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Electron energy-loss spectroscopy (EELS) is widely used in analyzing the electronic structure of inorganic materials at high spatial resolution. In this study, we use a monochromator to improve the energy resolution, allowing us to analyze the electronic structure of organic light-emitting diode (OLED) materials with greater precision. This study demonstrates the use of the energy-loss near-edge structure to map the nitrogen content of organic molecules and identify the distinct bonding characteristics of aromatic carbon and pyridinic nitrogen. Furthermore, we integrate EELS with time-of-flight secondary ion mass spectrometry for molecular mapping of three different bilayers composed of OLED materials. This approach allows us to successfully map functional groups in the by-layer OLED and measure the thickness of two OLED layers. This study introduces spatially resolved functional group analysis using electron beam spectroscopy and contributes to the development of methods for complete nanoscale analysis of organic multilayer architectures.
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RATIONALE: Exosomes contain biomarkers such as proteins and lipids that help in understanding normal physiology and diseases. Lipids, in particular, are infrequently studied using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) for biomarker discovery. In this study, MALDI was equipped with a high-resolution MS to investigate exosomal lipids from human serum. METHODS: Exosomal lipids were profiled using MALDI with Fourier-transform ion cyclotron resonance (FTICR)-MS. Four matrices (i.e., α-cyano-4-hydroxycinnamic acid [CHCA], 2,5-dihydroxybenzoic acid, sinapinic acid, and graphene oxide [GO]) and three sample preparation methods (i.e., dried droplet, thin layer, and two layer) were compared for the number of lipid species detected and the relative abundance of each lipid from human serum and human serum exosomes. RESULTS: In sum, 172 and 89 lipid species were identified from human serum and human serum exosomes, respectively, using all the methods. The highest number of exosome lipid species, 69, was detected using the CHCA matrix, whereas only 8 exosome lipid species were identified using the GO matrix. Among the identified lipid species, phosphatidylcholine was identified most frequently, probably due to the use of a positive ion mode. CONCLUSIONS: Exosomes and human serum showed comparable lipid profiles as determined using MALDI-FTICR-MS. These findings provide a new perspective on exosomal lipidomics analysis and may serve as a foundation for future lipidomics-based biomarker research using MALDI-FTICR-MS.
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Lípidos , Proteínas , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Lípidos/análisis , Rayos LáserRESUMEN
There are vast but uncharacterized microbial taxa and chemical metabolites (that is, dark matter) across the Earth's ecosystems. A lack of knowledge about dark matter hinders a complete understanding of microbial ecology and biogeochemical cycles. Here, we examine sediment bacteria and dissolved organic matter (DOM) in 300 microcosms along experimental global change gradients in subtropical and subarctic climate zones of China and Norway, respectively. We develop an indicator to quantify the importance of dark matter by comparing co-occurrence network patterns with and without dark matter in bacterial or DOM assemblages. In both climate zones, dark matter constitutes approximately 30-56% of bacterial taxa and DOM metabolites and changes connectivity within bacterial and DOM assemblages by between -15.5 and +61.8%. Dark matter is generally more important for changing network connectivity within DOM assemblages than those of microbes, especially in the subtropical zone. However, the importance of dark matter along global change gradients is strongly correlated between bacteria and DOM and consistently increased toward higher primary productivity because of increasing temperatures and nutrient enrichment. Our findings highlight the importance of microbial and chemical dark matter for changing biogeochemical interactions under global change.
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Materia Orgánica Disuelta , Ecosistema , Bacterias/metabolismo , Clima , ChinaRESUMEN
Alpine river and lake systems on the Tibetan Plateau are highly sensitive indicators and amplifiers of global climate change and important components of the carbon cycle. Dissolved organic matter (DOM) encompasses organic carbon in aquatic systems, yet knowledge about DOM variation throughout the river-lake aquatic continuum within alpine regions is limited. We used optical spectroscopy, ultrahigh-resolution mass spectrometry (Fourier transform ion cyclotron resonance mass spectrometry), and stable water isotopic measurements to evaluate linkages between DOM composition and hydrological connection. We investigated glacial influences on DOM composition throughout the watershed of Selin Co, including upstream glacier-fed rivers and downstream-linked lakes. We found that the dissolved organic carbon concentration increased, whereas specific ultraviolet absorbance (SUVA254) decreased along the river-lake continuum. Relative to rivers, the downstream lakes had low relative abundances of polyphenolic and condensed aromatic compounds and humic-like substances but increased relative abundances of aliphatics and protein-like compounds. SUVA254 decreased while protein-like components increased with enriched stable water isotope δ2H-H2O, indicating that DOM aromaticity declined while autochthonous production increased along the flow paths. Glacier meltwater contributed to elevated relative abundances of aliphatic and protein-like compounds in headwater streams, while increased relative abundances of aromatics and humic-like DOM were found in glacier-fed lakes than downstream lakes. We conclude that changes in hydrological conditions, including glacier melt driven by a warming climate, will significantly alter DOM composition and potentially their biogeochemical function in surface waters on the Tibetan Plateau.
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Materia Orgánica Disuelta , Hidrología , Lagos , Materia Orgánica Disuelta/análisis , Lagos/análisis , Lagos/química , Tibet , Movimientos del Agua , Ríos , Congelación , Espectrometría de Masas , Hidrógeno , Isótopos de Oxígeno , VolatilizaciónRESUMEN
The concentration of polycyclic aromatic hydrocarbons (PAHs) in the atmosphere has been continually monitored since their toxicity became known, whereas nitro-PAHs (NPAHs) and oxy-PAHs (OPAHs), which are derivatives of PAHs by primary emissions or secondary formations in the atmosphere, have gained attention more recently. In this study, a method for the quantification of 18 NPAH and OPAH congeners in the atmosphere based on combined applications of gas chromatography coupled with chemical ionization mass spectrometry is presented. A high sensitivity and selectivity for the quantification of individual NPAH and OPAH congeners without sample preparations from the extract of aerosol samples were achieved using negative chemical ionization (NCI/MS) or positive chemical ionization tandem mass spectrometry (PCI-MS/MS). This analytical method was validated and applied to the aerosol samples collected from three regions in Northeast Asia-namely, Noto, Seoul, and Ulaanbaatar-from 15 December 2020 to 17 January 2021. The ranges of the method detection limits (MDLs) of the NPAHs and OPAHs for the analytical method were from 0.272 to 3.494 pg/m3 and 0.977 to 13.345 pg/m3, respectively. Among the three regions, Ulaanbaatar had the highest total mean concentration of NPAHs and OPAHs at 313.803 ± 176.349 ng/m3. The contribution of individual NPAHs and OPAHs in the total concentration differed according to the regional emission characteristics. As a result of the aerosol samples when the developed method was applied, the concentrations of NPAHs and OPAHs were quantified in the ranges of 0.016~3.659 ng/m3 and 0.002~201.704 ng/m3, respectively. It was concluded that the method could be utilized for the quantification of NPAHs and OPAHs over a wide concentration range.
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Conventional dried droplet (DD) methods show poor reproducibility in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) due to the frequent induction of a heterogeneous sample distribution. Recently, a forced dried droplet (FDD) sample preparation method was introduced to form homogeneous samples; this method improves the reproducibility of MALDI-MS analysis and generates highly multiply charged ions compared to DD methods. The FDD method utilizes secondary nucleation to generate a homogeneous sample distribution by applying an external force such as fluid shear stress by stirring the sample using a micropipette tip. In this study, a 2-nitrophloroglucinol (2-NPG) matrix was used for the DD and FDD sample preparation methods, and the charge state and homogeneity were compared by detecting multiply charged ions of proteins including cytochrome c, myoglobin, and immunoglobulin G (IgG) and measuring the relative standard deviation (RSD). FDD with a 2-NPG matrix produced a more homogeneous sample distribution and higher charge state ions than the DD method. FDD with a 2-NPG matrix was applied in MALDI-MS analysis of IgG fragments obtained from sequential reduction of IgG. In addition, FDD with intentional scratching of the MALDI plate by rotating a micropipette tip was found to provide similar or better reproducibility, higher charge state ions, and more uniformly distributed sample morphology compared to FDD without scratching.
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Rayos Láser , Iones , Floroglucinol/análogos & derivados , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Dissolved organic matter (DOM) is a large and complex mixture of molecules that fuels microbial metabolism and regulates biogeochemical cycles. Individual DOM molecules have unique functional traits, but how their assemblages vary deterministically under global change remains poorly understood. Here, we examine DOM and associated bacteria in 300 aquatic microcosms deployed on mountainsides that span contrasting temperatures and nutrient gradients. Based on molecular trait dimensions of reactivity and activity, we partition the DOM composition into labile-active, recalcitrant-active, recalcitrant-inactive, and labile-inactive fractions and quantify the relative influences of deterministic and stochastic processes governing the assembly of each. At both subtropical and subarctic study sites, the assembly of labile or recalcitrant molecules in active fractions is primarily governed by deterministic processes, while stochastic processes are more important for the assembly of molecules within inactive fractions. Surprisingly, the importance of deterministic selection increases with global change gradients for recalcitrant molecules in both active and inactive fractions, and this trend is paralleled by changes in the deterministic assembly of microbial communities and environmental filtering, respectively. Together, our results highlight the shift in focus from potential reactivity to realized activity and indicate that active and inactive fractions of DOM assemblages are structured by contrasting processes, and their recalcitrant components are consistently sensitive to global change. Our study partitions the DOM molecular composition across functional traits and links DOM with microbes via a shared ecological framework of assembly processes. This integrated approach opens new avenues to understand the assembly and turnover of organic carbon in a changing world.
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Materia Orgánica Disuelta , Microbiota , Bacterias/metabolismo , Carbono/metabolismoRESUMEN
Lakes receive and actively process terrestrial dissolved organic matter (DOM) and play an important role in the global carbon cycle. Urbanization results in elevated inputs of nonpoint-source DOM to headwater streams. Retention of water in lakes allows time for alteration and transformation of the chemical composition of DOM by microbes and UV radiation. Yet, it remains unclear how anthropogenic and natural drivers impact the composition and biolability of DOM in non-pristine lakes. We used optical spectroscopy, Fourier transform ion cyclotron mass spectrometry, stable isotopic measurements, and laboratory bioincubations to investigate the chemical composition and biolability of DOM across two large data sets of lakes associated with a large gradient of urbanization in lowland Eastern China, encompassing a total of 99 lakes. We found that increased urban land use, gross domestic products, and population density in the catchment were associated with an elevated trophic level index, higher chlorophyll-a, higher bacterial abundance, and a higher amount of organic carbon with proportionally higher contribution of aliphatic and peptide-like DOM fractions, which can be highly biolabile. Catchment areas, water depth, lake area: catchment area, gross primary productivity, δ18O-H2O, and bacterial abundance, however, had comparatively little linkage with DOM composition and biolability. Urban land use is currently intensifying in many developing countries, and our results anticipate an increase in the level of biolabile aliphatic DOM from nonpoint sources and accelerated carbon cycling in lake ecosystems in such regions.
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Materia Orgánica Disuelta , Ecosistema , Carbono/análisis , Lagos/química , RíosRESUMEN
Although the particle phase state is an important property, there is scant information on it, especially, for real-world aerosols. To explore the phase state of fine mode aerosols (PM2.5) in two megacities, Seoul and Beijing, we collected PM2.5 filter samples daily from Dec 2020 to Jan 2021. Using optical microscopy combined with the poke-and-flow technique, the phase states of the bulk of PM2.5 as a function of relative humidity (RH) were determined and compared to the ambient RH ranges in the two cities. PM2.5 was found to be liquid to semisolid in Seoul but mostly semisolid to solid in Beijing. The liquid state was dominant on polluted days, while a semisolid state was dominant on clean days in Seoul. These findings can be explained by the aerosol liquid water content related to the chemical compositions of the aerosols at ambient RH; the water content of PM2.5 was much higher in Seoul than in Beijing. Furthermore, the overall phase states of PM2.5 observed in Seoul and Beijing were interrelated with the particle size distribution. The results of this study aid in a better understanding of the fundamental physical properties of aerosols and in examining how these are linked to PM2.5 in polluted urban atmospheres.
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Contaminantes Atmosféricos , Material Particulado , Beijing , Material Particulado/análisis , Ciudades , Contaminantes Atmosféricos/análisis , Tamaño de la Partícula , Seúl , Monitoreo del Ambiente/métodos , Estaciones del Año , Agua , Aerosoles/análisis , ChinaRESUMEN
Large reservoirs are hotspots for carbon emissions, and the continued input and decomposition of terrestrial dissolved organic matter (DOM) from upstream catchments is an important source of carbon emissions. Rainstorm events can cause a surge in DOM input; however, periodic sampling often fails to fully capture the impact of these discrete rainstorm events on carbon emissions. We conducted a set of frequent observations prior to and following a rainstorm event in a major reservoir Lake Qiandao (China; 580 km2) from June to July 2021 to investigate how rainstorms alter water chemistry and CO2 and CH4 emissions. We found that the mean CO2 efflux (FCO2) (13.2 ± 9.3 mmol m-2 d-1) and CH4 efflux (FCH4) (0.12 ± 0.02 mmol m-2 d-1) in the postrainstorm campaign were significantly higher than those in the prerainstorm campaign (-3.8 ± 3.0 and +0.06 ± 0.02 mmol m-2 d-1, respectively). FCO2 and FCH4 increased with increasing nitrogen and phosphorus levels, elevated DOM absorption (a350), specific UV absorbance SUVA254, and terrestrial humic-like fluorescence. Furthermore, FCO2 and FCH4 decreased with increasing chlorophyll-a (Chl-a), dissolved oxygen (DO), and pH. A five-day laboratory anoxic bioincubation experiment further revealed a depletion of terrestrial-DOM concurrent with increased CO2 and CH4 production. We conclude that rainstorms boost the emission of CO2 and CH4 fueled by the surge and decomposition of fresh terrestrially derived biolabile DOM in this and likely many other reservoir's major inflowing river mouths.
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Agua Potable , Ríos , Ríos/química , Carbono/análisis , Dióxido de Carbono/análisis , Lagos/química , ChinaRESUMEN
Endocrine-disrupting potential was evaluated during the sewage treatment process using in vitro bioassays. Aryl hydrocarbon receptor (AhR)-, androgen receptor (AR)-, glucocorticoid receptor (GR)-, and estrogen receptor (ER)-mediated activities were assessed over five steps of the treatment process. Bioassays of organic extracts showed that AhR, AR, and GR potencies tended to decrease through the sewage treatment process, whereas ER potencies did not significantly decrease. Bioassays on reverse-phase high-performance liquid chromatography fractions showed that F5 (log KOW 2.5-3.0) had great ER potencies. Full-scan screening of these fractions detected two novel ER agonists, arenobufagin and loratadine, which are used pharmaceuticals. These compounds accounted for 3.3-25% of the total ER potencies and 4% of the ER potencies in the final effluent. The well-known ER agonists, estrone and 17ß-estradiol, accounted for 60 and 17% of the ER potencies in F5 of the influent and primary treatment, respectively. Fourier transform ion cyclotron resonance mass spectrometry analysis showed that various molecules were generated during the treatment process, especially CHO and CHOS (C: carbon, H: hydrogen, O: oxygen, and S: sulfur). This study documented that widely used pharmaceuticals are introduced into the aquatic environments without being removed during the sewage treatment process.
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Estrógenos/metabolismo , Receptores de Estrógenos/metabolismo , Aguas del Alcantarillado/química , Contaminantes Químicos del Agua/análisis , Bioensayo/métodos , Carbono/metabolismo , Estradiol/metabolismo , Estrógenos/genética , Estrona/metabolismo , Hidrógeno/metabolismo , Oxígeno/análisis , Preparaciones Farmacéuticas , Receptores Androgénicos/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Glucocorticoides/metabolismo , Azufre , Instalaciones de Eliminación de Residuos/normas , Eliminación de Residuos Líquidos/métodos , Eliminación de Residuos Líquidos/normasRESUMEN
Toll-like receptor (TLR)/myeloid differentiation primary response protein (MYD88) signaling aggravates sepsis by impairing neutrophil migration to infection sites. However, the role of intracellular fatty acids in TLR/MYD88 signaling is unclear. Here, inhibition of fatty acid synthase by C75 improved neutrophil chemotaxis and increased the survival of mice with sepsis in cecal ligation puncture and lipopolysaccharide-induced septic shock models. C75 specifically blocked TLR/MYD88 signaling in neutrophils. Treatment with GSK2194069 that targets a different domain of fatty acid synthase, did not block TLR signaling or MYD88 palmitoylation. De novo fatty acid synthesis and CD36-mediated exogenous fatty acid incorporation contributed to MYD88 palmitoylation. The binding of IRAK4 to the MYD88 intermediate domain and downstream signal activation required MYD88 palmitoylation at cysteine 113. MYD88 was palmitoylated by ZDHHC6, and ZDHHC6 knockdown decreased MYD88 palmitoylation and TLR/MYD88 activation upon lipopolysaccharide stimulus. Thus, intracellular saturated fatty acid-dependent palmitoylation of MYD88 by ZDHHC6 is a therapeutic target of sepsis.
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Receptores Toll-Like/metabolismo , Animales , Línea Celular , Acido Graso Sintasa Tipo I , Humanos , Inflamación , Lipoilación , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide , FN-kappa B/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Bacillus spores are highly resistant to toxic chemicals and extreme environments. Because some Bacillus species threaten public health, spore inactivation techniques have been intensively investigated. We exposed Bacillus atrophaeus spores to a 266 nm Nd:YVO4 laser at a laser power of 1 W and various numbers of scans. As a result, the UV laser reduced the viability of Bacillus atrophaeus spores. Although the outer coat of spores remained intact after UV laser irradiation of 720 scans, damage inside the spores was observed. Spore proteins were identified by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry during the course of UV laser irradiation. Photochemical and photothermal processes are believed to be involved in the UV laser sterilization of Bacillus spores. Our findings suggest that a UV laser is capable of sterilizing Bacillus atrophaeus spores.
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Bacillus , Esporas Bacterianas , Rayos Láser , Esporas , EsterilizaciónRESUMEN
How motile bacteria recognize their environment and decide whether to stay or navigate toward more favorable location is a fundamental issue in survival. The flagellum is an elaborate molecular device responsible for bacterial locomotion, and the flagellum-driven motility allows bacteria to move themselves to the appropriate location at the right time. Here, we identify the polar landmark protein HubP as a modulator of polar flagellation that recruits the flagellar assembly protein FapA to the old cell pole, thereby controlling its activity for the early events of flagellar assembly in Vibrio vulnificus. We show that dephosphorylated EIIAGlc of the PEP-dependent sugar transporting phosphotransferase system sequesters FapA from HubP in response to glucose and hence inhibits FapA-mediated flagellation. Thus, flagellar assembly and motility is governed by spatiotemporal control of FapA, which is orchestrated by the competition between dephosphorylated EIIAGlc and HubP, in the human pathogen V. vulnificus.
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Quimiotaxis/fisiología , Flagelos/metabolismo , Vibrio vulnificus/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Polaridad Celular/genética , Polaridad Celular/fisiología , Quimiotaxis/genética , Flagelos/fisiología , Regulación Bacteriana de la Expresión Génica/genética , Glucosa/metabolismo , Vibrio vulnificus/genéticaRESUMEN
Exosomes are nanosized vesicles commonly found in biological fluids as a result of a secretion process involving endosomes and multivesicular bodies. The isolation and analysis of exosomes can be useful for noninvasive clinical diagnosis of a variety of human diseases. We investigated the utility of analyzing exosomal proteins, using matrix-assisted laser desorption/ionization combined with Fourier-transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS), as a means of determining the presence of exosomes. MALDI-FTICR-MS analyses of exosomes enriched from human serum via centrifugation in a mass range of m/z 1000-20â¯000 yielded a distinctive protein around m/z 7766. The high mass accuracy and resolution of MALDI-FTICR-MS allowed for reliable comparisons against a protein database, through which the protein was identified as platelet factor 4 (PLF4), whose singly charged protein peak has an elemental composition of C341H577N96O101S4+, with a theoretical most abundant isotopic peak at m/z 7765.194 and a theoretical average peak at m/z 7766. The MALDI-TOF MS analysis of exosomes from the serum of 27 patients with different states of liver diseases provided the most abundant PLF4 peak for each mass spectrum, along with several additional minor peaks. In conclusion, MALDI-MS is suitable as an alternative exosome detection method, serving as a valuable confirmation tool, greatly decreasing the time and workload associated with exosome identification.
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Exosomas/química , Factor Plaquetario 4/sangre , Biomarcadores/sangre , Humanos , Hepatopatías/sangre , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Serum is one of the most commonly used samples in many studies to identify protein biomarkers to diagnose cancer. Although conventional enzyme-linked immunosorbent assay (ELISA) or liquid chromatography-mass spectrometry (LC-MS)-based methods have been applied as clinical tools for diagnosing cancer, there have been troublesome problems, such as inferior multiplexing capabilities, high development costs and long turnaround times, which are inappropriate for high-throughput analytical platforms. Here, we developed a simple and robust cancer diagnostic method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based total serum protein fingerprinting. First, serum samples were simply diluted with distilled water and subsequently spotted onto a MALDI plate without prior chromatographic purification or separation. The sample preparation method was enough to collect reproducible total serum protein fingerprints and would be highly advantageous for high-throughput assay. Each of the integrated main spectrum profiles (MSPs), which are representative of liver cancer patients (n = 40) or healthy controls (n = 80), was automatically generated by the MALDI Biotyper 3 software. The reliability of the integrated MSPs was successfully evaluated in comparison with a blind test set (n = 31), which consisted of 13 liver cancer patients and 18 healthy controls. Additionally, our partial least squares discriminant analysis (PLS-DA) demonstrated a statistically significant difference in MALDI-TOF MS-based total serum protein fingerprints between liver cancer patients and healthy controls. Taken together, this work suggests that this method may be an effective high-throughput platform technology for various cancer diagnoses and disease evaluations.
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Proteínas Sanguíneas/análisis , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/diagnóstico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estudios de Casos y Controles , HumanosRESUMEN
Globally, alpine glaciers hold a large quantity of dissolved organic matter (DOM) and are headwaters of numerous rivers supporting downstream heterotrophic metabolism. However, it remains unclear how glacial coverage and distance from the glacial terminus affect the fate of DOM. Here, we elucidate DOM variability in glacial-fed streams on the Tibetan Plateau using field sampling and bioincubation experiments and compare our findings with the existing literature. We found that dissolved organic carbon, DOM absorption a(254), DOM aromaticity, and the relative abundance of lignin compounds in glacial-fed streams and rivers all increased with increasing distance from the glacial terminus and with decreasing glacial coverage. We also found that contribution of protein-like components, the relative abundance of aliphatic compounds, and DOM biolability increased with increasing glacial coverage and with decreasing distance from the glacial terminus. The ratio of glacial coverage to the logarithmic transformed distance from the glacial terminus was better than that of actual glacial coverage and distance from the glacial terminus in tracing the variability of glacial-fed stream DOM. Microbes in surface ice can produce biolabile DOM that is exported downstream with meltwater. This glacial-fed stream and river DOM is an important source of the highly bioavailable material fueling downstream heterotrophic activity.
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Ecosistema , Ríos , Carbono , Cubierta de Hielo , TibetRESUMEN
In Campylobacterales and related ε-proteobacteria with N-linked glycosylation (NLG) pathways, free oligosaccharides (fOS) are released into the periplasmic space from lipid-linked precursors by the bacterial oligosaccharyltransferase (PglB). This hydrolysis results in the same molecular structure as the oligosaccharide that is transferred to a protein to be glycosylated. This allowed for the general elucidation of the fOS-branched structures and monosaccharides from a number of species using standard enrichment and mass spectrometry methods. To aid characterization of fOS, hydrazide chemistry has often been used for chemical modification of the reducing part of oligosaccharides resulting in better selectivity and sensitivity in mass spectrometry; however, the removal of the unreacted reagents used for the modification often causes the loss of the sample. Here, we develop a more robust method for fOS purification and characterize glycostructures using complementary tandem mass spectrometry (MS/MS) analysis. A cationic cysteine hydrazide derivative was synthesized to selectively isolate fOS from periplasmic fractions of bacteria. The cysteine hydrazide nicotinamide (Cyhn) probe possesses both thiol and cationic moieties. The former enables reversible conjugation to a thiol-activated solid support, while the latter improves the ionization signal during MS analysis. This enrichment was validated on the well-studied Campylobacter jejuni by identifying fOS from the periplasmic extracts. Using complementary MS/MS analysis, we approximated data of a known structure of the fOS from Campylobacter concisus. This versatile enrichment technique allows for the exploration of a diversity of protein glycosylation pathways.
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Campylobacter/química , Cisteína/análogos & derivados , Oligosacáridos/análisis , Espectrometría de Masas en Tándem/métodos , Cisteína/química , Oligosacáridos/aislamiento & purificación , Extracción en Fase Sólida/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
In the large-quantity production of α2,3- and α2,6-sialyllactose (Neu5Ac(α2,3)Galß1,4Glc (3'-SL) and Neu5Ac(α2,6)Galß1,4Glc (6'-SL)) using sialyltransferases (STs), there are major hurdles to overcome for further improvement in yield and productivity of the enzyme reactions. Specifically, Pasteurella multocida α2,3-sialyltransferase (α2,3PST) forms a by-product to a certain extent, owing to its multifunctional activity at pH below 7.0, and Photobacterium damselae α2,6-sialyltransferase (α2,6PdST) shows relatively low ST activity. In this study, α2,3PST and α2,6PdST were successfully engineered using a hybrid approach that combines rational design with site-saturation mutagenesis. Narrowly focused on the substrate-binding pocket of the STs, putative functional residues were selected by multiple sequence alignment and alanine scanning, and subsequently subjected to site-saturation mutagenesis. In the case of α2,3PST, R313N single mutation improved its activity slightly (by a factor of 1.5), and further improvement was obtained by making the double mutants (R313N/T265S and R313H/T265S) resulting in an overall 2-fold improvement in its specific α2,3 ST activity, which is mainly caused by the increase in kcat. It was revealed that the R313 mutations to N, D, Y, H or T greatly reduced the α2,6 ST side-reaction activity of α2,3PST at below pH 7.0. In the case of α2,6PdST, single-mutation L433S/T and double-mutation I411T/L433T exhibited 3- and 5-fold enhancement of the α2,6 ST-specific activity compared with the wild-type, respectively, via increase in kcat values. Our results show a very good model system for enhancing ST activity and demonstrate that the generated mutants could be used efficiently for the mass production of 3'-SL and 6'-SL with enhanced productivity and yield.
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Lactosa/análogos & derivados , Ingeniería de Proteínas/métodos , Sialiltransferasas/metabolismo , Clonación Molecular , Lactosa/biosíntesis , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Organismos Modificados Genéticamente , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , Photobacterium/genética , Photobacterium/metabolismo , Análisis de Secuencia de ADN , Sialiltransferasas/genética , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
SUMMARY: In recent years, the improvement of mass spectrometry-based glycomics techniques (i.e. highly sensitive, quantitative and high-throughput analytical tools) has enabled us to obtain a large dataset of glycans. Here we present a database named Xeno-glycomics database (XDB) that contains cell- or tissue-specific pig glycomes analyzed with mass spectrometry-based techniques, including a comprehensive pig glycan information on chemical structures, mass values, types and relative quantities. It was designed as a user-friendly web-based interface that allows users to query the database according to pig tissue/cell types or glycan masses. This database will contribute in providing qualitative and quantitative information on glycomes characterized from various pig cells/organs in xenotransplantation and might eventually provide new targets in the α1,3-galactosyltransferase gene-knock out pigs era. AVAILABILITY: The database can be accessed on the web at http://bioinformatics.snu.ac.kr/xdb.