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Edible fungi, healthier for humans and sustainable for the planet, attract unprecedented attention. In the study, the genetically modified Pleurotus ostreatus overexpression phosphoglucomutase (PGM) was constructed. P. ostreatus overexpression PGM (Po::PGM) had 4.96-folds higher expression level of PGM. Po::PGM grew thicker mycelium and more mycelium branches. Additional Ca2+ can inhibit mycelium growth, and cyclic adenosine monophosphate completely inhibited their growth of Po::PGM. Secondly, Overexpression of PGM made P. ostreatus become more sensitive to cell wall disruptors, and caused 12.75 % reduction of ß-1, 3-glucan and 40.53 % increase of chitin in cell wall. In submerged fermentation, the mycelia biomass yield and endopolysaccharide (IPS) production of Po::PGM in basic PDB can reach 11.18 g/l and 2.55 g/l, increasing by 20.86 % and 28.79 %, respectively. Whereas exopolysaccharide (EPS) reduced by 3.28 %. After replacing potato and glucose in PDB by wheat bran, mycelia biomass and EPS production of Po::PGM were all improved. The additional lactose in wheat bran did not only furtherly enhance mycelia biomass yield of Po::PGM to 27.78 g/l by 199.03 %, but IPS production also increased by 277.99 % to 6.07 g/l. The results provided us key ideas and important research directions that at least manipulating the PGM gene could obtain high-efficient use of agricultural wastes producing more fungus-based foods.
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Exploring the vast extraterrestrial space is an inevitable trend with continuous human development. Water treatment and reuse are crucial in the limited and closed space that is available in spaceships or long-term use space bases that will be established in the foreseeable future. Dedicated water treatment technologies have experienced iterative development for more than 60 years since the first manned spaceflight was successfully launched. Herein, we briefly review the related wastewater characteristics and the history of water treatment in space stations, and we focus on future challenges and perspectives, aiming at providing insights for optimizing wastewater treatment technologies and closing the water cycle in future.
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Eliminación de Residuos Líquidos , Aguas Residuales , Purificación del Agua , Eliminación de Residuos Líquidos/métodos , Purificación del Agua/métodos , Vuelo EspacialRESUMEN
The extensive application of ceramic membranes in wastewater treatment draws increasing attention due to their ultra-long service life. A cost-effective treatment for high-strength swine wastewater is an urgent and current need that is a worldwide challenge. A pilot-scale sequencing batch flat-sheet ceramic membrane bioreactor (ScMBR) coupled with a short-cut biological nitrogen removal (SBNR) process was developed to treat high-strength swine wastewater. The ScMBR achieved stable and excellent removal of COD (95.3%), NH4+-N (98.3%), and TN (92.7%), though temperature went down from 20 °C, to 15 °C, to 10 °C stepwise along three operational phases. The COD and NH4+-N concentrations in the effluent met with the discharge standards (GB18596-2001). Microbial community diversity was high, and the genera Pseudomonas and Comamonas were dominant in denitritation, and Nitrosomonas was dominant in nitritation. Ceramic membrane modules of this pilot-scale reactor were separated into six layers (A, B, C, D, E, F) from top to bottom. The total filtration resistance of both the top and bottom membrane modules was relatively low, and the resistance of the middle ones was high. These results indicate that the spatial distribution of the membrane fouling degree was different, related to different aeration scour intensities demonstrated by computational fluid dynamics (CFD). The results prove that the membrane fouling mechanism can be attributed to the cake layer formation of the middle modules and pore blocking of the top and bottom modules, which mainly consist of protein and carbohydrates. Therefore, different cleaning measures should be adopted for membrane modules in different positions. In this study, the efficient treatment of swine wastewater shows that the ScMBR system could be applied to high-strength wastewater. Furthermore, the spatial distribution characteristics of membrane fouling contribute to cleaning strategy formulation for further full-scale MBR applications.
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FMRFamide, a member of the neuropeptide family, is involved in numerous physiological processes. FMRFamide-activated sodium channels (FaNaCs) are a family of non-voltage-gated, amiloride-sensitive, Na+-selective channels triggered by the neuropeptide FMRFamide. In the present study, the full-length cDNA of the FaNaC receptor of Sepiella japonica (SjFaNaC) was cloned. The cDNA of SjFaNaC was 3004 bp long with an open reading frame (ORF) of 1812 bp, encoding 603 amino acid residues with no signal peptide at the N-terminus. Sequence analysis indicated that SjFaNaC shared a high identity with other cephalopods FaNaCs and formed a sister clade with bivalves. The protein structure was predicted using SWISS-MODEL with AcFaNaC as the template. Quantitative real-time PCR (qRT-PCR) revealed that SjFaNaC transcripts were highly expressed in both female and male reproductive organs, as well as in the optic lobe and brain of the central nervous system (CNS). Results of in situ hybridisation (ISH) showed that SjFaNaC mRNA was mainly distributed in the medulla and deep retina of the optic lobe and in both the supraesophageal and subesophageal masses of the brain. Subcellular localisation indicated that the SjFaNaC protein was localised intracellularly and on the cell surface of HEK293T cells. In summary, these findings may lay the foundation for future exploration of the functions of SjFaNaC in cephalopods.
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FMRFamida , Animales , Masculino , Femenino , FMRFamida/metabolismo , Secuencia de Aminoácidos , Canales de Sodio/metabolismo , Canales de Sodio/genética , Cefalópodos/metabolismo , Cefalópodos/genética , Cefalópodos/crecimiento & desarrollo , Gónadas/metabolismo , Gónadas/crecimiento & desarrollo , Filogenia , Perfilación de la Expresión Génica , Humanos , Clonación Molecular , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Oriented towards the pressing needs for hypersaline wastewater desalination and zero liquid discharge (ZLD), the contrasting mixed scaling of thermal-driven vacuum membrane distillation (VMD) and pressure-driven nanofiltration (NF) were investigated in this work. Bulk crystallization was the main mechanism in VMD due to the high salinity and temperature, but the time-independent resistance by the adsorption of silicate and organic matter dominated the initial scaling process. Surface crystallization and the consequent pore-blocking were the main scaling mechanisms in NF, with the high permeate drag force, hydraulic pressure, and cross-flow rate resulting in the dense scaling layer mainly composed of magnesium-silica hydrate (MSH). Silicate enhanced NF scaling with a 75% higher initial flux decline rate attributed to the MSH formation and compression, but delayed bulk crystallization in VMD. Organic matter presented an anti-scaling effect by delaying bulk crystallization in both VMD and NF, but specifically promoted CaCO3 scaling in NF. Furthermore, the incipient scaling was intensified as silicate and organic matter coexisted. The scaling mechanism shifted from surface to bulk crystallization due to the membrane concentration in both VMD and NF. This work fills the research gaps on mixed scaling mechanisms in different membrane processes, which offers insights for scaling mitigation and thereby supports the application of ZLD.
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Destilación , Filtración , Membranas Artificiales , Destilación/métodos , Purificación del Agua/métodos , Salinidad , Aguas Residuales/químicaRESUMEN
Noncaloric or low-caloric sweeteners have become popular worldwide, although debates persist regarding their impact on health. To investigate whether the sweeteners are favorable for glucose homeostasis, our study assessed the effects of glycyrrhetinic acid monoglucuronide (GAMG) and several commonly used sweeteners [glycyrrhetinic acid (GA), stevioside, erythritol, sucralose, and aspartame] on glycometabolism and elucidated the underlying mechanisms. The C57BL/6J male mice were exposed to different sweeteners for 10 weeks, and our results showed that GAMG significantly reduced fasting blood glucose (FBG) levels (FBG-control: 3.81 ± 0.42 mmol/L; FBG-GAMG: 3.37 ± 0.38 mmol/L; p < 0.05) and the blood glucose levels 15 and 30 min after sucrose or maltose loading (p < 0.05). Furthermore, it improved glucose tolerance (p = 0.028) and enhanced insulin sensitivity (p = 0.044), while the other sweeteners had negligible or adverse effects on glucose homeostasis. Subsequent experiments showed that GAMG inhibited α-glucosidases potently (IC50 = 0.879 mg·mL-1), increased three SCFA-producing bacteria and SCFAs levels (p < 0.05), and promoted the gene expression of SCFA receptor GPR43 (p = 0.018). These results suggest that GAMG may regulate blood glucose by inhibiting α-glucosidases and modulating gut microbial SCFAs. Our findings prove that GAMG, beneficial to blood glucose regulation, is a promising natural sweetener for future utilization.
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Ácido Glicirretínico , Edulcorantes , Masculino , Animales , Ratones , Edulcorantes/análisis , Ácido Glicirretínico/metabolismo , Glucemia , alfa-Glucosidasas , Ratones Endogámicos C57BL , HomeostasisRESUMEN
Rapid advancement in aerospace technology has successfully enabled long-term life and economic activities in space, particularly in Low Earth Orbit (LEO), extending up to 2000 km from the mean sea level. However, the sustainance of the LEO Economy and its Environmental Control and Life Support System (ECLSS) still relies on a regular cargo supply of essential commodities (e.g., water, food) from Earth, for which there still is a lack of adequate and sustainable technologies. One key challenge in this context is developing water treatment technologies and standards that can perform effectively under microgravity conditions. Solving this technical challenge will be a milestone in providing a scientific basis and the necessary support mechanisms for establishing permanent bases in outer space and beyond. To identify clues towards solving this challenge, we looked back at relevant scientific research exploring novel technologies and standards for deep space exploration, also considering feedback for enhancing these technologies on land. Synthesizing our findings, we share our outlook for the future of drinking water treatment in microgravity. We also bring up a new concept for space aquatic chemistry, considering the closed environment of engineered systems operating in microgravity.
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BACKGROUND: Neuropeptide pedal peptide (PP) and orcokinin (OK), which are structurally related active peptides, have been widely discovered in invertebrates and constitute the PP/OK neuropeptide family. They have complex structures and play myriad roles in physiological processes. To date, there have been no related reports of PP/OK-type neuropeptide in cephalopods, which possess a highly differentiated multi-lobular brain. METHODS: Rapid Amplification of cDNA Ends (RACE) was employed to obtain the open reading frame (ORF) of PP/OK-type neuropeptide in Sepiella japonica (termed as Sj-PP/OK). Various software were used for sequence analysis. Semi-quantitative PCR was applied to analyze the tissue distribution profile, quantitative real-time PCR (qRT-PCR) was used to study spatio-temporal expression throughout the entire growth and development period, and in situ hybridization (ISH) was employed to observe the tissue location of Sj-PP/OK. RESULTS: in the present study, we identified the ORF of Sj-PP/OK. The putative precursor of Sj-PP/ OK encodes 22 mature peptides, of which only tridecapeptides could undergo post-translationally amidated at C-terminus. Each of these tridecapeptides possesses the most conserved and frequent N-terminus Asp-Ser-Ile (DSI). Sequence analysis revealed that Sj-PP/OK shared comparatively low identity with other invertebrates PP or OK. The tissue distribution profile showed differences in the expression level of Sj-PP/OK between male and female. qRT-PCR data demonstrated that Sj-PP/OK was widely distributed in various tissues, with its expression level increasing continuously in the brain, optic lobe, liver, and nidamental gland throughout the entire growth and development stages until gonad maturation. ISH detected that Sj-PP/OK positive signals existed in almost all regions of the optic lobe except the plexiform zone, the outer edge of all functional lobes in the brain, epithelial cells and the outer membrane layer of the accessory nidamental gland. These findings suggest that Sj-PP/OK might play a role in the regulation of reproduction, such as vitellogenin synthesis, restoration, and ova encapsulation. CONCLUSION: The study indicated that Sj-PP/OK may be involved in the neuroendocrine regulation in cephalopods, providing primary theoretical basis for further studies of its regulation role in reproduction.
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Secuencia de Aminoácidos , Decapodiformes , Hibridación in Situ , Neuropéptidos , Animales , Neuropéptidos/genética , Neuropéptidos/metabolismo , Neuropéptidos/química , Decapodiformes/genética , Decapodiformes/metabolismo , Hibridación in Situ/métodos , Filogenia , Sistemas de Lectura Abierta , Clonación Molecular , Secuencia de Bases , FemeninoRESUMEN
Large yellow croaker (Larimichthys crocea) is the most productive marine fish in China. Cryptocaryon irritans is an extremely destructive parasite that causes great economic losses in large yellow croaker aquaculture industry. Therefore, it is very necessary to study the immune response of large yellow croaker in response to C. irritans infection. In this study, the transcriptomic profiles of large yellow croaker were sequenced and analyzed in the brain and head kidney at 72 h after C. irritans infection. Cytokines and chemokines related terms were significantly enriched based on the GO enrichment of down-regulated differentially expressed genes (DEGs) from the head kidney. Meanwhile, cytokine-cytokine receptor interaction was significantly enriched based on the KEGG enrichment of up-regulated DEGs from the brain and down-regulated DEGs from the head kidney, respectively. Moreover, the majority of inflammation-related DEGs were significantly up-regulated in the brain, but distinctly down-regulated in the head kidney. These results showed that the brain and head kidney might play different roles against C. irritans infection, and the inflammatory response of large yellow croaker may be restrained during C. irritans infection. Taken together, the transcriptomic analyses will be helpful to more comprehensively understand the immune mechanism of teleost against C. irritans infection, and provide a theoretical basis for the prevention and treatment of Cryptosporidiosis.
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Infecciones por Cilióforos , Cilióforos , Enfermedades de los Peces , Hymenostomatida , Perciformes , Animales , Cilióforos/fisiología , Proteínas de Peces/genética , Perfilación de la Expresión Génica/veterinariaRESUMEN
Membrane distillation (MD) has promising potential in the water purification and wastewater treatment industries; however, fouling and wetting are the main obstacles to its commercialization, and higher fluxes and energy efficiencies are essential. Magneto-responsive membranes (MagMem) with integrated magnetic nanoparticles (MNPs) enable in situ fouling mitigation and switchable separation by nano-mixing or nano-heating, triggered by external magnetic fields, in a range of membrane processes, but not yet been demonstrated in MD. This perspective discussed the potential paths of MagMem utilization in MD based on the research status and dilemmas of MD. It can be envisioned that MagMem will lead to a paradigm shift in MD, especially by in situ fouling/wetting mitigation and enhancing energy efficiency via in-place actuation and localized heating by MNPs. Moreover, remotely controllable pore tuning and specific or switchable wettability can also be anticipated. Overall, MagMem provides attractive opportunities for advanced robust and efficient MD.
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Nanopartículas , Purificación del Agua , Humectabilidad , Destilación , Membranas ArtificialesRESUMEN
Nanofiltration (NF) will play a crucial role in salt fractionation and recovery, but the complicated and severe mixed scaling is not yet fully understood. In this work, the mixed scaling patterns and mechanisms of high-pressure NF in zero-liquid discharge (ZLD) scenarios were investigated by disclosing the role of key foulants. The bulk crystallization of CaSO4 and Mg-Si complexes and the resultant pore blocking and cake formation under high pressure were the main scaling mechanisms in hypersaline desalination. The incipient scalants were Mg-Si hydrates, CaF2, CaCO3, and CaMg(CO3)2. Si deposited by adsorption and polymerization prior to and impeded Ca scaling when Mg was not added, thus pore blocking was the main mechanism. The amorphous Mg-Si hydrates contribute to dense cake formation under high hydraulic pressure and permeate drag force, causing rapid flux decline as Mg was added. Humic acid has a high affinity to Ca2+by complexation, which enhances incipient scaling by adsorption or lowers the energy barrier of nucleation but improves the interconnectivity of the foulants layer and inhibits bulk crystallization due to the chelation and directional adsorption. Bovine serum albumin promotes cake formation due to the low electrostatic repulsion and acts as a cement to particles by adsorption and bridging in bulk. This work fills the research gaps in mixed scaling of NF, which is believed to support the application of ZLD and shed light on scaling in hypersaline/ultra-hypersaline wastewater desalination applications.
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Aguas Residuales , Purificación del Agua , Membranas Artificiales , Cloruro de Sodio , Sustancias HúmicasRESUMEN
The treatment and reuse of hygiene wastewater is crucial to "close the loop" in the controlled ecological life support system (CELSS), and to guarantee longer space missions or planetary habitation. In this work, anaerobic membrane bioreactor (AnMBR) was applied for hygiene wastewater treatment, focused on surfactant degradation and microbial community succession. The removal efficiency of COD and surfactants was 90%â¼97% and 80% with a urine source-separation strategy. The microbial community gradually shifted from methanogens to sulfur-metabolizing and surfactant-degradation bacteria, such as Aeromonas. Sulfate was a surfactant degradation product, which triggered sulfate reduction and methane inhibition. The activated carbohydrate and sulfur metabolism were the key mechanism of the microbial process for the excellent performance of AnMBR. This study analyzed the degradation mechanism from the perspective of microbial mechanism, offers a solution for CELSS hygiene wastewater treatment, and supports the future improvement and refinement of AnMBR technology.
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Microbiota , Purificación del Agua , Eliminación de Residuos Líquidos , Sistemas de Manutención de la Vida , Anaerobiosis , Tensoactivos , Higiene , Reactores Biológicos/microbiología , Metano , Sulfatos , Membranas Artificiales , Aguas del AlcantarilladoRESUMEN
Mineral scaling is one key obstacle to membrane distillation in hypersaline wastewater desalination, but the scaling or fouling mechanism is poorly understood. Addressing this challenge required revealing the foulants layer formation process. In this work, the scaling process was deconstructed with a cascade strategy by stepwise changing the composition of the synthetic desulfurization wastewater. The flux decline curves presented a 3-stage mode in vacuum membrane distillation (VMD). Heterogeneous nucleation of CaMg(CO3)2, CaF2, and CaCO3 was the main incipient scaling mechanism. Mg-Si complex was the leading foulant in 2nd-stage, during which the scaling mechanism shifted from surface to bulk crystallization. The flux decreased sharply for the formation of a thick and compacted scaling layer by the bricklaying of CaSO4 and Mg-Si-BSA complexes in the 3rd-stage. Bulk crystallization was identified as the key scaling mechanism in VMD for the high salinity and concentration multiple. The organic matter had an anti-scaling effect by changing the bulk crystallization. Humic acids (HA) and colloidal silica also contributed to incipient scaling for the high affinity to membrane, bovine serum albumin (BSA) acting as the cement of Mg-Si complexes. Mg altered the Si scaling from polymerization to Mg-Si complex formation, which significantly influence the mixed scaling mechanism. This work deconstructed the mixed scaling process and illuminated the role of main foulants, filling in the knowledge gap on the mixed scaling mechanism in VMD for hypersaline wastewater treatment and recovery.
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Aguas Residuales , Purificación del Agua , Destilación , Vacio , Membranas ArtificialesRESUMEN
Interfacial structure optimization is important to enhance the thermal boundary conductance (TBC) as well as the overall performance of thermal conductive composites. In this work, the effect of interfacial roughness on the TBC between copper and diamond is investigated with molecular dynamics (MD) simulations and time-domain thermoreflectance (TDTR) experiments. It is found from MD simulations that the thermal transport efficiency across a rough interface is higher, and the TBC can be improved 5.5 times to 133 MW/m2·K compared with that of the flat interface. Also, the TBC is only dominated by the actual contact area at the interface for larger roughness cases; thus, we conclude that the phonon scattering probability increases with the increase of roughness and becomes stable gradually. Finally, the TBC of the copper/diamond interface with different roughness is characterized by TDTR experiments, and the results also confirm the trend of MD simulations. This study demonstrates the feasibility of the roughness modification for interfacial thermal management from both theoretical analysis and experimental measurements and provides a new idea for enhancing the thermal conductivity of composites.
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As one of the key components of innate immune system, piscidins are likely to play pivotal role in the first defense line in fish. Piscidins own multiple resistance activity. A novel piscidin 5-like type 4 was excavated from Larimichthys crocea (termed Lc-P5L4) liver transcriptome immuned by Cryptocaryon irritans, and upregulated at 7 days post infection when secondary bacterial infection occurred. In the study, we characterized the antibacterial activity of Lc-P5L4. The liquid growth inhibition assay detected the recombinant Lc-P5L4 (rLc-P5L) had potent antibacterial activity to Photobacterium damselae. Scanning electron microscope (SEM) observed the cell surface of P. damselae collapsed to form pit, and membrane of some bacteria ruptured after co-incubation with rLc-P5L. Further, transmission electron microscope (TEM) was also employed to observe the intracellular microstructural damage, rLc-P5L4 caused cytoplasm contraction, pores formation and contents leakage. After knowing about its antibacterial effects, the preliminary antibacterial mechanism was also explored, western blot analysis showed rLc-P5L4 could bind to P. damselae through targeting to LPS. Agarose gel eletrophoresis analysis further showed rLc-P5L4 could also penetrate into cells and brought about genome DNA degradation. Therefore, rLc-P5L4 was of potential being a candidate to explore new antimicrobial drug or additive agent, especially to P. damselae.
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Infecciones por Cilióforos , Enfermedades de los Peces , Hymenostomatida , Perciformes , Animales , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Antibacterianos/farmacología , Antibacterianos/metabolismo , Proteínas de Peces/químicaRESUMEN
Neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide), specifically existing in invertebrates, plays pivotal roles in various physiological processes. The involvement in neuroendocrine-immune regulation was explored in recent years, and it could modulate nitric oxide (NO) production under immune stress. However, detailed knowledge is still little known. In this study, we identified FMRFamide as an inhibitory factor on NO production in the immune reaction of Sepiella japonica. Firstly, Vibrio harveyi incubation caused significantly upregulated expression of FMRFamide precursor and NO synthase (NOS) in just hatched cuttlefish with quantitative Real-time PCR (qRT-PCR), which indicated that both were likely to be involved in the immune defense. The whole-mount in situ hybridization (ISH) detected FMRFamide precursor and NOS-positive signals appeared colocalization, suggesting that at histological and anatomical levels FMRFamide might interact with NOS. Next, NOS mRNA was highly significantly upregulated at 72 h when FMRFamide precursor mRNA was knocked down effectively with the RNA interference (RNAi) method; the results hinted that FMRFamide was likely to regulate NO production. Continuously, the inflammatory model was constructed in RAW 264.7 cells induced by lipopolysaccharide (LPS), FMRFamide administration resulted in a highly significant reduction of the NO level in dose- and time-response manners. Although the addition of the selected inducible NOS (iNOS) inhibitor had inhibited the NO production induced by LPS, the additional FMRFamide could still furtherly sharpen the process. Collectively, it was concluded that neuropeptide FMRFamide could indeed inhibit NO production to serve as feedback regulation at the late stage of immune response to protect hosts from excessive immune cytotoxicity. The inhibitory effect on NO production could not only be mediated by the NOS pathway but also be implemented through other pathways that needed to be furtherly explored. The results will provide data for comparing the structure and immune function of neuroendocrine-immune system (NEIS) between "advanced" cephalopods and other invertebrates and will provide new information for understanding the NEIS of cephalopods.
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Neuropéptidos , Óxido Nítrico , Animales , Decapodiformes/genética , Decapodiformes/metabolismo , FMRFamida/genética , FMRFamida/metabolismo , Lipopolisacáridos/metabolismo , Neuropéptidos/metabolismo , Óxido Nítrico/metabolismo , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Neuropeptide FF (NPFF), an octapeptide of the RFamide-related peptides (FaRPs), is involved in regulatory function in various biological processes. The regulatory role of NPFF in the immune and inflammatory response was currently being revealed. METHODS: Neuropeptide FF-related gene (termed LpNPFF) and its two receptors, NPFF receptor 1 (LpNPFFR1) and NPFF receptor 2 (LpNPFFR2) were identified by PCR and Semi-quantitative RT-PCR assay. Effect of LpNPFF on the production of nitric oxide (NO) in macrophage RAW264.7 cell was divided into PBS group, lipopolysaccharide (LPS) group, LPS treated with LpNPFF group, and LPS treated with LpNPFF and receptor antagonist RF9 group. Then specimens were measured by color reaction at 570 nm absorbance value. RESULTS: Sequence analysis showed that LpNPFF cDNA consists of 835 nucleotides with a 5'- untranslated region (UTR) of 150 base pair (bp), an open reading frame (ORF) of 384 bp and a 3'-UTR of 300 bp (Accession No. MT012894). The ORF encodes 127 amino acid (aa) residues with a hydrophobic signal peptide at N-terminus and two presumptive peptides with -PQRFa structure, LpNPFF (1) and LpNPFF (2). LpNPFFR1 and LpNPFFR2 encode 427 and 444 aa residues respectively, which both have seven hydrophobic TMDs and identified as G protein coupled receptors (GPCRs). Results of tissue distribution showed that LpNPFF and receptors were highly expressed in the brain and gonad. Furtherly, in vitro assay found LpNPFF could inhibit NO production in RAW 264.7 macrophages under inflammatory stress with LPS, while its receptor antagonist RF9 caused the evoke of NO generation. CONCLUSIONS: These results contribute to the further study of neuropeptide evolution in marine organisms, and also provide a new research idea for exploring the related functions of NPFF gene.
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Lipopolisacáridos , Receptores de Neuropéptido , Animales , Antiinflamatorios , Lipopolisacáridos/farmacología , Óxido Nítrico , Oligopéptidos/farmacología , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismoRESUMEN
Myeloid differentiation factor 88 (MyD88), composed of an N-terminal death domain and a C-terminal Toll/interleukin (IL)-IR homology domain, is a key connector protein in the TLR signal transduction pathway. In this study a novel isoform of MyD88 in Nibea albiflora (named as NaMyD88) was identified and functionally characterized (GenBank accession no. MN384261.1). Its complete cDNA sequence was 1672 bp and contained an open reading frame of 879 bp encoding 292 amino acid residues, which was similar to its teleost fish counterparts in the length. The theoretical molecular mass was 33.63 kDa and the isoelectric point was 5.24. BLASTp analysis suggested that the deduced amino acids sequence of NaMyD88 shared high identity to the known MyD88, for instance, 94.77% identity with Collichthys lucidus. Sequence analysis showed that NaMyD88 protein was consistent with MyD88 protein of other species at three conserved domains, N-terminal DD, short middle domain and C-terminal TIR, and the TIR domain contained three highly conserved motifs: Box1, Box2, and Box3. NaMyD88 and red fluorescent protein (Dsred) were fused and expressed in the cytoplasm of the epithelioma papulosum cyprini (EPC cells). The NaTLR9-TIR-EGFP fusion protein, which was obtained in our previous studies, showed green fluorescence and mainly distributed in the cytoplasm. After co-transfection, NaMyD88-Dsred and NaTLR9-TIR-EGFP obviously overlapped and displayed orange-yellow color. The results showed that the homologous MyD88-Dsred could interact with NaTLR9-TIR-EGFP. Based on this result pcMV-NaMyD88-TIR-Myc plasmids and the pcDNA3.1-NaTLR9-TIR-flag were constructed and co-transfected into 293T cells for the immunoprecipitation test. According to Western blot, the protein eluted by Flag-beads could be detected by anti-Flag-tag antibody and anti-Myc tag antibody respectively, while the protein without NaTLR9-TIR could not be found, which further proved that TLR and MyD88 could interact each other. The prokaryotic plasmid of MyD88-TIR domain was constructed, expressed in BL21 (DE3) and purified by Ni-NAT super flow resin conforming to the expected molecular weight of 27 kDa with the corresponding active sites for its conferring protein-protein interaction functions. Real-time fluorescence quantitative PCR showed that NaMyD88 could be expressed in intestine, stomach, liver, kidney, gill, heart and spleen, with the highest in the kidney, and it was up-regulated after being infected with Polyinosinic:polycytidylic acid - Poly (I:C) and Pseudomonas plecoglossicida, which showed that NaMyD88 was involved in the immune response of N.albiflora. These data afforded a basis for understanding the role of NaMyD88 in the TLR signaling pathway of N.albiflora.
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Factor 88 de Diferenciación Mieloide , Perciformes , Secuencia de Aminoácidos , Animales , Factor 88 de Diferenciación Mieloide/metabolismo , Perciformes/genética , Filogenia , Poli I-CRESUMEN
Toll-like receptors (TLRs) are an important class of molecules involved in non-specific immunity, and they are also the bridge connecting between non-specific immunity and specific immunity. As a vital member of TLR family TLR9 can be activated by bacterial DNA and induce the production of inflammatory cytokines. In this study, a full length of TLR9 homologue of 3677 bp in Nibea albiflora (named as NaTLR9, GenBank accession no: MN125017.1) was characterized, and its ORF was 3180 bp encoding 1059 amino acid residues with a calculated molecular weight of 121.334 kDa (pI = 6.29). Several leucine-rich repeated sequences (LRR domain) and conservative TIR domain were found in NaTLR9, which was mainly expressed in dendritic cells and macrophages. The phylogenetic and synteny analysis further revealed high sequence identity of NaTLR9 with its counterparts of other teleost, confirming their correct nomenclature and conservative during evolution as an important pattern recognition receptor. The NaTLR9-TIR-pEGFP-N1 fusion protein showed green fluorescence and mainly distributed in the cytoplasm. After co-transfection of NaTLR9-TIR-pEGFP-N1 and NaMyD88-pDsRED-Monomer-N1, green fluorescence obviously overlapped with red and changed into yellowish-green, which suggested that there might be the interaction between homologous NaTLR9-TIR and MyD88. Based on this result the pCDNA3.1-NaTLR9-TIR-flag and pcMV-NaMyD88-TIR-Myc plasmids were co-transfected into 293T cells for the immunoprecipitation test. According to Western blot, TLR9 and MyD88 protein could interact with each other. Furthermore, NaTLR9 was ubiquitously expressed in all the investigated tissues, most abundantly in head kidney, followed by stomach, spleen, liver and gill, but lower in muscle. The vitro immune stimulation experiments revealed that Pseudomonas plecoglossicida and polyinosinic-polycytidylic acid [Poly (I:C)] induced higher levels of NaTLR9 mRNA expression with the peaks of 9.52 times at 2 h and 39.91 times at 24 h compared with the control group respectively. The functional domains (LRRs and TIR, named NaTLR9-TIR and NaTLR9-LRR respectively) of NaTLR9 were expressed and purified, the recombinant proteins both could bind three kinds of typical aquatic pathogenic bacteria (Vibrio. parahaemolyticus, Vibrio alginolyticus, and Vibrio harveyi), which showed that NaTLR9 could couple to bacteria by its function domains. The aforementioned results indicated that NaTLR9 played a significant role in the defense against pathogenic bacteria infection in innate immune response of sciaenidae fish, which may provide some further understandings of the regulatory mechanisms in the teleostean innate immune system.
Asunto(s)
Proteínas de Peces , Perciformes , Vibrio parahaemolyticus , Secuencia de Aminoácidos , Animales , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica , Inmunidad Innata/genética , Perciformes/genética , Perciformes/metabolismo , Filogenia , Poli I-C , Receptor Toll-Like 9/genéticaRESUMEN
Coagulation-based pre-treatment efficiency of high strength digestate of food waste (HSDFW) anaerobic digestion is negated by organic ligand-catalyzed decomposition of coagulants. In this study, an efficient HSDFW pre-treatment method, magnetic seeds (MS) coagulation, was employed by using highly stable Keggin Al30 nanocluster (PAC30), MS and polyacrylamide (PAM), and its operation was optimized by evaluating the performance of removing turbidity, total suspended solids (TSS), chemical oxygen demand (COD), and total phosphorous (TP) phosphate. Results showed that at the optimum dosage of 4.82 g/L, PAC30 demonstrated excellent removals as high as 98.93% ± 0.1% of turbidity, 98.04% ± 0.1% of TSS, 58.28% ± 0.3% of total COD, 99.98% ± 0.01% of TP and 99.50% ± 0.01% of dissolved phosphate, respectively. Apparent molecular weight (AMW) and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy analyses demonstrated more efficient removal of dissolved organic matter (DOM), particularly non-biodegradable and hydrophobic components by PAC30 than commercial coagulant. The sedimentation was much improved from 40 min by coagulation/flocculation to about 5 min settling by MS coagulation. The PAC30 based magnetic coagulation (MC) presents theoretical guidance on a cost-effective and much less footprint pre-treatment alternative for high strength wastewater.