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1. Effects of chemical preservation on bulk and amino acid isotope ratios of zooplankton, fish, and squid tissues

   https://doi.org/10.1002/rcm.8408  

   Hetherington, Elizabeth D. ; Kurle, Carolyn M. ; Ohman, Mark D. ; Popp, Brian N. ( April 2019 , Rapid Communications in Mass Spectrometry)

   It is imperative to understand how chemical preservation alters tissue isotopic compositions before using historical samples in ecological studies. Specifically, although compound‐specific isotope analysis of amino acids (CSIA‐AA) is becoming a widely used tool, there is little information on how preservation techniques affect amino acidδ¹⁵N values.

   We evaluated the effects of chemical preservatives on bulk tissueδ¹³C andδ¹⁵N and amino acidδ¹⁵N values, measured by gas chromatography/isotope ratio mass spectrometry (GC/IRMS), of (a) tuna (Thunnus albacares) and squid (Dosidicus gigas) muscle tissues that were fixed in formaldehyde and stored in ethanol for 2 years and (b) two copepod species,Calanus pacificusandEucalanus californicus, which were preserved in formaldehyde for 24–25 years.

   Tissues in formaldehyde‐ethanol had higher bulkδ¹⁵N values (+1.4,D. gigas; +1.6‰,T. albacares), higherδ¹³C values forD. gigas(+0.5‰), and lowerδ¹³C values forT. albacares(−0.8‰) than frozen samples. The bulkδ¹⁵N values from copepods were not different those from frozen samples, although theδ¹³C values from both species were lower (−1.0‰ forE. californicusand −2.2‰ forC. pacificus) than those from frozen samples. The mean amino acidδ¹⁵N values from chemically preserved tissues were largely within 1‰ of those of frozen tissues, but the phenylalanineδ¹⁵N values were altered to a larger extent (range: 0.5–4.5‰).

   The effects of preservation on bulkδ¹³C values were variable, where the direction and magnitude of change varied among taxa. The changes in bulkδ¹⁵N values associated with chemical preservation were mostly minimal, suggesting that storage in formaldehyde or ethanol will not affect the interpretation ofδ¹⁵N values used in ecological studies. The preservation effects on amino acidδ¹⁵N values were also mostly minimal, mirroring bulkδ¹⁵N trends, which is promising for future CSIA‐AA studies of archived specimens. However, there were substantial differences in phenylalanine and valineδ¹⁵N values, which we speculate resulted from interference in the chromatographic resolution of unknown compounds rather than alteration of tissue isotopic composition due to chemical preservation.

    

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2. Changes in stable isotope compositions during fasting in phocid seals

   https://doi.org/10.1002/rcm.8308  

   Habran, Sarah ; Damseaux, France ; Pomeroy, Paddy ; Debier, Cathy ; Crocker, Daniel ; Lepoint, Gilles ; Das, Krishna ( December 2018 , Rapid Communications in Mass Spectrometry)

   The grey seal,Halichoerus grypus(GS), and the northern elephant seal,Mirounga angustirostris(NES), come ashore for reproduction. This period involves intense physiological processes such as lactation in females and a developmental post‐weaning fast in juveniles. Previous studies have shown thatδ¹³C andδ¹⁵N values are affected by starvation, but the precise effects of fasting associated with lactation and post‐weaning fast in seals remain poorly understood.

   To examine the effect of lactation and post‐weaning fast on stable isotope ratios in GS and NES, blood and hair were sampled from 21 GS mother‐pup pairs on the Isle of May and on 22 weaned NES pups at Año Nuevo State Reserve during their respective breeding seasons. Milk samples were also collected from GS mothers. Stable isotope measurements were performed with an isotope ratio mass spectrometer coupled to an N‐C elemental analyser.

   Changes in stable isotope ratios in blood components during fasting were similar and weak between GS and NES mothers especially in blood cells (GS:Δ¹⁵N = 0.05‰,Δ¹³C = 0.02‰; NES:Δ¹⁵N = 0.1‰,Δ¹³C = 0.1‰). GS showed a¹⁵N discrimination factor between maternal and pup blood cells and milk, but not for¹³C. The strongest relationship between the isotopic compositions of the mother and the pup was observed in the blood cells.

   Isotopic consequences of lactation, fasting, and growth seem limited in NES and GS, especially in medium‐term integrator tissues of feeding activity such as blood cells. Stable isotope ratios in the blood of pups and mothers are correlated. We observed a subtle mother‐to‐pup fractionation factor. Our results suggest that pup blood cells are mostly relevant for exploring the ecology of female seals.

    

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3. Carbon and nitrogen isotope fractionation of amino acids in an avian marine predator, the gentoo penguin ( Pygoscelis papua )

   https://doi.org/10.1002/ece3.1437  

   McMahon, Kelton W. ; Polito, Michael J. ; Abel, Stephanie ; McCarthy, Matthew D. ; Thorrold, Simon R. ( February 2015 , Ecology and Evolution)

   Compound‐specific stable isotope analysis (CSIA) of amino acids (AA) has rapidly become a powerful tool in studies of food web architecture, resource use, and biogeochemical cycling. However, applications to avian ecology have been limited because no controlled studies have examined the patterns inAAisotope fractionation in birds. We conducted a controlledCSIAfeeding experiment on an avian species, the gentoo penguin (Pygoscelis papua), to examine patterns in individualAAcarbon and nitrogen stable isotope fractionation between diet (D) and consumer (C) (Δ¹³C_C‐Dand Δ¹⁵N_C‐D, respectively). We found that essentialAAδ¹³C values and sourceAAδ¹⁵N values in feathers showed minimal trophic fractionation between diet and consumer, providing independent but complimentary archival proxies for primary producers and nitrogen sources respectively, at the base of food webs supporting penguins. Variations in nonessentialAAΔ¹³C_C‐Dvalues reflected differences in macromolecule sources used for biosynthesis (e.g., protein vs. lipids) and provided a metric to assess resource utilization. The avian‐specific nitrogen trophic discrimination factor (TDF_Glu‐Phe= 3.5 ± 0.4‰) that we calculated from the difference in trophic fractionation (Δ¹⁵N_C‐D) of glutamic acid and phenylalanine was significantly lower than the conventional literature value of 7.6‰. Trophic positions of five species of wild penguins calculated using a multi‐TDF_Glu‐Pheequation with the avian‐specificTDF_Glu‐Phevalue from our experiment provided estimates that were more ecologically realistic than estimates using a singleTDF_Glu‐Pheof 7.6‰ from the previous literature. Our results provide a quantitative, mechanistic framework for the use ofCSIAin nonlethal, archival feathers to study the movement and foraging ecology of avian consumers.

    

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4. Membrane topology and identification of key residues of Ea DAcT, a plant MBOAT with unusual substrate specificity

   https://doi.org/10.1111/tpj.13636  

   Tran, Tam N. T. ; Shelton, Jennifer ; Brown, Susan ; Durrett, Timothy P. ( August 2017 , The Plant Journal)

   Euonymus alatusdiacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl‐CoA to thesn‐3 position of diacylglycerol to form 3‐acetyl‐1,2‐diacyl‐sn‐glycerol (acetyl‐TAG).EaDAcT belongs to a small, plant‐specific subfamily of the membrane bound O‐acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed thatEaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)‐specific marker. By mapping the membrane topology ofEaDAcT, we obtained an experimentally determined topology model for a plantMBOAT. TheEaDAcT model contains four transmembrane domains (TMDs), with both the N‐ and C‐termini orientated toward the lumen of theER. In addition, there is a large cytoplasmic loop between the first and secondTMDs, with theMBOATsignature region of the protein embedded in the thirdTMDclose to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl‐TAG‐producing plants. Among them, the acetyltransferase fromEuonymus fortuneipossessed the highest activityin vivoandin vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential forEaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity ofEaDAcT, suggesting that multiple amino acids are important for substrate recognition.

    

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5. A molecular fluorophore in citric acid/ethylenediamine carbon dots identified and quantified by multinuclear solid‐state nuclear magnetic resonance

   https://doi.org/10.1002/mrc.4985  

   Duan, Pu ; Zhi, Bo ; Coburn, Luke ; Haynes, Christy L. ; Schmidt‐Rohr, Klaus ( January 2020 , Magnetic Resonance in Chemistry)

   The composition of fluorescent polymer nanoparticles, commonly referred to as carbon dots, synthesized by microwave‐assisted reaction of citric acid and ethylenediamine was investigated by¹³C,¹³C{¹H},¹H─¹³C,¹³C{¹⁴N}, and¹⁵N solid‐state nuclear magnetic resonance (NMR) experiments.¹³C NMR with spectral editing provided no evidence for significant condensed aromatic or diamondoid carbon phases.¹⁵N NMR showed that the nanoparticle matrix has been polymerized by amide and some imide formation. Five small, resolved¹³C NMR peaks, including an unusual ═CH signal at 84 ppm (¹H chemical shift of 5.8 ppm) and ═CN₂at 155 ppm, and two distinctive¹⁵N NMR resonances near 80 and 160 ppm proved the presence of 5‐oxo‐1,2,3,5‐tetrahydroimidazo[1,2‐a]pyridine‐7‐carboxylic acid (IPCA) or its derivatives. This molecular fluorophore with conjugated double bonds, formed by a double cyclization reaction of citric acid and ethylenediamine as first shown by Y. Song, B. Yang, and coworkers in 2015, accounts for the fluorescence of the carbon dots. Cross‐peaks in a¹H─¹³C HETCOR spectrum with brief¹H spin diffusion proved that IPCA is finely dispersed in the polyamide matrix. From quantitative¹³C and¹⁵N NMR spectra, a high concentration (18 ± 2 wt%) of IPCA in the carbon dots was determined. A pronounced gradient in¹³C chemical‐shift perturbations and peak widths, with the broadest lines near the COO group of IPCA, indicated at least partial transformation of the carboxylic acid of IPCA by amide or ester formation.

    

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