The deep-fried meatball type most susceptible to oxidation was used as the model meat product for testing the lipid oxidation inhibiting capacity of 11 plant materials and extracts, in two concentrations ), measured after 14 days of storage using TBARS. Summer savory lyophilized powder was the most efficient plant material, lowering lipid oxidation to 13.8% and 21.8% at the 200 and 100 mg kg−1 concentration, respectively, followed by a sea buckthorn leaf extract, lowering lipid oxidation to 22.9% at 100 mg kg−1, compared to the meatball without added antioxidants. The lipid oxidation was thus successfully reduced using these natural antioxidants.The underlying mechanism(s) behind the potential carcinogenicity of processed meat is a popular research subject of which the lipid oxidation is a common suspect. Different formulations and cooking parameters of a processed meat product were evaluated for their capacity to induce lipid oxidation. Meatballs made of beef or pork, containing different concentrations of fat (10 or 20 g 100 g Since the International Agency for Research on Cancer (IARC) released their monograph stating that consumption of red and processed meats are linked to colorectal cancer (CRC) in 2015, research regarding this topic has increased, particularly since the mechanisms leading to these links remain partly unknown .−1), onion, egg, breadcrumbs, and salt (ranging between 2 and 4 g 100 g−1). Meatballs were chosen as the study material due to the large market and the fact that the formulation and cooking process covers most of the main components of processed meat products in general.Processed meat refers to products typically made of red meat that have undergone curing, salting, or smoking, often containing high amounts of fatty tissues together with endogenous phospholipids . These fThe addition of antioxidants to meat, meat products, and meat model systems has been widely studied for oxidation preventing purposes . SynthetThe aim of the present study was (I) to develop a relevant oxidized processed meatball model to study the effects of supplemented antioxidants, and (II) investigate lipid oxidation in meatballs without and with a range of plant materials and extracts at different concentrations.Hippophae rhamnoides L.) leaves and bilberry leaves (Vaccinium myrtillus L.), respectively. The static extraction time was 1 min for both samples. After PHWE, the extracts were filtered and lyophilized. Estonian phenol-rich extracts were prepared using a pilot-scale solid-liquid Naviglio extractor  with 20 mL 100 mL−1 ethanol (aq). Extracts of rhubarb root (Rheum rhabarbarum L.) and black currant (Ribes nigrum L.) leaves were concentrated to half their volume and were lyophilized to a powder. The ethanol of the Swedish extracts (Satureja hortensis L.) powder was added non-extracted to the meat batter with the same amount of tap water as the extracts. The extracts were dissolved in MilliQ water prior to dilution in tap water and addition to the meat batter. All samples were analyzed for their total phenols content using Folin-Ciocalteu reagent [Eleven plant materials and extracts were collected from Denmark, Estonia, Finland, Latvia, and Sweden , i.e., fextracts  was evap reagent .−1 (TBA), 1,1,3,3-tetramethoxypropane 99 mL 100 mL−1 (TMP), trichloroacetic acid ≥99.0 g 100 g−1 (TCA), and ethanol 96 mL 100 mL−1 were obtained from Sigma-Aldrich Inc., St. Louis, MO, USA. Hydrochloric acid (40 mM L−1) and 85 mL 100 mL−1 ortho-phosphoric acid (H3PO4) were obtained from Merck KGaA, Darmstadt, Germany.2-Thiobarbituric acid ≥98 g 100 g−1), fat (10 and 20 g 100 g−1) from two types of meat (pork and beef obtained from Atria Sverige AB) and were either pan-fried or deep-fried, and stored for 1, 7, and 14 days (Musculus trapezius) (fat content 4.3 g 100 g−1) was adjusted to fat contents of 10 and 20 g 100 g−1, respectively, during mincing using pork belly  (fat content 28.2 g 100 g−1). Lean meat from boneless beef knuckle (M. semitendinosus) (fat content 5.5 g 100 g−1) was adjusted to the same fat contents using a mixture of cuts from beef chuck (M. deltoideus) and clod  (fat content 27.5 g 100 g−1). After fat adjustment, NaCl was added and blended to the mince before vacuum packing and freezing at −18 °C until trials begun. The mince was thawed in a refrigerator (4 °C) overnight before meatballs were manufactured for the trial. Meatballs were either pan-fried or deep-fried in randomized order in refined rapeseed oil , containing 7.5 g 100 g−1 saturated fatty acids, 62.5 g 100 g−1 mono-unsaturated fatty acids, and 30 g 100 g−1 poly-unsaturated fatty acids, where the temperature in the pan was kept stable at 175 °C ± 1 °C controlled by a laser thermometer  and the deep-frying temperature was kept stable at 160 °C ± 0.5 °C. When the meatballs had reached a 72 °C inner temperature, they were removed from heating and rested on paper towels until room temperature was reached. The meatballs were then stored in a refrigerator (4 °C) in sealed polyethylene bags for 1, 7, and 14 days before the level of lipid oxidation was measured using thiobarbituric reactive substances (TBARS).Meatballs were produced in triplicates with two concentrations of NaCl , respectively, based on their total phenols content , the level of lipid oxidation was measured using thiobarbituric reactive substances (TBARS).The 11 antioxidant powders and extracts were added in 100 and 200 mg kg content  to the m−1 TCA and 0.75 g 100 mL−1 TBA in 0.25 M HCl. Samples from meatballs were weighed to 6 ± 0.1 g and crushed by mortar and pestle prior to being added to a 50-mL falcon tube containing 22.5 mL Milli-Q H2O together with 1.5 mL of 10 g 100 mL−1 TCA solution. The samples were then vortexed for 60 s prior to heating at 40 °C for 5 min to precipitate proteins, before 2 mL 96 mL 100 mL−1 ethanol was added to solubilize fats. The mixtures were then filtered through a filter paper  to obtain a clear filtrate for TBARS analysis. In total, 2.5 mL of the TBA reagent was added to 0.5 mL of the filtrates, which were then heated to 90 °C for 10 min before being cooled in tap water to end the reaction. All samples were centrifuged at 3600 g for 20 min  before the absorbance of the supernatants was measured at 534 nm and 600 nm, respectively . If the coefficient of variation (cv%) between the sample replicates exceeded 10%, the analyses were re-run. Results are reported as µM malondialdehyde (MDA) g−1 meatball for meatball parameter evaluations, and as a percentage oxidation of the blank sample (without added antioxidants) for antioxidant evaluation.The lipid oxidation was measured in triplicates for each meatball using TBARS based on the method of Buege and Aust , where t−1 meatball). In the antioxidant study, species and concentrations of antioxidants were fixed factors in the full-factorial model of the dependent variable percent lipid oxidation of meatballs compared to meatballs without antioxidants. In both trials, meatball samples were made in triplicates of which TBARS was measured in 3 technical replicates; statistical analyses were conducted on an average of the results of the meatball triplicates. Post-hoc tests were performed using the Scheffe method. A Pearson correlation analysis was conducted using SPSS on previous antioxidant activity data for the samples included in this study together with the lipid oxidation inhibiting capacity to study if, and if so, which antioxidant mode of action gave rise to the lipid oxidation inhibiting capacity.Statistical analyses were carried out using SPSS Statistics 25.0  and R for Windows GUI front-end version 3.5.3 (R version 3.5.2 (2018-12-20)—“Eggshell Igloo” Copyright © 2018 The R Foundation for Statistical Computing Platform: x86_64-w64-mingw32/x64 (64-bit). Box-Cox transformations were used to achieve normal distribution of data. A univariate general linear model (GLM) was performed on logarithmic values of oxidation data in both the preliminary trial and antioxidant study in order to ensure normal distribution of samples . In thep < 0.001) as did the fat content (p < 0.01). Nearly all interactions had significant effects on lipid oxidation (p < 0.05) except for interactions between: Salt × cooking, meat × salt × storage, meat × salt × fat × cooking, and meat × salt × fat × storage (2) showed the effect sizes of most interactions were minor. The factors affecting lipid oxidation the most were meat type and storage time (p < 0.001)  , where p< 0.001) a. There < 0.001) b, where  14 days  (Table A 14 days .−1) in triplicates after 14 days of storage where the results were calculated as a percentage of oxidation compared to samples without added antioxidants  * 100) (p < 0.001)  at 200 mg kg−1 and 100 mg kg−1, water extracted sea buckthorn  at 100 mg kg−1, and olive polyphenols  at 200 mg kg−1 were the most efficient antioxidants, lowering lipid oxidation to 13.8%, 21.8%, 22.9%, and 26.1%, respectively (−1) and the lipid oxidation inhibition capacity of the antioxidants  µM g) * 100) . Statist< 0.001)  of which< 0.001) . The sumectively , compareoxidants .This study was divided into two parts, where the aim of the first part was to select a meatball model prone to oxidize. The aim of the second part of this work was to include and test various types of natural antioxidants for lipid oxidation inhibiting purposes. Both parts of the study will be discussed separately hereunder.p < 0.001) more prone to oxidize than those of beef meat (−1) than beef meat (8 mg g−1) [−1) and muscle tissue (14.2 g 100 g−1) than beef adipose tissue (1.1 g 100 g−1) and muscle tissue (2.4 g 100 g−1) [We found that meat type was the most important factor affecting lipid oxidation  where theef meat . This wa mg g−1) , and sin mg g−1) ,17,18. H100 g−1) . This di−1 fat, and 2 g 100 g−1 salt, and had been deep-fried and stored for 14 days    was expep < 0.001) (−1 (p < 0.001), where species had the largest effect size (−1 than at 100 mg kg−1 (−1 ethanol (SBT), which was significantly more effective at 100 mg kg−1, and the sea buckthorn sample extracted with H2O (SBTH2O), which showed tendencies to be more efficient at 100 mg kg−1. Radenkovs et al. [−1 of these meatballs.Synthetic antioxidants have typically been included into meat products to increase, e.g., shelf-life and nutritional value, and now the industry is demanding new natural sources of antioxidant ingredients . Numerou< 0.001) , where t001) (−1 . The staect size . Overall mg kg−1  except fs et al.  previous−1 fat and 2 g 100 g−1 salt, and had been stored for 14 days. This meatball type was then manufactured without and with 11 different plant materials and extracts at two concentrations, 100 mg kg−1 and 200 mg kg−1 GAE, and was stored for 14 days. All samples inhibited lipid oxidation effectively in both tested concentrations, where the summer savory powder was the most efficient in both the 100 mg kg−1 and 200 mg kg−1 concentration, lowering lipid oxidation to 21.8% and 13.8%, respectively, compared to meatballs with no added antioxidants. Thus, antioxidant rich plant materials and extracts could efficiently prevent lipid oxidation in processed meat products, such as meatballs.The aim of the current study was to evaluate the lipid oxidation inhibiting capacity of natural antioxidants in a readily oxidized meat product. Various meatball properties were studied in order to find the combination that gave rise to the most lipid oxidation. The meatball most prone to oxidize was deep-fried, made of pork, contained 20 g 100 g


During subsequent ex vitro culture, the use of MS medium resulted in a higher growth rate, compared to Hoagland’s medium. Thus, a higher starch content of L. punctata in MS medium was previously hypothesized. Here, L. punctata strain 5632 was isolated and characterized using morphological characteristics and the atpF-atpH intergenic region. During early cultivation stage, fresh weight and relative growth rate in MS medium were lower than Hoagland’s medium. Conversely, starch content in MS medium was considerably higher than in Hoagland’s medium. Medium effects on expression of genes coding for starch-biosynthesis ADP-glucosepyrophosphorylase (AGPase) were determined. Genomic fragments of small (LeAPS) and large (LeAPL1) AGPase subunits were characterized. Differential expression between each AGPase subunit genes was observed in both media. Additionally, in MS medium, the highest correlation coefficients between starch content and gene expression was found with LeAPS (0.81) and followed by LeAPL3 (0.67), LeAPL2 (0.65) and LeAPL1 (0.28). In Hoagland’s medium, the coefficients of LeAPL3 (0.83) and LeAPL2 (0.62) were higher than LeAPS (0.18) and LeAPL1 (−0.62). This suggested different levels of contributions of these genes in starch biosynthesis in both media.Murashige & Skoog (MS) and Hoagland’s media were previously used for  During photosynthesis, carbon compounds are generated and converted into glucose that serves as the precursor for starch formation1. There are three committed steps in the process1. The first one is the generation of ADP-glucose. Secondly, the glucosyl moiety of ADP-glucose is linked to an existing glucan chain through the formation of the α(1-4) linkage, resulting in an extension of the starch chain. Finally, branching of the chain is formed through the formation of the α(1-6) linkage between glucosyl moieties and the chain. ADP-glucose pyrophophorylase (AGPase) is responsible for the formation of ADP-glucose that was proposed as the rate-limiting step2. In higher plants, AGPase is heterotetrameric and consists of two small and two large subunits4. Small subunits play the catalytic role, while large subunits mainly function in regulating the enzyme activity6. Genes that code for large and small subunits were previously identified in various dicotyledonous and monocotyledonous plants, e.g., Arabidopsis7, potato9, chickpea10, maize12 and wheat14. Additionally, overexpression of AGPase large subunit genes increased starch content in maize and wheat grains15. Another study showed that a transgenic wheat line, overexpressing an AGPase small subunit gene, accumulated starch at a level substantially higher than the wild-type cultivar6.Starch functions as an important energy reserve in plants16. Thus far, 37 duckweed species were affiliated with the family and classified into five genera, including Spirodela, Landoltia, Lemna, Wolffiella and Wolffia17. Fronds are leaf-like and function as both vegetative and reproductive organs18. L. punctata has gained the interest in research communities, because of its high starch content that could be used in bioethanol production21. Several advantages of L. punctata over other energy crops were also cited22. These included rapid growth rate, high starch content and low levels of fiber and lignin content22. Additionally, the use of L. punctata for the energy industry did not compete for land with food crops23. Various external and internal factors affected starch biosynthesis of L. punctata. Nutrient starvation induced starch accumulation4, while phytohormones were internal regulators of starch biosynthesis25. Expression of L. punctata AGPase subunit genes, including LeAPS, LeAPL1, LeAPL2 and LeAPL3, were also affected by growth conditions25. Expression of LeAPL2 under nitrogen starvation was relatively lower than that under phosphorus starvation4. In contrast, nitrogen deficiency induced an early response of LeAPL3, while phosphorus deprivation resulted in an upregulation of LeAPL3 at a later phase4. A transcriptomic study showed that, when L. punctata was treated with uniconazole, expression of two AGPase large subunit genes and starch content were concurrently increased25.Duckweeds are small aquatic plants that belong to the family Lemnaceae27. Medium compositions had various physiological impacts on duckweeds. In Spirodela polyrhiza, exogenous addition of abscisic acid (ABA) in culture medium induced a morphological transition of fronds to turions, a starch accumulating structure28. SpAPL2 and SpAPL3, encoding two different AGPase large subunits, were also upregulated under such conditions, whereas SpAPL1 expression was not significantly increased28. For L. punctata, biomass production was higher when plants were cultivated in Hoagland’s medium for 13 days, compared to MS medium26. However, after transferred to nutrient-limited pond water for ex vitro culture, L. punctata obtained from MS medium grew 17.1% faster than those from Hoagland’s medium. It was previously hypothesized that cultivation in MS medium resulted in higher starch accumulation that subsequently promoted ex vitro growth26. This hypothesis was tested here. L. punctata strain 5632 was newly isolated and characterized, based on its morphologies and the atpF-atpH intergenic region sequence. Strain 5632 was long-term (35 days) cultured in MS and Hoagland’s media to observe its biomass production and starch accumulation. The cDNA fragments of LeAPS and LeAPL1, coding for AGPase large and small subunits, respectively, were cloned and compared with the reference sequences of L. punctata strain 02024. Genomic fragments of both genes were also cloned, and their introns and exons were characterized. Expression levels of four AGPase subunit genes, including LeAPS, LeAPL1, LeAPL2 and LeAPL3, in both media were analysed. Correlation coefficients, between AGPase gene expression levels and starch content, were calculated to determine the contribution of each gene in starch biosynthesis, during the cultivation in MS and Hoagland’s media.Synthetic media were generally used for culturing duckweeds under controlled environments29. Originally, two different macronutrient solutions were described for the preparation of Hoagland’s medium29. The main difference between them was the nitrogen source. While one solution contained only NO3−, the other one was supplemented with both NO3− and NH4+ 29. Hoagland’s solution, used in this study, had nutrient compositions 29 and this study (6 mM). Additionally, iron was supplied as iron tartate in the original publication29, whereas it was given here as chelated iron. Basic morphological characteristics of strain 5632 were examined  was found between the sequences of strain 5632 and L. punctata strains DW2701-1 (accession number: KJ630554) and DW2701-4 (KJ630555). To understand the phylogenetic relationship between strain 5632 and other L. punctata strains, the phylogenetic tree was reconstructed based on the atpF-atpH intergenic sequences. It showed that strain 5632 was closely related to L. punctata strains DW2701-1, DW2701-4 and 20d  became significantly higher (P = 0.038) than that in Hoagland’s medium (3.04 ± 0.04 g). On day 35, it became 1.2 times of that in Hoagland’s medium.A previous study showed that the growth rate of ays Fig. . It was ays Fig.  and in t−1. During the same period, the fresh weight in Hoagland’s medium rose from 1.02 ± 0.07 g to 2.23 ± 0.12 g, and yielded relative growth rate at 0.11 ± 0.002 day−1.Relative growth rate of strain 5632 in both media were calculated and found relatively consistent with fresh weight analysis Fig. . During Starch content of strain 5632 in MS and Hoagland’s media was also determined throughout the cultivation period Fig. . In MS m4+ and NO3− in MS and Hoagland’s media (Table 4+ in MS medium (20.6 mM). A previous study showed that NH4+ was preferable over NO3− for the uptake by L. punctata35. Thus, cultivation of strain 5632 in the medium likely caused excessive accumulation of NH4+ in strain 5632. This resulted in lower biomass production, compared to the use of Hoagland’s medium, that was supplemented with 1 mM NH4+. When present at a high concentration, NH4+ was known to negatively affect plant growth36. This was also demonstrated in other duckweed species. Relative growth rate of S. polyrhiza decreased upon the increase of NH4+ concentration37. Growth inhibition and frond chlorosis were also found in Lemna minor, grown in Hoagland’s medium supplemented with 280 and 840 mg l−1 NH4+-N, which equaled to 20 and 60 mM NH4+, respectively38. During the later cultivation period, growth of strain 5632 in Hoagland’s medium declined more rapidly, compared to that in MS medium. This may be attributed to the NO3− concentration in Hoagland’s medium (10 mM) that was only 25.4% of that in MS medium (39.4 mM). The result suggested that this level of NO3−, in Hoagland’s medium, was insufficient to sustain growth of strain 5632 for the entire cultivation period. Additionally, another nutrient that may cause the distinction in growth of strain 5632 in the two media was iron. It is an important microelement in various plant metabolic processes39. Generally, FeSO4 was added together with Na2EDTA to generate chelated iron that ensured its transport into plant cells40. Here, the concentrations of FeSO4 and Na2EDTA, in Hoagland’s medium, were approximately 9% of those in MS medium and may be insufficient to sustain growth during the extended cultivation period. On the other hand, starch biosynthesis was previously known to be upregulated under nutrient starvation conditions, which also resulted in growth retardation42. This was somewhat consistent with the increase in starch content of strain 5632 in MS medium, where growth was relatively lower than in Hoagland’s medium. However, as mentioned above, both NH4+ and NO3− in MS medium were supplied at much higher levels than Hoagland’s medium. This suggested that differential starch accumulation of strain 5632 in the two media was likely a secondary effect that resulted from growth modulation by the nitrogen sources.Growth and starch content of strain 5632 were likely affected by the differences between the concentrations of both NHia Table . During LeAPS and LeAPL1 cDNA were previously sequenced in L. punctata strain 0202 that was collected in China4. However, strain 5632 was obtained from a local pond in Thailand. Thus, cDNA sequences of both genes were determined to investigate nucleotide variations that may exist between the two genes of these strains. Full-length LeAPS  and LeAPL1  cDNA of strain 5632 were sequenced and compared with their corresponding sequences of strain 0202 4. For LeAPS, three nucleotide variations were found at positions 27, 575 and 769 . Thus, these amino acids of strain 5632 likely represented the actual residues of LeAPS and LeAPL1. The only exception was the amino acid position 252 of LeAPL1 that was derived from its corresponding codon containing the nucleotide variant position 754. The amino acid sequences of strain 5632 and other orthologs carried methionine at this position, as opposed to valine found in L. punctata 0202 and S. tuberosum sequences  was obtained using the CLeAPL1-F and CLeAPL1-R primers. The genomic sequence consisted of 14 exons and 13 introns , LeAPL2 (1.92 ± 0.88) and LeAPL3 (1.48 ± 0.77) were observed on day 21 and approximately 8.2 times of the level on day 0. In contrast, LeAPL1 expression increased and reached the maximum level (0.66 ± 0.14) on day 14, which was approximately 2.6 times of that on day 0. Similar to LeAPS, the highest levels of LeAPL2 and LeAPL3 expression occurred on day 21 and were 4.0 and 8.1 folds of day 0, respectively. In Hoagland’s medium, expression of LeAPS (0.72 ± 0.10) and LeAPL1 (1.02 ± 0.15) similarly peaked on day 14 and were 10.3 and 4.2 folds of day 0, respectively. LeAPL2 expression became the highest on day 28 (2.38 ± 0.80) and was 9.5 times of day 0. In contrast, expression of LeAPL3 continuously increased throughout the cultivation period and was the highest on day 35 (1.48 ± 0.61) and 3.6 folds of day 0. Significant differences were also observed between expression levels of some AGPase subunit genes in both media. On day 21, LeAPS in MS medium was expressed at a level significantly higher (P = 0.049) than in Hoagland’s medium. On the other hand, LeAPL1 in MS medium was expressed at levels significantly lower than in Hoagland’s medium on day 14 (P = 0.039) and 21 (P = 0.029). However, its expression in MS medium became significantly higher than in Hoagland’s medium on day 28 (P = 0.03) and 35 (P = 0.014). In contrast, differences in LeAPL2 and LeAPL3 expression levels observed in MS and Hoagland’s media were not substantial throughout the cultivation period. The result observed here indicated differential regulation of each AGPase subunit gene. This was also previously found with L. punctata strain 0202, that was grown under nitrogen and phosphorus starvation conditions4. In response to nitrogen starvation, upregulation of LeAPL1 and LeAPL3 was observed earlier than LeAPL24. Under phosphorus deprivation, a dramatic increase in LeAPL1 and LeAPL2 expression occurred more rapidly than LeAPL24. In contrast, LeAPS was highly expressed throughout the nitrogen starvation period and during the early stage of phosphorus starvation4. ABA may play a role in regulating expression of AGPase genes in this study, since its application on L. punctata was previously shown to increase starch content and AGPase activity45. Consistently, a transcriptomic study demonstrated that both ABA content and expression levels of AGPase large subunit genes were elevated, when L. punctata was treated with uniconazole25. Reports in S. polyrhiza28 and rice cell culture46 suggested that each AGPase subunit gene may respond to ABA differently. Expression of SpAPL2 and SpAPL3 was upregulated by the ABA treatment28. In contrast, the expression level of SpAPL1 remained relatively unchanged and was much lower than SpAPL2 and SpAPL3. On the other hand, application of ABA to rice cell culture resulted in elevated expression of OsAPL3 and OsAPS1, while its effects on other rice AGPase subunit genes were much less pronounced46. These previous observations suggested that ABA effects on expression of AGPase genes may also vary among different plant species.Expression of AGPase subunit genes, including LeAPS had the highest correlation coefficient (0.81) and was followed by LeAPL3 (0.67) and LeAPL2 (0.65). A small correlation (0.28) was observed with LeAPL1. In Hoagland’s medium, expression of LeAPL3 was the most correlated (0.83) with starch content. An intermediate level of correlation was found with LeAPL2 (0.62). In contrast, the correlation coefficient of LeAPS was only 0.18, while LeAPL1 was negatively correlated (−0.62). The intermediate and high correlation coefficients of LeAPL2 and LeAPL3 were consistent with their expression levels that were found comparable in both media  physiological and molecular responses were demonstrated in L. punctata (G. Mey.) Les & D. J. Crawford was collected from a local pond in Bangkok, Thailand. To establish in vitro culture, fronds were surface-sterilized, using 10% (v/v) NaClO solution and a few drops of Tween-20 for two minutes with vigorous shaking. They were washed in sterilized distilled water three times. Surface-sterilized fronds were grown on Hoagland’s solid medium , containing 2% (w/v) sucrose and 0.7% (w/v) agar, pH 5.7. Growth conditions were 25 ± 2 °C and 16-hour-light/8-hour-dark photoperiods. Photosynthetically active radiation (PAR) flux was 65 µmol m−2 s−1. Daughter fronds derived from a single mother frond were obtained. Morphological characteristics were examined. This duckweed strain was registered to the Rutgers Duckweed Stock Cooperative as strain 5632.−2 s−1. Samples were collected on day 0, 7, 14, 21, 28 and 35 of cultivation and rinsed thoroughly with distilled water. Excessive water was removed by blotting on tissue paper. Total fresh weight was determined. Relative growth rate on day 7, 14, 21, 28 and 35 was calculated as follows:Wt = fresh weight on day 7, 14, 21, 28 and 35 of cultivation; Wt-1 = fresh weight of prior sample collection and time = the time interval (7 days).Twenty colonies of strain 5632 were randomly selected and transferred to glass bottles containing 100 ml of either MS or Hoagland’s liquid media , supplemented with 2% sucrose, pH 5.7. The cultures were grown for 35 days under 25 ± 2 °C and 16-hour-light/8-hour-dark photoperiods. PAR flux was 65 µmol m−1 D-glucose was used as the standard. The experiment was done in triplicate. Two-tailed student’s t-test was used to determine statistically significant differences (P < 0.05) between fresh weight, relative growth rate, and starch content, in MS and Hoagland’s media on each day.One-hundred milligrams of fresh samples collected on each day were used for the analysis of starch content with Total Starch Assay Kit , according to the manufacturer’s protocol. Water was used as the blank control, and 1 mg mlatpF-atpH intergenic region were done with the atpF-atpH forward (5′-ACTCGCACACACTCCCTTTCC-3′) and reverse (5′-GCTTTTATGGAAGCTTTAACAAT-3′) primers, according to the previous study33. The sequence was analysed with blastn. Multiple alignment of the sequence with those of other duckweed strains in the GenBank database used the CLUSTAL W program version 1.8147. Gaps were manually removed and adjusted. A phylogenetic tree was reconstructed, using unweighted pair group method with arithmetic mean (UPGMA), in MEGA 7.048. Bootstrap analysis49 with 1,000 re-samplings determined the confidence level of each clade.Fronds from axenic culture were used for DNA extraction with a FavorPrep Plant Genomic DNA Extraction Mini Kit , according to the manufacturer’s protocol. Amplification and sequencing of the LeAPS cDNA, while CLeAPL1-F (5′-ATGGCGCTGCGGATTGAG-3′) and CLeAPL1-R (5′-TCAGATGACAAGGCCATCCTT-3′) primers were used for LeAPL1 cDNA. The primer sequences and amplification cycles were according to the previous study4. Q5 High-Fidelity DNA polymerase  was used for the amplification of full-length cDNA. The nucleotide sequences were compared with those of L. punctata strain 0202, using blastn. Multiple alignment analysis was performed between the deduced amino acid sequences of LeAPS and LeAPS and their homologs, using Clustal Omega .Total RNA was extracted from 100 mg of fronds, using a FavorPrep Plant Total RNA Mini Kit , according to the manufacturer’s protocol. Genomic DNA was removed with DNaseI  by the on-column method, described in the protocol. cDNA of all samples was synthesized from 500 ng of total RNA, using iScript cDNA Synthesis Kit . Oligo-dT supplied with the kit was used as the primer for the first-strand synthesis. CLeAPS-F (5′-ATGGCGGCGACGAGCTTC-3′) and CLeAPS-R (5′-TCATATGATGGTTCCGCTAGGG-3′) primers were used for amplification of LeAPL1 and LeAPS genomic fragments. CLeAPS-F and LeAPS-R (5′-GCCCAATCCGAGCATTCT TAT-3′)4 primers were used for amplification of partial LeAPS genomic fragment, while CLeAPL1-F and CLeAPL1-R primers were for genomic LeAPL1. The amplified cycles were: 95 °C for 5 min; 40 cycles of 95 °C for 30 sec, 60 °C for 30 sec, and 72 °C for 90 sec; 72 °C for 5 min. The amplified fragments were cut from an agarose gel and purified using FavorPrep GEL/PCR Purification Kit . Subsequently, the fragments were ligated with the pGEM-T vector  and cloned in Escherichia coli strain DH5-α. Recombinant plasmids were extracted, using FavorPrep Plasmid DNA Extraction Mini Kit . Universal primers M13F (−20) and M13R (−40) were used for initial DNA sequencing. Additional primers were designed based on the sequences and used to complete the sequences. The genomic sequences of LeAPS (accession number: MK878513) and LeAPL1 (MK878512) were deposited in GenBank database.Q5 High-Fidelity DNA polymerase  was used for the amplification of LeAPS and LeAPL1 expression were designed. APSQ-F1 (5′-TCCCAGATTTCAGCTTCTATGATCGG-3′) and APSQ-R1 (5′ TGAATCTTGCAGTTCTTAATCACGC-3′) primers were for LeAPS, while APL1Q-F1 (5′-AGAACTCGAAGATCAGGAACTGC-3′) and APL1Q-R1 (5′-TCTTTCGGCTTCTTGGATTCCCTC-3′) primers were for LeAPL1. Previously described primers were used to quantify expression of LeAPL2 (LeAPL2-F/LeAPL2-R) and LeAPL3 (LeAPL3-F/LeAPL3-R)4. Gene expression levels were determined according to a previously described method28. Briefly, five μl of cDNA of all samples were combined. A standard dilution series of 1x, 10−1X, 10−2X and 10−3X concentrations were made. The quantitation cycle (Cq) values of the standards and all samples were determined. Standard curves of standard cDNA concentrations and Cq values were generated. Gene expression levels of all samples were determined by normalizing the sample Cq values against the standard curves. The experiment was performed in triplicate. Two-tailed student’s t-test was used to determine statistically significant differences (P < 0.05) between expression levels of each AGPase subunit genes in MS and Hoagland’s media on each day. Correlation coefficients between the averages of starch content and expression levels were determined for each gene.Total RNA was extracted from 100 mg of strain 5632 fronds, collected at each time point. The extraction and cDNA synthesis followed the methods mentioned above. Quantitative reverse transcription PCR was done using SensiFast Real-Time PCR kit , on CFX96 Touch Real-Time PCR Detection System . New primers for quantification of


Besides, the CoSe/NC composites show a distinct incremental behavior of capacity.As a typical metal selenide, CoSe is a kind of foreground anode material for lithium-ion batteries (LIBs) because of its two-dimensional layer structure, good electrical conductivity, and high theoretical capacity. In this work, the original CoSe/N-doped carbon (CoSe/NC) composites were synthesized using ZIF-67 as a precursor, in which the CoSe nanoparticles are encapsulated in NC nanolayers and they are connected through C–Se bonds. The coating structure and strong chemical coupling make the NC nanolayers could better effectively enhance the lithium storage properties of CoSe/NC composites. As a consequence, the CoSe/NC composites deliver a reversible capacity of 310.11 mAh g Lithium-ion batteries (LIBs) stand out from many new energy sources because of their high energy density, long cycle life, and environmental friendliness , 2. They of LIBs –6. There of LIBs –9, trans of LIBs –13, meta of LIBs –17, meta of LIBs –21, and  of LIBs –25.Selenium has higher density and conductivity, so metal selenide has higher energy density and rate performance than transition metal oxides and sulfides . Compare2+ and 2-methylimidazole and has a porous structure similar to zeolite. 2-Methylimidazole has a nitrogen-containing functional group that carbonizes to form N-doped carbon (NC) through pyrolysis [3O4 [Metal-organic frameworks (MOFs) are a class of porous materials formed by the attachment of metal ions to organic compounds via coordinate bonds –30. Becayrolysis , 33. On yrolysis . Besidessis [3O4 , 35, CoSsis [3O4 , 37, CoPsis [3O4 –40, and 2+ within ZIF-67 reacts with selenium to form CoSe nanoparticles, which are enfolded by NC nanolayers that originate from the carbonization of 2-methylimidazole. The NC nanolayers could enhance the conductivity of CoSe and suppress the volume expansion, and offer additional lithium storage as active sits. The porous structure from ZIF-67 could shorten the diffusion path of electrons and lithium ions. More importantly, the C–Se bonds between CoSe nanoparticles and NC nanolayers; the unique chemical connection could not only better promote the electrical active connection between CoSe and NC, but also better mitigate the volume variation. As a result, the CoSe/NC composites show superb lithium storage properties.Here, we report a facile method to prepare CoSe/N-doped carbon (CoSe/NC) composites through the selenization of ZIF-67 with selenium powder at inert atmosphere. Figure 3)2·6H2O and 3.244 g of 2-methylimidazole were dissolved in 100 ml of methanol solution, respectively. Subsequently, the Co(NO3)2·6H2O solution was poured into 2-methylimidazole solution with stirring for 12 min, and then aged for 20 h. Finally, the resulting purple precipitates were collected by centrifugation, washed with methanol several times, and dried in air at 60 °C.All chemicals are analytical grade and used without further purification. In a typical synthesis, 1.436 g of Co at a scan rate of 0.05 s−1. The Raman spectrums were recorded on a LabRAM HR800 Raman spectrometer with 532-nm laser light. TGA was carried out on a STA 449 F3 thermoanalyser under ambient atmosphere and at a heating rate of 10 °C min−1 from ambient temperature to 900 °C. XPS measurements were performed on a Thermo ESCALAB 250XI X-ray photoelectron spectrometer with monochromatic Al Kα radiation (hν = 1486.6 eV). Nitrogen adsorption/desorption isotherms were collected at 77 K using a BELSORP-Max instrument, which were used to evaluate the specific surface area (BET) and pore size distribution (BJH). FESEM images were collected through Quanta 250 scanning electron microscopy and SIGMA 500 scanning electron microscopy, respectively. TEM images and SAED were taken on a Tacnai G2 F20 transmission electron microscopy.The powder XRD patterns of the samples were obtained on a TD-3500X X-ray diffractometer with Cu Kα radiation (N-methyl-2-pyrrolidone (NMP) to form a slurry. Subsequently, the slurry was uniformly coated on Cu foil and dried at 80 °C for 4 h in air and then punched in disks with a dimeter of 14 mm and dried at 120 °C for 12 h under vacuum. The metallic lithium foil was used as both counter electrode and reference electrode. The electrolyte was 1.00 M LiPF6 in ethylene carbonate and diethyl carbonate (EC: DEC = 1:1) and the separator was Celgard 2500 film. The all cells were assembled in an argon-filled glovebox with the content of oxygen and moisture below 1 ppm. The galvanostatic charge-discharge tests were conducted on a Neware CT-3008W battery test system between 0.01 and 3.0 V at room temperature. The cyclic voltammetry (CV) measurements between 0.01 and 3.0 V at a scan rate of 0.2 mV s−1 and electrochemical impedance spectroscopy (EIS) with the frequency range from 0.01 to 100 kHz were all performed on a CHI 760E electrochemical workstation.All electrochemical behaviors of as-synthesized samples were performed using CR2032 coin-type cell. The working electrodes were prepared by mixing 80 wt% of active materials, 10 wt% of acetylene black, and 10 wt% of polyvinylidene fluoride (PVDF) binder in an appropriate The crystal structure and morphology of as-synthesized ZIF-67 were confirmed by XRD and SEM, respectively, as shown in Fig. −1 are severally attributed to the defects (D-band) and ordered graphitic carbon (G-band), indicating the presence of carbon materials in CoSe/NC [−1, which is related with C–Se bond [2 adsorption/desorption isotherm of CoSe/NC with a typical type-IV isotherm and a type-H3 hysteresis loop, suggesting the mesoporous feature. The CoSe/NC exhibits a large specific surface area (BET) of 49.958 m2 g−1. However, the specific surface area of CoSe/NC is far less than that of the precursor ZIF-67, which may be caused by the structure collapse of ZIF-67 during calcination process and the volume expansion due to the formation of CoSe. The pore size distribution (BJH) displays the primary pores in a range of 1 to 10 nm with an average size of 7.238 nm. The large specific surface area and mesoporous structure are advantageous to the penetration of electrolyte and the fast transport of Li+.Figure  CoSe/NC . The val–Se bond , 43. The1/2 and Co 2p3/2 could be observed at 796.92 and 780.94 eV in Co 2p spectrum, which could be assigned to CoSe . The three Nyquist plots exhibit same features, a semicircle and an inclined line. The diameter of semicircle is related to the charge-transfer resistance and internal resistance. As can be seen, the semicircle in CoSe/NC is clearly smaller than that of CoSe, suggesting CoSe/NC possesses better electrical conductivity, which should be assigned to the NC nanolayers. Besides, after 100 cycles at 0.1 A g−1, the diameter of CoSe/NC declines than before, which may be associated with the constitution of SEI layer on the appearance of electrode materials and the pulverization of CoSe nanoparticles to increase the contact area between electrode and electrolyte.Figure −1 after 100 cycles at 0.1 A g−1  versus log (i). According to the above-mentioned method, the b values of peaks I, II, and III in CV curves are quantified to 0.71, 0.62, and 0.77, separately, suggesting that the capacity is contributed by a hybrid controlling process, as shown in Fig. where a and b are adjustable values. If f b, Eq. . The valk1 and k2 are adjustable values. The current (i) is composed of capacitive-controlled (k1ν) and diffusion-controlled process (k2ν1/2). In order to calculate the values of k1ν and k2ν1/2, Eq. (k1 and k2 correspond to the slope and intercept of i(V)/ν1/2 versus ν1/2, respectively. According to this method, k1ν and k2ν1/2 at various potential could be gained. As shown in Fig. −1. The contribution ratios at other scan rates are also conducted (Fig. −1, respectively. Above results demonstrate the electrochemical process of CoSe/NC is a hybrid controlling process.where 1/2, Eq.  is conve1/2, Eq. . The valted Fig. d. The co−1 after 190 cycles at 0.1 A g−1. Even at a large current density of 1.0 A g−1, it can remain 310.11 mAh g−1 after 500 cycles. Besides, according to the results of electrochemical kinetics, the electrochemical process of CoSe/NC is a hybrid controlling process. These results indicate that preparing metal compounds/carbonaceous composites by using MOFs, as precursor is valid strategy to enhance the lithium storage properties of metal compounds.In summary, the original CoSe/NC composites were prepared by using ZIF-67 as precursor, in which the CoSe nanoparticles are coated by NC nanolayers and they are connected through C–Se bonds. In the as-prepared composites, the NC nanolayers could improve the conductivity of CoSe, buffer the volume expansion, and take part into the lithium storage reaction as active sits. The coating structure and C–Se bonds make the connection between CoSe nanoparticles and NC nanolayers closer, which are beneficial for the work of NC nanolayers to improve the electrochemical properties. Therefore, the CoSe/NC composites exhibit outstanding cyclic capability and rate performance as anode materials for LIBs. The CoSe/NC composites could deliver a reversible capacity of 1244 mAh gAdditional file 1. Figure S1. SEM image of as-synthesized ZIF-67. Figure S2. C 1s spectrum of CoSe/NC. Figure S3. The EDS spectrum of CoSe/NC. Figure S4. CV curves of pure CoSe at 0.2 mV s-1. Table S1. Percentages of elements in CoSe/NC.


Trace elements, like Cu, Zn, Fe, or Se, are important for the proper functioning of antioxidant enzymes. However, in excessive amounts, they can also act as pro-oxidants. Accordingly, trace elements influence redox-modulated signaling pathways, such as the Nrf2 pathway. Vice versa, Nrf2 target genes belong to the group of transport and metal binding proteins. In order to investigate whether Nrf2 directly regulates the systemic trace element status, we used mice to study the effect of a constitutive, whole-body Nrf2 knockout on the systemic status of Cu, Zn, Fe, and Se. As the loss of selenoproteins under Se-deprived conditions has been described to further enhance Nrf2 activity, we additionally analyzed the combination of Nrf2 knockout with feeding diets that provide either suboptimal, adequate, or supplemented amounts of Se. Experiments revealed that the Nrf2 knockout partially affected the trace element concentrations of Cu, Zn, Fe, or Se in the intestine, liver, and/or plasma. However, aside from Fe, the other three trace elements were only marginally modulated in an Nrf2-dependent manner. Selenium deficiency mainly resulted in increased plasma Zn levels. One putative mediator could be the metal regulatory transcription factor 1, which was up-regulated with an increasing Se supply and downregulated in Se-supplemented Nrf2 knockout mice. Essential trace elements (TEs) are micronutrients with indispensable roles in enzymatic reactions, which consequently modify signaling pathways. The effects of TEs are mostly attributed to their redox-modulatory properties. TEs, such as Cu, Zn, and Fe but also Se, can act as pro-oxidants if present in excess or if available as free unbound ions. Otherwise, antioxidant and protective enzymes, such as the selenoproteins, glutathione peroxidases (GPX) and thioredoxin reductases (TXNRD) and also Cu/Zn superoxide dismutase SOD1), catalase, and metallothioneins (MT), depend on the supply with specific TEs. Via both ways, TEs have the potential to influence redox-modulated signaling pathways. Until now, many transcription factors have been shown to be sensitive towards the cellular redox status. Among them, nuclear factor erythroid 2 p45-related factor 2 (Nrf2) is a better characterized one [, catalasUnder basal conditions the transcription factor Nrf2 is kept in the cytosol by its binding partner, Kelch-like ECH-associated protein 1 (Keap1), which is anchored to the actin cytoskeleton and acts as a scaffold for the cullin3-dependent E3 ubiquitin ligase complex. After poly-ubiquitination, Nrf2 is degraded via the proteasome. There are several ways to induce the nuclear translocation and DNA binding of Nrf2. Of those, the best understood mechanism is the redox-dependent modification of thiol groups in the Keap1 protein, which results in a conformational change locking Nrf2 at Keap1. Newly synthesized Nrf2 can no longer be degraded and translocates to the nucleus. Besides Keap1, caveolin 1, the ubiquitin ligase Skp, cullin, F-box containing complex (SCF), and the retinoid X receptor α (RXRα) also interact with and repress Nrf2. Nrf2 target genes contain so-called antioxidant-responsive elements (ARE) within their promoter regions , nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), tumor suppressor protein p53, and Notch making Nrf2, an important factor in regulating immune defense, differentiation, and tissue regeneration, as well as cell death . AnotherIn most previous studies, the authors focused on high concentrations and pro-oxidant effects of overloading single TEs, e.g., Se. However, we have previously shown that Nrf2 becomes activated under conditions of a suboptimal Se status in the duodenum  and liveBased on the results obtained for single TEs, we aimed to study the effect of a whole-body Nrf2 knockout (KO) in mice on the systemic TE status of Fe, Se, Cu, and Zn. A suboptimal Se status results in limited expression of Se-sensitive selenoproteins. As reduced expression of selenoproteins, such as TXNRD1, under Se-deprived conditions further enhances Nrf2 activity , we alsoAnimal experiments were approved by the ethics committee of the Ministry of Agriculture and Environment  and all methods were carried out in accordance to permission number V3-2437-29-2012. Nrf2 KO mice on a C57BL/6J background were kindly provided by Masayuki Yamamoto (Tohoku University Graduate School of Medicine) and genotyped as previously described [g and 4 °C). In the second experiment, male WT and Nrf2 KO mice were weaned onto a diet based on torula yeast , 50 µL of a solution containing 100 µg Rh/L  as the internal standard, and 950 µL ultrapure water were added. For feed samples, 900 µL of concentrated HNO3, 250 µL of H2O2 , and 810 µL of ultrapure water were added. In addition, 20 µL of a solution containing 1000 µg Rh/L and 20 µL of 10,000 µg 77Se/L , purchased from Eurisotop SAS , were added as internal standard or isotope dilution standard, respectively. The samples were then digested in a Mars 6 microwave digestion system  by heating to 200 °C over a period of 10 min and holding this temperature for 20 min. In each digestion, two blank samples and 50 mg of certified reference material ERM-BB 422 (fish muscle) or ERM BB 186  were carried along to ensure accuracy of results. Samples were repeated if the recovery for any analyzed element deviated by more than 10% from the reference value and/or was outside the error range of the material. After digestion, samples were quantitatively transferred to 15-mL polypropylene tubes combined with two times 475 µL (tissue) or 1 mL (feed) of ultrapure water from vessel rinsing. The samples were kept at 4 °C until one day prior to measurement, when they were further diluted 1 + 4 in 15-mL polypropylene tubes to give a final concentration of 2.93% HNO3, as well as either 2.5 µg Rh/L (tissue) or 1 µg Rh/L and 10 µg 77Se/L (feed). Mixed-element calibration standards were made to match the concentration of HNO3 and the internal standard in the diluted digests from 1000 mg/L single-element stock solutions (Carl Roth). Calibration ranges were Fe: 5–1000 µg/L, Cu: 0.5–100 µg/L, Zn: 2.5–500 µg/L, Se: 0.05–10 µg/L. For Se isotope dilution analysis (IDA), a solution containing 10 µg 77Se/L, as well as a 1 + 1 mixture of 10 µg 77Se/L and 10 µg naturally distributed Se/L was prepared. Solutions were then analyzed via ICP-MS/MS  monitoring the following mass to charge ratios (Q1→Q2): He-mode: Fe (56→56), Cu (63→63), Zn (66→66), Rh (103→103); O2-mode: Se (77→93), Se (80→96), Rh (103→103). Elements in He-mode were determined via external calibration after internal standard correction using Rh and Se was also determined either via external calibration (tissue) or via isotope dilution analysis (IDA) (feed) as described previously [156(CeO)+/140Ce+), and a doubly charged ratio of <2% (140Ce2+/140Ce+), as well as a background of <0.1 CPS prior to measurement.Frozen tissue (liver or duodenum) was pulverized using a TissueLyser  for 2 × 30 s at maximum speed. Feed samples were pulverized by mortar and pestle. About 50 mg of each sample were weighed precisely into polytetrafluoroethylene (PTFE) microwave vessels. For mouse tissue, the variance was found to be far below 5% between replicates of the same mouse (data not shown); therefore, only one replicate was analyzed to preserve tissue for other experiments. However, in the case of obvious outliers, the sample was digested and analyzed again. Due to high in-batch variance in the chow diets from some manufacturers, at least three independent replicates were prepared in the case of feed samples. For tissue samples, 1000 µL of concentrated HNOeviously . The ins® III, pro analysis, Merck), 0.05 vol.-% Triton™ X-100 (Merck Sigma-Aldrich), and 0.25 vol.-% ammonium hydroxide ), as well as internal standards . The diluted sample was then subjected to analysis for Fe, Cu, Zn, and Se (IDA) via ICP MS/MS.The applied method for the analysis of TEs in murine plasma has been described previously . In brie® , and 180 U Moloney murine leukemia virus reverse transcriptase  in a total volume of 45 μL. Real-time PCR was performed in a total volume of 25 μL with 1 μL of 1 + 9 diluted cDNA measured in triplicates using a Mx3005P QPCR System (Agilent). SYBR Green I  served as the fluorescent reporter. The annealing temperature was 60 °C for all PCR reactions and specificity was confirmed by a melting curve analysis. All PCR products were quantified with a standard curve to correct for differences in PCR efficiencies. Primer sequences  are listed in Epcam and Rpl13a, and used for normalization.The mRNA was isolated from frozen and pulverized  tissues with the Dynabeads mRNA DIRECT Kit  according to the manufacturer’s protocol. Reverse transcription (RT) was performed with 150 ng mRNA, 0.15 pmol oligo(dT)15 primers, 1× RT buffer, 700 μM dNTPs, 0.1 mg/mL BSA, 30 U RNasinFerritin and transferrin were measured in plasma samples using Mouse Ferritin and Transferrin ELISA  following the manufacturer’s instruction. Therefore, plasma samples were either diluted 1:20 or 1:200,000, respectively.g, 15 min, 4 °C) and protein concentrations were determined by Bradford analysis . After SDS polyacrylamide gel electrophoresis, gels were immunoblotted to nitrocellulose and blots were blocked in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 at room temperature for 1 h. The following antibodies were used: Rabbit anti-Ferritin-H , rabbit anti-MT , rabbit anti-Ctr1 , rabbit anti-MTF-1 antibody , and rabbit anti-β-Actin . Horseradish peroxidase-conjugated goat anti-rabbit IgG  served as secondary antibody. Intensities of identified bands were quantified densitometrically with the Luminescent Image Analyzer LAS-3000 system . Protein expression was normalized to β-actin expression or Ponceau staining.To obtain protein lysates, frozen liver samples were homogenized in Tris buffer ) using a TissueLyser (Qiagen) for 2 × 30 s at maximum speed. Cellular debris was removed by centrifugation  using two-way analysis of variance (ANOVA) with Bonferroni’s post-test as indicated in the figure legends. A To address the question of whether Nrf2 not only modulates the status of single TEs, such as Fe, but also of several TEs in parallel, we analyzed Se, Fe, Cu, and Zn in male and female Nrf2 KO mice fed a standard chow diet. TE concentrations were assessed in the intestine, liver, and plasma. Fe was retained more in the liver and small intestine of Nrf2 KO than in WT mice A,B. ConsNo changes were observed concerning the Fe markers, ferritin and transferrin . In paraTo analyze the role of the Se status on other TEs in combination with loss of Nrf2, both WT and Nrf2 KO mice were weaned onto one of three diets containing suboptimal (0.03 ppm), adequate (0.15 ppm), or supplemented (0.6 ppm) amounts of Se. The experimental set-up was chosen according to previous feeding experiments to efficiently reduce the Se status in the –Se group. For better comparability with previous experiments, only male mice were studied . In addiAs expected, the dietary approach successfully modulated the Se status of the different feeding groups A,B. The In order to confirm that loss of Nrf2 resulted in diminished expression of classical Nrf2 target genes, NQO1 activity A was anap < 0.09; Besides Nrf2 target genes, selenoprotein expression was also studied. Classical biomarkers of the murine Se status, such as TXNRD and GPX activity, already reached a plateau in the +Se groups and could not be further increased by the ++Se supply C,D. TotaAs shown before , Fe, ferFirst, we tested Hamp expression in the liver, because Hamp is the major regulator of Fe homeostasis, which is upregulated in response to an increase in Fe levels. Recently, it has been shown that this upregulation is partially impaired in Nrf2 KO livers . Herein,As in the previous experiment , there wAs seen before, hepatic Zn levels were neither affected by Nrf2 nor by Se status E. HoweveIt is well established that the transcription factor Nrf2 is an important mediator of Fe homeostasis . In thisWe observed an increase in Fe tissue levels upon loss of Nrf2 D. Vice vIn parallel to Fe, intracellular levels of Se and Zn were affected by loss of Nrf2 as well. In this case, both were reduced. Also, plasma Cu levels were slightly reduced in Nrf2 KO mice  showed a comparable expression pattern, being induced specifically under –Se Nrf2 KO conditions, the question arose if there is a common regulator. One possibility would be MTF1, which regulates MT expression in response to Zn or Cu [Comparable to Zip14, mRNA levels of Atp7b, the essential ATPase for Cu export into the bile , were upZn or Cu . RecentlZn or Cu . In addiZn or Cu . HoweverBesides DMT1, Ctr1 is the main universal Cu importer in mammalian cells. A KO of Ctr1 in the intestine resulted in peripheral Cu hypoaccumulation. In parallel, hepatic Fe levels were upregulated . ExpressHmox1 in whole blood samples as compared to healthy controls, indicating potential Nrf2 activation in humans under Se deficient conditions [ferritin H was clearly upregulated in –Se WT livers and was almost lost in Nrf2 KO mice (We and others have shown previously that a suboptimal selenium supply results in Nrf2 activation predominantly in the liver and intestine of mice ,57. Thisnditions . Howevernditions ,26. Only KO mice K,L. ThisTherefore, the initial aim of studying the crosstalk between Nrf2 and selenium status in modulating three other TEs is difficult to address under the present conditions. Herein, it is relatively clear that Se effects observed on Zn appear to be regulated independently of Nrf2. However, when only considering WT mice, we could still draw conclusions towards the role of the Se status on TE status of Zn, Fe, and Cu. Most strikingly, Zn plasma levels were higher in mice with low Se status and vice versa D. In comOverall, only Fe was substantially regulated in response to Nrf2 while the impact of Nrf2 on homeostasis of Se, Cu, and Zn appeared to be rather marginal. Nevertheless, crosstalk between Se and MTF1 is a promising idea that needs to be followed up in the future and might provide an explanation for the observed counter regulation of plasma Se and Zn levels.The mammalian ionome has been evaluated in 26 species and across several tissues . In this


The Cu(i)-binding protein NosL functions specifically as an assembly factor for the unique CuZ centre of nitrous oxide reductase (N2OR). 2OR) is the terminal enzyme of the denitrification pathway of soil bacteria that reduces the greenhouse gas nitrous oxide (N2O) to dinitrogen. In addition to a binuclear CuA site that functions in electron transfer, the active site of N2OR features a unique tetranuclear copper cluster bridged by inorganic sulfide, termed CuZ. In copper-limited environments, N2OR fails to function, resulting in truncation of denitrification and rising levels of N2O released by cells to the atmosphere, presenting a major environmental challenge. Here we report studies of nosL from Paracoccus denitrificans, which is part of the nos gene cluster, and encodes a putative copper binding protein. A Paracoccus denitrificans ΔnosL mutant strain had no denitrification phenotype under copper-sufficient conditions but failed to reduce N2O under copper-limited conditions. N2OR isolated from ΔnosL cells was found to be deficient in copper and to exhibit attenuated activity. UV-visible absorbance spectroscopy revealed that bands due to the CuA center were unaffected, while those corresponding to the CuZ center were significantly reduced in intensity. In vitro studies of a soluble form of NosL without its predicted membrane anchor showed that it binds one Cu(i) ion per protein with attomolar affinity, but does not bind Cu(ii). Together, the data demonstrate that NosL is a copper-binding protein specifically required for assembly of the CuZ center of N2OR, and thus represents the first characterised assembly factor for the CuZ active site of this key environmental enzyme, which is globally responsible for the destruction of a potent greenhouse gas.Nitrous oxide reductase (N The main contributor within this sector is the soil microbial community, which produces 40% of these emissions.2O from 270 ppb to 324 ppb over the last 100 years correlates strongly with the use of anthropogenic nitrogen-based fertilisers in farming to improve crop yield.2) gas via the free intermediates nitrite, nitric oxide and N2O using different metallo-enzymes. Environmental factors such as soil pH, moisture, carbon to nitrogen ratio, temperature and a lack of copper2O emissions from these microbes.Nitrous oxide NO is a sinosZ gene, the cupro-enzyme nitrous oxide reductase (N2OR or NosZ) catalyses the 2-electron reduction of N2O to N2. Two distinct and approximately equally abundant clades of N2OR-containing bacteria and archaea have been identified.2O sink, while members of clade I, such as α-, β- and γ-proteobacteria, are able to produce and remove N2O under optimum conditions. In order to assist with future strategies for the control of emissions from soil ecosystems, a key task is to explore how the enzyme is produced and matured in N2O emitting bacteria.3Encoded by the 2OR in the denitrifying α-proteobacterium Paracoccus denitrificans (PdN2OR) is exported to the periplasm via the twin-arginine translocation (TAT) pathway,A and CuZ, are assembled. Several crystal structures are now available for N2OR,A site is housed in a C-terminal cupredoxin domain, while the CuZ site lies within the N-terminal seven bladed β-propeller domain. The CuA center contains two copper ions that are bridged by two conserved Cys residues and further coordinated by Met, His and Trp ligands to form a site that closely resembles the electron transfer CuA site present in subunit II of cytochrome c oxidase.2OR the CuA site also acts as an electron shuttle, accepting electrons from small electron donors such as cytochrome c550  or two ([4Cu:2S]) sulfides, depending on the presence or absence of O2, respectively, during purification.Nome c550  or pseud2O emissions from soil is to understand how the cofactor sites of N2OR are assembled and, in doing so, identify assembly/chaperone systems that are involved. For clade I organisms, including P. denitrificans, any such systems must be periplasmic, as mutations in the TAT leader sequence of N2OR results in the protein remaining in the cytoplasm, in a folded but copper-free state.c oxidase have identified the proteins Cox17 and ScoB, which are involved in the assembly of the CuA site,AC and SenC have been implicated in the maturation of the CuA site of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides.19A major challenge that is particularly important for addressing NZ site. The nos gene cluster of clade I N2O reducing bacteria vary between denitrifying phyla but contain, in addition to nosZ, five other core genes (nosRZDFYL), while predominantly α- and β-proteobacteria members also contain a further two genes (nosC and nosX). nosDFY are predicted to encode an ABC-type transporter homologous to ATM1 from eukaryotes, which transports a sulfur-containing species out of the mitochondrion for Fe–S cluster assembly in the cytoplasm,Z center. In P. denitrificans, downstream of nosDFY are two further accessory genes that are part of the same operon, nosL and nosX. The only mutation analysis of nosL has been in P. stutzerinosX in P. denitrificans,2O reduction. However, nosL is part of the denitrification core gene cluster, its expression is responsive to cellular copper status,2OR.In comparison, little is known about the assembly of the CuP. denitrificans nosL/NosL. The data demonstrate that NosL is a Cu(i)-binding protein that is required for efficient assembly of the N2OR CuZ center, and thus represents the first characterised assembly factor for this unique metal center in biology.Here, we report genetic and biochemical studies of nosZ (lacking the gene encoding N2OR) and ΔnosL strains , Δ Table S1 were cul Table S1. For thenditions . Growth Z strain .nosL deletion on N2OR activity in vivo was assessed by measuring N2O levels in the headspace of cultures. Consistent with previous studies, no N2O was accumulated by the WT strain under Cu-sufficient conditions and only a transient low level of N2O (<1 mM at ∼16 h) was observed under Cu-limited conditions (nosZ under these conditions.nosZ mutant exhibited a marked Nos-negative phenotype (Nos–) regardless of copper levels, with N2O emitted from cultures to >4 mM after 24 h  with a C-terminal Strep-II tag was overproduced in P. denitrificans strains grown under Cu-sufficient conditions and purified. N2OR from ΔnosL cells contained on average 4 Cu atoms per monomer, compared to ∼6 for the enzyme from ΔnosZ cells   .2OR was carried out aerobically, the enzyme from ΔnosZ cells contained the   form of the active site, which is catalytically inactive.2OR to the fully reduced form, the enzyme was incubated with excess reduced methyl viologen at room temperature2O reductase activity had reached a maximum. N2OR from ΔnosL and ΔnosZL cells exhibited significantly lower maximum activities than N2OR from ΔnosZ, see As purification of N2OR purified from ΔnosZ P. denitrificans displayed bands at 480, 535 and 645 nm (2OR enzymes from a range of bacteria.2– to Cu(ii) charge-transfer transitions and transitions due to interactions between Cu(i) and Cu(ii) ions.A centre, while that at 645 nm is characteristic of the CuZ centre (in its  form).29The UV-visible absorbance spectrum of as isolated air-oxidized  strep-tagged Nd 645 nm  and S2†,2OR enzymes purified from ΔnosL and ΔnosZL strains were very similar in the 450–550 nm region, but exhibited significantly reduced intensity beyond 550 nm (A (maximum at 535 nm). Reduction with sodium dithionite resulted in the spectra shown in A center to its colorless diamagnetic Cu(i)/Cu(i) state. The remaining band is characteristic of the CuZ center following addition of dithionite.2OR from both ΔnosL and ΔnosZL strains exhibited a much less intense CuZ absorbance than that from ΔnosZ, indicating diverse occupancies of the center. Ferricyanide-oxidized minus dithionite-reduced difference spectra  specifically and XAFS data were consistent with a Cu(i) coordination consisting of S and N/O ligands.P. denitrificans NosL, the protein lacking its periplasmic export signal sequence and its predicted N-terminal Cys residueA. cycloclastes.23NosL contains a Type-II signal peptidase recognition sequence that, when cleaved, produces a protein with an N-terminal Cys residue that is predicted to bind lipid and anchor NosL into the outer membrane.i) resulted in the series of spectra shown in i) per NosL (i) coordinated to a cysteine thiolate.i)-binding was also investigated by CD spectroscopy, which confirmed the tight association of one Cu(i) per protein but also suggested that further Cu(i) can associate with NosL, albeit weakly -NosL  binding does not significantly affect the association state equilibrium.Titration of apo-NosL with Cu followed by absorbance spectroscopy resulted in spectra very similar to those for Cu(i)  may undergo auto-reduction upon binding to NosL. This possibility was investigated using EPR  to NosL resulted in a characteristic S = 1/2 Cu(ii) signal that, by comparison with a Cu(ii) standard, corresponded to only ∼8% of the Cu(ii) initially added. The same sample in the presence of EDTA (a Cu(ii) chelator), which prevents Cu(ii) ions in solution dimerizing to form EPR-inactive species, resulted in a Cu(ii) concentration of ∼11%. Similar experiments but with the addition of Cu(i) in place of Cu(ii) resulted in 4% and 8% in the absence and presence of EDTA, respectively (Table S2ii). Together, these data indicate that Cu(ii) undergoes auto-reduction to EPR silent Cu(i) upon binding to NosL.Equivalent titrations with Cu Fig. S6, suggest Table S2. As isoli)-binding to NosL, ESI-MS under non-denaturing conditions, where non-covalent interactions are preserved, was also employed. via buffer exchange, changes in pH and ionic strength, were unsuccessful. NosL was loaded with a single Cu(i) ion and the peak envelope of the resulting deconvoluted mass spectrum was at +63 Da relative to that of the apo-NosL spectrum (i) ion.To further characterise Cu-binding to NosL. Cu(i)-NosL was titrated with BCS, and the partition of Cu(i) between NosL and BCS determined from measured A483 nm, due to [Cu(BCS)2]3–, together with the well-characterised formation constant for Cu(BCS2)3–, β2 = 1019.8  to NosL.Competition binding experiments using the high-affinity chelator BCS were used to determine the dissociation constant for Cu8 Fig. S833. From . From i-nosL being a core component of the nos gene clusters in a range of microorganisms, mutational studies2O reduction. A nosL mutant of Pseudomonas stutzeri (containing an insertion towards the 3′ end of the gene) exhibited a slightly lower growth rate but produced active, holo-N2OR.Sinorhizobium melilotiP. stutzeri protein is not conserved.Despite nosL deletion mutant of P. denitrificans was generated and compared to the WT and nosZ deletion strains, as growth and N2O benchmarks, under both Cu-limited and Cu-sufficient conditions. The nosL mutant exhibited a Cu-limited growth phenotype relative to WT, associated with a deficiency in the activity of N2OR, leading to accumulation of N2O in the culture headspace. The phenotype was complemented in trans by nosL and also under copper replete conditions, strongly suggesting that NosL functions in an aspect of copper metabolism. The reason why the previously reported P. stutzeri nosL mutant did not present a phenotype is unclear; one possibility is that there was sufficient copper in the growth medium to mask the phenotype, but growth conditions were not reported.9Here, a 2OR enzymes purified from ΔnosZ, ΔnosL and ΔnosZL mutants exhibited clear differences. Under Cu-sufficient conditions, N2OR from ΔnosL cells contained only ∼4 Cu per N2OR monomer and was substantially less active (though it was apparently sufficiently active in vivo to mask any obvious in vivo phenotype). Importantly, spectroscopic characterisation revealed that the N2OR protein from ΔnosL was specifically deficient in its CuZ center, demonstrating that NosL functions in the assembly of this unique biological metal center required for N2O destruction.Strep-tagged N2OR purified from the ΔnosZ mutant contained nearly 5 Cu/monomer, while those from ΔnosL or ΔnosZL mutant strains lacked copper entirely, indicating complete failure to assemble either N2OR copper center in the absence of NosL. Thus, although the CuA can be reconstituted using copper alone,A.Under Cu-limited conditions, tagged Nvia a triacylated N-terminal Cys residue  with attomolar affinity, a characteristic of many copper chaperones.The N-terminal sequence of NosL contains a periplasmic export signal and a lipobox that is predicted to be processed by Lgt, Lsp and Lnt enzymes, resulting in a mature protein that is membrane-anchored A. cycloclastes NosL using thiol-specific reagents and EXAFS, with data from the latter consistent with a three or four coordinate Cu(i) center. The best fit was obtained for three coordinate Cu(i) with (O/N)S2 ligands.2O reducing bacteria identifies two conserved Cys residues: one through which the protein is believed to be anchored to the membrane -binding.et al., there is no absolutely conserved His residue . P. stutzeri NosL is the exception to this, as it comes from a Clade I organism but is more similar to Clade II NosL proteins.Separation of NosL proteins according to the clade to which the organism belongs may shed some further light on this, as it reveals residues that are conserved within, but not between, clades. Clade I NosL proteins, which include Z center assembly. Our data indicate that NosL, which binds Cu(i) with attomolar affinity, is a key part of a high-affinity Cu trafficking pathway that enables assembly of the CuZ center of N2OR. The pathway functions under Cu-sufficient conditions, but becomes essential under Cu-limited conditions. During copper limitation, the NosL pathway may also play a key role in supplying Cu for the CuA center. How NosL delivers Cu to N2OR is unknown; it is likely that NosL functions directly in the transfer of Cu(i) to N2OR, and is thus a copper chaperone, but further studies are now needed to investigate this. The identification of NosL as a key component of a Cu-trafficking pathway for the assembly of the active holoreductase N2OR, the sole pivotal enzyme for N2O destruction, is a significant advance towards the long-term aim of mitigating microbial emissions of N2O into the atmosphere.In summary, we identify here the first Cu chaperone with a specific role in CunosL in P. denitrificans wild type or ΔnosZ backgrounds  and kanamycin (50 μg ml–1). Primary SpecR/KmR transconjugants were grown to stationary phase in Luria Bertani broth (LB) with no antibiotic. Double cross over events were selected for using a high-salt modified LB agar supplemented with 6% (w/v) sucrose. Sucrose resistant colonies with SpecR were screened using colony PCR and gene deletion confirmed by sequencing. Deletion strains were named PD2501 (ΔnosL) and PD2505 (ΔnosZL). For complementation of ΔnosL cells the coding sequence of Pden_4215 was synthesised (Genscript) with flanking 5′ NdeI and 3′ EcoRI restriction sites and sub-cloned into a taurine-inducible modified pLMB509 plasmid with gentamicin resistance (20 μg ml–1) to generate pSPBN2. The complementation plasmid was conjugated into the mutant strain using the helper E. coli pRK2013 strain and successful conjugants were SpecR/GmR. Expression of nosL from the plasmid was induced by adding 1 mM taurine to the medium at the start of the growth experiment.Unmarked deletions of  Table S1 were pro Table S4 were clo–1 Vishniac and Santer trace element solution600 nm of 0.02 and incubated at 30 °C. Samples of the liquid culture were taken in 1 ml aliquots and OD600 nm measured. 3 ml gas samples were removed from the headspace of the cultures and stored in pre-evacuated 3 ml Exetainer® vials. A 50 μl gas sample was injected into a Clarus 500 gas chromatograph (PerkinElmer) with an Elite-PLOT Q  and an electron capture detector. Carrier gas was N2, make-up gas was 95% (v/v) argon, 5% (v/v) methane. Standards containing N2O at 0.4, 5, 100, 1000, 5000, and 10 000 ppm  were measured and total N2O was determined as previously described.22Anaerobic minimal media batch cultures (400 ml) were grown in sealed Duran flasks , fitted with a septum to allow for gas-tight sample extraction. Minimal media consisted of: 30 mM succinate, 20 mM nitrate, 11 mM dihydrogen orthophosphate, 29 mM di-sodium orthophosphate, 0.4 mM magnesium sulfate, 1 mM ammonium chloride, pH 7.5. The minimal media was supplemented with a 2 ml l2OR (NosZ) was expressed in trans in P. denitrificans using pLMB511, a derivative plasmid of the taurine-inducible expression vector pLMB509 for α-proteobacteria  was synthesised (GenScript) and cloned into pLMB511 as a NdeI–XmaI fragment, yielding pMSL002 from which N2OR (NosZ) with a C-terminal Strep-tag II was overproduced. The pMSL002 plasmid with GenR (20 μl ml–1) was conjugated into PdΔnosZ (PD2303), PdΔnosL (PD2501) and PdΔnosZL (PD2505) using the E. coli pRK2013 helper strain. Conjugants were screened for both GenR/SpecR and first cultured in LB and subsequently in 4 L minimal media supplemented with 2 ml l–1 trace element solution, at 30 °C. Overproduction of strep-tagged N2OR was initiated by the addition of 10 mM taurine when the culture reached OD600 nm ∼ 0.6 and cultures were incubated at 30 °C for 24 h. Cells were harvested by centrifugation at 5000 × g and resuspended in binding buffer  with a protease inhibitor  and lysed using a French pressure cell at 1000 psi. The cell lysate was centrifuged at 205 000 × g for 1 h at 4 °C and the supernatant applied to a Hi-Trap HP Strep II affinity column . N2OR-Strep-tag II was eluted using elution buffer  and exchanged back into binding buffer using a 30 kDa MWCO Centricon filter unit. Purity of the sample was confirmed using SDS-PAGE analysis and LC-MS. Protein concentrations were determined using the Bradford assay (BioRad)N Table S1.39 The E2OR-Strep-tag II were recorded on a Jasco V-550 spectrophotometer. Circular dichroism spectra were recorded using Jasco J-810 Spectropolarimeter. Samples were made anaerobic by sparging with nitrogen gas for 5 min and oxidised or reduced with 5 mg ml–1 stocks of potassium ferricyanide and sodium dithionite, respectively, in 20 mM HEPES, 150 mM NaCl, pH 7.2, by titrating concentration equivalents. Total copper content of the protein was determined using a colorimetric bathocuproinedisulfonic acid (BCS) assay. A 100 μl protein sample was heated to 95 °C for 1 h with an equal volume of 20% (v/v) HNO3. The reaction was cooled and neutralised using 0.6 ml saturated ammonium sulfate solution. Copper was reduced using 100 μl hydroxylamine (100 mM) and 100 μL BCS (10 mM) added. The absorbance at 483 nm was recorded after 30 min. A standard curve was produced using a standard copper sulfate solutions (Sigma).UV-visible absorbance spectra of N2OR-Strep-tag II enzymes was determined using an adapted methyl viologen assay2O saturated buffer and the oxidation of blue (reduced) methyl viologen to its oxidised colourless form was followed at 600 nm as a function of time and data converted to specific activity using ε600 nm = 13 600 M–1 cm–1 for the reduced methyl viologen cation radical.41Activities of NPdNosL (Pden_4215) was synthesised (Genscript) and sub-cloned into pET-21a(+) using 5′ NdeI and 3′ EcoRI restriction sites to generate pSPBN3. The truncation, which resulted in the replacement of the first 16 residues with a Met, was designed to simplify the expression and maturation of the protein in E. coli as it yielded a soluble protein located in the cytoplasmic fraction. The pSPBN3 plasmid was used to transform E. coli BL21 (DE3) to ampicillin (100 μg ml–1) resistance. Typically, 2 L flasks containing 500 ml LB supplemented with 100 μg ml–1 ampicillin were inoculated with 1% (v/v) of an overnight culture and grown for 2 h, 180 rpm, 37 °C until OD600 nm reached ∼0.6. Expression of the NosL-encoding gene was induced by addition of 500 μM IPTG and cultures were subsequently incubated at 37 °C, 180 rpm for 5 h. Cells were harvested by centrifugation at 4000 × g for 15 min at 4 °C, resuspended with buffer A  and lysed by three rounds of sonication, each for 8 min 20 s , on ice. The cell lysate was centrifuged at 40 000 × g for 45 min at 4 °C and the supernatant applied to a DEAE column  equilibrated in buffer A. NosL was eluted using a 0–50% gradient of buffer B . Fractions containing NosL were buffer exchanged using a 10 kDa MWCO Centricon into buffer A and applied to a Q-sepharose column  and eluted using a 20–50% gradient of buffer B. NosL-containing fractions (as determined by SDS-PAGE) were pooled and subsequently applied to an S-100 gel filtration column  equilibrated in 100 mM MOPS, 100 mM NaCl, pH 7.5 (buffer C) and eluted in the same buffer. Fractions containing pure NosL were combined and dialysed overnight against buffer C containing 1 mM EDTA at 4 °C, and subsequently back into buffer C.A codon-optimised gene encoding an N-terminally truncated version of –1 cm–1, determined using a guanidine hydrochloride assay,i)Cl (in 1 M NaCl and 0.1 M HCl) or CuCl2 dissolved in water. The protein was titrated with copper to give increases in the ratio of Cu : NosL of 0.1 per addition and spectra were recorded between 240 and 600 nm after each addition. Competition assays between Cu(i)-NosL and BCS were carried out to measure the dissociation constant, Kd, for Cu(i)-binding to NosL, using the extinction coefficient ε483nm = 13 300 M–1 cm–1 to determine the concentration of [CuBCS2]3–, as previously described,An extinction coefficient at 280 nm of 11 923 ± 5.2 Mm/z range 300–2000 using ESI-L Low Concentration Tuning Mix (Agilent Technologies). Samples were prepared by ten-fold dilution of 50 μM protein solution with 2% (v/v) acetonitrile and 0.1% (v/v) formic acid to 0.5 ml. Samples were loaded into the LC-MS via an autosampler using an UltiMate 3000 HPLC system (Dionex). A 20 μl injection volume of the protein was applied to a ProSwift reversed phase RP-1S column  at 25 °C. A gradient elution was performed at a flow rate of 200 ml min–1 using solvents A (0.1% formic acid) and B . Once loaded the following chromatographic method was used: isocratic wash , linear gradient from 2–100% B (2–12 min), followed by an isocratic wash  and column re-equilibration . Mass spectra were acquired throughout using the following parameters: dry gas flow 8 l min–1, nebuliser gas pressure 0.8 bar, dry gas 240 °C, capillary voltage 4500 V, offset 500 V, collision RF 650 Vpp. Mass spectra from manually chosen elution volumes were averaged and deconvoluted using a maximum entropy deconvolution algorithm in Compass DataAnalysis version 4.1 .LC-MS was conducted using a Bruker microQTof-QIII electrospray ionisation time of flight (TOF) mass spectrometer calibrated in the 2 was removed and protein samples were loaded into a Hamilton syringe and directly infused into the ESI source at a rate of 300 μl h–1. Data was acquired in 5 min increments with ion scans between 500 and 3000 m/z. NosL mass spectra (m/z 1000–3000) were recorded with acquisition controlled by Bruker qTOF Control software, with parameters as follows: dry gas flow 4 l min–1, nebulise gas pressure 0.8 Bar, dry gas 180 °C, capillary voltage 4000 V, offset 500 V, quadrupole voltage 5 V, collision RF 1000 Vpp, collision cell voltage 20 V. Spectra were deconvoluted as above. Exact masses are reported from peak centroids representing the isotope average neutral mass. Predicted masses are given as the isotope average of the neutral protein or protein complex, in which Cu(i)-binding is expected to be charge compensated.43Samples for non-denaturing ESI-MS were prepared in volatile buffer, ammonium acetate, 50 mM, pH 7.8. Lines were washed with anaerobic buffer prior to sample loading to ensure all O–1; single scan per spectrum. A 98 μM Cu(ii)–EDTA standard was used to estimate Cu(ii) concentrations for protein samples by spin integration of signal area for spectra versus that of the standard, where all spectra were recorded under non-saturating power conditions.Continuous wave X-band electronic paramagnetic resonance (EPR) measurements were recorded using a Bruker EMX EPR Spectrometer equipped with an ESR-900 liquid helium flow cryostat (Oxford Instruments). Spectra were recorded at 10 K with the following instrumental settings: microwave frequency, 9.4652 GHz; microwave power, 3.18 mW; modulation frequency, 100 kHz; modulation amplitude, 5 G; time constant, 82 ms; scan rate, 22.6 G sS. P. B., D. J. R., A. J. G. and N. L. B. conceived the study. S. P. B., M. J. S. L., J. M. B. and D. A. S. performed the experiments. S. P. B., D. A. S., A. J. G. and N. L. B. analysed the data. S. P. B., D. J. R., A. J. G. and N. L. B. wrote the manuscript.The authors declare no competing financial interests.Supplementary informationClick here for additional data file.


Crh) acting at cognate type 1 receptors  modulates inhibitory and excitatory synaptic transmission in the CeA. Here, we used CRF1:GFP reporter mice to characterize the morphological, neurochemical and electrophysiological properties of CRF1-expressing (CRF1+) and CRF1-non-expressing (CRF1–) neurons in the CeA. We assessed these two neuronal populations for distinctions in the expression of GABAergic subpopulation markers and neuropeptides, dendritic spine density and morphology, and excitatory transmission. We observed that CeA CRF1+ neurons are GABAergic but do not segregate with calbindin (CB), calretinin (CR), parvalbumin (PV), or protein kinase C-δ (PKCδ). Among the neuropeptides analyzed, Penk and Sst had the highest percentage of co-expression with Crhr1 in both the medial and lateral CeA subdivisions. Additionally, CeA CRF1+ neurons had a lower density of dendritic spines, which was offset by a higher proportion of mature spines compared to neighboring CRF1– neurons. Accordingly, there was no difference in basal spontaneous glutamatergic transmission between the two populations. Application of CRF increased overall vesicular glutamate release onto both CRF1+ and CRF1– neurons and does not affect amplitude or kinetics of EPSCs in either population. These novel data highlight important differences in the neurochemical make-up and morphology of CRF1+ compared to CRF1– neurons, which may have important implications for the transduction of CRF signaling in the CeA.The central nucleus of the amygdala (CeA) is a brain region implicated in anxiety, stress-related disorders and the reinforcing effects of drugs of abuse. Corticotropin-releasing factor (CRF,  Our novel identification of specific molecular, morphologic and functional properties to distinguishing CRF receptor type 1 (CRFCrh) plays an important role in emotional regulation via hypothalamic and amygdalar circuits under normal physiologic conditions (1) antagonists on mood disorders have been unsuccessful  expressing neurons are located in several brain regions including the central nucleus of the amygdala (CeA), which functions as the main output nucleus for amygdala functions  subset. Thus, we used a bacterial artificial chromosome (BAC) transgenic mouse line expressing the green fluorescent protein (GFP) under the Crhr1 promoter (CRF1:GFP) to readily identify neurons expressing CRF1  neurons display no ongoing tonic inhibition  co-expression patterns of CRF1 (via GFP reporter) with CBPs parvalbumin (PV), calretinin (CR), and calbindin (CB); (2) co-expression patterns of Crhr1 with Penk, Pdyn, Sst, Npy, Crh, and Prkcd; (3) dendritic spine morphology and density; and (4) basal and CRF-modulated glutamatergic transmission. We found that Penk and Sst have the highest percentage of co-expression with Crhr1 in both the CeM and CeL, and that Penk is enriched in CeM Crhr1+ neurons compared to their Crhr1– neighbors. We also show that CeM CRF1+ neurons exhibit an overall lower spine density and differential fractions of mature versus immature spines compared to CRF1– neurons. Consistent with a comparable density of mature spines in these two populations, we found no difference in spontaneous EPSCs (sEPSCs) or miniature EPSCs (mEPSCs). Importantly, acute CRF application increased overall CeM glutamatergic transmission and does not affect amplitude or kinetics of EPSCs in either population. These data provide important information about the neurobiology of CeA CRF1+ neurons that may have critical implications for their functional role under physiological and pathological conditions.Given the critical role of CRF and CRF1:GFP mice were generated on a mixed C57BL/6J x BALB/c background using BAC recombination  were anesthetized (isoflurane) and perfused with cold PBS followed by 4% paraformaldehyde (PFA). Brains were dissected and immersion fixed in PFA for 24 h at 4°C, cryoprotected in sterile 30% sucrose in PBS for 24–48 h at 4°C or until brains sank, flash frozen in pre-chilled isopentane on dry ice, and stored at –80°C. Free floating 35-µm brain sections were obtained using a cryostat and kept at 4°C in PBS containing 0.01% sodium azide.Mice , mouse anti-PV , mouse anti-CR , and mouse anti-CB . Next, sections were triple washed in PBS for 10 min at RT with gentle agitation followed by a 1-h secondary antibody incubation in PBS (in the dark). The following secondary antibodies were used: Alexa Fluor 488 goat anti-chicken  and Alexa Fluor 568 goat anti-mouse . Sections were then washed  and mounted in Vectashield .Sections were washed in PBS for 10 min at room temperature (RT) with gentle agitation and then blocked for 1 h at RT in blocking solution . Primary antibody was incubated at 4Sections were imaged on a Zeiss LSM 780 laser scanning confocal microscope . All microscope settings were kept the same within experiments during image acquisition. Analyst was blind to the identity of the red fluorescent signal (CBP) when performing cell counts, and analysis was performed manually in an unbiased manner at four anterior-posterior levels (equidistant sections located –1.00 through –1.70 mm from bregma). Data are presented as mean ± SEM.n = 3–4) were anesthetized (isoflurane) and perfused with cold PBS/Z-fix . Brains were dissected and immersion fixed in Z-fix for 24 h at 4°C, cryoprotected in sterile 30% sucrose in PBS for 24–48 h at 4°C or until brains sank, flash frozen in pre-chilled isopentane on dry ice, and stored at –80°C. Brains were then sliced on a cryostat in 20-µm-thick sections, mounted on SuperFrost Plus slides  and stored at –80°C.Mice  in RNase-free conditions. To perform the RNAscope in situ hybridization, a target retrieval pretreatment protocol was performed as outlined in the manual . Briefly, slides were submersed in target retrieval buffer  at 95–98°C for 10 min, immediately washed in distilled water, dehydrated with 100% ethanol (storage at –80°C if required), and lastly digested with Protease IV for 20 min at 40°C. Following this pretreatment the RNAScope Fluorescent Multiplex Reagent kit User Manual  was followed and slides were mounted with DAPI-containing Vectashield . A negative control  was run in tandem for each experiment. The probes used from ACD Biotechne were as follows: Crhr1 , Gad2 (439371-C3), Slc17a7 (416631-C2), Penk (318761), Pdyn (318771), Sst (404631), Npy (313321), Prkcd (441791), Crh (316091), and eGFP (400281).Slides were imaged on a Zeiss LSM 780 laser scanning confocal microscope . All microscope settings were kept the same within experiments during image acquisition. To perform quantification, ImageJ  was usedCrhr1+ nuclei expressing a marker of interest was determined by dividing the number of co-labeled nuclei by the total number of Crhr1+ nuclei. The percentage of Crhr1– nuclei co-expressing a marker of interest was determined by dividing the number of cells expressing the marker of interest but not Crhr1 by the total number of Crhr1– nuclei per image. Crhr1+ compared to Crhr1– patterns of co-expression was assessed and normalized to the Crhr1– cells.Next, the percentage of nuclei positive for one or both probes and the percentage of signal co-expression were calculated. Percentage of total nuclei was determined by dividing the total number of nuclei expressing that marker by the total number of DAPI positive nuclei per image. The percentage of t test was used to assess significance of the CeL versus CeM expression difference for each gene, as well as the Crhr1+ to Crhr1– percentage co-expression of each gene. Analysis and statistics were performed using R programming  were anesthetized (isoflurane) and perfused with cold PBS/4% PFA. Brains were extracted and immersion fixed in 4% PFA at 4°C for 2 h before being sectioned coronally into 100-μm slices on a vibrating microtome .Mice . The slices were triple washed with PBS for 10 min followed by secondary antibody incubation for 1 h at RT in blocking solution . The slices were triple washed in PBS and mounted on slides with Prolong Diamond .Sections were biolistically labeled with DiI -coated 1.1-μm tungsten particles delivered with a Bio-Rad gene gun and incubated in PBS overnight at 4°C before immunostaining. Slices were permeabilized in 0.01% Triton X-100 for 15 min at RT with gentle agitation and blocked for 30 min at RT with gentle agitation in blocking solution (10% NGS in 0.01% Triton X-100). Primary antibody incubation was performed overnight at 4°C in PBS  and CRF1– neurons . The experimenter was blind to cell type (CRF1+ vs CRF1–) when performing spine quantification. Data obtained in each mouse were averaged for each cell type, such that the number of mice was used as the n in statistical analyses. Spine densities and spine type proportions in CRF1– and CRF1+ neurons were compared using paired t tests. Data are presented as mean ± standard error. In all cases, p < 0.05 was the criterion for statistical significance.Slides were imaged for CeA on a Zeiss LSM 710 laser scanning confocal microscope . All microscope settings were kept the same within experiments during image acquisition. ImageJ  was usedn = 12) were briefly anesthetized with 3–5% isoflurane and decapitated. Brains were rapidly removed and placed in an ice-cold high-sucrose solution (pH 7.3–7.4) that contained: 206.0 mM sucrose, 2.5 mM KCl, 2.5 mM CaCl2, 7 mM MgCl2, 1.2 mM NaH2PO4, 26 mM NaHCO3, 5 mM glucose, and 5 mM HEPES. Brains were cut into coronal sections (300 µm) using a Leica 1200s vibratome (Leica Microsystems) and placed in oxygenated (95% O2/5% CO2) artificial CSF (aCSF) solution composed of the following: 130 mM NaCl, 3.5 mM KCl, 2 mM CaCl2, 1.25 mM NaH2PO4, 24 mM NaHCO3, and 10 mM glucose. Slices were incubated in aCSF for 30 min at 37°C, followed by a minimum 30-min equilibration at RT (21–22°C) before use.Mice  optics and CCD camera . A 60× magnification water immersion objective (Olympus) was used for identifying and approaching neurons. To avoid photolytic damage, initial exposure to episcopic fluorescence illumination was brief (<2 s). We detected fluorescent neurons using the Lumen 300 LED system (Prior Scientific) and captured images using Luminera software (Lumenera Corp.). Whole-cell current-clamp and voltage-clamp recordings were obtained with patch pipettes  using a Multiclamp 700B amplifier (Molecular Devices), sampled at 20 kHz, low pass filtered at 10 kHz, digitized , and stored on a computer using pClamp 10 software (Molecular Devices). Series resistance was not compensated but monitored throughout the experiment, and cells with a series resistance >15 MΩ or a >20% change were excluded.2, 10 mM HEPES, 2 mM Na-ATP, and 0.2 mM Na-GTP. Drugs were dissolved in aCSF and applied by bath perfusion. To isolate sEPSC and mEPSC, recordings were performed in the presence of GABAA (30 µM bicuculline) and GABAB (1 µM CGP 55845A) receptor antagonists, and tetrodotoxin  was included for mEPSCs. CeA neurons were held at –60 mV for voltage-clamp recordings and maintained at –70 mV for current-clamp recordings.The intracellular solution used for voltage and current clamp recordings was composed of 145 mM potassium gluconate, 0.5 mM EGTA, 2 mM MgClWe purchased CGP 55845A (1 µM) and CRF (200 nM) from Tocris Bioscience, bicuculline methiodide (30 µM) and TTX (0.5 µM) from Sigma.t test or one-sample t test using Prism 6 (GraphPad Prism). In all cases, p < 0.05 was the criterion for statistical significance, and n represents the number of cells or mice as indicated.Frequency, amplitude, rise, and decay time of EPSCs were analyzed and visually confirmed using a semi-automated threshold based mini detection software . Average EPSC characteristics were determined during baseline and experimental drug conditions in 3 min bins containing a minimum of 60 events. All detected events were used for event frequency analysis, but superimposed events were eliminated for amplitude and kinetic analysis. Data are presented as mean ± standard error, and statistical significance was assessed using a two-tailed 1+ neurons  and vesicular glutamate transporter 1 (Slc17a7) were used as GABAergic and glutamatergic neuronal markers, respectively. In situ hybridization in both the CeL , Prkcd , Pdyn , and Sst ] except Penk, which was expressed at equivalent levels in CeM and CeL (–0.07-fold), and Npy , whose mRNA density was higher in CeM. Additionally, we observed that Crhr1 density was higher in the CeM compared to the CeL  neurons was lower than in CRF1– (16 spines/10 μm) neurons  = –3.3, p < 0.05; 1+  and CRF1– neurons  = 4.9, p < 0.05) while the proportion of thin spines was significantly lower (t(3) = –4.9, p < 0.05) in CRF1+ neurons compared to CRF1– neurons. There were no differences in the proportion of stubby spines (t(3) = 0.4, n.s.) and filopodia (t(3) = –1.0, n.s.) between the two cell types.Higher levels of GFP and  the CeL –4, as prthe CeL –. We therstaining A,B. Spin1+ and CRF1– CeM neurons. Before recording glutamatergic activity, whole-cell current clamp recordings with a step protocol consisting of hyperpolarizing to depolarizing current injections were obtained from each cell to determine cell type based on spiking characteristics. As previously described, CeA neurons are composed of three principal cell types: regular spiking, low threshold bursting, and late spiking neurons  = 1.3, n.s.) and amplitude (t(26) = 1.23, n.s.) in CRF1+  and CRF1– neurons  = 0.75, n.s.) and amplitude (t(26) = 0.70, n.s.) in CRF1+  and CRF1– neurons  in CRF neurons C–H. We o1+ and CRF1– populations to CRF, we tested the effect of CRF  on mEPSCs (1+ (t(10) = 2.78, p < 0.05) and CRF1– (t(8) = 2.68, p < 0.05) CeM neurons (1+: t(10) = 0.50, n.s.; CRF1–: t(8) = 1.12, n.s.; 1+: t(10) = 1.28, n.s.; CRF1–: t(8) = 0.95, n.s.; t(10) = 1.32, n.s.; CRF1–: t(8) = 0.85, n.s.; To assess differences in the functional responsivity of CRFn mEPSCs I–N. We f neurons J but did1+ neurons in the CeA of adult male mice. We found that CB is the predominant CBP expressed in the CeA but overall there is no specific enrichment or exclusion of CBPs in Crhr1+ neurons. Co-expression analysis using in situ hybridization revealed Crhr1 is co-expressed mostly with Penk and Sst and least with Npy. In the CeM, Penk is significantly enriched in CRF1+ neurons compared to CRF1– neurons. Morphologically, CRF1+ neurons have a lower spine density but a higher proportion of mushroom spines compared to CRF1– neurons. Accordingly, basal excitatory transmission between CRF1+ and CRF1– neurons are similar. Additionally, acute CRF application increases glutamatergic transmission in both CRF1+ and CRF1– neurons. The lack of enrichment or exclusion of any particular CBP or neuropeptide in this population contributes to the difficulty in identifying and studying this subpopulation in this heterogenous region.Here, we investigated the neurochemistry, morphology, and physiology of CRF1–expressing neurons project out to the dorsolateral BNST  are associated with increased synaptic strength, whereas immature spines (thin/filopodia) may sustain limited synaptic signaling  spines, resulting in a comparable density of functional spines in CRF1+ and CRF1– neurons.We characterized the dendritic morphology of CeA CRFignaling . Abnormaignaling . Previouignaling . Most neignaling . Here, w1 activity  and drives appropriate behavioral outputs. Previous work has demonstrated a major role of the CRF system, and particularly the CRF1 receptors, within the amygdala complex to influence cellular functions that produce maladaptive behavior in animal models. The amygdalar CRF1 system represents a common pathway for the convergence of stress, addiction, pain, depression, memory formation and anxiety-related signaling  under pathologic conditions, such as alcohol dependence and anxiety disorders. The multiplicity of neuronal subpopulations and the complexity of local microcircuits provide numerous targets for potential dysregulation by drugs of abuse  and stress, and their potential clinical relevance.Our new evidence on the CRF