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1.
Physiol Plant ; 176(3): e14338, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38740528

RESUMO

Bacteria can be applied as biofertilizers to improve crop growth in phosphorus (P)-limited conditions. However, their mode of action in a soil environment is still elusive. We used the strain ALC_02 as a case study to elucidate how Bacillus subtilis affects dwarf tomato cultivated in soil-filled rhizoboxes over time. ALC_02 improved plant P acquisition by increasing the size and P content of P-limited plants. We assessed three possible mechanisms, namely root growth stimulation, root hair elongation, and solubilization of soil P. ALC_02 produced auxin, and inoculation with ALC_02 promoted root growth. ALC_02 promoted root hair elongation as the earliest observed response and colonized root hairs specifically. Root and root hair growth stimulation was associated with a subsequent increase in plant P content, indicating that a better soil exploration by the root system improved plant P acquisition. Furthermore, ALC_02 affected the plant-available P content in sterilized soil differently over time and released P from native P pools in the soil. Collectively, ALC_02 exhibited all three mechanisms in a soil environment. To our knowledge, bacterial P biofertilizers have not been reported to colonize and elongate root hairs in the soil so far, and we propose that these traits contribute to the overall effect of ALC_02. The knowledge gained in this research can be applied in the future quest for bacterial P biofertilizers, where we recommend assessing all three parameters, not only root growth and P solubilization, but also root hair elongation. This will ultimately support the development of sustainable agricultural practices.


Assuntos
Bacillus subtilis , Fósforo , Raízes de Plantas , Solo , Solanum lycopersicum , Fósforo/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Raízes de Plantas/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Solo/química , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/microbiologia , Solanum lycopersicum/metabolismo , Microbiologia do Solo , Solubilidade , Ácidos Indolacéticos/metabolismo , Fertilizantes
2.
Foot Ankle Surg ; 30(1): 44-49, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37718148

RESUMO

BACKGROUND: Different coatings of the metal implants of STAR prostheses have been used since 1999. In Europe metal implants with a double calcium-phosphate coating (BONIT) on a titanium sprayed surface have been available since 1999. METHODS: We present a 2-17 year follow-up of a consecutive series from a single center with 474 STAR ankle replacements where the BONIT type of coating has been used. RESULTS: 55 prostheses (12%) have been revised, the majority of them due to fracture of the mobile bearing. 22 prostheses (5%) have been converted to an arthrodesis. Analysis of survival of the specific components showed an estimated 10-year survival rate of the tibia component, talus component and polyethylene mobile bearing of 99%, 98% and 84%, respectively. The corresponding estimated 15-year survival was 98%, 98% and 74%, respectively. CONCLUSION: This study showed an extraordinary high survival rate of the metal implants. LEVEL OF EVIDENCE: Level III, prospective cohort series.


Assuntos
Artroplastia de Substituição do Tornozelo , Prótese Articular , Humanos , Seguimentos , Tornozelo/cirurgia , Articulação do Tornozelo/cirurgia , Estudos Prospectivos , Reoperação , Polietileno , Desenho de Prótese
3.
Opt Lett ; 48(16): 4225-4228, 2023 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-37581998

RESUMO

Variation of the brain temperature is strongly affected by blood flow, oxygen supply, and neural cell metabolism. Localized monitoring of the brain temperature is one of the most effective ways to correlate brain functions and diseases such as stroke, epilepsy, and mood disorders. While polymer optical fibers (POFs) are considered ideal candidates for temperature sensing in the brain, they have never been used so far in vivo. Here, we developed for the first, to the best of our knowledge, time an implantable probe based on a microstructured polymer optical fiber Bragg grating (FBG) sensor for intracranial brain temperature mapping. The temperature at different depths of the brain (starting from the cerebral cortex) and the correlation between the brain and body core temperature of a rat were recorded with a sensitivity of 33 pm/°C and accuracy <0.2°C. Our in vivo experimental results suggest that the proposed device can achieve real-time and high-resolution local temperature measurement in the brain, as well as being integrated with existing neural interfaces.


Assuntos
Fibras Ópticas , Termografia , Animais , Ratos , Temperatura , Polímeros , Encéfalo
4.
Metab Eng ; 67: 153-163, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34174425

RESUMO

Filamentous fungi secrete protein with a very high efficiency, and this potential can be exploited advantageously to produce therapeutic proteins at low costs. A significant barrier to this goal is posed by the fact that fungal N-glycosylation varies substantially from that of humans. Inappropriate N-glycosylation of therapeutics results in reduced product quality, including poor efficacy, decreased serum half-life, and undesirable immune reactions. One solution to this problem is to reprogram the glycosylation pathway of filamentous fungi to decorate proteins with glycans that match, or can be remodeled into, those that are accepted by humans. In yeast, deletion of ALG3 leads to the accumulation of Man5GlcNAc2 glycan structures that can act as a precursor for remodeling. However, in Aspergilli, deletion of the ALG3 homolog algC leads to an N-glycan pool where the majority of the structures contain more hexose residues than the Man3-5GlcNAc2 species that can serve as substrates for humanized glycan structures. Hence, additional strain optimization is required. In this report, we have used gene deletions in combination with enzymatic and chemical glycan treatments to investigate N-glycosylation in the model fungus Aspergillus nidulans. In vitro analyses showed that only some of the N-glycan structures produced by a mutant A. nidulans strain, which is devoid of any of the known ER mannose transferases, can be trimmed into desirable Man3GlcNAc2 glycan structures, as substantial amounts of glycan structures appear to be capped by glucose residues. In agreement with this view, deletion of the ALG6 homolog algF, which encodes the putative α-1,3- glucosyltransferase that adds the first glucose residue to the growing ER glycan structure, dramatically reduces the amounts of Hex6-7HexNAc2 structures. Similarly, these structures are also sensitive to overexpression of the genes encoding the heterodimeric α-glucosidase II complex. Without the glucose caps, a new set of large N-glycan structures was formed. Formation of this set is mostly, perhaps entirely, due to mannosylation, as overexpression of the gene encoding mannosidase activity led to their elimination. Based on our new insights into the N-glycan processing in A. nidulans, an A. nidulans mutant strain was constructed in which more than 70% of the glycoforms appear to be Man3-5GlcNAc2 species, which may serve as precursors for further engineering in order to create more complex human-like N-glycan structures.


Assuntos
Aspergillus nidulans , Glicosilação , Polissacarídeos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Glucosiltransferases , Humanos , Manosiltransferases/metabolismo , Proteínas de Membrana , Microrganismos Geneticamente Modificados , Polissacarídeos/genética
5.
Fungal Genet Biol ; 139: 103377, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32251730

RESUMO

Certain Aspergillus species such as Aspergillus flavus and A. parasiticus are well known for the formation of sclerotia. These developmental structures are thought to act as survival structures during adverse environmental conditions but are also a prerequisite for sexual reproduction. We previously described an A. niger mutant (scl-2) which formed sclerotium-like structures, suggesting a possible first stage of sexual development in this species. Several lines of evidence presented in this study support the previous conclusion that the sclerotium-like structures of scl-2 are indeed sclerotia. These included the observations that: (i) safranin staining of the sclerotia-like structures produced by the scl-2 mutant showed the typical cellular structure of a sclerotium; (ii) metabolite analysis revealed specific production of indoloterpenes, which have previously been connected to sclerotium formation; (iii) formation of the sclerotium-like structures is dependent on a functional NADPH complex, as shown for other fungi forming sclerotia. The mutation in scl-2 responsible for sclerotium formation was identified using parasexual crossing and bulk segregant analysis followed by high throughput sequencing and subsequent complementation analysis. The scl-2 strain contains a mutation that introduces a stop codon in the putative DNA binding domain of a previously uncharacterized Zn(II)2Cys6 type transcription factor (An08g07710). Targeted deletion of this transcription factor (sclB) confirmed its role as a repressor of sclerotial formation and in the promotion of asexual reproduction in A. niger. Finally, a genome-wide transcriptomic comparison of RNA extracted from sclerotia versus mycelia revealed major differences in gene expression. Induction of genes related to indoloterpene synthesis was confirmed and also let to the identification of a gene cluster essential for the production of aurasperones during sclerotium formation. Expression analysis of genes encoding other secondary metabolites, cell wall related genes, transcription factors, and genes related to reproductive processes identified many interesting candidate genes to further understand the regulation and biosynthesis of sclerotia in A. niger. The newly identified SclB transcription factor acts as a repressor of sclerotium formation and manipulation of sclB may represent a first prerequisite step towards engineering A. niger strains capable of sexual reproduction. This will provide exciting opportunities for further strain improvement in relation to protein or metabolite production in A. niger.


Assuntos
Aspergillus niger/genética , Proteínas Fúngicas/genética , Micélio/genética , Fatores de Transcrição/genética , Aspergillus niger/patogenicidade , Mutação/genética , Micélio/crescimento & desenvolvimento , Domínios Proteicos/genética , Reprodução Assexuada/genética , Esporos Fúngicos/genética , Zinco/química
6.
J Nat Prod ; 81(4): 785-790, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29488766

RESUMO

Cyclopiamines C (1) and D (2) were isolated from the extract of Penicillium sp. CML 3020, a fungus sourced from an Atlantic Forest soil sample. Their structures and relative configuration were determined by 1D and 2D NMR, HRMS, and UV/vis data analysis. Cyclopiamines C and D belong to a small subset of rare spiroindolinone compounds containing an alkyl nitro group and a 4,5-dihydro-1 H-pyrrolo[3,2,1- ij]quinoline-2,6-dione ring system. NMR and MS/HRMS data confirmed the presence of an epoxide unit (C-17-O-C-18) and a hydroxy group at C-5, not observed for their known congeners. Cytotoxic and antimicrobial activities were evaluated.


Assuntos
Antibacterianos/química , Compostos de Epóxi/química , Alcaloides Indólicos/química , Penicillium/química , Compostos de Espiro/química , Antibacterianos/isolamento & purificação , Compostos de Epóxi/isolamento & purificação , Alcaloides Indólicos/isolamento & purificação , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Compostos de Espiro/isolamento & purificação
7.
Appl Microbiol Biotechnol ; 102(19): 8477-8491, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29995241

RESUMO

Cereals are vulnerable substrates for fungal growth and subsequent mycotoxin contamination. One of the major fungal genera to colonize the ecosystem of stored grain is Penicillium, especially species in the series of Viridicata and Verrucosa. Culturing these species on grains, we hoped to induce the production of relevant secondary metabolites produced by these fungi in the early stage of cereal breakdown. In a multivariate setup six different cereal grains (wheat, rye, barley, oat, rice, and maize), one kind of white beans, and two standard fungal media, Yeast Extract Sucrose agar (YES agar) and Czapek Yeast Autolysate agar (CYA agar), were inoculated with the ten most important cereal-associated species from Penicillium (P. aurantiogriseum, P. cyclopium, P. freii, P. melanoconidium, P. neoechinulatum, P. polonicum, P. tricolor, P. viridicatum, P. hordei, and P. verrucosum). P. nordicum is a meat-associated species, which was included due to its chemical association with P. verrucosum, in addition to see if a substrate change would alter the profile of known chemistry. We found that cereals function very well as substrates for secondary metabolite production, but did not present significantly different secondary metabolite profiles, concerning known chemistry, as compared to standard laboratory agar media. However, white beans altered the semi-quantitative secondary metabolite profiles for several species. Correlations between substrates and certain metabolites were observed, as illuminated by principal component analysis. Many bioactive secondary metabolites were observed for the first time in the analyzed fungal species, including ergot type alkaloids in P. hordei.


Assuntos
Grão Comestível/microbiologia , Penicillium/metabolismo , Metabolismo Secundário , Meios de Cultura , Contaminação de Alimentos , Microbiologia de Alimentos , Hordeum/microbiologia , Micotoxinas , Triticum/microbiologia
8.
Appl Environ Microbiol ; 83(9)2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28213548

RESUMO

Only 1% of marine bacteria are currently culturable using standard laboratory procedures, and this is a major obstacle for our understanding of the biology of marine microorganisms and for the discovery of novel microbial natural products. Therefore, the purpose of this study was to investigate if improved cultivation conditions, including the use of an alternative gelling agent and supplementation with signaling molecules, improve the culturability of bacteria from seawater. Replacing agar with gellan gum improved viable counts 3- to 40-fold, depending on medium composition and incubation conditions, with a maximum of 6.6% culturability relative to direct cell counts. Through V4 amplicon sequencing we found that culturable diversity was also affected by a change in gelling agent, facilitating the growth of orders not culturable on agar-based substrates. Community analyses showed that communities grown on gellan gum substrates were significantly different from communities grown on agar and that they covered a larger fraction of the seawater community. Other factors, such as incubation temperature and time, had less obvious effects on viable counts and culturable diversity. Supplementation with acylated homoserine lactones (AHLs) did not have a positive effect on total viable counts or a strong effect on culturable diversity. However, low concentrations of AHLs increased the relative abundance of sphingobacteria. Hence, with alternative growth substrates, it is possible to significantly increase the number and diversity of cultured marine bacteria.IMPORTANCE Serious challenges to human health, such as the occurrence and spread of antibiotic resistance and an aging human population in need of bioactive pharmaceuticals, have revitalized the search for natural microbial products. The marine environment, representing the largest ecosystem in the biosphere, harbors an immense and virtually untapped microbial diversity producing unique bioactive compounds. However, we are currently able to cultivate only a minute fraction of this diversity. The lack of cultivated microbes is hampering not only bioprospecting efforts but also our general understanding of marine microbes. In this study, we present a means to increase the number and diversity of cultured bacteria from seawater, showing that relatively simple changes to medium components may facilitate the isolation and growth of hitherto unknown bacterial orders.


Assuntos
Organismos Aquáticos/efeitos dos fármacos , Organismos Aquáticos/crescimento & desenvolvimento , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Técnicas Bacteriológicas/métodos , Meios de Cultura/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Biodiversidade , Viabilidade Microbiana , Polissacarídeos Bacterianos/metabolismo
9.
Opt Lett ; 42(11): 2209-2212, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28569883

RESUMO

An endlessly single-mode doped microstructured poly(methyl methacrylate) (PMMA) optical fiber is produced for effective fiber Bragg grating (FBG) photo-inscription by means of a 400 nm femtosecond pulsed laser and the phase mask technique. The fiber presents a uniform benzyl dimethyl ketal (BDK) distribution in its core without drastic loss increase. It was produced using the selected center hole doping technique, and the BDK dopant acts as a photoinitiator. In this Letter, we report a rapidly growing process of the grating reflection band. For an 11 mW mean laser power, the FBG reflectivity reaches 83% in only 40 s.

10.
Opt Lett ; 42(6): 1161-1164, 2017 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-28295073

RESUMO

In this Letter, we report for the first time, to the best of our knowledge, the fabrication and characterization of a Zeonex/PMMA microstructured polymer optical fiber (mPOF) Bragg grating sensor for simultaneous monitoring of relative humidity (RH) and temperature. The sensing element (probe) is based on two separate in-line fiber Bragg gratings (FBGs) inscribed in the fabricated mPOF. A root mean square deviation of 0.8% RH and 0.6°C in the range of 10%-90% RH and 20°C-80°C was found. The developed mPOFBG sensor constitutes an efficient route toward low-cost, easy-to-fabricate and compact multi-parameter sensing solutions.

11.
Int J Syst Evol Microbiol ; 67(11): 4559-4564, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28984543

RESUMO

Four heterotrophic, antimicrobial, motile, marine bacterial strains, 27-4T, 8-1, M6-4.2 and S26, were isolated from aquaculture units in Spain, Denmark and Greece. All four strains produced the antibiotic compound tropodithietic acid, which is a key molecule in their antagonism against fish pathogenic bacteria. Cells of the strains were Gram-reaction-negative, rod-shaped and formed star-shaped aggregates in liquid culture and brown-coloured colonies on marine agar. The predominant cellular fatty acids were C18 : 1ω7c, C16 : 0, C11 methyl C18 : 1ω7c and C16 : 0 2-OH, and the polar lipids comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an aminolipid, a phospholipid and an unidentified lipid. The strains grew optimally at 31-33 °C. Growth was observed at a salt concentration between 0.5 and 5-6 % NaCl with an optimum at 2-3 %. The pH range for growth of the strains was from pH 6 to 8-8.5 with an optimum at pH 7. Based on 16S rRNA gene sequence analysis, the strains are affiliated with the genus Phaeobacter. The genome sequences of the strains have a DNA G+C content of 60.1 % and share an average nucleotide identity (ANI) of more than 95 %. The four strains are distinct from the type strains of the closely related species Phaeobactergallaeciensis and Phaeobacterinhibens based on an ANI of 90.5-91.7 and 89.6-90.4 %, respectively, and an in silico DNA-DNA hybridization relatedness of 43.9-46.9 and 39.8-41.9 %, respectively. On the basis of phylogenetic analyses as well as phenotypic and chemotaxonomic properties, the isolates are considered to represent a novel species, for which the name Phaeobacter piscinae sp. nov. is proposed. The type strain is 27-4T (=DSM 103509T=LMG 29708T).


Assuntos
Aquicultura , Filogenia , Rhodobacteraceae/classificação , Água do Mar/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Dinamarca , Ácidos Graxos/química , Peixes , Grécia , Processos Heterotróficos , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Rhodobacteraceae/genética , Rhodobacteraceae/isolamento & purificação , Análise de Sequência de DNA , Espanha , Tropolona/análogos & derivados , Tropolona/química
12.
J Nat Prod ; 80(5): 1287-1293, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28379705

RESUMO

Marine algae from the genus Karlodinium are known to be involved in fish-killing events worldwide. Here we report for the first time the chemistry and bioactivity of a natural product from the newly described mixotrophic dinoflagellate Karlodinium armiger. Our work describes the isolation and structural characterization of a new polyhydroxy-polyene named karmitoxin. The structure elucidation work was facilitated by use of 13C enrichment and high-field 2D NMR spectroscopy, where 1H-13C long-range correlations turned out to be very informative. Karmitoxin is structurally related to amphidinols and karlotoxins; however it differs by containing the longest carbon-carbon backbone discovered for this class of compounds, as well as a primary amino group. Karmitoxin showed potent nanomolar cytotoxic activity in an RTgill-W1 cell assay as well as rapid immobilization and eventual mortality of the copepod Acartia tonsa, a natural grazer of K. armiger.


Assuntos
Aminas/química , Dinoflagellida/química , Toxinas Marinhas/química , Polienos/química , Polienos/farmacologia , Animais , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Polienos/isolamento & purificação
13.
Mar Drugs ; 15(9)2017 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-28858210

RESUMO

Being able to quantify ichthyotoxic metabolites from microalgae allows for the determination of ecologically-relevant concentrations that can be simulated in laboratory experiments, as well as to investigate bioaccumulation and degradation. Here, the ichthyotoxin karmitoxin, produced by Karlodinium armiger, was quantified in laboratory-grown cultures using high-performance liquid chromatography (HPLC) coupled to electrospray ionisation high-resolution time-of-flight mass spectrometry (HRMS). Prior to the quantification of karmitoxin, a standard of karmitoxin was purified from K. armiger cultures (80 L). The standard was quantified by fluorescent derivatisation using Waters AccQ-Fluor reagent and derivatised fumonisin B1 and fumonisin B2 as standards, as each contain a primary amine. Various sample preparation methods for whole culture samples were assessed, including six different solid phase extraction substrates. During analysis of culture samples, MS source conditions were monitored with chloramphenicol and valinomycin as external standards over prolonged injection sequences (>12 h) and karmitoxin concentrations were determined using the response factor of a closely eluting iturin A2 internal standard. Using this method the limit of quantification was 0.11 µg·mL-1, and the limit of detection was found to be 0.03 µg·mL-1. Matrix effects were determined with the use of K. armiger cultures grown with 13C-labelled bicarbonate as the primary carbon source.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dinoflagellida/química , Fumonisinas/análise , Toxinas Marinhas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Fumonisinas/isolamento & purificação , Limite de Detecção , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos
14.
Mar Drugs ; 15(8)2017 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-28805711

RESUMO

A marine-derived Stilbella fimetaria fungal strain was screened for new bioactive compounds based on two different approaches: (i) bio-guided approach using cytotoxicity and antimicrobial bioassays; and (ii) dereplication based approach using liquid chromatography with both diode array detection and high resolution mass spectrometry. This led to the discovery of several bioactive compound families with different biosynthetic origins, including pimarane-type diterpenoids and hybrid polyketide-non ribosomal peptide derived compounds. Prefractionation before bioassay screening proved to be a great aid in the dereplication process, since separate fractions displaying different bioactivities allowed a quick tentative identification of known antimicrobial compounds and of potential new analogues. A new pimarane-type diterpene, myrocin F, was discovered in trace amounts and displayed cytotoxicity towards various cancer cell lines. Further media optimization led to increased production followed by the purification and bioactivity screening of several new and known pimarane-type diterpenoids. A known broad-spectrum antifungal compound, ilicicolin H, was purified along with two new analogues, hydroxyl-ilicicolin H and ilicicolin I, and their antifungal activity was evaluated.


Assuntos
Produtos Biológicos/isolamento & purificação , Diterpenos/isolamento & purificação , Hypocreales/química , Antifúngicos/química , Produtos Biológicos/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Diterpenos/química , Biologia Marinha , Espectrometria de Massas/métodos , Estrutura Molecular
15.
Anal Chem ; 88(24): 12461-12469, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-28193030

RESUMO

Dynamic cluster analysis (DCA) is an automated, unbiased technique which can identify Cl, Br, S, and other A + 2 element containing metabolites in liquid chromatographic high-resolution mass spectrometric data. DCA is based on three features, primarily the previously unutilized A + 1 to A + 2 isotope cluster spacing which is a strong classifier in itself but improved with the addition of the monoisotopic mass, and the well-known A:A+2 intensity ratio. Utilizing only the A + 1 to A + 2 isotope cluster spacing and the monoisotopic mass it was possible to filter a chromatogram for metabolites which contain Cl, Br, and S. Screening simulated isotope patterns of the Antibase Natural Products Database it was determined that the A + 1 to A + 2 isotope cluster spacing can be used to correctly classify 97.4% of molecular formulas containing these elements, only misclassifying a few metabolites which were either over 2800 u or metabolites which contained other A + 2 elements, such as Cu, Ni, Mg, and Zn. It was determined that with an interisotopic mass accuracy of 1 ppm, in a fully automated process, using all three parameters, it is possible to specifically filter a chromatogram for S containing metabolites with monoisotopic masses less than 825 u. Furthermore, it was possible to specifically filter a chromatogram for Cl and Br containing metabolites with monoisotopic masses less than 1613 u. Here DCA is applied on (i) simulated isotope patterns of the Antibase natural products databases, (ii) LC-QTOF data of reference standards, and (iii) LC-QTOF data of crude extracts of 10 strains of laboratory grown cultures of the microalga Prymnesium parvum where it identified known metabolites of the prymnesin series as well as over 20 previously undescribed prymnesin-like molecular features.

16.
Curr Genet ; 62(4): 799-807, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26936154

RESUMO

Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) approach to generate knockout mutants of two candidate non-ribosomal peptide synthetases (NRPS29 and NRPS30). Comparative studies of secondary metabolites in the two deletion mutants and wild type confirmed the absence of sansalvamide in the NRPS30 deletion mutant, implicating this synthetase in the biosynthetic pathway for sansalvamide. Sansalvamide is structurally related to the cyclic hexadepsipeptide destruxin, which both contain an α-hydroxyisocaproic acid (HICA) unit. A gene cluster responsible for destruxin production has previously been identified in Metarhizium robertsii together with a hypothetical biosynthetic pathway. Using comparative bioinformatic analyses of the catalytic domains in the destruxin and sansalvamide NRPSs, we were able to propose a model for sansalvamide biosynthesis. Orthologues of the gene clusters were also identified in species from several other genera including Acremonium chrysogenum and Trichoderma virens, which suggests that the ability to produce compounds related to destruxin and sansalvamide is widespread.


Assuntos
Depsipeptídeos/biossíntese , Depsipeptídeos/farmacologia , Fusarium/genética , Fusarium/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Antineoplásicos , Depsipeptídeos/química , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Metaboloma , Metabolômica , Modelos Biológicos , Família Multigênica , Filogenia , Metabolismo Secundário , Deleção de Sequência , Transcrição Gênica
17.
Appl Environ Microbiol ; 82(2): 502-9, 2016 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-26519388

RESUMO

Tropodithietic acid (TDA) is an antibacterial compound produced by some Phaeobacter and Ruegeria spp. of the Roseobacter clade. TDA production is studied in marine broth or agar since antibacterial activity in other media is not observed. The purpose of this study was to determine how TDA production is influenced by substrate components. High concentrations of ferric citrate, as present in marine broth, or other iron sources were required for production of antibacterially active TDA. However, when supernatants of noninhibitory, low-iron cultures of Phaeobacter inhibens were acidified, antibacterial activity was detected in a bioassay. The absence of TDA in nonacidified cultures and the presence of TDA in acidified cultures were verified by liquid chromatography-high-resolution mass spectrometry. A noninhibitory TDA analog (pre-TDA) was produced by P. inhibens, Ruegeria mobilis F1926, and Phaeobacter sp. strain 27-4 under low-iron concentrations and was instantaneously converted to TDA when pH was lowered. Production of TDA in the presence of Fe(3+) coincides with formation of a dark brown substance, which could be precipitated by acid addition. From this brown pigment TDA could be liberated slowly with aqueous ammonia, and both direct-infusion mass spectrometry and elemental analysis indicated a [Fe(III)(TDA)2]x complex. The pigment could also be produced by precipitation of pure TDA with FeCl3. Our results raise questions about how biologically active TDA is produced in natural marine settings where iron is typically limited and whether the affinity of TDA to iron points to a physiological or ecological function of TDA other than as an antibacterial compound.


Assuntos
Antibacterianos/biossíntese , Ferro/metabolismo , Rhodobacteraceae/metabolismo , Tropolona/análogos & derivados , Antibacterianos/química , Espectrometria de Massas , Estrutura Molecular , Rhodobacteraceae/genética , Tropolona/química , Tropolona/metabolismo
18.
Appl Environ Microbiol ; 82(13): 4035-4044, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27129958

RESUMO

UNLABELLED: Many factors, such as the substrate and the growth phase, influence biosynthesis of secondary metabolites in microorganisms. Therefore, it is crucial to consider these factors when establishing a bioprospecting strategy. Mimicking the conditions of the natural environment has been suggested as a means of inducing or influencing microbial secondary metabolite production. The purpose of the present study was to determine how the bioactivity of Vibrionaceae was influenced by carbon sources typical of their natural environment. We determined how mannose and chitin, compared to glucose, influenced the antibacterial activity of a collection of Vibrionaceae strains isolated because of their ability to produce antibacterial compounds but that in subsequent screenings seemed to have lost this ability. The numbers of bioactive isolates were 2- and 3.5-fold higher when strains were grown on mannose and chitin, respectively, than on glucose. As secondary metabolites are typically produced during late growth, potential producers were also allowed 1 to 2 days of growth before exposure to the pathogen. This strategy led to a 3-fold increase in the number of bioactive strains on glucose and an 8-fold increase on both chitin and mannose. We selected two bioactive strains belonging to species for which antibacterial activity had not previously been identified. Using ultrahigh-performance liquid chromatography-high-resolution mass spectrometry and bioassay-guided fractionation, we found that the siderophore fluvibactin was responsible for the antibacterial activity of Vibrio furnissii and Vibrio fluvialis These results suggest a role of chitin in the regulation of secondary metabolism in vibrios and demonstrate that considering bacterial ecophysiology during development of screening strategies will facilitate bioprospecting. IMPORTANCE: A challenge in microbial natural product discovery is the elicitation of the biosynthetic gene clusters that are silent when microorganisms are grown under standard laboratory conditions. We hypothesized that, since the clusters are not lost during proliferation in the natural niche of the microorganisms, they must, under such conditions, be functional. Here, we demonstrate that an ecology-based approach in which the producer organism is allowed a temporal advantage and where growth conditions are mimicking the natural niche remarkably increases the number of Vibrionaceae strains producing antibacterial compounds.


Assuntos
Anti-Infecciosos/metabolismo , Produtos Biológicos/metabolismo , Carbono/metabolismo , Metabolismo Secundário , Vibrionaceae/metabolismo , Quitina/metabolismo , Glucose/metabolismo , Manose/metabolismo
19.
Opt Express ; 24(2): 1206-13, 2016 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-26832503

RESUMO

The effect of humidity on annealing of poly (methyl methacrylate) (PMMA) based microstructured polymer optical fiber Bragg gratings (mPOFBGs) and the resulting humidity responsivity are investigated. Typically annealing of PMMA POFs is done in an oven without humidity control around 80°C and therefore at low humidity. We demonstrate that annealing at high humidity and high temperature improves the performances of mPOFBGs in terms of stability and sensitivity to humidity. PMMA POFBGs that are not annealed or annealed at low humidity level will have a low and highly temperature dependent sensitivity and a high hysteresis in the humidity response, in particular when operated at high temperature. PMMA mPOFBGs annealed at high humidity show higher and more linear humidity sensitivity with negligible hysteresis. We also report how annealing at high humidity can blue-shift the FBG wavelength more than 230 nm without loss in the grating strength.

20.
Opt Express ; 24(2): 1253-60, 2016 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-26832507

RESUMO

We have fabricated the first single-mode step-index and humidity insensitive polymer optical fiber operating in the 850 nm wavelength ranges. The step-index preform is fabricated using injection molding, which is an efficient method for cost effective, flexible and fast preparation of the fiber preform. The fabricated single-mode step-index (SI) polymer optical fiber (POF) has a 4.8µm core made from TOPAS grade 5013S-04 with a glass transition temperature of 134°C and a 150 µm cladding made from ZEONEX grade 480R with a glass transition temperature of 138°C. The key advantages of the proposed SIPOF are low water absorption, high operating temperature and chemical inertness to acids and bases and many polar solvents as compared to the conventional poly-methyl-methacrylate (PMMA) and polystyrene based POFs. In addition, the fiber Bragg grating writing time is short compared to microstructured POFs.

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