimicrobianos/farmacología , Caspofungina , Equinocandinas/farmacología , Humanos , Lipopéptidos , Ratones , Pruebas de Sensibilidad Microbiana/métodos , Plancton/efectos de los fármacosRESUMEN
SUMMARY: We present PIUS, a tool that identifies peptides from tandem mass spectrometry data by analyzing the six-frame translation of a complete genome. It differs from earlier studies that have performed such a genomic search in two ways: (i) it considers a larger search space and (ii) it is designed for natural peptide identification rather than proteomics. Differently from other peptidomics tools designed for genome-wide searches, PIUS does not limit the analysis to a set of sequences that match a list of de novo reconstructions. AVAILABILITY: Source code, executables and a detailed technical report are freely available at http://dtai.cs.kuleuven.be/ml/systems/pius. CONTACT: eduardo.costa@cs.kuleuven.be SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Péptidos/química , Programas Informáticos , Espectrometría de Masas en Tándem , Algoritmos , Animales , Línea Celular , Bases de Datos de Proteínas , Genoma , Genómica , Ratones , Péptidos/análisis , Proteómica/métodos , Análisis de Secuencia de ProteínaRESUMEN
This study was conducted to isolate and identify the bioactive compounds from the ethanolic extract of Syzygium cumini leaf against Vibrio species through a bioassay-guided fractionation. The ethanol extract was exposed to silica gel chromatography followed by reversed phase HPLC to isolate the most effective fraction against V. parahaemolyticus. Using further UHPLC-orbitrap-ion trap mass spectrometry, five compounds were isolated with broad-spectrum potency against a range of Vibrio species viz. V. parahaemolyticus, V. alginolyticus, V. harveyi, V. vulnificus and V. anguillarum. The IC50 values for the compounds ranged from 8 to 48 µg/mL against the most sensitive species V. vulnificus and 58 to >400 µg/mL against V. alginolyticus. The results of the toxicity tests demonstrated that the compounds were not harmful for shrimp. The study's findings indicate that S. cumini leaf extract may contain bioactive molecules that are able to be substituted for antibiotics to treat vibriosis in shrimp farming.
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BACKGROUND: In Chinese Pharmacopeia, Picrasma quassioides (PQ) stems and leaves are recorded as Kumu with antimicrobial, anti-cancer, anti-parasitic effects, etc. However, thick stems are predominantly utilized as medicine in many Asian countries, with leaves rarely used. By now, the phytochemistry and bioactivity of PQ leaves are not well investigated. METHODS: An Orbitrap Elite mass spectrometer was employed to comprehensively investigate PQ stems and leaves sourced from 7 different locations. Additionally, their bioactivities were evaluated against 5 fungi, 6 Gram-positive bacteria and 9 Gram-negative bacteria, a tumor cell line (A549), a non-tumor cell line (WI-26 VA4) and N2 wild-type Caenorhabditis elegans. RESULTS: Bioassay results demonstrated the efficacy of both leaves and stems against tumor cells, several bacteria and fungi, while only leaves exhibited anthelmintic activity against C. elegans. A total of 181 compounds were identified from PQ stems and leaves, including 43 ß-carbolines, 20 bis ß-carbolines, 8 canthinone alkaloids, 56 quassinoids, 12 triterpenoids, 13 terpenoid derivatives, 11 flavonoids, 7 coumarins, and 11 phenolic derivatives, from which 10 compounds were identified as indicator components for quality evaluation. Most alkaloids and triterpenoids were concentrated in PQ stems, while leaves exhibited higher levels of quassinoids and other carbohydrate (CHO) components. CONCLUSION: PQ leaves exhibit distinct chemical profiles and bioactivity with the stems, suggesting their suitability for medicinal purposes. So far, the antibacterial, antifungal, and anthelmintic activities of PQ leaves were first reported here, and considering PQ sustainability, the abundant leaves are recommended for increased utilization, particularly for their rich content of PQ quassinoids.
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Caenorhabditis elegans , Fitoquímicos , Picrasma , Hojas de la Planta , Tallos de la Planta , Hojas de la Planta/química , Picrasma/química , Animales , Tallos de la Planta/química , Caenorhabditis elegans/efectos de los fármacos , Fitoquímicos/farmacología , Fitoquímicos/aislamiento & purificación , Humanos , Línea Celular Tumoral , Estructura Molecular , Antineoplásicos Fitogénicos/farmacología , Alcaloides/farmacología , Cuassinas/farmacología , Cuassinas/química , Cuassinas/aislamiento & purificación , Antihelmínticos/farmacología , Antihelmínticos/química , Hongos/efectos de los fármacos , Flavonoides/farmacología , Flavonoides/análisisRESUMEN
Three previously unidentified dihydrostilbene glycosides, named oleiferaside A (1), oleiferaside B (2), and oleiferaside C (3), were discovered through a phytochemical exploration on Camellia oleifera Abel. leaves. Additionally, nine known secondary metabolites (4-12) were also identified. The undescribed secondary metabolites 1-3 were elucidated as 3,5-dimethoxydihydrostilbene 4'-O-α-l-arabinofuranosyl-(1 â 6)-ß-d- glucopyranoside, 3,5-dimethoxydihydrostilbene 4'-O-α-l-arabinopyranosyl-(1 â 6)-ß-d- glucopyranoside and 3,5-dimethoxydihydrostilbene 4'-O-ß-d-apiofuranosyl-(1 â 6)-ß-d- glucopyranoside, respectively. HR-MS and NMR spectroscopy were utilized for determining the structures of the isolates. The natural products were assessed for their anti-inflammatory effect using RAW264.7 macrophage stimulated by LPS. The findings demonstrated that compounds 1-4 exhibited inhibitory activities on NO and PGE2 production without causing cytotoxicity. These observations suggest that these compounds may have potential anti-inflammatory properties.
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Tandem mass spectrometry (MS/MS) combined with bioinformatics tools have enabled fast and systematic protein identification based on peptide-to-spectrum matches. However, it remains challenging to obtain accurate identification of endogenous peptides, such as neuropeptides, peptide hormones, peptide pheromones, venom peptides, and antimicrobial peptides. Since these peptides are processed at sites that are difficult to predict reliably, the search of their MS/MS spectra in sequence databases needs to be done without any protease setting. In addition, many endogenous peptides carry various post-translational modifications, making it essential to take these into account in the database search. These characteristics of endogenous peptides result in a huge search space, frequently leading to poor confidence of the peptide characterizations in peptidomics studies. We have developed a new MS/MS spectrum search tool for highly accurate and confident identification of endogenous peptides by combining two different fragmentation methods. Our approach takes advantage of the combination of two independent fragmentation methods (collision-induced dissociation and electron transfer dissociation). Their peptide spectral matching is carried out separately in both methods, and the final score is built as a combination of the two separate scores. We demonstrate that this approach is very effective in discriminating correct peptide identifications from false hits. We applied this approach to a spectral data set of neuropeptides extracted from mouse pituitary tumor cells. Compared to conventional MS-based identification, i.e., using a single fragmentation method, our approach significantly increased the peptide identification rate. It proved also highly effective for scanning spectra against a very large search space, enabling more accurate genome-wide searches and searches including multiple potential post-translational modifications.
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Electrones , Neuropéptidos/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificación , Hipófisis/química , Secuencia de Aminoácidos , Animales , Transporte de Electrón , Ratones , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Neuropéptidos/química , Fragmentos de Péptidos/química , Hipófisis/citología , Proteolisis , Proteómica , Espectrometría de Masas en Tándem , Células Tumorales CultivadasRESUMEN
BACKGROUND: It was long assumed that proteins are at least 100 amino acids (AAs) long. Moreover, the detection of short translation products (e.g. coded from small Open Reading Frames, sORFs) is very difficult as the short length makes it hard to distinguish true coding ORFs from ORFs occurring by chance. Nevertheless, over the past few years many such non-canonical genes (with ORFs < 100 AAs) have been discovered in different organisms like Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster. Thanks to advances in sequencing, bioinformatics and computing power, it is now possible to scan the genome in unprecedented scrutiny, for example in a search of this type of small ORFs. RESULTS: Using bioinformatics methods, we performed a systematic search for putatively functional sORFs in the Mus musculus genome. A genome-wide scan detected all sORFs which were subsequently analyzed for their coding potential, based on evolutionary conservation at the AA level, and ranked using a Support Vector Machine (SVM) learning model. The ranked sORFs are finally overlapped with ribosome profiling data, hinting to sORF translation. All candidates are visually inspected using an in-house developed genome browser. In this way dozens of highly conserved sORFs, targeted by ribosomes were identified in the mouse genome, putatively encoding micropeptides. CONCLUSION: Our combined genome-wide approach leads to the prediction of a comprehensive but manageable set of putatively coding sORFs, a very important first step towards the identification of a new class of bioactive peptides, called micropeptides.