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Browne, Thomas R., George K. Szabo, Alfred Ajami y David Wagner. "Performance of Human Mass Balance/Metabolite Identification Studies Using Stable Isotope (13C,15N) Labeling and Continuous-Flow Isotope-Ratio Mass Spectrometry as an Alternative to Radioactive Labeling Methods". Journal of Clinical Pharmacology 33, n.º 3 (marzo de 1993): 246–52. http://dx.doi.org/10.1002/j.1552-4604.1993.tb03951.x.
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Dusny, Christian y Andreas Schmid. "The Metabolic Flux Probe (MFP)—Secreted Protein as a Non-Disruptive Information Carrier for 13C-Based Metabolic Flux Analysis". International Journal of Molecular Sciences 22, n.º 17 (30 de agosto de 2021): 9438. http://dx.doi.org/10.3390/ijms22179438.
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Novel cultivation technologies demand the adaptation of existing analytical concepts. Metabolic flux analysis (MFA) requires stable-isotope labeling of biomass-bound protein as the primary information source. Obtaining the required protein in cultivation set-ups where biomass is inaccessible due to low cell densities and cell immobilization is difficult to date. We developed a non-disruptive analytical concept for 13C-based metabolic flux analysis based on secreted protein as an information carrier for isotope mapping in the protein-bound amino acids. This “metabolic flux probe” (MFP) concept was investigated in different cultivation set-ups with a recombinant, protein-secreting yeast strain. The obtained results grant insight into intracellular protein turnover dynamics. Experiments under metabolic but isotopically nonstationary conditions in continuous glucose-limited chemostats at high dilution rates demonstrated faster incorporation of isotope information from labeled glucose into the recombinant reporter protein than in biomass-bound protein. Our results suggest that the reporter protein was polymerized from intracellular amino acid pools with higher turnover rates than biomass-bound protein. The latter aspect might be vital for 13C-flux analyses under isotopically nonstationary conditions for analyzing fast metabolic dynamics.
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Dashper, Stuart G., Ching-Seng Ang, Paul D. Veith, Helen L. Mitchell, Alvin W. H. Lo, Christine A. Seers, Katrina A. Walsh et al. "Response of Porphyromonas gingivalis to Heme Limitation in Continuous Culture". Journal of Bacteriology 191, n.º 3 (21 de noviembre de 2008): 1044–55. http://dx.doi.org/10.1128/jb.01270-08.
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ABSTRACT Porphyromonas gingivalis is an anaerobic, asaccharolytic, gram-negative bacterium that has essential requirements for both iron and protoporphyrin IX, which it preferentially obtains as heme. A combination of large-scale quantitative proteomic analysis using stable isotope labeling strategies and mass spectrometry, together with transcriptomic analysis using custom-made DNA microarrays, was used to identify changes in P. gingivalis W50 protein and transcript abundances on changing from heme-excess to heme-limited continuous culture. This approach identified 160 genes and 70 proteins that were differentially regulated by heme availability, with broad agreement between the transcriptomic and proteomic data. A change in abundance of the enzymes of the aspartate and glutamate catabolic pathways was observed with heme limitation, which was reflected in organic acid end product levels of the culture fluid. These results demonstrate a shift from an energy-efficient anaerobic respiration to a less efficient process upon heme limitation. Heme limitation also resulted in an increase in abundance of a protein, PG1374, which we have demonstrated, by insertional inactivation, to have a role in epithelial cell invasion. The greater abundance of a number of transcripts/proteins linked to invasion of host cells, the oxidative stress response, iron/heme transport, and virulence of the bacterium indicates that there is a broad response of P. gingivalis to heme availability.
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Bhatia, Muskan, Jyotika Thakur, Shradha Suyal, Ruchika Oniel, Rahul Chakraborty, Shalini Pradhan, Monika Sharma et al. "Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism". Journal of Biological Chemistry 295, n.º 47 (15 de septiembre de 2020): 16037–57. http://dx.doi.org/10.1074/jbc.ra120.015129.
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Methylenetetrahydrofolate reductase (MTHFR) links the folate cycle to the methionine cycle in one-carbon metabolism. The enzyme is known to be allosterically inhibited by SAM for decades, but the importance of this regulatory control to one-carbon metabolism has never been adequately understood. To shed light on this issue, we exchanged selected amino acid residues in a highly conserved stretch within the regulatory region of yeast MTHFR to create a series of feedback-insensitive, deregulated mutants. These were exploited to investigate the impact of defective allosteric regulation on one-carbon metabolism. We observed a strong growth defect in the presence of methionine. Biochemical and metabolite analysis revealed that both the folate and methionine cycles were affected in these mutants, as was the transsulfuration pathway, leading also to a disruption in redox homeostasis. The major consequences, however, appeared to be in the depletion of nucleotides. 13C isotope labeling and metabolic studies revealed that the deregulated MTHFR cells undergo continuous transmethylation of homocysteine by methyltetrahydrofolate (CH3THF) to form methionine. This reaction also drives SAM formation and further depletes ATP reserves. SAM was then cycled back to methionine, leading to futile cycles of SAM synthesis and recycling and explaining the necessity for MTHFR to be regulated by SAM. The study has yielded valuable new insights into the regulation of one-carbon metabolism, and the mutants appear as powerful new tools to further dissect out the intersection of one-carbon metabolism with various pathways both in yeasts and in humans.
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Ermolaev, Stanislav, Aino Skasyrskaya y Aleksandr Vasiliev. "A Radionuclide Generator of High-Purity Bi-213 for Instant Labeling". Pharmaceutics 13, n.º 6 (21 de junio de 2021): 914. http://dx.doi.org/10.3390/pharmaceutics13060914.
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A new two-column 225Ac/213Bi generator was developed specifically for using 225Ac containing an impurity of long lived 227Ac. The parent 225Ac was retained on the first Actinide Resin column, while 213Bi was accumulated on the second column filled with AG MP-50 resin via continuous elution and decay of intermediate 221Fr. The 213Bi accumulation was realized in circulation mode which allowed a compact generator design. It was demonstrated that 213Bi could be quickly and effectively extracted from AG MP-50 in form of complexes with various chelating agents including DTPA and DOTA. The performance of the generator presented and a conventional single-column generator on the base of AG MP-50 was tested and both generators were loaded with 225Ac containing 227Ac impurity. The 213Bi generation efficiencies were comparable and greater than 70%, whereas the developed generator provided a deeper degree of purification of 213Bi from Ac isotopes and decay products of 227Ac.
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Rambal, Corinne, Christiane Pachiaudi, Sylvie Normand, Jean-Paul Riou, Pierre Louisot y Ambroise Martin. "Effects of specific dietary sugars on the incorporation of 13C label from dietary glucose into neutral sugars of rat intestine and serum glycoproteins". British Journal of Nutrition 73, n.º 3 (marzo de 1995): 443–54. http://dx.doi.org/10.1079/bjn19950046.
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Although theoretically all glycoprotein sugars can be derived from glucose, it may be hypothesized that specific dietary sugars could be preferential substrates for glycoprotein synthesis. To test this hypothesis, groups of rats received either continuously (continuous-labelling experiment) or for a single nutritional period (pulse-labelling experiment) a 13C-rich diet containing either maize starch or artificially labelled [13C]glucose. Some groups of rats were also provided during a single nutritional period with low amounts (20–200 mg/animal) of low-13C dietary sugars (mannose, galactose, fucose or fructose). If specific dietary sugars were preferentially incorporated into glycoproteins instead nf glucose-derived labelled sugars, a decrease would be expected in the intestinal or serum glycoprotein-sugar 13C enrichment monitored by gas chromatography-isotope-ratio mass spectrometry (GC-IRMS). Contrary to this hypothesis the results showed no significant decrease with any of the specific dietary sugars. Furthermore, with dietary low-13C mannose or galactose, a significant increase in 13C enrichment of glycoprotein-sugars was observed compared with some other nutritional groups. Moreover, in the pulse-labelling experiment, dietary mannose and galactose induced similar patterns of 13C enrichment in intestinal and serum glycoprotein-sugars. Therefore, although specific dietary sugars do not appear to be preferential substrates for glycosylation under conditions and doses relevant to current concepts of nutrition, regulatory roles of some specific dietary sugars in relation to glycoprotein-sugar metabolism might be hypothesized. These findings could lead to similar studies using stable-isotope methodology in man which could have practical consequences, especially in parenteral nutrition where glucose is the only sugar provided to the metabolism.
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Wasinger, Valerie C., Ming Zeng y Yunki Yau. "Current Status and Advances in Quantitative Proteomic Mass Spectrometry". International Journal of Proteomics 2013 (6 de marzo de 2013): 1–12. http://dx.doi.org/10.1155/2013/180605.
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The accurate quantitation of proteins and peptides in complex biological systems is one of the most challenging areas of proteomics. Mass spectrometry-based approaches have forged significant in-roads allowing accurate and sensitive quantitation and the ability to multiplex vastly complex samples through the application of robust bioinformatic tools. These relative and absolute quantitative measures using label-free, tags, or stable isotope labelling have their own strengths and limitations. The continuous development of these methods is vital for increasing reproducibility in the rapidly expanding application of quantitative proteomics in biomarker discovery and validation. This paper provides a critical overview of the primary mass spectrometry-based quantitative approaches and the current status of quantitative proteomics in biomedical research.
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Holm, Lars, Søren Reitelseder, Kasper Dideriksen, Rie H. Nielsen, Jacob Bülow y Michael Kjaer. "The single-biopsy approach in determining protein synthesis in human slow-turning-over tissue: use of flood-primed, continuous infusion of amino acid tracers". American Journal of Physiology-Endocrinology and Metabolism 306, n.º 11 (1 de junio de 2014): E1330—E1339. http://dx.doi.org/10.1152/ajpendo.00084.2014.
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Muscle protein synthesis (MPS) rate is determined conventionally by obtaining two or more tissue biopsies during a primed, continuous infusion of a stable isotopically labeled amino acid. The purpose of the present study was to test whether tracer priming given as a flooding dose, thereby securing an instantaneous labeling of the tissue pools of free tracee amino acids, followed by a continuous infusion of the same tracer to maintain tracer isotopic steady state, could be used to determine the MPS rate over a prolonged period of time by obtaining only a single tissue biopsy. We showed that the tracer from the flood prime appeared immediately in the muscle free pool of amino acids and that this abundance could be kept constant by a subsequent continuous infusion of the tracer. When using phenylalanine as tracer, the flood-primed, continuous infusion protocol does not stimulate the MPS rate per se. In conclusion, the flood-primed, continuous infusion protocol using phenylalanine as tracer can validly be used to measure the protein synthesis rate in human in vivo experiments by obtaining only a single tissue biopsy after a prolonged infusion period.
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Kuzyakov, Y. "How to link soil C pools with CO<sub>2</sub> fluxes?" Biogeosciences Discussions 8, n.º 1 (28 de febrero de 2011): 1947–83. http://dx.doi.org/10.5194/bgd-8-1947-2011.
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Abstract. Despite the importance of carbon (C) pools and CO2 fluxes in terrestrial ecosystems and especially in soils, as well as many attempts to assign fluxes to specific pools, this challenge remains unsolved. Interestingly, scientists investigating pools are not closely linked with scientists studying fluxes. This mini-review therefore focused on experimental approaches enabling soil C pools to be linked with CO2 flux from the soil. The background, advantages and shortcomings of uncoupled approaches (measuring only pools or fluxes) and of coupled approaches (measuring both pools and fluxes) were evaluated and their prerequisites – steady state of pools and isotopic steady state – described. The uncoupled approaches include: (i) monitoring the decrease of C pools in long-term fallow bare soil lacking C input over decades, (ii) analyzing components of CO2 efflux dynamics by incubating soil without new C input over months or a few years, and (iii) analyzing turnover rates of C pools based on their 13C and 14C isotopic signature. The uncoupled approaches are applicable for non steady state conditions only and have limited explanatory power. The more advantageous coupled approaches partition simultaneously pools and fluxes and are based on one of three types of changes in the isotopic signature of input C compared to soil C: (i) abrupt permanent, (ii) gradual permanent, and (iii) abrupt temporary impacts. I show how the maximal sensitivity of the approaches depends on the differences in the isotopic signature of pools with fast and slow turnover rates. The promising coupled approaches include: (a) &delta13C of C pools and CO2 efflux from soil after C3/C4 vegetation changes or in FACE experiments (both corresponding to continuous labeling), (b) addition of 13C or 14C labeled organics (corresponding to pulse labeling), and (c) bomb-14C. I show that physical separation of soil C pools is not a~prerequisite to estimate pool size or to link pools with fluxes. The future challenges include combining two or more promising approaches to elucidate more than two C sources for CO2 fluxes, and linking scientific communities investigating the pools with those investigating the fluxes.
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AlSalka, Yamen, Osama Al-Madanat, Amer Hakki y Detlef W. Bahnemann. "Boosting the H2 Production Efficiency via Photocatalytic Organic Reforming: The Role of Additional Hole Scavenging System". Catalysts 11, n.º 12 (23 de noviembre de 2021): 1423. http://dx.doi.org/10.3390/catal11121423.
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The simultaneous photocatalytic H2 evolution with environmental remediation over semiconducting metal oxides is a fascinating process for sustainable fuel production. However, most of the previously reported photocatalytic reforming showed nonstoichiometric amounts of the evolved H2 when organic substrates were used. To explain the reasons for this phenomenon, a careful analysis of the products and intermediates in gas and aqueous phases upon the photocatalytic hydrogen evolution from oxalic acid using Pt/TiO2 was performed. A quadrupole mass spectrometer (QMS) was used for the continuous flow monitoring of the evolved gases, while high performance ion chromatography (HPIC), isotopic labeling, and electron paramagnetic resonance (EPR) were employed to understand the reactions in the solution. The entire consumption of oxalic acid led to a ~30% lower H2 amount than theoretically expected. Due to the contribution of the photo-Kolbe reaction mechanism, a tiny amount of formic acid was produced then disappeared shortly after the complete consumption of oxalic acid. Nevertheless, a much lower concentration of formic acid was generated compared to the nonstoichiometric difference between the formed H2 and the consumed oxalic acid. Isotopic labeling measurements showed that the evolved H2, HD, and/or D2 matched those of the solvent; however, using D2O decreased the reaction rate. Interestingly, the presence of KI as an additional hole scavenger with oxalic acid had a considerable impact on the reaction mechanism, and thus the hydrogen yield, as indicated by the QMS and the EPR measurements. The added KI promoted H2 evolution to reach the theoretically predictable amount and inhibited the formation of intermediates without affecting the oxalic acid degradation rate. The proposed mechanism, by which KI boosts the photocatalytic performance, is of great importance in enhancing the overall energy efficiency for hydrogen production via photocatalytic organic reforming.
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Kuzyakov, Y. "How to link soil C pools with CO<sub>2</sub> fluxes?" Biogeosciences 8, n.º 6 (14 de junio de 2011): 1523–37. http://dx.doi.org/10.5194/bg-8-1523-2011.
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Abstract. Despite the importance of carbon (C) pools and CO2 fluxes in terrestrial ecosystems and especially in soils, as well as many attempts to assign fluxes to specific pools, this challenge remains unsolved. Interestingly, scientists investigating pools are not closely linked with scientists studying fluxes. This review therefore focused on experimental approaches enabling soil C pools to be linked with CO2 flux from the soil. The background, advantages and shortcomings of uncoupled approaches (measuring only pools or fluxes) and of coupled approaches (measuring both pools and fluxes) were evaluated and their prerequisites – steady state of pools and isotopic steady state – described. The uncoupled approaches include: (i) monitoring the decrease of C pools in long-term fallow bare soil lacking C input over decades, (ii) analyzing components of CO2 efflux dynamics by incubating soil without new C input over months or years, and (iii) analyzing turnover rates of C pools based on their 13C and 14C isotopic signature. The uncoupled approaches are applicable for non-steady state conditions only and have limited explanatory power. The more advantageous coupled approaches partition simultaneously pools and fluxes based on one of three types of changes in the isotopic signature of input C compared to soil C: (i) abrupt permanent, (ii) gradual permanent, and (iii) abrupt temporary impacts. I show how the maximal sensitivity of the approaches depends on the differences in the isotopic signature of pools with fast and slow turnover rates. The promising coupled approaches include: (a) δ13C of C pools and CO2 efflux from soil after C3/C4 vegetation changes or in FACE experiments (both corresponding to continuous labeling), (b) addition of 13C or 14C labeled organics (corresponding to pulse labeling), and (c) bomb-14C. I show that physical separation of soil C pools is not a prerequisite to estimate pool size or to link pools with fluxes. Based on simple simulation of C aging in soil after the input, the discordance of MRT of C in pools and of C released in CO2 was demonstrated. This discordance of MRT between pools and fluxes shows that the use of MRT of pools alone underestimates the fluxes at least for two times. The future challenges include combining two or more promising approaches to elucidate more than two C sources for CO2 fluxes, and linking scientific communities investigating the pools with those investigating the fluxes.
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Bequette, B. J., F. R. C. Backwell, M. S. Dhanoa, A. Walker, A. G. Calder, D. Wray-Cahen, J. A. Metcalf et al. "Kinetics of blood free and milk casein-amino acid labelling in the dairy goat at two stages of lactation". British Journal of Nutrition 72, n.º 2 (agosto de 1994): 211–20. http://dx.doi.org/10.1079/bjn19940025.
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The kinetics of blood free amino acids (AA) transfer into milk casein were compared in goats (n 4) at 61 (SE5)d (Expt 1; post-peak, 4.51 (SE 0.26) kg milk/d) and at 180 (SE 6) d (Expt 2; late, 2.36 (SE 0.16) kg milk/d) of lactation during non-primed, continuous (Expt 1, 12 h; Expt 2, 16 h) intravenous infusions of mixtures of L-[1-13C]leucine and L-[1-13C]phenylalanine with either L-[1-13C]valine (Expt 1) or L-[5-13Cmethionine (Expt 2). The 13C enrichments of blood free and casein-bound AA were fitted to a single exponential model to estimate isotopic plateaux and the fractional rate constant for milk casein labelling. Milk protein output and its contribution to whole-body flux was higher in Expt 1 (post-peak) than in Expt 2 (late lactation), but the kinetics of 13C labelling of the casein-bound AA were similar for all AA tracers in both experiments. At both stages of lactation the delay (6–8 h) between the attainment of isotopic plateau for the blood free AA and the corresponding attainment of plateau for the casein-bound AA indicated that the blood free pool was not the immediate precursor pool for milk casein biosynthesis. Plateau enrichments of casein-bound AA were generally higher than those for the corresponding blood free AA in both experiments. These results indicate that the relative contributions of different AA sources to the immediate precursor pool for milk casein biosynthesis are similar at different stages of lactation despite major changes in the partitioning of whole-body flux towards milk protein output. Non-milk protein fluxes were also similar in post-peak and late lactation.
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Fuller, Malcolm F. y Daniel Tomé. "In vivo Determination of Amino Acid Bioavailability in Humans and Model Animals". Journal of AOAC INTERNATIONAL 88, n.º 3 (1 de mayo de 2005): 923–34. http://dx.doi.org/10.1093/jaoac/88.3.923.
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Abstract Because the digestion of many dietary proteins is incomplete, and because there is a continuous (but variable) entry into the intestinal lumen of endogenous protein and amino acid nitrogen that is also subject to digestion, the fluxes of nitrogen, amino acids, and protein in the gut exhibit a rather complicated pattern. Methods to distinguish and quantitate the endogenous and dietary components of nitrogen and amino acids in ileal chyme or feces include the use of a protein-free diet, the enzyme-hydrolyzed protein method, different levels of protein intake, multiple regression methods, and stable-isotope labelling of endogenous or exogenous amino acids. Assessment of bioavailability can be made, with varying degrees of difficulty, in man directly but, for routine evaluation of foods, the use of model animals is attractive for several reasons, the main ones being cost and time. Various animals and birds have been proposed as models for man but, in determining their suitability as a model, their physiological, enzymological, and microbiological differences must be considered. Fecal or ileal digestibility measurements, as well as apparent and true nitrogen and amino acid digestibility measurements, have very different nutritional significance and can, thus, be used for different objectives. Measurements at the ileal level are critical for determining amino acid losses of both dietary and endogenous origin, whereas measurements at the fecal level are critical in assessing whole-body nitrogen losses. A complementary and still unresolved aspect is to take into account the recycling of intestinal nitrogen and bacterial amino acids to the body.
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Chisanga, Malama, Howbeer Muhamadali, David I. Ellis y Royston Goodacre. "Surface-Enhanced Raman Scattering (SERS) in Microbiology: Illumination and Enhancement of the Microbial World". Applied Spectroscopy 72, n.º 7 (23 de marzo de 2018): 987–1000. http://dx.doi.org/10.1177/0003702818764672.
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The microbial world forms a huge family of organisms that exhibit the greatest phylogenetic diversity on Earth and thus colonize virtually our entire planet. Due to this diversity and subsequent complex interactions, the vast majority of microorganisms are involved in innumerable natural bioprocesses and contribute an absolutely vital role toward the maintenance of life on Earth, whilst a small minority cause various infectious diseases. The ever-increasing demand for environmental monitoring, sustainable ecosystems, food security, and improved healthcare systems drives the continuous search for inexpensive but reproducible, automated and portable techniques for detection of microbial isolates and understanding their interactions for clinical, environmental, and industrial applications and benefits. Surface-enhanced Raman scattering (SERS) is attracting significant attention for the accurate identification, discrimination and characterization and functional assessment of microbial cells at the single cell level. In this review, we briefly discuss the technological advances in Raman and Fourier transform infrared (FT-IR) instrumentation and their application for the analysis of clinically and industrially relevant microorganisms, biofilms, and biological warfare agents. In addition, we summarize the current trends and future prospects of integrating Raman/SERS-isotopic labeling and cell sorting technologies in parallel, to link genotype-to-phenotype in order to define community function of unculturable microbial cells in mixed microbial communities which possess admirable traits such as detoxification of pollutants and recycling of essential metals.
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NAKSHABENDI, I. M., S. DOWNIE, R. I. RUSSELL y M. J. RENNIE. "Small-intestinal mucosal protein synthesis and whole-body protein turnover in alcoholic liver disease". Clinical Science 97, n.º 6 (29 de octubre de 1999): 633–38. http://dx.doi.org/10.1042/cs0970633.
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We used stable-isotope-labelled amino acids to measure the effects of alcoholic liver disease (ALD) on whole-body protein turnover and small-intestinal mucosal protein synthesis. Groups comprising eight patients with ALD and eight healthy control subjects were studied. They received primed, continuous intravenous infusions of L-[1-13C]leucine after an overnight fast; after 4 h, duodenal biopsies were obtained via endoscopy. Protein synthesis was calculated from protein labelling relative to intracellular leucine enrichment. Rates of duodenal mucosal protein synthesis were 2.58±0.32%·h-1 (mean±S.D.) in the normal subjects and 2.04±0.18%·h-1 in the ALD patients (P< 0.003), despite the fact that the protein synthetic capacity (μg of RNA/mg of protein) was higher in ALD patients (160±14 compared with 137±6 μg/mg; P < 0.003). The mucosal cell size (protein/DNA ratio) was lower in ALD patients (9.23±0.91 compared with 13±2.2 μg/mg; P< 0.002). Although the mean rates of whole-body protein turnover were not significantly different between the two groups (204±18 and 196±44 μmol leucine·h-1·kg-1 for ALD and control subjects respectively), there was, in the ALD patients, an inverse relationship between the rate of small-intestinal mucosal protein synthesis and the severity of ALD; furthermore, there was a direct relationship between the rate of whole-body protein turnover and the severity of ALD. Thus there was an inverse relationship between the rate of small-intestinal mucosal protein synthesis and the rate of whole-body protein turnover in ALD patients, which was not seen in the normal subjects.
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Gomes, J., N. Brüggemann, D. P. Dick, G. M. Pedroso, M. Veloso y C. Bayer. "Urea and legume residues as 15N-N2O sources in a subtropical soil". Soil Research 57, n.º 3 (2019): 287. http://dx.doi.org/10.1071/sr18300.
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In this work, we used the 15N labelling technique to identify the sources of N2O emitted by a subtropical soil following application of mineral nitrogen (N) fertiliser (urea) and residues of a legume cover crop (cowpea). For this purpose, a 45-day incubation experiment was conducted by subjecting undisturbed soil cores from a subtropical Acrisol to five different treatments: (1) control (no crop residue or fertiliser-N application); (2) 15N-labelled cowpea residue (200 μg N g–1 soil); (3) 15N-labelled urea (200 μg N g–1 soil); (4) 15N-labelled cowpea residue (100 μg N g–1 soil) + unlabelled urea (100 μg N g–1 soil); and (5) unlabelled cowpea residue (100 μg N g–1 soil) + 15N-labelled urea (100 μg N g–1 soil). Cores were analysed for total N2O formation, δ15N-N2O and δ18O-N2O by continuous flow isotope ratio mass spectrometry, as well as for total NO3–-N and NH4+-N. Legume crop residues and mineral fertiliser increased N2O emissions from soil to 10.5 and 9.7 µg N2O-N cm–2 respectively, which was roughly six times the value for control (1.5 µg N2O-N cm–2). The amount of 15N2O emitted from labelled 15N-urea (0.40–0.45% of 15N applied) was greater than from 15N-cowpea residues (0.013–0.015% of 15N applied). Unlike N-poor crop residues, urea in combination with N-rich residues (cowpea) failed to reduce N2O emissions relative to urea alone. Legume cover crops thus provide an effective mitigation strategy for N2O emissions in relation to mineral N fertilisation in climate-smart agriculture. Judging by our inconclusive results, however, using urea in combination with N-rich residues provides no clear-cut environmental advantage.
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Errante, Paolo Ruggero, Pâmela Carolina Cruz Ebbing, Francisco Sandro Menezes Rodrigues, Renato Ribeiro Nogueira Ferraz y Neusa Pereira Da Silva. "Flow cytometry: a literature review". Revista de Ciências Médicas e Biológicas 14, n.º 2 (18 de febrero de 2016): 221. http://dx.doi.org/10.9771/cmbio.v14i2.12182.
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Introduction: flow cytometry is a technique that employs an optical-electronic detection apparatus to analyze the physical and chemical properties of microscopic particles suspend in a liquid medium. Objective: to review the literature in search of the main studies that used flow cytometry as the main methodology. Method: Articles were selected according to their impact factor in the Journal of Citation Reports. Literature review: a light beam is direct to a continuous flow of suspended particles marked with fluorescent substances. The light is scattered differently from the beam by the particles and is captured by sensors in line and perpendicular to the light beam. These microscopic particles are conjugated with fluorescent substances that, once excited, emit light of lower frequency than the light source. The emitted light is captured by sensors and the particles are analyzed according to fluctuations in brightness of each detector and/or fluorescence emission. The result of this process is the formation of images in real time for each cell fluorescence, scattering and transmission of light. A major problem of flow cytometry is to determine whether a subset of cells labeled with fluorochrome-conjugated monoclonal antibodies is positive or negative. Gains compensation should be determined and applied correctly, and controls should be conducted concisely with the adoption of a biological control, isotype control or Fluorescence Minus One (FMO). None of these controls are considered ideal, and must be chosen according the number of different labeling done, rarity of molecule expression on surface or intracellularly in certain cell subsets, overlap of wavelengths or unspecific binding of the fluorochrome-conjugated antibodies. Conclusion: due to its great potential, flow cytometry has been expanded to diverse fields of biological sciences, and is routinely used in clinical diagnostic, biotechnology, and basic and applied research.
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Van Voorhis, Wesley C., Kasey L. Rivas, Pravin Bendale, Laxman Nallan, Carolyn Hornéy, Lynn K. Barrett, Kevin D. Bauer et al. "Efficacy, Pharmacokinetics, and Metabolism of Tetrahydroquinoline Inhibitors of Plasmodium falciparum Protein Farnesyltransferase". Antimicrobial Agents and Chemotherapy 51, n.º 10 (2 de julio de 2007): 3659–71. http://dx.doi.org/10.1128/aac.00246-07.
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ABSTRACT New antimalarials are urgently needed. We have shown that tetrahydroquinoline (THQ) protein farnesyltransferase (PFT) inhibitors (PFTIs) are effective against the Plasmodium falciparum PFT and are effective at killing P. falciparum in vitro. Previously described THQ PFTIs had limitations of poor oral bioavailability and rapid clearance from the circulation of rodents. In this paper, we validate both the Caco-2 cell permeability model for predicting THQ intestinal absorption and the in vitro liver microsome model for predicting THQ clearance in vivo. Incremental improvements in efficacy, oral absorption, and clearance rate were monitored by in vitro tests; and these tests were followed up with in vivo absorption, distribution, metabolism, and excretion studies. One compound, PB-93, achieved cure when it was given orally to P. berghei-infected rats every 8 h for a total of 72 h. However, PB-93 was rapidly cleared, and dosing every 12 h failed to cure the rats. Thus, the in vivo results corroborate the in vitro pharmacodynamics and demonstrate that 72 h of continuous high-level exposure to PFTIs is necessary to kill plasmodia. The metabolism of PB-93 was demonstrated by a novel technique that relied on double labeling with a radiolabel and heavy isotopes combined with radiometric liquid chromatography and mass spectrometry. The major liver microsome metabolite of PB-93 has the PFT Zn-binding N-methyl-imidazole removed; this metabolite is inactive in blocking PFT function. By solving the X-ray crystal structure of PB-93 bound to rat PFT, a model of PB-93 bound to malarial PFT was constructed. This model suggests areas of the THQ PFTIs that can be modified to retain efficacy and protect the Zn-binding N-methyl-imidazole from dealkylation.
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Abdallah, Nadine, Francis Buadi, Patricia T. Greipp, Morie A. Gertz, Prashant Kapoor, Angela Dispenzieri, Linda Baughn et al. "Cytogenetic abnormalities in MM: Association with disease characteristics and treatment response." Journal of Clinical Oncology 38, n.º 15_suppl (20 de mayo de 2020): e20520-e20520. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e20520.
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e20520 Background: Cytogenetic abnormalities detected by FISH are found in the majority of multiple myeloma (MM) patients. Although their prognostic value has been studied extensively, less is known about their association with disease characteristics and treatment response. Methods: To address these questions, we designed a retrospective study including 2031 Mayo Clinic patients diagnosed with MM from 2004 to 2018. We compared baseline characteristics and treatment outcomes between primary cytogenetic groups: t(11;14), t(4;14), t(14;16), (14;20), t(6;14), unknown IgH translocation/del and trisomy (without IgH translocation). These included 373, 177, 78, 20, 18, 228 and 791 patients respectively. Kruskal-Wallis and Fisher’s exact tests were used for categorical and continuous variables respectively. Time to next treatment (TTNT) was estimated using Kaplan-Meier method and compared using Log-Rank test. Results: t(4;14), t(14;16), t(6;14) and t(14;20) groups were associated with hemoglobin < 10 g/dL, beta2microglobulin > 5.5 µg/ml, ISS stage 3 and ≥50% bone marrow plasma cells. The latter 3 groups were also associated with renal dysfunction (Cr ≥2 mg/dL) and higher urinary monoclonal protein. t(4;14) was associated with IgA isotype, serum monoclonal protein ≥1g/dL and plasma cell labeling index ≥1%. Light chain myeloma was more prevalent in patients with t(11;14). Overall response rate (ORR) to proteasome inhibitor (PI) induction was higher for those with IgH translocations (any) compared to trisomies (85% vs 77% P = 0.02), while ORR was higher for those with trisomies with immunomodulatory drug (IMiD) induction (90% vs 78% P < 0.01). The rate of ≥ very good partial response was higher for patients with high risk IgH translocations [t(4;14), t(14;16) or t(14;20)] compared to standard risk with PI-IMiD combination treatment (88% vs 65% P < 0.01). Otherwise, response rates did not differ between these 2 groups. TTNT was longer in patients with trisomies compared to those with IgH translocation with IMiD or PI-IMiD treatments (32.2 vs 19 and 44 vs 27.4 months, respectively P < 0.01). For all cytogenetic groups, better treatment responses and longer TTNT were seen with PI-IMiD combinations compared to other treatments. Conclusions: t(4;14), t(14;16), t(6;14), and t(14;20) are associated with high risk disease characteristics. Patients with IgH translocations may have better response to PI induction compared to those with trisomies, while those with trisomies may have better response to IMiD treatment, with best outcomes for both seen with PI-IMiD combinations.
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Minya, Ferenc, Ádám Mészáros, Eszter Csizmadia, Dávid Suskó, Mounir Raji y Gellért Sipos. "Raney Nickel‐Catalyzed Deuterium Labeling of Nitrogen‐Containing Heterocycles and Pharmaceuticals under Continuous Flow Conditions". Advanced Synthesis & Catalysis, 14 de mayo de 2024. http://dx.doi.org/10.1002/adsc.202400168.
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Deuterium‐labeled compounds play a pivotal role in physical organic chemistry, life sciences, and materials science. This has resulted in a surge of interest in deuterium‐labeled active pharmaceutical ingredients in recent years. In this study, we present a continuous flow Raney nickel‐catalyzed hydrogen isotope exchange process that boasts compatibility with a wide spectrum of nitrogen‐containing heterocycles and pharmaceutical compounds. The broad applicability of the developed method was demonstrated through successful labeling of various purine bases, imidazoles, pyridines, and active pharmaceutical ingredients, including complex structures like abacavir and remdesivir. Control experiments revealed Raney nickel's crucial role in the exchange process, showcasing the superiority of the continuous flow approach over batch reactions. Furthermore, a scaled‐up experiment demonstrated the robustness of the catalyst.
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Soong, Jennifer L., Dan Reuss, Colin Pinney, Ty Boyack, Michelle L. Haddix, Catherine E. Stewart y M. Francesca Cotrufo. "Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling". Journal of Visualized Experiments, n.º 83 (16 de enero de 2014). http://dx.doi.org/10.3791/51117.
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Dreydoppel, Matthias, Roman J. Lichtenecker, Mikael Akke y Ulrich Weininger. "1H R1ρ relaxation dispersion experiments in aromatic side chains". Journal of Biomolecular NMR, 12 de septiembre de 2021. http://dx.doi.org/10.1007/s10858-021-00382-w.
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AbstractAromatic side chains are attractive probes of protein dynamic, since they are often key residues in enzyme active sites and protein binding sites. Dynamic processes on microsecond to millisecond timescales can be studied by relaxation dispersion experiments that attenuate conformational exchange contributions to the transverse relaxation rate by varying the refocusing frequency of applied radio-frequency fields implemented as either CPMG pulse trains or continuous spin-lock periods. Here we present an aromatic 1H R1ρ relaxation dispersion experiment enabling studies of two to three times faster exchange processes than achievable by existing experiments for aromatic side chains. We show that site-specific isotope labeling schemes generating isolated 1H–13C spin pairs with vicinal 2H–12C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmann–Hahn matching due to the 3JHH couplings and limited chemical shift differences among 1H spins in phenylalanine, tyrosine and the six-ring moiety of tryptophan. This labeling pattern is sufficient in that remote protons do not cause additional complications. We validated the approach by measuring ring-flip kinetics in the small protein GB1. The determined rate constants, kflip, agree well with previous results from 13C R1ρ relaxation dispersion experiments, and yield 1H chemical shift differences between the two sides of the ring in good agreement with values measured under slow-exchange conditions. The aromatic1H R1ρ relaxation dispersion experiment in combination with the site-selective 1H–13C/2H–12C labeling scheme enable measurement of exchange rates up to kex = 2kflip = 80,000 s–1, and serve as a useful complement to previously developed 13C-based methods.
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Möbius, Klaus y Anton Savitsky. "High-field/High-frequency EPR Spectroscopy in Protein Research: Principles and Examples". Applied Magnetic Resonance, 13 de diciembre de 2022. http://dx.doi.org/10.1007/s00723-022-01511-w.
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AbstractDuring the last decades, the combined efforts of biologists, chemists, and physicists in developing high-field/high-frequency EPR techniques and applying them to functional proteins have demonstrated that this type of magnetic resonance spectroscopy is particularly powerful for characterizing the structure and dynamics of stable and transient states of proteins in action on biologically relevant time scales ranging from nanoseconds to hours. The review article describes how high-field EPR methodology, in conjunction with site-specific isotope and spin-labeling strategies, is capable of providing new insights into fundamental biological processes. Specifically, we discuss the theoretical and instrumental background of continuous-wave and pulse high-field EPR and the multiple-resonance extensions EDNMR, ENDOR, TRIPLE, ESEEM, PELDOR, and RIDME. Some emphasis is placed on a balanced description of both the historical spadework and the achieved performance of advanced EPR at 95 GHz and 360 GHz. This culminates in a coherent treatment of state-of-the-art research of high-field EPR in terms of both instrumentation development and application to representative protein complexes such as cofactor binding sites in photosynthesis.
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Rao, Nalini R., Arun Upadhyay y Jeffrey N. Savas. "Derailed protein turnover in the aging mammalian brain". Molecular Systems Biology, 5 de enero de 2024. http://dx.doi.org/10.1038/s44320-023-00009-2.
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AbstractEfficient protein turnover is essential for cellular homeostasis and organ function. Loss of proteostasis is a hallmark of aging culminating in severe dysfunction of protein turnover. To investigate protein turnover dynamics as a function of age, we performed continuous in vivo metabolic stable isotope labeling in mice along the aging continuum. First, we discovered that the brain proteome uniquely undergoes dynamic turnover fluctuations during aging compared to heart and liver tissue. Second, trends in protein turnover in the brain proteome during aging showed sex-specific differences that were tightly tied to cellular compartments. Next, parallel analyses of the insoluble proteome revealed that several cellular compartments experience hampered turnover, in part due to misfolding. Finally, we found that age-associated fluctuations in proteasome activity were associated with the turnover of core proteolytic subunits, which was recapitulated by pharmacological suppression of proteasome activity. Taken together, our study provides a proteome-wide atlas of protein turnover across the aging continuum and reveals a link between the turnover of individual proteasome subunits and the age-associated decline in proteasome activity.
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Bessler, Larissa, Jason Sirleaf, Christopher J. Kampf, Katarzyna Frankowska, Grażyna Leszczyńska, Till Opatz y Mark Helm. "Esterification of Cyclic N6‐threonylcarbamoyladenosine during RNA Sample Preparation". ChemMedChem, 17 de abril de 2024. http://dx.doi.org/10.1002/cmdc.202400115.
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The continuous deciphering of crucial biological roles of RNA modifications and their involvement in various pathological conditions, together with their key roles in the use of RNA‐based therapeutics, has reignited interest in studying the occurrence and identity of non‐canonical ribonucleoside structures during the past years. Discovery and structural elucidation of new modified structures is usually achieved by combination of liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) at the nucleoside level and stable isotope labeling experiments. This approach, however, has its pitfalls as demonstrated in the course of the present study: we structurally elucidated a new nucleoside structure that showed significant similarities to the family of (c)t6A modifications and was initially considered a genuine modification, but turned out to be an in vitro formed glycerol ester of t6A. This artifact is generated from ct6A during RNA hydrolysis upon addition of enzymes stored in glycerol containing buffers in a mildly alkaline milieu, and was moreover shown to undergo an intramolecular transesterification reaction. This warrants extra caution in the discovery of new RNA modifications and in quantification of known modified structures, in particular chemically labile modifications that might suffer from exposure to putatively harmless reagents during the diverse steps of sample preparation.
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Li, Pengfa, Jia Liu, Muhammad Saleem, Guilong Li, Lu Luan, Meng Wu y Zhongpei Li. "Reduced chemodiversity suppresses rhizosphere microbiome functioning in the mono-cropped agroecosystems". Microbiome 10, n.º 1 (16 de julio de 2022). http://dx.doi.org/10.1186/s40168-022-01287-y.
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Abstract Background Rhizodeposits regulate rhizosphere interactions, processes, nutrient and energy flow, and plant-microbe communication and thus play a vital role in maintaining soil and plant health. However, it remains unclear whether and how alteration in belowground carbon allocation and chemodiversity of rhizodeposits influences microbiome functioning in the rhizosphere ecosystems. To address this research gap, we investigated the relationship of rhizosphere carbon allocation and chemodiversity with microbiome biodiversity and functioning during peanut (Arachis hypogaea) continuous mono-cropping. After continuously labeling plants with 13CO2, we studied the chemodiversity and composition of rhizodeposits, along with the composition and diversity of active rhizosphere microbiome using metabolomic, amplicon, and shotgun metagenomic sequencing approaches based on DNA stable-isotope probing (DNA-SIP). Results Our results indicated that enrichment and depletion of rhizodeposits and active microbial taxa varied across plant growth stages and cropping durations. Specifically, a gradual decrease in the rhizosphere carbon allocation, chemodiversity, biodiversity and abundance of plant-beneficial taxa (such as Gemmatimonas, Streptomyces, Ramlibacter, and Lysobacter), and functional gene pathways (such as quorum sensing and biosynthesis of antibiotics) was observed with years of mono-cropping. We detected significant and strong correlations between rhizodeposits and rhizosphere microbiome biodiversity and functioning, though these were regulated by different ecological processes. For instance, rhizodeposits and active bacterial communities were mainly governed by deterministic and stochastic processes, respectively. Overall, the reduction in carbon deposition and chemodiversity during peanut continuous mono-cropping tended to suppress microbial biodiversity and its functions in the rhizosphere ecosystem. Conclusions Our results, for the first time, provide the evidence underlying the mechanism of rhizosphere microbiome malfunctioning in mono-cropped systems. Our study opens new avenues to deeply disentangle the complex plant-microbe interactions from the perspective of rhizodeposits chemodiversity and composition and will serve to guide future microbiome research for improving the functioning and services of soil ecosystems.
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Zheng, Chunyu, Christina Khoo y Frank M. Sacks. "Abstract 675: Contrasting Effects of Dietary Carbohydrate Compared to Mono-unsaturated Fat on Hepatic Production of ApoE and ApoC-III Containing VLDL, and Formation of LDL". Circulation 116, suppl_16 (16 de octubre de 2007). http://dx.doi.org/10.1161/circ.116.suppl_16.ii_125-b.
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The effects of substituting dietary carbohydrate (CHO) with mono-unsaturated fat (MUFA) on plasma apoB metabolism were evaluated in 12 adults: 6 with normal and 6 with high plasma triglyceride levels. They consumed for 3 weeks each time a high CHO diet (48% complex CHO, 8% MUFA) and a high MUFA diet (31% complex CHO, 24% MUFA). ApoB100 kinetic studies were performed at the end of each dietary intervention using stable isotope labeling with a bolus and a primed continuous infusion. Multiple VLDL, IDL, and LDL fractions were prepared according to their apoE and apoC-III content. Compared to the CHO diet, the MUFA diet increased the percentage of VLDL and IDL secreted with both apoE and apoC-III (45% on MUFA vs. 14% on CHO, p < 0.01) and reduced the percentage of VLDL and IDL secreted without either apoE or apoC-III (MUFA 19% vs. CHO 40%, p = 0.02). Total liver secretion rates of apoB100 lipoproteins were similar between diets (MUFA 11.6 vs. CHO 11.3 mg·day −1 ·kg −1 , p = NS). The dietary change did not affect the fractional catabolic rates and flux patterns of the lipoproteins. On both diets, VLDL and IDL that had apoE were rapidly cleared from the circulation, limiting LDL formation; whereas lipoproteins that did not have apoE or apoCIII mostly underwent lipolysis with little direct clearance, and were the main precursors of LDL. As a result, increased secretion of VLDL and IDL containing apoE and apoC-III caused by the MUFA diet was associated with higher direct clearance and lower LDL production rates (p = 0.02 vs. CHO), while the CHO diet increased LDL production due to increased secretion of VLDL without apoE or apoC-III. In conclusion, our results reveal a strong dietary effect on the secretion pattern of apoB100 lipoproteins. Substituting dietary complex carbohydrate with mono-unsaturated fat selectively promotes liver secretion of VLDL and IDL containing both apoE and apoC-III while suppressing the secretion of VLDL and IDL without apoE or apoC-III. This leads to significant downstream effects on LDL formation due to differential effects of apoE and apoC-III on apoB lipoprotein metabolism, resulting in enhanced particle clearance and reduced LDL formation with the MUFA diet compared to the CHO diet.
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Li, Juan, Yu Xia, Xiaowei Song, Bolei Chen y Richard N. Zare. "Continuous ammonia synthesis from water and nitrogen via contact electrification". Proceedings of the National Academy of Sciences 121, n.º 4 (17 de enero de 2024). http://dx.doi.org/10.1073/pnas.2318408121.
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We synthesized ammonia (NH 3 ) by bubbling nitrogen (N 2 ) gas into bulk liquid water (200 mL) containing 50 mg polytetrafluoroethylene (PTFE) particles (~5 µm in diameter) suspended with the help of a surfactant (Tween 20, ~0.05 vol.%) at room temperature (25 °C). Electron spin resonance spectroscopy and density functional theory calculations reveal that water acts as the proton donor for the reduction of N 2 . Moreover, isotopic labeling of the N 2 gas shows that it is the source of nitrogen in the ammonia. We propose a mechanism for ammonia generation based on the activation of N 2 caused by electron transfer and reduction processes driven by contact electrification. We optimized the pH of the PTFE suspension at 6.5 to 7.0 and employed ultrasonic mixing. We found an ammonia production rate of ~420 μmol L −1 h −1 per gram of PTFE particles for the conditions described above. This rate did not change more than 10% over an 8-h period of sustained reaction.
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Wang, Mengle, Stefan Asam, Jianqi Chen, Matthias Ehrmann y Michael Rychlik. "Production of Four 15N-Labelled Cobalamins via Biosynthesis Using Propionibacterium freudenreichii". Frontiers in Microbiology 12 (13 de agosto de 2021). http://dx.doi.org/10.3389/fmicb.2021.713321.
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Cobalamins (vitamin B12) are required by humans for their essential roles as enzyme cofactors in diverse metabolic processes. The four most common cobalamin vitamers are hydroxocobalamin (OHCbl), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), and cyanocobalamin (CNCbl). Humans are not able to synthesise cobalamins de novo and thus must acquire them from external sources. Therefore, a reliable and robust analytical method to determine the cobalamins in dietary sources is highly required. For such a purpose, stable isotope dilution assays (SIDAs) with LC-MS/MS are most suited due to their superior sensitivity, specificity, and ability to compensate for matrix effects and analyte loss during sample work-up. However, a critical bottleneck for developing a SIDA method for cobalamins is the availability of stable isotope-labelled internal standards. In the present study, we harnessed the potential of Propionibacterium (P.) freudenreichii for the biosynthesis of 15N-labelled cobalamins. First, we developed a chemically defined medium (CDM) containing ammonium sulphate as a single nitrogen source except three essential vitamins that supported long-term stable growth of P. freudenreichii throughout continuous transfers. The CDM was further optimised for cobalamin production under different incubation schemes. With the optimised CDM and incubation scheme, fully 15N-labelled cobalamins were obtained in P. freudenreichii with a final yield of 312 ± 29 μg/L and 635 ± 102 μg/L, respectively, for [15N]-OHCbl and [15N]-AdoCbl. Additionally, an optimised incubation process under anaerobic conditions was successfully employed to produce specifically labelled [15N, 14N2]-cobalamins, with a yield of 96 ± 18 μg/L and 990 ± 210 μg/L, respectively, for [15N, 14N2]-OHCbl and [15N, 14N2]-AdoCbl. The labelled substances were isolated and purified by solid phase extraction and semi-preparative HPLC. Chemical modifications were carried out to produce [15N]-CNCbl and [15N]-MeCbl. Eventually, 15N-labelled compounds were obtained for the four cobalamin vitamers in high chromatographic and isotopic purity with desired 15N-enrichment and labelling patterns, which are perfectly suited for future use in SIDAs or other applications that require isotopologues.
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Sarri, Laura, Joaquim Balcells, Ahmad Reza Seradj y Gabriel de la Fuente. "Protein turnover in pigs: A review of interacting factors". Journal of Animal Physiology and Animal Nutrition, 17 de noviembre de 2023. http://dx.doi.org/10.1111/jpn.13906.
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AbstractProtein turnover defines the balance between two continuous and complex processes of protein metabolism, synthesis and degradation, which determine their deposition in tissues. Although the liver and intestine have been studied extensively for their important roles in protein digestion, absorption and metabolism, the study of protein metabolism has focused mainly on skeletal muscle tissue to understand the basis for its growth. Due to the high adaptability of skeletal muscle, its protein turnover is greatly affected by different internal and external factors, contributing to carcass lean‐yield and animal growth. Amino acid (AA) labelling and tracking using isotope tracer methodology, together with the study of myofiber type profiling, signal transduction pathways and gene expression, has allowed the analysis of these mechanisms from different perspectives. Positive stimuli such as increased nutrient availability in the diet (e.g., AA), physical activity, the presence of certain hormones (e.g., testosterone) or a more oxidative myofiber profile in certain muscles or pig genotypes promote increased upregulation of translation and transcription‐related genes, activation of mTORC1 signalling mechanisms and increased abundance of satellite cells, allowing for more efficient protein synthesis. However, fasting, animal aging, inactivity and stress, inflammation or sepsis produce the opposite effect. Deepening the understanding of modifying factors and their possible interaction may contribute to the design of optimal strategies to better control tissue growth and nutrient use (i.e., protein and AA), and thus advance the precision feeding strategy.
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Wang, Ting-Hua y Jean Phillipe Merlio. "Purification of Megakaryocytes for flow cytometric analysis". New Cell, 1 de agosto de 2023, 1–6. http://dx.doi.org/10.61958/ncwj7398.
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Objectives: Megakaryocytes (MKs) represent only a limited cell number in normal bone marrow, which hampers the analysis of MKs by flow cytometric technique. To optimize the usage of flow cytometry for the analysis of megakaryopoiesis, we established a simple method to enrich MKs. Methods: Bone marrow tissues were taken from mice (n=5) and made into cell suspensions. The cell suspension was processed by velocity sedimentation with a continuous BSA solution (3%-6%) for 1h at room temperature from top to bottom of the gradient to purify MKs. Each layer (1 ml) gradient from top to bottom was collected and the number of MKs and other cells was counted under a microscope. The enriched MKs gradients were located in the lower 5 ml between layer 7 and layer 11 following sedimentation. They were then collected, centrifuged and labeled by antibody anti-CD41-FITC, and their DNA was also labeled by propidium iodide. Then, cells were performed isotype antibody labeling instead of CD41 for flow cytometric analysis. Another group underwent the same procedures at the beginning of cell suspension. Results: Under the microscope, The MKs were enriched mainly in the lower 7~11 layers, while other small cells were largely enriched in the upper 1~6 layer following sedimentation. The ploidy analysis by flow cytometric analysis showed that there was a typical peak which can represent various DNA content in the enriched MKs group, while the peak was not typical in the group without MK isolation. Conclusions: The results showed that the purification of MKs was effective in this experimental condition, providing a possibility to analyze megakaryopoiesis by flow cytometry instrument.
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Voss, Caroline M., Lene Arildsen, Jakob D. Nissen, Helle S. Waagepetersen, Arne Schousboe, Pierre Maechler, Peter Ott, Hendrik Vilstrup y Anne B. Walls. "Glutamate Dehydrogenase Is Important for Ammonia Fixation and Amino Acid Homeostasis in Brain During Hyperammonemia". Frontiers in Neuroscience 15 (16 de junio de 2021). http://dx.doi.org/10.3389/fnins.2021.646291.
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Impaired liver function may lead to hyperammonemia and risk for hepatic encephalopathy. In brain, detoxification of ammonia is mediated mainly by glutamine synthetase (GS) in astrocytes. This requires a continuous de novo synthesis of glutamate, likely involving the action of both pyruvate carboxylase (PC) and glutamate dehydrogenase (GDH). An increased PC activity upon ammonia exposure and the importance of PC activity for glutamine synthesis has previously been demonstrated while the importance of GDH for generation of glutamate as precursor for glutamine synthesis has received little attention. We therefore investigated the functional importance of GDH for brain metabolism during hyperammonemia. To this end, brain slices were acutely isolated from transgenic CNS-specific GDH null or litter mate control mice and incubated in aCSF containing [U-13C]glucose in the absence or presence of 1 or 5 mM ammonia. In another set of experiments, brain slices were incubated in aCSF containing 1 or 5 mM 15N-labeled NH4Cl and 5 mM unlabeled glucose. Tissue extracts were analyzed for isotopic labeling in metabolites and for total amounts of amino acids. As a novel finding, we reveal a central importance of GDH function for cerebral ammonia fixation and as a prerequisite for de novo synthesis of glutamate and glutamine during hyperammonemia. Moreover, we demonstrated an important role of the concerted action of GDH and alanine aminotransferase in hyperammonemia; the products alanine and α-ketoglutarate serve as an ammonia sink and as a substrate for ammonia fixation via GDH, respectively. The role of this mechanism in human hyperammonemic states remains to be studied.