Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 752
Filtrar
1.
Methods Mol Biol ; 2246: 207-224, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576991

RESUMO

Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) is an imaging method used to identify microorganisms in environmental samples based on their phylogeny. CARD-FISH can be combined with nano-scale secondary ion mass spectrometry (nanoSIMS) to directly link the cell identity to their activity, measured as the incorporation of stable isotopes into hybridized cells after stable isotope probing. In environmental microbiology, a combination of these methods has been used to determine the identity and growth of uncultured microorganisms, and to explore the factors controlling their activity. Additionally, FISH-nanoSIMS has been widely used to directly visualize microbial interactions in situ. Here, we describe a step-by-step protocol for a combination of CARD-FISH, laser marking, and nanoSIMS analysis on samples from aquatic environments.


Assuntos
Hibridização in Situ Fluorescente/métodos , Espectrometria de Massa de Íon Secundário/métodos , Isótopos de Carbono/metabolismo , Microbiologia Ambiental , Marcação por Isótopo/métodos , Microbiota/genética , Microbiota/fisiologia , Isótopos de Nitrogênio/metabolismo , Filogenia
2.
Proc Natl Acad Sci U S A ; 117(33): 20044-20051, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747534

RESUMO

Carbon and nitrogen isotope ratios in hair sampled from 65 communities across the central and intermountain regions of the United States and more intensively throughout 29 ZIP codes in the Salt Lake Valley, Utah, revealed a dietary divergence related to socioeconomic status as measured by cost of living, household income, and adjusted gross income. Corn-fed, animal-derived proteins were more common in the diets of lower socioeconomic status populations than were plant-derived proteins, with individual estimates of animal-derived protein diets as high as 75%; United States towns and cities averaged 57%. Similar patterns were seen across the socioeconomic status spectrum in the Salt Lake Valley. It is likely that corn-fed animal proteins were associated with concentrated animal-feeding operations, a common practice for industrial animal production in the United States today. Given recent studies highlighting the negative impacts of animal-derived proteins in our diets, hair carbon isotope ratios could provide an approach for scaling assessments of animal-sourced foods and health risks in communities across the United States.


Assuntos
Isótopos de Carbono/análise , Dieta/economia , Proteínas na Dieta/análise , Cabelo/química , Isótopos de Nitrogênio/análise , Animais , Proteínas na Dieta/economia , Proteínas na Dieta/metabolismo , Cabelo/metabolismo , Humanos , Isótopos de Nitrogênio/metabolismo , Classe Social , Estados Unidos , Utah
3.
PLoS One ; 15(6): e0235114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574188

RESUMO

Nursing and weaning periods are poorly understood in cetaceans due to the difficulty of assessing underwater behaviour in the wild. However, the onset and completion of weaning are critical turning points for individual development and survival, with implications for a species' life history including reproductive potential. δ15N and δ13C deposited in odontocete teeth annuli provide a lifetime record of diet, offering an opportunity to investigate variation and trends in fundamental biology. While available reproductive parameters for beaked whales have largely been inferred from single records of stranded or hunted animals and extrapolated across species, here we examine the weaning strategy and nursing duration in northern bottlenose whales (Hyperoodon ampullatus) by measuring stable isotopes deposited in dentine growth layer groups (GLGs). Using a collection of H. ampullatus teeth taken from whales killed during the whaling era (N = 48) and from two stranded specimens, we compared ontogenetic variation of δ15N and δ13C found in annual GLGs across all individuals, by sex and by region. We detected age-based trends in both δ15N and δ13C that are consistent across regions and males and females, and indicate that nursing is prolonged and weaning does not conclude until whales are 3-4 years old, substantially later than previous estimates of 1 year. Incorporating a prolonged period of maternal care into H. ampullatus life history significantly reduces their reproductive potential, with broad implications for models of beaked whale life history, energetics and the species' recovery from whaling.


Assuntos
Dentina/metabolismo , Marcação por Isótopo/métodos , Reprodução/fisiologia , Baleias/fisiologia , Animais , Animais Recém-Nascidos , Isótopos de Carbono/metabolismo , Feminino , Geografia , Islândia , Masculino , Terra Nova e Labrador , Isótopos de Nitrogênio/metabolismo , História Reprodutiva
4.
J Med Chem ; 63(10): 5360-5366, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32374601

RESUMO

Noninvasive evaluation of tertiary structures is fundamental to the research, development, and use of the biologics. However, few methodologies are currently available for evaluating large molecular weight (MW) biologics, such as therapeutic monoclonal antibodies (mAbs; 150 kDa). Here, we have newly developed a 15N direct detection nuclear magnetic resonance (NMR) technique, the 15N direct detection CRINEPT, which allows the observation of the main chain amide resonances of a nondeuterated protein with MW 150 kDa. The technique not only substantially expands the range of proteins applicable to solution NMR studies but also allows the noninvasive structural analyses of intact mAbs in a wide range of temperature and solvent conditions. As a proof of principle, we successfully acquired the 15N-detected CRINEPT spectra of an intact mAb in its formulated solution at 4 °C. The technique was able to discriminate heterogeneous galactosylation states, demonstrating the benefit of high resolution of the 15N direct detection.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Composição de Medicamentos/métodos , Espectroscopia de Ressonância Magnética/métodos , Mapeamento de Peptídeos/métodos , Anticorpos Monoclonais/metabolismo , Armazenamento de Medicamentos/métodos , Células HEK293 , Humanos , Isótopos de Nitrogênio/química , Isótopos de Nitrogênio/metabolismo , Estrutura Secundária de Proteína
5.
Sci Rep ; 10(1): 6803, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321946

RESUMO

Aedes aegypti is the main vector of arboviral diseases such as dengue, chikungunya and Zika. A key feature for disease transmission modeling and vector control planning is adult mosquito dispersal. We studied Ae aegypti adult dispersal by conducting a mark-capture study of naturally occurring Ae. aegypti from discarded containers found along a canal that divided two residential communities in Donna, Texas, USA. Stable isotopes were used to enrich containers with either 13C or 15N. Adult mosquitoes were collected outdoors in the yards of households throughout the communities with BG Sentinel 2 traps during a 12-week period. Marked mosquito pools with stable isotopes were used to estimate the mean distance travelled using three different approaches (Net, Strip or Circular) and the probability of detecting an isotopically marked adult at different distances from the larval habitat of origin. We consistently observed, using the three approaches that male (Net: 220 m, Strip: 255 m, Circular: 250 m) Ae. aegypti dispersed further in comparison to gravid (Net: 135 m, Strip: 176 m, Circular: 189 m) and unfed females (Net: 192 m, Strip: 213 m, Circular: 198 m). We also observed that marked male capture probability slightly increased with distance, while, for both unfed and gravid females, such probability decreased with distance. Using a unique study design documenting adult dispersal from natural larval habitat, our results suggest that Ae. aegypti adults disperse longer distances than previously reported. These results may help guide local vector control authorities in their fight against Ae. aegypti and the diseases it transmits, suggesting coverage of 200 m for the use of insecticides and innovative vector control tools.


Assuntos
Isótopos de Carbono/metabolismo , Ecossistema , Meio Ambiente , Controle de Mosquitos/métodos , Isótopos de Nitrogênio/metabolismo , Algoritmos , Animais , Febre de Chikungunya/prevenção & controle , Febre de Chikungunya/transmissão , Febre de Chikungunya/virologia , Dengue/prevenção & controle , Dengue/transmissão , Dengue/virologia , Feminino , Humanos , Inseticidas/farmacologia , Masculino , Modelos Teóricos , Controle de Mosquitos/instrumentação , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/metabolismo , Mosquitos Vetores/virologia , Texas , Infecção por Zika virus/prevenção & controle , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia
6.
Sci Rep ; 10(1): 6415, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286483

RESUMO

Drought stress is the most pervasive threat to plant growth, which predominantly encumbers turf grass growth by causing alterations in plant functions. This study appraised the role of nitrogen isotopes in providing a theoretical basis for developing and improving Kentucky bluegrass cultivar performance under drought stress. Nitrogen isotopes labelled 15NH4Cl and K15NO3 were prepared to replace KNO3 in Hoagland's solution at concentrations of 15NH4+ and 15NO3 at 1.5, 15, and 30 mM; the solutions were imposed on stressed plants under glasshouse conditions. Nitrogenous nutrition reduced oxidative stress by elevating the enzymatic activities and proline contents of all three clonal ramet leaves, particularly under stress conditions. Apart from nitrogen content, nitrogen isotope abundance, relative water content and water potential within controls were enhanced in treated with 15NH4+ than in with 15NO3 in both the roots and leaves of Kentucky bluegrass. Nevertheless, an application of 15NH4Cl and K15NO3 at 30 mM had a positive influence to some extent on these attributes under drought stress. Overall, our results suggested that nitrogen isotopes contributed to drought tolerance in all three clonal ramets of Kentucky bluegrass by maintaining a better osmoprotectant and antioxidant defence system, which helped the plants eliminate reactive oxygen species.


Assuntos
Secas , Isótopos de Nitrogênio/metabolismo , Poa/enzimologia , Poa/fisiologia , Estresse Fisiológico , Água/metabolismo , Catalase/metabolismo , Peroxidase/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Prolina/metabolismo , Superóxido Dismutase/metabolismo
7.
Sci Rep ; 10(1): 3387, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32099047

RESUMO

The extent to which commercially important Nephrops norvegicus lobsters feed on particulates in the wild is unknown, even though this could be an important way for burrow-dwelling females to avoid starvation during the long breeding season. This was investigated using δ13C and δ15N isotopic signatures in tissues with long and short turnover rates to provide diet discrimination and compare this between males and females. Secondary objectives examined size-related differences and calculated the trophic position based on the new results. Almost half the diet (47%) was made up of suspended particulate organic matter (POMsusp) alone. Fish was another important item in the diet, with plankton and invertebrate sources coming much lower down in dietary importance. Significantly more suspension feeding was observed in small or medium sized individuals than large ones in both sexes. However, there were no sex-related patterns, despite females being restricted to burrows for part of the analysis period. Female diet was almost identical to males and POMsusp comprised a large component of the diet in both sexes. The trophic position was estimated at 2.94 ± 0.16 (mean ± SD), which was at the lower end of the range reported in previous studies (2.60 to 4.32).


Assuntos
Dieta , Nephropidae/metabolismo , Material Particulado/metabolismo , Animais , Teorema de Bayes , Isótopos de Carbono/química , Isótopos de Carbono/metabolismo , Dieta/veterinária , Feminino , Masculino , Isótopos de Nitrogênio/química , Isótopos de Nitrogênio/metabolismo , Material Particulado/análise , Estações do Ano
8.
Sci Rep ; 10(1): 2522, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054911

RESUMO

Natural abundances of stable nitrogen and carbon isotopes (δ15N and δ13C) can vary with both dietary intake and metabolic (specifically catabolic) state. In low-income countries, weaning is a period of dietary transition from milk to plant-based foods and a high-risk period for malnutrition. We explored how diet and malnutrition impact hair δ15N and δ13C in young children by an observational, cross-sectional study in Cox's Bazar District, Bangladesh [255 children, 6-59 months with 19.6% wasted (7.1% severely) and 36% stunted (9.8% severely)]. Hair δ15N and δ13C exhibited exponential decreases with age, with the loss of one trophic level (3.3‰ and 0.8‰, respectively) from 6 to 48 months, which we associate with the shift from exclusive breastfeeding to complete weaning. After adjustment for age and breastfeeding status, hair isotopic values were unaffected by wasting but lower in severe stunting (-0.45‰ to -0.6‰, P < 0.01). In this population of young children, whose isotopic values in hair primarily depended on age, we failed to observe any effect of wasting, likely due to opposite, compensating effects between dietary and metabolic changes involved. In contrast, we evidenced low δ15N and δ13C values in severely stunted children that likely indicate chronic exposure to diets low in animal products.


Assuntos
Isótopos de Carbono/análise , Transtornos do Crescimento/metabolismo , Cabelo/química , Isótopos de Nitrogênio/análise , Síndrome de Emaciação/metabolismo , Desmame , Bangladesh/epidemiologia , Isótopos de Carbono/metabolismo , Pré-Escolar , Estudos Transversais , Dieta , Feminino , Transtornos do Crescimento/epidemiologia , Cabelo/metabolismo , Humanos , Lactente , Masculino , Isótopos de Nitrogênio/metabolismo , Síndrome de Emaciação/epidemiologia
9.
PLoS One ; 15(2): e0229740, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32106263

RESUMO

Bacterial production has been often estimated from DNA synthesis rates by using tritium-labeled thymidine. Some bacteria species cannot incorporate extracellular thymidine into their DNA, suggesting their biomass production might be overlooked when using the conventional method. In the present study, to evaluate appropriateness of deoxyribonucleosides for evaluating bacterial production of natural bacterial communities from the viewpoint of DNA synthesis, incorporation rates of four deoxyribonucleosides (thymidine, deoxyadenosine, deoxyguanosine and deoxycytidine) labeled by nitrogen stable isotope (15N) into bacterial DNA were examined in both ocean (Sagami Bay) and freshwater (Lake Kasumigaura) ecosystems in July 2015 and January 2016. In most stations in Sagami Bay and Lake Kasumigaura, we found that incorporation rates of deoxyguanosine were the highest among those of the four deoxyribonucleosides, and the incorporation rate of deoxyguanosine was approximately 2.5 times higher than that of thymidine. Whereas, incorporation rates of deoxyadenosine and deoxycytidine were 0.9 and 0.2 times higher than that of thymidine. These results clearly suggest that the numbers of bacterial species which can incorporate exogenous deoxyguanosine into their DNA are relatively greater as compared to the other deoxyribonucleosides, and measurement of bacterial production using deoxyguanosine more likely reflects larger numbers of bacterial species productions.


Assuntos
DNA Bacteriano/biossíntese , DNA Bacteriano/química , Isótopos de Nitrogênio/metabolismo , Baías/microbiologia , Biomassa , Desoxiadenosinas/metabolismo , Desoxicitidina/metabolismo , Desoxiguanosina/metabolismo , Ecossistema , Japão , Cinética , Lagos/microbiologia , Consórcios Microbianos/fisiologia , Timidina/metabolismo
10.
J Biol Chem ; 295(8): 2324-2335, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31953322

RESUMO

Defects in energy metabolism in either the retina or the immediately adjacent retinal pigment epithelium (RPE) underlie retinal degeneration, but the metabolic dependence between retina and RPE remains unclear. Nitrogen-containing metabolites such as amino acids are essential for energy metabolism. Here, we found that 15N-labeled ammonium is predominantly assimilated into glutamine in both the retina and RPE/choroid ex vivo [15N]Ammonium tracing in vivo show that, like the brain, the retina can synthesize asparagine from ammonium, but RPE/choroid and the liver cannot. However, unless present at toxic concentrations, ammonium cannot be recycled into glutamate in the retina and RPE/choroid. Tracing with 15N-labeled amino acids show that the retina predominantly uses aspartate transaminase for de novo synthesis of glutamate, glutamine, and aspartate, whereas RPE uses multiple transaminases to utilize and synthesize amino acids. Retina consumes more leucine than RPE, but little leucine is catabolized. The synthesis of serine and glycine is active in RPE but limited in the retina. RPE, but not the retina, uses alanine as mitochondrial substrates through mitochondrial pyruvate carrier. However, when the mitochondrial pyruvate carrier is inhibited, alanine may directly enter the retinal mitochondria but not those of RPE. In conclusion, our results demonstrate that the retina and RPE differ in nitrogen metabolism and highlight that the RPE supports retinal metabolism through active amino acid metabolism.


Assuntos
Nitrogênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Aminoácidos/metabolismo , Compostos de Amônio/farmacologia , Animais , Corioide/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Isótopos de Nitrogênio/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Piruvatos/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Retinaldeído/metabolismo
11.
J Biol Chem ; 295(10): 3362-3370, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31988244

RESUMO

Ammonium transporters (AMT), methylamine permeases (Mep), and the more distantly related rhesus factors (Rh) are trimeric membrane proteins present in all domains of life. AMT/Mep/Rhs are highly selective membrane proteins required for ammonium uptake or release, and they efficiently exclude the similarly sized K+ ion. Previously reported crystal structures have revealed that each transporter subunit contains a unique hydrophobic but occluded central pore, but it is unclear whether the base (NH3) or NH3 coupled with an H+ are transported. Here, using expression of two plant AMTs (AtAMT1;2 and AMT2) in budding yeast, we found that systematic replacements in the conserved twin-histidine motif, a hallmark of most AMT/Mep/Rh, alter substrate recognition, transport capacities, N isotope selection, and selectivity against K+ AMT-specific differences were found for histidine variants. Variants that completely lost ammonium N isotope selection, a feature likely associated with NH4 + deprotonation during passage, substantially transported K+ in addition to NH4 + Of note, the twin-histidine motif was not essential for ammonium transport. However, it conferred key AMT features, such as high substrate affinity and selectivity against alkali cations via an NH4 + deprotonation mechanism. Our findings indicate that the twin-His motif is the core structure responsible for substrate deprotonation and isotopic preferences in AMT pores and that decreased deprotonation capacity is associated with reduced selectivity against K+ We conclude that optimization for ammonium transport in plant AMT represents a compromise between substrate deprotonation for optimal selectivity and high substrate affinity and transport rates.


Assuntos
Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Histidina/metabolismo , Proteínas de Plantas/metabolismo , Compostos de Amônio/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Histidina/química , Íons/química , Cinética , Metilaminas/metabolismo , Mutagênese Sítio-Dirigida , Isótopos de Nitrogênio/química , Isótopos de Nitrogênio/metabolismo , Oócitos/metabolismo , Proteínas de Plantas/genética , Potássio/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Especificidade por Substrato , Xenopus laevis/crescimento & desenvolvimento
12.
Sci Rep ; 10(1): 1211, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988309

RESUMO

Nitrogen (N) fertilizers can potentially alter spatial distribution of soil organic carbon (SOC) and total nitrogen (TN)  concentrations in croplands such as switchgrass (SG: Panicum virgatum L.) and gamagrass (GG: Tripsacum dactyloides L.), but it remains unclear whether these effects are the same between crops and under different rates of fertilization. 13C and 15N are  two important proxy measures of soil biogeochemistry, but they were rarely examined as to their spatial distributions in soil. Based on a three-year long fertilization experiment in Middle Tennessee, USA, the top mineral horizon soils (0-15 cm) were collected using a spatially explicit design within two 15-m2 plots under three fertilization treatments in SG and GG croplands. A total of 288 samples were collected based on 12 plots and 24 samples in each plot. The fertilization treatments were no N input (NN), low N input (LN: 84 kg N ha-1 in urea) and high N input (HN: 168 kg N ha-1 in urea). The SOC, TN, SOC/TN (C: N), δ13C and δ15N were quantified and their within-plot variations and spatial distributions were achieved via descriptive and geostatistical methods. Results showed that SG generally displayed 10~120% higher plot-level variations in all variables than GG, and the plot-level variations were 20~77% higher in NN plots than LN and HN plots in SG but they were comparable in unfertilized and fertilized plots in GG. Relative to NN, LN and HN showed more significant surface trends and spatial structures in SOC and TN in both croplands, and the fertilization effect appeared more pronounced in SG. Spatial patterns in C: N, δ13C and δ15N were comparable among different fertilization treatments in both croplands. The descending within-plot variations were also identified among variables (SOC > TN > δ15N > C: N > δ13C). This study demonstrated that N fertilizations generally reduced the plot-level variance and simultaneously re-established spatial structures of SOC and TN in bioenergy croplands, which little varied with fertilization rate but was more responsive in switchgrass cropland.


Assuntos
Isótopos de Carbono/metabolismo , Produtos Agrícolas/metabolismo , Fertilizantes , Isótopos de Nitrogênio/metabolismo , Compostos Orgânicos/metabolismo , Panicum/metabolismo , Solo/química , Ciclo do Carbono , Isótopos de Carbono/química , Isótopos de Nitrogênio/química , Concentração Osmolar , Tennessee
13.
J Sci Food Agric ; 100(4): 1608-1615, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31773732

RESUMO

BACKGROUND: The possible escape from the rumen of undegraded-feed soluble proteins (SP) was investigated using nitrogen-15 (15 N) infusions (25 mg) in three rumen and duodenum cannulated wethers. Animals were fed three isoproteic diets differing in SP content and including protein concentrates either untreated (control) or treated with solutions of either malic acid (MHT) or orthophosphoric acid (OHT) and heat. RESULTS: Compared with control, MHT and OHT diets reduced ruminal concentrations of ammonia-nitrogen (NH3 -N) by 35.9% (P = 0.007), non-ammonia nitrogen (NAN) by 36.8% (P = 0.007), and SP-nitrogen (SP-N) by 45.2% (P = 0.072) over the post-feeding period. Both NAN and SP-N were lower (P ≤ 0.040) for OHT than for MHT diet. The 15 N enrichment of NAN and SP-N did not vary either among diets or with time, and both values were closely related (R2 = 0.965; P < 0.001). Estimations, either using solid-associated bacteria (SAB) or liquid-associated bacteria (LAB) as a reference, indicated that 0.983 and 0.894 of SP-N (values averaged across diets) was of microbial origin, respectively, which would indicate a practically negligible ruminal escape of feed SP-N. Values of 15 N-enrichment in SAB and LAB fitted well to previously published SAB-LAB relationships, indicating a 22% underevaluation of the N supply from SAB when only LAB is used as a reference. CONCLUSION: Both the negligible ruminal escape of feed SP and the underevaluation of the bacterial nutrient supply as a consequence of the use of LAB as the only bacterial reference should be considered to improve ruminant protein feeding systems. © 2019 Society of Chemical Industry.


Assuntos
Bactérias/metabolismo , Proteínas/metabolismo , Rúmen/microbiologia , Amônia/análise , Amônia/metabolismo , Ração Animal/análise , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Duodeno/metabolismo , Duodeno/microbiologia , Masculino , Isótopos de Nitrogênio/análise , Isótopos de Nitrogênio/metabolismo , Nutrientes/análise , Nutrientes/metabolismo , Proteínas/análise , Rúmen/metabolismo
14.
J Nutr ; 150(3): 450-457, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31825068

RESUMO

BACKGROUND: In the context of developing plant protein sources for humans, sunflower is a good candidate in its form as an oilseed coproduct. OBJECTIVES: We aimed to compare the real digestibility in rats of a sunflower isolate to that of goat whey protein. We also studied the efficiency of 15N and 2H intrinsic labeling in this assessment. METHODS: Sunflower seeds and goat milk were labeled with 15N and 2H. Male Wistar rats (10 wk old) were fed a meal containing 12% of either sunflower isolate (n = 8) or whey (n = 8). Six hours after meal ingestion, protein and amino acid digestibility were assessed by measuring nitrogen, hydrogen, and amino acids in the digesta, as well as isotope enrichments in the bulk and individual amino acids. The differences between groups and isotopes were respectively tested with an unpaired and a paired t test. RESULTS: Protein isolate purity was 87% for whey and 94% for sunflower. 2H and 15N enrichments were, respectively, 0.12 atom % (AP) and 1.06 AP in sunflower isolate and 0.18 AP and 0.95 AP in whey. Fecal 15N protein digestibility was 97.2 ± 0.2% for whey and 95.1 ± 0.5% for sunflower isolate. The use of 2H resulted in a lower digestibility estimate than 15N for whey (96.9 ± 0.2%, P < 0.05) and sunflower (94.2 ± 0.5%, P < 0.01). For both isotopes, protein digestibility was about 2% higher for whey than for sunflower isolate. Mean 15N amino acid caecal digestibility was 97.5 ± 0.2% for whey and 96.3 ± 0.2% for sunflower isolate. The values obtained with 15N and 2H resulted in significant differences ranging from -0.1% to 3.5%. The DIAAS was >1.0 for whey and 0.84 for sunflower (lysine). CONCLUSIONS: The protein and amino acid digestibility of sunflower isolate was high but its DIAAS reflected a moderate lysine imbalance. Despite slight differences with 15N, deuterium produced comparable results, making it suitable for in vivo digestion studies.


Assuntos
Aminoácidos/metabolismo , Deutério/metabolismo , Proteínas na Dieta/metabolismo , Digestão , Helianthus/metabolismo , Isótopos de Nitrogênio/metabolismo , Proteínas de Plantas/metabolismo , Soro do Leite/metabolismo , Animais , Cabras , Masculino , Ratos , Ratos Wistar
15.
Elife ; 82019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31820737

RESUMO

The lenticular fiber cells are comprised of extremely long-lived proteins while still maintaining an active biochemical state. Dysregulation of these activities has been implicated in diseases such as age-related cataracts. However, the lenticular protein dynamics underlying health and disease is unclear. We sought to measure the global protein turnover rates in the eye using nitrogen-15 labeling of mice and mass spectrometry. We measured the 14N/15N-peptide ratios of 248 lens proteins, including Crystallin, Aquaporin, Collagen and enzymes that catalyze glycolysis and oxidation/reduction reactions. Direct comparison of lens cortex versus nucleus revealed little or no 15N-protein contents in most nuclear proteins, while there were a broad range of 14N/15N ratios in cortex proteins. Unexpectedly, like Crystallins, many enzymes with relatively high abundance in nucleus were also exceedingly long-lived. The slow replacement of these enzymes in spite of young age of mice suggests their potential roles in age-related metabolic changes in the lens.


Assuntos
Cristalino/enzimologia , Cristalino/metabolismo , Espectrometria de Massas , Coloração e Rotulagem , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Matriz Extracelular/metabolismo , Proteínas do Olho/metabolismo , Glicólise , Camundongos Endogâmicos C57BL , Isótopos de Nitrogênio/metabolismo , Oxirredução , Biossíntese de Proteínas , Proteoma/metabolismo
16.
Nat Commun ; 10(1): 5318, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31754221

RESUMO

Stimulated Raman scattering (SRS) microscopy allows for high-speed label-free chemical imaging of biomedical systems. The imaging sensitivity of SRS microscopy is limited to ~10 mM for endogenous biomolecules. Electronic pre-resonant SRS allows detection of sub-micromolar chromophores. However, label-free SRS detection of single biomolecules having extremely small Raman cross-sections (~10-30 cm2 sr-1) remains unreachable. Here, we demonstrate plasmon-enhanced stimulated Raman scattering (PESRS) microscopy with single-molecule detection sensitivity. Incorporating pico-Joule laser excitation, background subtraction, and a denoising algorithm, we obtain robust single-pixel SRS spectra exhibiting single-molecule events, verified by using two isotopologues of adenine and further confirmed by digital blinking and bleaching in the temporal domain. To demonstrate the capability of PESRS for biological applications, we utilize PESRS to map adenine released from bacteria due to starvation stress. PESRS microscopy holds the promise for ultrasensitive detection and rapid mapping of molecular events in chemical and biomedical systems.


Assuntos
Lasers , Microscopia Óptica não Linear/métodos , Imagem Individual de Molécula/métodos , Análise Espectral Raman/métodos , Staphylococcus aureus/metabolismo , Adenina/metabolismo , Elétrons , Isótopos de Nitrogênio/metabolismo , Reprodutibilidade dos Testes , Imagem Individual de Molécula/instrumentação , Estresse Fisiológico
17.
PLoS One ; 14(9): e0222754, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31539419

RESUMO

Sharks are top predators across ocean food webs and have a major ecological role in marine ecosystems. Investigating the trophic ecology of this group of species is thus essential to understand ecosystem functioning and inform specific management actions aimed at shark conservation. The Galapagos Islands represent one of the last ocean wildernesses, where populations of sharks and other top marine predators come close to a pristine status. Here we provide the first study on the trophic ecology of the tiger shark (Galeocerdo cuvier) within the Galapagos Marine Reserve (GMR), using a combination of stable isotope analysis, satellite tracking, and passive acoustic telemetry to investigate ontogenetic and spatial variations at two regions. The mean estimated δ13C and δ15N at Isabela island (western region) were -13.9 ± 0.5‰ and 13.7 ± 0.7‰; and for Santa Cruz island (central region) were -13.8 ± 0.3‰ and 13.4 ± 0.7‰, respectively. Green sea turtles (Chelonia mydas) were the main prey item for large tiger sharks (>280 cm TL), while smaller sharks mainly fed on squid and pelagic fish. Tiger sharks exhibited a high degree of philopatry around green sea-turtle nesting areas, with the majority of sharks detected around green sea-turtle nesting areas for at least 10 months after their capture date, and some individuals were even present during the entire three-year study period. Although we did not report statistically significant differences between the two regions, isotopic and electronic tagging data suggest that tiger sharks in the Galapagos could be segregated into specific populations separated by geographical scales of <100 km. The high productivity of the archipelago, along with the protection from industrial fishing granted by the GMR, result in abundant and predictable sources of prey. This high food abundance, combined with the presence of suitable habitats throughout the tiger shark life cycle, might result in a reduction of migratory behaviours when compared to movement patterns of tiger sharks in other ocean basins. Additional studies using genetic tools could provide further evidence on the presence of separate management units, as it has been recently revealed for other shark species inhabiting the GMR.


Assuntos
Migração Animal/fisiologia , Ecologia/métodos , Ecossistema , Cadeia Alimentar , Comportamento Predatório/fisiologia , Tubarões/fisiologia , Animais , Isótopos de Carbono/metabolismo , Equador , Feminino , Geografia , Ilhas , Masculino , Biologia Marinha/métodos , Isótopos de Nitrogênio/metabolismo , Paladar , Telemetria/métodos
18.
FEMS Microbiol Ecol ; 95(10)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504453

RESUMO

The endophytic, insect pathogenic fungus, Metarhizium, exchanges insect-derived nitrogen for photosynthate as part of a symbiotic association similar to well-known mycorrhizal relationships. However, little is known about this nitrogen transfer in soils where there is an abundance of nitrogen and/or carbon. Here, we applied D-glucose and ammonium nitrate to soil to examine the effect on root colonization and transfer of labelled nitrogen (15N) from an insect (injected with 15N-ammonium sulfate) to Metarhizium robertsii, into leaves of the common bean, Phaseolus vulgaris, over the course of 28 days. Application of exogenous carbon and/or nitrogen to soils significantly reduced detectable 15N in plant leaves. Metarhizium root colonization, quantified with real-time PCR, revealed colonization persisted under all conditions but was significantly greater on roots in soil supplemented with glucose and significantly lower in soil supplemented with ammonium nitrate. Fungal gene expression analysis revealed differential expression of sugar and nitrogen transporters (mrt, st3, nrr1, nit1, mep2) when Metarhizium was grown in pure broth culture or in co-culture with plant roots under various carbon and nitrogen conditions. The observation that Metarhizium maintained root colonization in the absence of nitrogen transfer, and without evidence of plant harm, is intriguing and indicates additional benefits with ecological importance.


Assuntos
Carbono/metabolismo , Insetos/microbiologia , Metarhizium/metabolismo , Isótopos de Nitrogênio/metabolismo , Phaseolus/metabolismo , Raízes de Plantas/microbiologia , Animais , Carbono/análise , Insetos/metabolismo , Metarhizium/crescimento & desenvolvimento , Micorrizas/crescimento & desenvolvimento , Micorrizas/metabolismo , Isótopos de Nitrogênio/análise , Phaseolus/química , Phaseolus/microbiologia , Folhas de Planta/química , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Solo/química
19.
Methods Mol Biol ; 2046: 71-87, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31407297

RESUMO

Chip-SIP is a stable isotope probing (SIP) method for linking microbial identity and function in mixed communities and is capable of analyzing multiple isotopes (13C, 15N, and 18O) simultaneously. This method uses a high-density microarray to separate taxon-specific 16S (or 18S) rRNA genes and a high sensitivity magnetic sector secondary ion mass spectrometer (SIMS) to determine the relative isotope incorporation of the rRNA at each probe location. Using a maskless array synthesizer (MAS), we synthesize multiple unique sequences to target hundreds of taxa at the ribosomal operational taxonomic unit (OTU) level on an array surface, and then analyze it with a NanoSIMS 50, using its high-spatial resolution imaging capability to generate isotope ratios for individual probes. The Chip-SIP method has been used in diverse systems, including surface marine and estuarine water, rhizosphere, and peat soils, to quantify taxon-specific relative incorporation of different substrates in complex microbial communities. Depending on the hypothesis and experimental design, Chip-SIP allows the user to compare the same community incorporating different substrates, different communities incorporating the same substrate(s), or quantify how a community responds to treatment effects, such as temperature or nutrient concentrations.


Assuntos
DNA Bacteriano/genética , Marcação por Isótopo/métodos , Microbiota/genética , RNA Ribossômico/genética , Bactérias/genética , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Classificação , Espectrometria de Massas , Análise em Microsséries , Isótopos de Nitrogênio/análise , Isótopos de Nitrogênio/metabolismo , Isótopos de Oxigênio/análise , Isótopos de Oxigênio/metabolismo , Filogenia , RNA Ribossômico/metabolismo , Software
20.
Methods Mol Biol ; 2046: 95-107, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31407299

RESUMO

Microorganisms play a key role in driving the global element (C, N, H, P, and S) cycling. However, the function and activity of environmental microbes remain largely elusive because the vast majority of them are yet uncultured. Recent achievements in single cell stable isotope-labeled Raman spectroscopy enable direct investigation of function and activity of individual microbes in complex environmental communities. Here, this protocol describes a workflow to investigate environmental microbes in soil and water by combining 15N, 2D, and 13C stable isotope labeling with different single-cell Raman techniques, including normal Raman, resonance Raman (RR), and surface-enhanced Raman spectroscopy (SERS). Their applications in investigating functional bacteria driving the N and C cycles, and metabolically active cells are described.


Assuntos
Microbiologia Ambiental , Marcação por Isótopo/métodos , Análise de Célula Única/métodos , Análise Espectral Raman/métodos , Azotobacter/isolamento & purificação , Azotobacter/metabolismo , Isótopos de Carbono/metabolismo , Deutério/metabolismo , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Nanopartículas Metálicas/química , Microbiota/fisiologia , Isótopos de Nitrogênio/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...