RESUMO
An animal's choice of foraging habitat reflects its response to environmental cues and is likely to vary among individuals in a population. Analyzing the magnitude of individual habitat selection can indicate how resilient populations may be to anthropogenic habitat change, where individually varying, broadly generalist populations have the potential to adjust their behavior. We collected GPS point data from 39 European nightjars (Caprimulgus europaeus) at a UK breeding site where restoration measures have altered large areas of habitat between breeding seasons. We calculated individual habitat selection over four breeding seasons to observe changes that might align with change in habitat. We also analyzed change in home range size in line with change in habitat availability, to examine functional relationships that can represent trade-offs made by the birds related to performance of the habitat. Individual explained more of the variation in population habitat selection than year for most habitat types. Individuals differed in the magnitude of their selection for different habitat types, which created a generalist population composed of both generalist and specialist individuals. Selection also changed over time but only significantly for scrub habitat (60% decrease in selection over 4 years). Across the population, individual home range size was 2% smaller where availability of cleared habitat within the home range was greater, but size increased by 2% where the amount of open water was higher, indicating the presence of trade-offs related to habitat availability. These results highlight that using individual resource selection and specialization measures, in conjunction with functional responses to change, can lead to better understanding of the needs of a population. Pooling specialist and generalist individuals for analysis could hide divergent responses to change and consequently obscure information that could be important in developing effective conservation strategies.
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It is well known that at the interface between neuronal tissue and recording electrode low electrical impedance is required. However, if simultaneous optical detection or stimulation is an issue, good optical transmittance of the electrode material is desirable as well. State-of-the-art titanium nitride electrodes provide superior low impedance compared to gold or iridium, but are nontransparent. Transparent electrode materials like the transparent conducting oxide, indium tin oxide (ITO), or graphene offer high light transmittance (>80%) but reveal relatively high impedance. In this paper, the authors propose the conducting polymer poly(3,4-ethylenedioxythiophene) with the counter ion NO3- as the electrode material for low impedance and good optical transmittance properties. The polymer is electrochemically deposited onto ITO improving the relatively high impedance of ITO. This multilayer electrode allows not only for electrophysiological recordings of cardiomyocytes but also for monitoring of cell contraction under the microscope. Electrochemical impedance spectroscopy and action potential recordings reveal that the new transparent electrodes are a good compromise in terms of low impedance and transparency if deposition parameters are optimized.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Fenômenos Químicos , Fenômenos Eletrofisiológicos , Microeletrodos , Polímeros/química , Potenciais de Ação , Células Cultivadas , Espectroscopia Dielétrica , Impedância Elétrica , Humanos , Microscopia , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Compostos de EstanhoRESUMO
Terrestrial Trunked Radio (TETRA) is a worldwide common mobile communication standard, used by authorities and organizations with security tasks. Previous studies reported on health effects of TETRA, with focus on the specific pulse frequency of 17.64Hz, which affects calcium efflux in neuronal cells. Likewise among others, it was reported that TETRA affects heart rate variability, neurophysiology and leads to headaches. In contrast, other studies conclude that TETRA does not affect calcium efflux of cells and has no effect on people's health. In the present study we examine whether TETRA short- and long-term exposure could affect the electrophysiology of neuronal in vitro networks. Experiments were performed with a carrier frequency of 395MHz, a pulse frequency of 17.64Hz and a differential quaternary phase-shift keying (π/4 DQPSK) modulation. Specific absorption rates (SAR) of 1.17W/kg and 2.21W/kg were applied. In conclusion, the present results do not indicate any effect of TETRA exposure on electrophysiology of neuronal in vitro networks, neither for short-term nor long-term exposure. This applies to the examined parameters spike rate, burst rate, burst duration and network synchrony.
Assuntos
Cálcio , Neurônios , Ondas de Rádio , Campos Eletromagnéticos , Humanos , Neurônios/fisiologiaRESUMO
Sphingolipids are well known to promote keratinocyte differentiation and to induce ceramide production. In addition, they show anti-inflammatory and antimicrobial activities. Thus, the aim of this study is to investigate the potential effect of sphinganine on prolonging the hair anagen rate and improving the overall hair quality and scalp health. The inhibitory potential of sphinganine toward 5-α-reductase was studied using an in vitro assay. The stimulation of the antimicrobial peptide HBD2 by sphinganine was measured by real-time polymerase chain reaction and immunostaining. Sphinganine bioavailability was studied ex vivo using a pig skin model. A placebo-controlled, double-blind study was designed to evaluate the efficacy of sphinganine on hair loss and hair/scalp quality in vivo. In vitro results showed that sphinganine is a potent inhibitor of 5-α-reductase type I that prevents the conversion of testosterone to dihydrotestosterone, a key factor of androgenetic male baldness. In vivo results demonstrated efficacy in reducing non-illness-related hair loss among males. In terms of expert rating, all hair quality and scalp parameters improved after application of sphinganine. Improved scalp health might be linked to the observed increase of the antimicrobial peptide HBD2. Thus, sphinganine is well suited as a topical alternative for the improvement of scalp health and hair quality and anti-hair loss application.
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Termite gut flagellates are typically colonized by specific bacterial symbionts. Here we describe the phylogeny, ultrastructure and subcellular location of 'Candidatus Adiutrix intracellularis', an intracellular symbiont of Trichonympha collaris in the termite Zootermopsis nevadensis. It represents a novel, deep-branching clade of uncultured Deltaproteobacteria widely distributed in intestinal tracts of termites and cockroaches. Fluorescence in situ hybridization and transmission electron microscopy localized the endosymbiont near hydrogenosomes in the posterior part and near the ectosymbiont 'Candidatus Desulfovibrio trichonymphae' in the anterior part of the host cell. The draft genome of 'Ca. Adiutrix intracellularis' obtained from a metagenomic library revealed the presence of a complete gene set encoding the Wood-Ljungdahl pathway, including two homologs of fdhF encoding hydrogenase-linked formate dehydrogenases (FDHH ) and all other components of the recently described hydrogen-dependent carbon dioxide reductase (HDCR) complex, which substantiates previous claims that the symbiont is capable of reductive acetogenesis from CO2 and H2 . The close phylogenetic relationship between the HDCR components and their homologs in homoacetogenic Firmicutes and Spirochaetes suggests that the deltaproteobacterium acquired the capacity for homoacetogenesis via lateral gene transfer. The presence of genes for nitrogen fixation and the biosynthesis of amino acids and cofactors indicate the nutritional nature of the symbiosis.
Assuntos
Deltaproteobacteria/classificação , Deltaproteobacteria/isolamento & purificação , Hypermastigia/microbiologia , Intestinos/microbiologia , Isópteros/parasitologia , Animais , Deltaproteobacteria/genética , Desulfovibrio/genética , Formiato Desidrogenases/genética , Transferência Genética Horizontal/genética , Hibridização in Situ Fluorescente , Fixação de Nitrogênio/genética , Filogenia , SimbioseRESUMO
The gut microbiota of termites plays critical roles in the symbiotic digestion of lignocellulose. While phylogenetically 'lower termites' are characterized by a unique association with cellulolytic flagellates, higher termites (family Termitidae) harbour exclusively prokaryotic communities in their dilated hindguts. Unlike the more primitive termite families, which primarily feed on wood, they have adapted to a variety of lignocellulosic food sources in different stages of humification, ranging from sound wood to soil organic matter. In this study, we comparatively analysed representatives of different taxonomic lineages and feeding groups of higher termites to identify the major drivers of bacterial community structure in the termite gut, using amplicon libraries of 16S rRNA genes from 18 species of higher termites. In all analyses, the wood-feeding species were clearly separated from humus and soil feeders, irrespective of their taxonomic affiliation, offering compelling evidence that diet is the primary determinant of bacterial community structure. Within each diet group, however, gut communities of termites from the same subfamily were more similar than those of distantly related species. A highly resolved classification using a curated reference database revealed only few genus-level taxa whose distribution patterns indicated specificity for certain host lineages, limiting any possible cospeciation between the gut microbiota and host to short evolutionary timescales. Rather, the observed patterns in the host-specific distribution of the bacterial lineages in termite guts are best explained by diet-related differences in the availability of microhabitats and functional niches.
Assuntos
Bactérias/classificação , Dieta , Trato Gastrointestinal/microbiologia , Isópteros/microbiologia , Microbiota , Animais , Análise por Conglomerados , República Democrática do Congo , Guiana Francesa , Isópteros/classificação , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Solo , África do Sul , Simbiose , MadeiraRESUMO
Recent developments in sequencing technology have given rise to a large number of studies that assess bacterial diversity and community structure in termite and cockroach guts based on large amplicon libraries of 16S rRNA genes. Although these studies have revealed important ecological and evolutionary patterns in the gut microbiota, classification of the short sequence reads is limited by the taxonomic depth and resolution of the reference databases used in the respective studies. Here, we present a curated reference database for accurate taxonomic analysis of the bacterial gut microbiota of dictyopteran insects. The Dictyopteran gut microbiota reference Database (DictDb) is based on the Silva database but was significantly expanded by the addition of clones from 11 mostly unexplored termite and cockroach groups, which increased the inventory of bacterial sequences from dictyopteran guts by 26%. The taxonomic depth and resolution of DictDb was significantly improved by a general revision of the taxonomic guide tree for all important lineages, including a detailed phylogenetic analysis of the Treponema and Alistipes complexes, the Fibrobacteres, and the TG3 phylum. The performance of this first documented version of DictDb (v. 3.0) using the revised taxonomic guide tree in the classification of short-read libraries obtained from termites and cockroaches was highly superior to that of the current Silva and RDP databases. DictDb uses an informative nomenclature that is consistent with the literature also for clades of uncultured bacteria and provides an invaluable tool for anyone exploring the gut community structure of termites and cockroaches.
Assuntos
Bactérias/classificação , Bactérias/genética , Baratas/microbiologia , Microbioma Gastrointestinal , Isópteros/microbiologia , Animais , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
While the gut microbiota of termites and its role in symbiotic digestion have been studied for decades, little is known about the bacteria colonizing the intestinal tract of the distantly related wood-feeding cockroaches (Blaberidae: Panesthiinae). Here, we show that physicochemical gut conditions and microbial fermentation products in the gut of Panesthia angustipennis resemble that of other cockroaches. Microsensor measurements confirmed that all gut compartments were anoxic at the center and had a slightly acidic to neutral pH and a negative redox potential. While acetate dominated in all compartments, lactate and hydrogen accumulated only in the crop. The high, hydrogen-limited rates of methane emission from living cockroaches were in agreement with the restriction of F420-fluorescent methanogens to the hindgut. The gut microbiota of both P. angustipennis and Salganea esakii differed strongly between compartments, with the highest density and diversity in the hindgut, but similarities between homologous compartments of both cockroaches indicated a specificity of the microbiota for their respective habitats. While some lineages were most closely related to the gut microbiota of omnivorous cockroaches and wood- or litter-feeding termites, others have been encountered also in vertebrates, reinforcing the hypothesis that strong environmental selection drives community structure in the cockroach gut.
Assuntos
Baratas/microbiologia , Intestinos/microbiologia , Metano/metabolismo , Microbiota/genética , Acetatos/metabolismo , Animais , Sequência de Bases , Ecossistema , Hidrogênio/metabolismo , Isópteros/microbiologia , Lignina/metabolismo , Oxirredução , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Madeira/metabolismoRESUMO
Mixed infections of insect larvae with different baculoviruses are occasionally found. They are of interest from an evolutionary as well as from a practical point of view when baculoviruses are applied as biocontrol agents. Here, we report mixed-infection studies of neonate larvae of the common cutworm, Agrotis segetum, with two baculoviruses, Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) and Agrotis segetum granulovirus (AgseGV). By applying quantitative PCR (qPCR) analysis, coinfections of individual larvae were demonstrated, and occlusion body (OB) production within singly infected and coinfected larvae was determined in individual larvae. Mixtures of viruses did not lead to changes in mortality rates compared with rates of single-virus treatments, indicating an independent action within host larvae under our experimental conditions. AgseNPV-B-infected larvae showed an increase in OB production during 2 weeks of infection, whereas the number of AgseGV OBs did not change from the first week to the second week. Fewer OBs of both viruses were produced in coinfections than in singly infected larvae, suggesting a competition of the two viruses for larval resources. Hence, no functional or economic advantage could be inferred from larval mortality and OB production from mixed infections of A. segetum larvae with AgseNPV-B and AgseGV.
Assuntos
Granulovirus/fisiologia , Mariposas/virologia , Nucleopoliedrovírus/fisiologia , Animais , Agentes de Controle Biológico/normas , Coinfecção/virologia , Larva/crescimento & desenvolvimento , Larva/virologia , Mariposas/crescimento & desenvolvimentoRESUMO
Termites digest wood and other lignocellulosic substrates with the help of their intestinal microbiota. While the functions of the symbionts in the digestive process are slowly emerging, the origin of the bacteria colonizing the hindgut bioreactor is entirely unknown. Recently, our group discovered numerous representatives of bacterial lineages specific to termite guts in a closely related omnivorous cockroach, but it remains unclear whether they derive from the microbiota of a common ancestor or were independently selected by the gut environment. Here, we studied the bacterial gut microbiota in 34 species of termites and cockroaches using pyrotag analysis of the 16S rRNA genes. Although the community structures differed greatly between the major host groups, with dramatic changes in the relative abundances of particular bacterial taxa, we found that the majority of sequence reads belonged to bacterial lineages that were shared among most host species. When mapped onto the host tree, the changes in community structure coincided with major events in termite evolution, such as acquisition and loss of cellulolytic protists and the ensuing dietary diversification. UniFrac analysis of the core microbiota of termites and cockroaches and construction of phylogenetic tree of individual genus level lineages revealed a general host signal, whereas the branching order often did not match the detailed phylogeny of the host. It remains unclear whether the lineages in question have been associated with the ancestral cockroach since the early Cretaceous (cospeciation) or are diet-specific lineages that were independently acquired from the environment (host selection).
Assuntos
Biota , Baratas/microbiologia , Isópteros/microbiologia , Animais , Análise por Conglomerados , DNA Ribossômico/química , DNA Ribossômico/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Trato Gastrointestinal/microbiologia , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual "handshaking" mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Qa-SNARE/metabolismo , Vesículas Secretórias/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ligação Competitiva , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Membrana Celular/metabolismo , Immunoblotting , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Mutação , Plantas Geneticamente Modificadas , Ligação Proteica , Proteínas Qa-SNARE/genética , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismoRESUMO
The Wickerhamomyces ciferrii strain NRRL Y-1031 F-60-10A is a well-known producer of tetraacetylphytosphingosine (TAPS) and used for the biotechnological production of sphingolipids and ceramides. It was our aim to gain new biological insights into the sphingolipid metabolism by employing a dual platform mass spectrometry strategy. The first step comprised metabolic (15)N-labeling in combination with label-free proteomics using high resolution LTQ Orbitrap mass spectrometry. Then selected reaction monitoring tandem mass spectrometry served for the targeted quantification of sphingoid base biosynthesis enzymes. The non-producer strain NRRL Y-1031-27 served as a reference strain. In total, 1697 proteins were identified, and 123 enzymes of main metabolic pathways were observed as differentially expressed. Major findings were: 1) the likely presence of higher carbon flux in NRRL Y-1031 F-60-10A, resulting in higher availability of pyruvate and acetyl-CoA; 2) indications of oleaginous yeast-like behavior in NRRL Y-1031 F-60-10A; and 3) approx. 2-fold higher abundance of eight sphingolipid biosynthesis enzymes in NRRL Y-1031 F-60-10A. Taken together, in NRRL Y-1031 F-60-10A, the levels of glycolytic enzymes apparently gear carbon flux towards higher acetyl-CoA synthesis rates, while simultaneously reducing protein biosynthesis in the process. Thereby, C2 building blocks for acyl-moieties and downstream sphingoid base acetylation are provided to maintain TAPS production. BIOLOGICAL SIGNIFICANCE: First quantitative proteome study for a phytosphingosine-producing yeast.
Assuntos
Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica/fisiologia , Proteoma/biossíntese , Proteômica/métodos , Saccharomycetales/metabolismo , Esfingolipídeos/biossíntese , Acetilcoenzima A/biossíntese , Acetilação , Ceramidas/biossíntese , Marcação por Isótopo/métodos , Isótopos de Nitrogênio/farmacologiaRESUMO
Wickerhamomyces ciferrii secretes tetraacetyl phytosphingosine (TAPS), and in this study, the catalyzing acetyltransferases were identified using mass spectrometry-based proteomics. The proteome of wild-type strain NRRL Y-1031 served as control and was compared to the tetraacetyl phytosphingosine defective mating type NRRL Y-1031-27. Acetylation of phytosphingosine in W. ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p, encoded by genes similar to Saccharomyces cerevisiae YGR212W and YGR177C, respectively. Ablation of SLI1 resulted in an almost complete loss of tri- and tetraacetyl phytosphingosines, whereas the loss ATF2 resulted in an 15-fold increase in triacetyl phytosphingosine. Most likely, it is the concerted action of these two acetyltransferases that yields tetraacetyl phytosphingosine, in which Sli1p catalyzes initial O- and N-acetylation, producing triacetyl phytosphingosine. Finally, Atf2p catalyzes final O-acetylation to yield tetraacetyl phytosphingosine. The current study demonstrates that mass spectrometry-based proteomics can be employed to identify key steps in ill-explored metabolite biosynthesis pathways of nonconventional microorganisms. Furthermore, the identification of phytosphingosine as substrate for alcohol acetyltransferase Atf2p broadens the known substrate range of this enzyme. This interesting property of Atf2p may be exploited to enhance the secretion of heterologous compounds.
Assuntos
Acetiltransferases/metabolismo , Saccharomycetales/enzimologia , Saccharomycetales/metabolismo , Esfingosina/análogos & derivados , Acetiltransferases/química , Acetiltransferases/genética , Deleção de Genes , Espectrometria de Massas , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Homologia de Sequência de Aminoácidos , Esfingosina/metabolismoRESUMO
Wickerhamomyces ciferrii is a microorganism characterized by the production and secretion of large amounts of acetylated sphingoid bases, in particular tetraacetyl phytosphingosine. Here, we present the 15.90-Mbp draft genome sequence of W. ciferrii NRRL Y-1031 F-60-10 generated by pyrosequencing and de novo assembly. The draft genome sequence comprising 364 contigs in 150 scaffolds was annotated and covered 6,702 protein-coding sequences. This information will contribute to the metabolic engineering of this yeast to improve the yield and spectrum of acetylated sphingoid bases in biotechnological production.
Assuntos
Genoma Fúngico , Pichia/genética , Sequência de Bases , Mapeamento de Sequências Contíguas , Bases de Dados Genéticas , Anotação de Sequência Molecular , Dados de Sequência MolecularRESUMO
Termite gut flagellates are colonized by host-specific lineages of ectosymbiotic and endosymbiotic bacteria. Previous studies have shown that flagellates of the genus Trichonympha may harbour more than one type of symbiont. Using a comprehensive approach that combined cloning of SSU rRNA genes with fluorescence in situ hybridization and electron microscopy, we investigated the phylogeny and subcellular locations of the symbionts in a variety of Trichonympha species from different termites. The flagellates in Trichonympha Cluster I were the only species associated with 'Endomicrobia', which were located in the posterior part of the cell, confirming previous results. Trichonympha species of Cluster II from the termite genus Incisitermes (family Kalotermitidae) lacked 'Endomicrobia' and were associated with endosymbiotic Actinobacteria, which is highly unusual. The endosymbionts, for which we suggest the name 'Candidatus Ancillula trichonymphae', represent a novel, deep-branching lineage in the Micrococcineae that consists exclusively of clones from termite guts. They preferentially colonized the anterior part of the flagellate host and were highly abundant in all species of Trichonympha Cluster II except Trichonympha globulosa. Here, they were outnumbered by a Desulfovibrio species associated with the cytoplasmic lamellae at the anterior cell pole. Such symbionts are present in both Trichonympha clusters, but not in all species. Unlike the intracellular location reported for the Desulfovibrio symbionts of Trichonympha agilis (Cluster I), the Desulfovibrio symbionts of T. globulosa (Cluster II) were situated in deep invaginations of the plasma membrane that were clearly connected to the exterior of the host cell.
Assuntos
Actinobacteria/classificação , Desulfovibrio/classificação , Trato Gastrointestinal/microbiologia , Hypermastigia/classificação , Isópteros/microbiologia , Simbiose , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Actinobacteria/ultraestrutura , Animais , Clonagem Molecular , Desulfovibrio/genética , Desulfovibrio/isolamento & purificação , Desulfovibrio/ultraestrutura , Genes de RNAr , Hypermastigia/isolamento & purificação , Hypermastigia/fisiologia , Hypermastigia/ultraestrutura , Filogenia , Especificidade da Espécie , Simbiose/genéticaRESUMO
To follow endocytosis in BY-2 cells we made use of fluorescent nano beads. Beads with 20nm in diameter were internalised rapidly and accumulated partially in compartments also labelled by the endocytic marker FM4-64. Studies in BY-2 cells and protoplasts revealed that larger beads (100nm) were excluded from uptake into turgescent and plasmolysed cells while protoplasts were able to internalise beads with a diameter of up to 1000nm. Endocytosis of beads was only partially inhibited by the clathrin-specific inhibitor Ikarugamycin and strongly blocked by wortmannin. These results imply that uptake of beads involves clathrin-dependent and clathrin-independent endocytic mechanisms and supports the hypothesis that clathrin-independent endocytosis plays a general role in plants.
Assuntos
Clatrina/metabolismo , Endocitose , Corantes Fluorescentes/metabolismo , Microesferas , Nanopartículas , Nicotiana/citologia , Proteínas de Plantas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Nanopartículas/química , Tamanho da Partícula , Protoplastos/metabolismoRESUMO
Higher termites are characterized by a purely prokaryotic gut microbiota and an increased compartmentation of their intestinal tract. In soil-feeding species, each gut compartment has different physicochemical conditions and is colonized by a specific microbial community. Although considerable information has accumulated also for wood-feeding species of the genus Nasutitermes, including cellulase activities and metagenomic data, a comprehensive study linking physicochemical gut conditions with the structure of the microbial communities in the different gut compartments is lacking. In this study, we measured high-resolution profiles of H(2), O(2), pH, and redox potential in the gut of Nasutitermes corniger termites, determined the fermentation products accumulating in the individual gut compartments, and analyzed the bacterial communities in detail by pyrotag sequencing of the V3-V4 region of the 16S rRNA genes. The dilated hindgut paunch (P3 compartment) was the only anoxic gut region, showed the highest density of bacteria, and accumulated H(2) to high partial pressures (up to 12 kPa). Molecular hydrogen is apparently produced by a dense community of Spirochaetes and Fibrobacteres, which also dominate the gut of other Nasutitermes species. All other compartments, such as the alkaline P1 compartment (average pH, 10.0), showed high redox potentials and comprised small but distinct populations characteristic for each gut region. In the crop and the posterior hindgut compartments, the community was even more diverse than in the paunch. Similarities in the communities of the posterior hindgut and crop suggested that proctodeal trophallaxis or coprophagy also occurs in higher termites. The large sampling depths of pyrotag sequencing in combination with the determination of important physicochemical parameters allow cautious conclusions concerning the functions of particular bacterial lineages in the respective gut sections to be drawn.
Assuntos
Bactérias/classificação , Bactérias/genética , Biodiversidade , Isópteros/microbiologia , Animais , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Dieta , Trato Gastrointestinal/química , Trato Gastrointestinal/microbiologia , Hidrogênio/análise , Concentração de Íons de Hidrogênio , Oxirredução , Oxigênio/análise , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Madeira/metabolismoRESUMO
The study describes the identification of sphingolipid biosynthesis genes in the non-conventional yeast Pichia ciferrii, the development of tools for its genetic modification as well as their application for metabolic engineering of P. ciferrii with the goal to generate strains capable of producing the rare sphingoid bases sphinganine and sphingosine. Several canonical genes encoding ceramide synthase (encoded by PcLAG1 and PcLAF1), alkaline ceramidase (PcYXC1) and sphingolipid C-4-hydroxylase(PcSYR2), as well as structural genes for dihydroceramide Δ(4)-desaturase (PcDES1) and sphingolipid Δ(8)-desaturase (PcSLD1) were identified, indicating that P. ciferrii would be capable of synthesizing desaturated sphingoid bases, a property not ubiquitously found in yeasts. In order to convert the phytosphingosine-producing P. ciferrii wildtype into a strain capable of producing predominantly sphinganine, Syringomycin E-resistant mutants were isolated. A stable mutant almost exclusively producing high levels of acetylated sphinganine was obtained and used as the base strain for further metabolic engineering. A metabolic pathway required for the three-step conversion of sphinganine to sphingosine was implemented in the sphinganine producing P. ciferrii strain and subsequently enhanced by screening for the appropriate heterologous enzymes, improvement of gene expression and codon optimization. These combined efforts led to a strain capable of producing 240mgL(-1) triacetyl sphingosine in shake flask, with tri- and diacetyl sphinganine being the main by-products. Lab-scale fermentation of this strain resulted in production of up to 890mgkg(-1) triacetyl sphingosine. A third by-product was unequivocally identified as triacetyl sphingadienine. It could be shown that inactivation of the SLD1 gene in P. ciferrii efficiently suppresses triacetyl sphingadienine formation. Further improvement of the described P. ciferrii strains will enable a biotechnological route to produce sphinganine and sphingosine for cosmetic and pharmaceutical applications.
Assuntos
Engenharia Metabólica/métodos , Pichia/enzimologia , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Ceramidase Alcalina/genética , Ceramidase Alcalina/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Pichia/genética , Esfingosina/genéticaRESUMO
The non-conventional yeast Pichia ciferrii is known to secrete the sphingoid long-chain base phytosphingosine in a tetraacetylated form (TAPS). Sphingolipids are important ingredients in cosmetic applications as they play important roles in human skin. Our work aimed to improve TAPS production by genetic engineering of P. ciferrii. In the first step we improved precursor availability by blocking degradation of L-serine, which is condensed with palmitoyl-CoA by serine palmitoyltransferase in the first committed step of sphingolipid biosynthesis. Successive deletion of two genes, SHM1 and SHM2, encoding L-serine hydroxymethyltransferases, and of CHA1 encoding L-serine deaminase, resulted in a strain producing 65 mg((TAPS))g(-1)((cdw)), which is a threefold increase in comparison with the parental strain. Attempts to increase the metabolic flux into and through the L-serine biosynthesis pathway did not improve TAPS production. However, genetic engineering of the sphingolipid pathway further increased secretion of TAPS. Blocking of sphingoid long-chain base phosphorylation by deletion of the LCB kinase gene PcLCB4 resulted in a further increase in TAPS production by 78% and significant secretion of the direct precursor of phytosphingosine, sphinganin, in a triacetylated form (TriASa). Overproduction of two serine palmitoyltransferase subunits, Lcb1 and Lcb2, together with a deletion of the gene ORM12 encoding a putative negative regulator of sphingolipid synthesis resulted in a strain producing 178 mg((TAPS))g(-1)((cdw)). Additional overproduction of the C4-hydroxylase Syr2 converting sphinganine to phytosphingosine reduced TriASa production and further improved TAPS production. The final recombinant P. ciferrii strain produced up to 199 mg((TAPS))g(-1)((cdw)) with a maximal production rate of 8.42 mg×OD(600nm)(-1)h(-1) and a titer of about 2 g L(-1), and should be applicable for industrial TAPS production.
Assuntos
Engenharia Metabólica , Pichia/metabolismo , Serina/metabolismo , Esfingolipídeos/biossíntese , Esfingosina/análogos & derivados , Deleção de Genes , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Humanos , Pichia/genética , Serina/genética , Esfingolipídeos/genética , Esfingosina/biossíntese , Esfingosina/genéticaRESUMO
Uneven skin pigmentation is a significant cosmetic concern, and the identification of topically applicable molecules to address this issue is of general interest. We report that the tetrapeptide PKEK (Pro-Lys-Glu-Lys) can exert skin whitening effects based on one in vitro and four double-blinded vehicle-controlled in vivo studies. (i) Treatment of human keratinocytes with PKEK significantly reduced UVB-stimulated mRNA expression of interleukin (IL)-6, IL-8 and TNF-α and, most importantly, proopiomelanocorticotropin (POMC), i.e. a gene encoding the pigmentation-inducing soluble mediator α- (α-MSH). (ii) PKEK treatment significantly inhibited UVB-induced upregulation of genes encoding for IL-1α, IL-6, IL-8, TNF-α as well as POMC and tyrosinase in 10 healthy volunteers pretreated with PKEK for 4 weeks once daily. (iii) In a study enrolling 39 Caucasian women, facial pigment spots significantly faded after 6 weeks when PKEK was combined with the skin whitener sodium ascorbyl phosphate (SAP), whereas PKEK or SAP alone led to less pronounced fading of the pigment spots. (iv) Addition of PKEK enhanced the skin whitening potency of a SAP-containing preparation if applied for 8 weeks to the back of hands of 19 Caucasians. (v) 27 Japanese women were treated on their faces twice daily with an SAP only or a PKEK+SAP-containing formulation for 8 weeks. Application of PKEK+SAP significantly reduced skin pigmentation by 26% and by 18% according to SCINEXA score. We demonstrate that PKEK has the capacity to reduce UVB-induced skin pigmentation and may be suited to serve as a skin tone-modulating agent in cosmetic products.