The structures of both compounds were elucidated using spectroscopic methods, including extensive 1D and 2D NMR analysis, high resolution mass spectrometry (HRMS), and tandem mass spectrometry. Both compounds exhibited potent antibacterial activities against Gram-positive strains with minimum inhibitory concentration (MIC) values of 0.05−0.8 μg/mL and did not show cytotoxicity in the MTT assay up to a concentration of 10 μM. This study adds a new promising member, micrococcin P3, to the family of thiopeptide antibiotics, which shows potential for the development of new antibiotics targeting Gram-positive bacteria.A new thiopeptide (micrococcin P3,  Natural product-derived antibiotics have provided humankind with an effective weapon to fend off bacterial infections and diseases since their discovery in 1929. However, the popularity of antibiotics, especially broad-spectrum antibiotics, has quickly led to their overuse and misuse in people and animals, which has accelerated the emergence and development of antibiotic resistance in pathogens. Increasing resistance to antibiotics reduces the treatment options available for patients and imposes significant economic burdens on patients due to prolonged stays in hospitals. Moreover, medical procedures, such as major surgery and cancer chemotherapy, would become very risky without effective antibiotics to prevent and treat infections. According to the World Health Organization 20D −65.4 ; UV (MeOH) λmax (log ε) 220 (5.02), 349 (4.30); 1H NMR and 13C NMR data, m/z 1144 [M + H]+, 1166 [M + Na]+; HRESIMS m/z 1144.2185 [M + H]+ .Micrococcin P3 (2): white, amorphous powder; [α]20D −65.9 ; UV (MeOH) λmax (log ε) 220 (5.05), 349 (4.29); 1H NMR, 13C NMR data, m/z 1144 [M + H]+, 1166 [M + Na]+.Micrococcin P1  and 100-μL aliquots of the compounds and positive controls (vancomycin and ampicillin) at a concentration of 410 μg/mL in DMSO were added to different wells of column 1 of a 96-well microtiter plate. The wells of other columns were added with 50 μL of Mueller Hinton broth. Blank sample, the compounds, and positive controls were serially diluted by pipetting 50 μL of the sample in the wells of column 1 into the next wells of column 2, and the wells of column 2 into the next wells of column 3, and so on. Then, each well was inoculated with 50 μL of the test bacteria (5 × 105 cfu/mL). Thus, final concentrations of 205, 103, 51, 26, 13, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025 μg/mL of samples were made containing 50%, 25%, 13%, 6.3%, 3.1%, 1.6%, 0.8% 0.4%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005% DMSO (v/v), respectively. The plates were incubated under static conditions at 37 °C for 24 h. MIC was measured as the concentration at which no growth was observed.The following six microorganisms were obtained from the stock culture collection at the Korean Collection for Type Cultures: 4 cells/well) were cultured in 96-well plates at 37 °C for 24 h, and then each well was treated with DMSO or the compounds at the indicated concentration. After 2 days, 10 μL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated at 37 °C for 3 h. The optical density values were determined at 560 nm by a standard microplate reader .Cell viability was measured at 48 h post exposure to DMSO or indicated compounds by assessing the conversion of yellow MTT to purple formazan. In short, the CV-1 cells (1 × 101) and a known one (2) were obtained from a marine-derived strain of Bacillus stratosphericus. These compounds strongly inhibited the growth of all three tested Gram-positive strains without significant cytotoxicity up to a concentration of 10 µM, as evidenced by the MTT assay. This study adds a new promising member to the family of thiopeptide antibiotics, showing potential for the development of new antibiotics targeting Gram-positive bacteria.A new thiopeptide (


Candida, Galactomyces, Hanseniaspora, Guehomyces, Zygosaccharomyces, Trichosporon, Rhodosporidium, Penicillium, and Blastobotrys. Candida and Galactomyces were predominant genera, and their relative abundances were 57.59% and 34.95%, respectively. A total of 50 yeast strains were isolated and identified. Statistical analysis indicated that P kudriavzevii and Geo. silvicola affiliated with Ascomycota were the dominant yeasts in acidic gruel, accounting for 28% and 22%, respectively. This study provides an unequivocal theoretical basis for the study of fungal diversity and the identification and preservation of yeasts in traditional fermented cereals. It also provides validated strain resources for further exploration of the effect of yeasts on acidic gruel quality.Traditional fermented cereals are a rich source of naturally derived, diverse microorganisms. Illumina MiSeq high‐throughput sequencing was used to investigate thoroughly fungal microflora in Western Inner Mongolian acidic gruel. A total of 589,495 sequences were obtained from 16 acidic gruel samples. Ascomycota was found to be the predominant phylum with a relatively abundance of 97.54%, followed by Basidiomycota (2.26%) and Chytridiomycota (0.1%). The dominant genera obtained from the acidic gruel were  Diverse fungal species were abundant in traditional fermented acidic gruel, and significant differences between different samples were seen; The core fungal genera in acidic gruel were Candida and Pichia; Pichia kudriavzevii and Geotrichum silvicola were the dominant yeasts in acidic gruel. The integrity of genomic DNA was analyzed using 0.8% (w/v) agarose gel electrophoresis in 1 × TAE buffer. DNA concentration was measured using a Micro‐ultraviolet spectrophotometer .2.22O to a total volume of 20 µl. Seven barcodes were added to the forward primer. PCR amplifications used the following protocol: predenaturation at 95°C for 3 min, then 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, and a final extension for 10 min at 72°C. PCR products were analyzed using 0.8% (w/v) agarose gel electrophoresis, diluted to 100 nmol/L, and then sequenced using the MiSeq Sequencing Platform .The total volume of the 26S rDNA polymerase chain reaction (PCR) amplification was 20 μl. This comprised 4 μl 5 × PCR Mix, 2.5 μl dNTP mix (2.5 mmol/L), 0.8 μl SSU0817F (5’‐TTAGCATGGAATAATRRAATAGGA‐3’) (5 μmol/L), 0.8 μl SSU1196R (5′‐TCTGGACCTGGTGAGTTTCC‐3′) (5 μmol/L), 0.4 μl Taq DNA polymerase (5 U/μl), and 10 ng DNA, made up with ddH2.3Raw data were initially filtered and merged to remove barcodes, primers, adapters, and failed fragments  were coated on PDA agar  . Three gradients . The project number was mgp91622. Nucleotide sequences of evaluated yeasts have been deposited at GenBank with accession numbers MH880134‐MH880183.Raw sequence data have been made publically available online through MG‐RAST , Simpson index (representing species diversity), and Shannon index (Table Candida (57.59%), Galactomyces (34.95%), Hanseniaspora (2.43%), Guehomyces (2.43%), Zygosaccharomyces (1.06%), Trichosporon (0.75%), Rhodosporidium (0.26%), Penicillium (0.22%), and Blastobotrys (0.19%). Candida was the dominant fungal genus in samples A1, A2, A3, A8, A11, A12, A14, A15, and A16, with relative abundance of 97.12, 99.87, 99.40, 99.93, 94.91, 98.51, 99.85, and 99.95%, respectively.Galactomyces was the dominant fungal genus in the sample of A4, A5, A6, A7, A9, A10, and A13, with relative abundance of 74.31, 49.37, 81.29, 38.75, 94.58, 89.99, and 95.40%, respectively. The average relative abundance of Pichia was 0.09%, which was detected in all samples, as was candida. At the OTU level, a total of 10,895 OTUs were obtained in acidic gruel; OTU6112, OTU3445, OTU3125, OTU1398, OTU1273, and OTU10189 were the most dominant OTUs (relative abundance more than 1.0%) in the acidic gruel; and their average relative abundance was 38.56, 33.70, 8.54, 1.75, 1.36, and 1.35%, respectively. However, the relative abundance of each dominant OTUs in different samples was different. OTU6112, OTU3125, and OTU10189 were similar to Candida, OTU1398 was similar to Hanseniaspora, while OTU1237 was similar to Tausonia. These relationships were consistent with the results of the analysis shown in Figure All of sequences were classified into 6 phyla, 15 classes, 30 orders, 52 families, and 85 genera. Ascomycota, Basidiomycota, and Chytridiomycota were identified as dominant fungal phyla , Galactomyces (11.86%), and Trichosporon (3.45%) to be the dominant genera. These findings were similar to those of the present study. Candida found in natural fermentation products  and Basidiomycota (3.53%)   Table .3.3Pichia, eleven strains belonged to Geotrichum, seven strains belonged to Candida, six strains belonged to Kazachstania, two strains belonged to Cryptococcus, and two strains belonged Saccharomyces. The remaining four strains belonged to Geotrichum, Saturnispora, Yarrowia, and Zygoascus, respectively  plays a crucial role in the production of traditional fermented food, and it may have potential probiotic effects , S cerevisiae (14.29%), Geotrichum sp. (10.71%), C pararugosa (7.14%), C parapsilosis (7.14%), T asahii (7.14%), T coremiiforme (3.57%), C tropicalis (3.57%), and Cla. lusitaniae (3.57%). Using DGGE technology, T asahii, S cerevisiae, Geotrichum sp., and I orientalis were detected in acidic gruel from western Inner Mongolia , I orientalis (100%), and S cerevisiae (99%). Such studies indicate that elucidation of fungal diversity within acidic gruel and the accurate of yeast has been a longstanding research interest and that clear differences arise from analysis of different regional samples and the use of different research methods. This is a principal reason for using MiSeq high‐throughput sequencing technology, so that the fungal diversity in acidic gruel can be analyzed rigorously and completely. As well as acidic gruel, the researchers also investigated fungi and yeasts found in similar traditional fermented cereal foods from different regions and countries. Idil is a traditional fermented food from India and Sri Lanka, S cereuisiae, Debaryomyces hansenii, Hansenula anomala, and T beigelii were common yeasts involved in the fermentation of Idil (Durgadevi & Shetty, Ogi, made from maize, sorghum, or millet and produced by fermenting maize grains, is popular in several West African countries (Adegoke & Babalola, S cerevisiae, C krusei, Geo. candidum, Geo. fermentans, C tropicalis, and R graminis were isolated during ogi fermentation (Omemu, Oyewole, & Bankole, S cerevisiae, C pelliculosa, and Candida tropicalis have also been isolated from togwa, a Tanzanian fermented food (Mugula, Nnko, Narvhus, & Sørhaug, I orientalis and S cerevisiae. These may be caused by differences in local geography, and climate, as well as grain types and producers.Traditional methods used to produce fermented food are greatly affected by weather, temperature, and microorganisms in the environment, resulting in the inconsistent taste and flavor of food. To understand its diversity and function of microorganisms in the production of acidic gruel, several researchers have studied yeast. Bai (Bai et al., a Zhang, . In a rea Zhang, . Physiol4Pichia and Candia were the core fungal genera. Some in the isolated yeasts were found to have potential utilization value. Future research will further characterize these and other yeast strains, while also exploring the mechanism underlying how these and other fungi influence the physical and chemical properties of acidic gruel.To summarize, this work was a first approach to analyze the fungal community structure of traditional fermented Inner Mongolian acidic gruel with high‐throughput sequencing and pure culture technology. Multivariate statistical analysis indicated that the fungi in acidic gruel were abundant. Although there were certain differences between the samples, The authors have declared no conflict of interest.


The mRNA and protein levels of nuclear factor (NF)-κB and inducible nitric oxide synthase (iNOS) in synovial tissue were also determined. Anserine plus glucosamine significantly increased the catalase, SOD, Gpx, GSH, and zinc levels compared to the control, anserine, and glucosamine groups. Also, anserine plus glucosamine significantly reduced the PGE2, MMP-3, TNF-α, IL-1β, and IL-6 levels compared to the control, anserine, and glucosamine groups. Furthermore, anserine plus glucosamine significantly reduced the mRNA and protein levels of NF-κB and iNOS compared to the control, anserine, and glucosamine groups. Therefore, supplementation of anserine plus glucosamine shows therapeutic potential for RA.Rheumatoid arthritis (RA) is an autoimmune disorder that affects the joint synovium. Anserine is a functional dipeptide containing methylhistidine and β-alanine, and is present in the brain and skeletal muscle of birds and mammals. Glucosamine is an amino sugar used in the synthesis of glycosylated proteins and lipids. We evaluated the effects of anserine and glucosamine on RA. Rats were assigned into the control group, RA group, anserine group (1 mg/kg), glucosamine group (200 mg/kg), or anserine plus glucosamine group . Treatment was continued for 45 consecutive days and was administered orally. The serum levels of catalase, glutathione peroxidase (Gpx), superoxide dismutase (SOD), reduced glutathione (GSH), lipid peroxidation, uric acid, nitric oxide, ceruloplasmin, zinc, copper, prostaglandin E Therefore, induction of apoptosis and inhibition of cell proliferation are the primary therapeutic approaches for RA.Rheumatoid arthritis (RA) is an autoimmune disorder that affects the joint synovium. Swelling, inflamed joints, stiffness, and pain are the primary symptoms of RA  were purchased from the Animal House of the Department of Orthopaedics, The Second Affiliated Hospital of Nanchang University, No 1 minde road, Donghu district, Nanchang city, JiangXi province, China and maintained in rat cages under a 12/12 h light/dark cycle at 60 ± 5% relative humidity and 25 ± 0.5 °C. All procedures performed in studies involving animals were in accordance with the ethical standards of the Second Affiliated Hospital of Nanchang University at which the studies were conducted. Research proposal was approved on 03/02/2019.RA was induced according to Dai et al. . BrieflyRats were assigned into the normal control group, RA group, anserine  group (1 mg/kg), glucosamine (Sigma-Aldrich) group (200 mg/kg), or anserine plus glucosamine  group. Treatment was continued for 45 consecutive days and was administered orally. Each group contained six rats.2, MMP-3, TNF-α, IL-1β, and IL-6 were determined as described previously , SOD, and reduced glutathione (GSH) were determined as described previously (Weydert and Cullen The mRNA and protein levels of nuclear factor (NF)-κB and inducible nitric oxide synthase (iNOS) in synovial tissue were determined according to Dai et al. . BrieflySynovial tissue proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto a membrane, and incubated with antibodies against iNOS  and NF-κB  for 12 h. Finally, the blot was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody  for 60 min and the protein levels were assayed by enhanced chemiluminescence (ECL)  was applied to inhibit endogenous peroxidase activity and bovine serum albumin (2%) to block non-specific binding. Synovial tissue was treated with anti-iNOS  and anti-NF-κB  antibodies overnight and incubated with an HRP-conjugated antibody for 1 h  with the Tukey post hoc test. The difference was taken as significant when P < 0.05). Lipid peroxidation, NO, uric acid, ceruloplasmin, and copper were increased by 431.3# 288%, 200%, 126.7%, and 92.3%, respectively (Table P < 0.05), while the zinc level was decreased by 158.3% (Table P < 0.05). The MMP-3, PGE2, TNF-α, IL-1β, and IL-6 levels were increased by 328.6%, 251.3%, 68.9%, 126.7%, and 166.7%, respectively (Table P < 0.05). The mRNA and protein levels of NF-κB and iNOS were also significantly increased .We evaluated the effect of anserine and glucosamine on RA. The SOD, catalase, Gpx, and GSH levels were reduced by 63.6%, 74.2%, 73.3%, and 63.4%, respectively, in the RA group compared to the normal control group (Table P < 0.05). Lipid peroxidation, NO, uric acid, ceruloplasmin, and copper were reduced by 35.3%, 24.7%, 24.6%, 23.5%, and 8%, respectively, while the zinc level was increased by 25% (Table P < 0.05). The MMP-3, PGE2, TNF-α, IL-1β, and IL-6 levels were reduced by 23.8%, 24.1%, 16.3%, 20.6%, and 15.3%, respectively (Table P < 0.05). The mRNA levels of NF-κB and iNOS were reduced by 18.5% and 13% respectively, whereas protein levels of NF-κB and iNOS were reduced by > 9% compared to the control group .The SOD, catalase, Gpx, and GSH levels were increased by 48.3%, 73.5%, 75%, and 46.7%, respectively, in the anserine group compared to the control group (Table P < 0.05). Lipid peroxidation, NO, uric acid, ceruloplasmin, and copper were reduced by 47%, 36.1%, 21.7%, 8.8%, and 20%, respectively, while the zinc level was increased by 16.7% (Table P < 0.05). The MMP-3, PGE2, TNF-α, IL-1β, and IL-6 levels were reduced by 30.7%, 25%, 20.4%, 26.5%, and 29.2%, respectively (Table P < 0.05). The mRNA levels of NF-κB and iNOS were reduced by 25.9% and 17.4% respectively, whereas protein levels of NF-κB and iNOS were reduced by > 13% in the glucosamine group compared to the control group .The SOD, catalase, Gpx, and GSH levels were increased by 60.2%, 82.4%, 108.3%, and 60%, respectively, in the glucosamine group compared to the control group (Table P < 0.05). Lipid peroxidation, NO, uric acid, ceruloplasmin, and copper were reduced by 70%, 54.6%, 49.3%, 50%, and 32%, respectively, while the zinc level was increased by 100% (Table P < 0.05). The MMP-3, PGE2, TNF-α, IL-1β, and IL-6 levels were significantly reduced in the anserine plus glucosamine group (Table P < 0.05). The mRNA and protein levels of NF-κB and iNOS were also significantly reduced in the anserine plus glucosamine group compared to the control, anserine, and glucosamine groups .The SOD, catalase, Gpx, and GSH levels were increased by 135.5%, 211.8%, 216.7%, and 140%, respectively, in the anserine plus glucosamine group compared to the control group (Table We evaluated the effect of anserine and glucosamine on RA. A low level of cellular antioxidants and increased production of free radicals are implicated in RA (van Vugt et al. 2 and MMP-3 in patients with RA are reduced by treatment with glucosamine (Nakamura et al. 2 and MMP-3 levels in neonatal rats (Dai et al. Oxidative injury and inflammation increase the level of prostaglandin (Bae et al. Antioxidants are closely correlated with the levels of oxidants and lipid peroxidation (Gupta et al. In this study, anserine plus glucosamine restored the levels of copper, ceruloplasmin, and zinc to almost the normal ranges. Taken together, our findings suggest that anserine plus glucosamine supplementation shows therapeutic potential for RA.


Grazing as one of the most important disturbances affects the abundance, diversity and community composition of arbuscular mycorrhizal (AM) fungi in ecosystems, but the AM fungi in response to grazing in wetland ecosystems remain poorly documented. Here, we examined AM fungi in roots and soil in grazing and non-grazing plots in Zoige wetland on the Qinghai-Tibet plateau. Grazing significantly increased AM fungal spore density and glomalin-related soil proteins, but had no significant effect on the extra radical hyphal density of AM fungi. While AM fungal richness and community composition differed between roots and soil, grazing was found to influence only the community composition in soil. This study shows that moderate grazing can increase the biomass of AM fungi and soil carbon sequestration, and maintain the AM fungal diversity in the wetland ecosystem. This finding may enhance our understanding of the AM fungi in response to grazing in the wetland on the Qinghai-Tibet plateau. Wetlands cover about 6% of the land surface on the earth and have high species diversity, including many endemic species . In ChinArbuscular mycorrhizal (AM) fungi, as one of the key components of soil microorganisms, form symbiotic associations with most terrestrial plant species . In the Previous studies have demonstrated that the effect of grazing on AM fungi is depended on grazing intensity . For insZoige wetland is a typical representative of the alpine wetland ecosystem on the Qinghai-Tibet plateau in China, and has high plant species diversity and an important C sink function . HoweverOverall, the effects of intense grazing on mycorrhizal symbiosis may lead to a shortage on C from the plant to the fungi, what has been related to fungal response with a different production of glomalin and spores and extra radical hyphae of fungal biomass . BesidesIn order to reveal the AM fungi in response to grazing in wetland ecosystem, we established non-grazing  and moderate grazing plots in Zoige wetland on the Qinghai-Tibet plateau. AM fungal spore density, extra radical hyphal density and GRSP content were examined in grazing and non-grazing plots. We examined the communities of AM fungi in roots and soil by Illumina MiSeq sequencing of 18S rDNA region. We aimed to explore the effect of moderate grazing on AM fungal spore density, extra radical hyphal density, GRSP content, richness and community composition in the Zoige wetland.Blysmus sinocompressus, Potentilla anserina, Carex enervis, Caltha scaposa, Elymus nutans and Leontopodium wilsonii in the site . The site has a plateau cold temperate humid monsoon climate, with a mean annual temperature (MAT) of 1.1 °C, and a mean annual precipitation (MAP) of 660 mm . The sitthe site .ca. 31 cm in the non-grazing plots and 5.4 ± 0.255 and ca. seven cm in the grazing plots. This site was mainly grazed by yaks from June to September, as the growing season is from May to August. The grazing intensity (ca. 1.8 yak/ha) in this study site was described as moderate, as previous study showed that there are three different grazing intensities by yaks  in alpine meadow on the eastern Tibetan Plateau (ca. 300 g) and mixed into one composite sample from each plot. A total of 40 samples were obtained, packed in an ice box and transported to our laboratory. Soil samples were sieved (1-mm sieve) to remove debris and roots. Subsoil samples were kept at −80 °C until the extraction of fungal hyphae and DNA, and the remaining subsoil samples were air dried and kept at 10 °C until the analysis of AM fungal spore density, GRSP content and soil properties. We manually collected the mixed roots (< two mm in diameter) from each sieved root sample, washed with sterilized deionized water and kept at −80 °C until DNA extraction.The Sichuan Zoige Wetland National Nature Reserve Authority approved the collection of soil and root samples in the Zoige Wetland National Nature Reserve. We established 20 plots (each 1 m ×1 m), > 20 m away from each other, in non-grazing  and grazing area, respectively . The ave Plateau . In July4-H2SO4 digestion method and determined with a spectrophotometer .We weighed a certain amount of soil from each sample before and after drying for 24 h in an oven at 105 °C, then calculated the percentage of soil moisture. Soil pH was measured at a ratio of 1:2.5  with a glass electrode . Soil total nitrogen (N) and C were determined by CHNOS Elemental Analyser . Soil total phosphorus (P) was extracted using the HClOTotal GRSP (T-GRSP) and easily extracted GRSP (EE-GRSP) were measured according to the method of −1) and 100 mL distilled deionized water in a flask, and then blended for 30 s, settled for 30 min and sieved (38-µm sieve). The fungal hyphae on the sieve were washed into a flask with 200 mL distilled water, and then 2 mL aliquot was filtered through a 25-µm Millipore filter. The fungal hyphae on the filter were stained with 1% acid fuchsine and distinguished into AM and non-AM fungi on the basis of morphological characteristics and staining color  in accordance with the manufacturer’s instructions, and measured the DNA concentration using a NanoDrop 1000 Spectrophotometer . We amplified the fungal 18S rDNA region using a two-step PCR procedure. The first PCR using primers AML2 , 1.5 mM MgSO4, and 2.5 µL 10 × buffer. The thermal cycling conditions were performed as follows: an initial denaturation at 95 °C for 5 min, 30 cycles for denaturation at 94 °C for 1 min, annealing at 58 °C for 50 s and extension at 68 °C for 1 min, and a final extension at 68 °C for 10 min. The products of the first amplification were diluted 100 times, and 1 µL of the diluted DNA template was used for the second amplification. The thermal cycling conditions for the second amplification were the same as first amplification, except that the primers NS31 (−1. We constructed a sequencing library by addition of an Illumina sequencing adaptor (5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCT- CGTATGCCGTCTTCTGCTTG-3′) to the products using the Illumina TruSeq DNA PCR-Free LT Library Prep Kit  according to the manufacturer’s instructions. We sequenced the library by an Illumina MiSeq PE 250 platform using the paired-end (2 × 250 bp) option in the Chengdu Institute of Biology, Chinese Academy of Sciences, China.We extracted DNA from 0.2 g frozen roots and soil using the PowerSoilers AML2  and GeoAers AML2  was conders NS31  and AMDGers NS31  linked wAM database  nt database. We identified OTUs as the AM fungi based on the closest BLAST hit annotated as ‘Glomeromycotina’ and E values < e−50. Furthermore, we normalized the sequence number of each sample to the smallest sample size using the ‘sub.sample’ command in Mothur. We have submitted the representative sequence of each AM fungal OTU to the European Molecular Biology Laboratory (EMBL) database (accession no. LR736402-LR736557). The identified AM fungi are shown in We filtered the raw sequences using Quantitative Insights into Microbial Ecology (QIIME) v.1.7.0  to elimidatabase  using thdatabase . High qudatabase . Using adatabase , we seleP < 0.05. Generalized linear model (GLM) with Poisson error structure and log link function was conducted to evaluate the effect of grazing on AM fungal spore density, extra radical hyphal density and T-GRSP, as these data did not meet normal distribution, while GLM with Gaussian error structure and identity link function was conducted to evaluate the effect of grazing on EE-GRSP, and then Conover’s test was used to examine the significant difference between grazing and non-grazing treatments at P < 0.05 using the post-hoc.kruskal.conover.test function in the PMCMR package  in roots and soil were established by the Bray–Curtis method . Nested Soil pH was significantly lower in grazing treatment than in non-grazing treatment . Soil mo−1 (mean  ± SE) and 25.71  ± 2.26 µg g−1 in grazing and non-grazing treatments, respectively. The T-GRSP content was 96.7  ± 18.82 µg g−1 and 71.87   ± 12.87 µg g−1 in grazing and non-grazing treatments, respectively. GLM showed that grazing significantly influenced EE-GRSP  and 15.03 ± 5.88 g−1 in grazing and non-grazing treatments, respectively. The extra radical hyphal density of AM fungi was 4.00  ± 2.51 m g−1 and 3.10  ± 1.56 m g−1 in grazing and non-grazing treatments, respectively. GLM revealed that grazing significantly affected AM fungal spore density  belonged to AM fungi. As the sequence number of AM fungi varied from 20,408 to 48,572 in the 80 samples, the number of sequence was normalized to 20,408. The normalized dataset contained 156 AM fungal OTUs . Of the 156 AM fungal OTUs obtained, 154 were from soil, 152 from roots, and 150 shared both soil and roots. Among 156 AM fungal OTUs, 153 were detected from more than three samples (frequency ≥ 3.75%) . FurtherP < 0.001; P = 0.662; AM fungal OTU richness in grazing and non-grazing treatments was 123.70  ± 2.96 (mean  ± SE) and 122.85  ± 3.01 in soil, and 117.55  ± 2.26 and 118.00  ± 4.38 in roots, respectively. GLM revealed that AM fungal OTU richness was influenced by sample type , and sample type had significant effect on the relative abundance of abundant AM fungal OTU4, OTU5, OTU7, OTU12, OTU14, OTU18, OTU25 and OTU141 , OTU8 and OTU17  and OTU23   ; S4.GLM revealed that sample type significantly influenced the relative abundance of Glomerales, Diversisporales and Archaeosporales, and grazing significantly affected the relative abundance of Diversisporales . The relF = 7.2836, R2 = 0.157, P = 0.001; F = 2.339, R2 = 0.025, P = 0.012; F = 2.639, R2 = 0.055, P = 0.001; F = 0.998, R2 = 0.025, P = 0.419; The PerMANOVA demonstrated that the community composition of AM fungi was significantly influenced by sample type  vegetation in grazing plots, (b) vegetation in non-grazing plots.Click here for additional data file.10.7717/peerj.9375/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.9375/supp-3Supplemental Information 3SN, soil non-grazing; SG, soil grazing; RN, root non-grazing; RG, root grazing.Click here for additional data file.10.7717/peerj.9375/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.9375/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.9375/supp-6Supplemental Information 6G, grazing; NG, non-grazing; ST, sample type.Click here for additional data file.10.7717/peerj.9375/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.9375/supp-8Supplemental Information 8G, grazing; NG, non-grazing; ST, sample type.Click here for additional data file.10.7717/peerj.9375/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.9375/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.9375/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.9375/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.9375/supp-13Supplemental Information 13Click here for additional data file.10.7717/peerj.9375/supp-14Supplemental Information 14Click here for additional data file.10.7717/peerj.9375/supp-15Supplemental Information 15Click here for additional data file.10.7717/peerj.9375/supp-16Supplemental Information 16Click here for additional data file.10.7717/peerj.9375/supp-17Supplemental Information 17Click here for additional data file.10.7717/peerj.9375/supp-18Supplemental Information 18Click here for additional data file.10.7717/peerj.9375/supp-19Supplemental Information 19Click here for additional data file.10.7717/peerj.9375/supp-20Supplemental Information 20Click here for additional data file.10.7717/peerj.9375/supp-21Supplemental Information 21Click here for additional data file.10.7717/peerj.9375/supp-22Supplemental Information 22Click here for additional data file.10.7717/peerj.9375/supp-23Supplemental Information 23Click here for additional data file.10.7717/peerj.9375/supp-24Supplemental Information 24Click here for additional data file.10.7717/peerj.9375/supp-25Supplemental Information 25Click here for additional data file.10.7717/peerj.9375/supp-26Supplemental Information 26Click here for additional data file.10.7717/peerj.9375/supp-27Supplemental Information 27Click here for additional data file.10.7717/peerj.9375/supp-28Supplemental Information 28Click here for additional data file.


Wild blueberry (WBB) powder can be added to the formulation of foods to encourage consumption of health-promoting polyphenolics, but the stability of polyphenolics throughout storage is important. We determined the stability of polyphenolics in five products  prepared with WBB powder. Samples stored at 21 °C, 4.4 °C, or −20 °C (ice pops only) were analyzed at 0, 2, 4, 6, and 8 weeks for polyphenolic content and percent polymeric color. Total anthocyanins decreased over storage and storage temperatures in all products. However, the ice pop and the refrigerated juice both retained over 90% of their initial total anthocyanin content. The refrigerated oatmeal bar also showed good retention of anthocyanins (86%), but the gummy product retained only 43% and 51% when stored at 4.4 °C or 21 °C, respectively. The lower amount of polyphenolic compounds recovered in the gummies stored at 4.4 °C compared to 21 °C may be attributed to reduced extraction efficiency as a result of gel hardening at refrigerated temperature. Chlorogenic acid and flavonols were generally more stable than anthocyanins throughout storage. Vaccinium angustifolium Ait) are considered a nutrient-rich healthy food, due in large part to their exceptional phenolic content [Lowbush “wild” blueberries . In addThe mechanism responsible for loss of anthocyanins during storage is unknown, but anthocyanin losses are commonly accompanied by increased polymeric color values, suggesting that anthocyanins form polymers with proanthocyanidins . In addiThis study was undertaken to determine the stability of anthocyanins, flavonols, chlorogenic acid, and percent polymeric color in five blueberry products prepared with freeze-dried WBB powder. Gummy, oatmeal bar, graham cracker cookie, and juice were stored at 21 °C and 4.4 °C and evaluated for anthocyanin, flavonol, and chlorogenic acid content and percent polymeric color over eight weeks of storage. An ice pop product stored at −20 °C was evaluated for its anthocyanin and chlorogenic acid content over eight weeks of storage.® North American wild blueberry powder was purchased from FutureCeuticals, Inc. . Rutin, chlorogenic acid, high performance liquid chromatography (HPLC) grade methanol, HPLC grade acetonitrile, potassium metabisulfite, formic acid, acetic acid, chlorogenic acid and rutin were purchased from Sigma-Aldrich . A standard mixture of delphinidin, cyanidin, petunidin, peonidin, pelargonidin, and malvidin glucosides was purchased from Polyphenols . HiActiveSamples of juice, ice pop, gummy, oatmeal bar, and graham cracker cookie, each containing 15 g of WBB powder per serving, were prepared and packaged as previously described . One serg. The pellet was re-extracted two additional times with 25 mL of extraction solution and centrifuged for 5 min at 10,864 × g. The filtrates were pooled and adjusted to 100 mL with extraction solvent in a volumetric flask. Prior to HPLC analysis, 5 mL of extract were dried in a Thermo Savant Speed Vac Plus SC210A  and reconstituted in 1 mL 5% formic acid in water. All samples were passed through 0.45 µm nylon syringe filters  into 1 mL HPLC vials prior to HPLC analysis. The ice pop and juice samples did not undergo extraction due to prior extraction of anthocyanins to make the concentrate used in the formulation but were filtered using the 0.45 µm nylon syringe filters prior to HPLC analysis.Polyphenolics were extracted by homogenizing 5 g of WBB-containing food product or 1 g of WBB powder in 25 mL of extraction solution containing methanol/water/formic acid (60:37:3 v/v/v), to the smallest particle size using a Euro Turrax T18 Tissuemizer  for 1 min. Homogenates were centrifuged for 5 min at 10,864 × 18 column  preceded by a 3.9 mm × 20 mm Symmetry C18 guard column. The mobile phase was a linear gradient of 5% formic acid (A) and methanol (B) from 2% B to 60% B for 60 min at a flow rate of 1 mL/min. The system was equilibrated for 20 min at the initial gradient prior to each injection. Detection wavelengths of 320 nm and 510 nm were used to monitor chlorogenic acid and anthocyanin peaks, respectively. Individual anthocyanin monoglucosides and acylated anthocyanin derivatives were quantified as delphinidin, cyanidin, petunidin, peonidin, and malvidin glucoside equivalents using external calibration curves  of a mixture of authentic standards . Chlorogenic acid was quantified using external calibration curves  of an authentic standard . Results are expressed as mg of anthocyanin or chlorogenic acid per g of WBB powder.Anthocyanins and chlorogenic acid were analyzed by HPLC using the method of Cho and others (2004) . Samples18 column  preceded by a 3.0 mm × 4.0 mm ODS C18 guard column. The mobile phase was a linear gradient of 2% acetic acid (A) and 0.5% acetic acid in water and acetonitrile (50:50 v/v) (B) from 10% B to 55% B in 50 min and from 55% B to 100% B in 10 min at a flow rate of 1 mL/min. The system was equilibrated for 20 min at the initial gradient prior to each injection. A detection wavelength of 360 nm was used to monitor flavonol peaks. Flavonols were quantified as rutin equivalents using an external calibration curve  of an authentic standard , with results expressed as mg of rutin equivalents per g of WBB powder.Flavonols were analyzed by HPLC using the same HPLC system described above according to the method of Cho et al. (2005) . Separatm/z 50−1000 at 1.0 s per cycle. Characteristic ions (m/z) were used for peak assignment. For compounds where chemical standards were commercially available, retention times were also used to confirm the identification of components.An analytical Hewlett Packard 1100 series HPLC instrument  equipped with an autosampler, binary HPLC pump, and UV/Vis detector was used. For HPLC/MS analysis, the HPLC apparatus was interfaced to a Bruker model Esquire-LC/MS ion trap mass spectrometer . Mass spectral data were collected with the Bruker software , which also controlled the instrument and collected the signal at 520 nm. Typical conditions for mass spectral analysis conducted in positive-ion electrospray mode for anthocyanins and negative-ion electrospray mode for flavonols included a capillary voltage of 4000 V, a nebulizing pressure of 30.0 psi, a drying gas flow of 9.0 mL/min, and a temperature of 300 °C. Data were collected in full scan mode over a mass range of Percent polymeric color (% PC) of extracts was determined using the spectrophotometric assay of Giusti and Wrolstad (2001) . Sample Polymeric color was determined using the bisulfite-bleached sample using the following formula:Percent polymeric color was calculated using the formula:n = 3) of each sample per time point and using the correlation method. Correlations among total anthocyanins and percent polymeric color were determined by pairwise correlations method in the multivariate platform of JMP.The effect of storage time  on anthocyanins, flavonols, chlorogenic acid, and % polymeric color in each blueberry product was evaluated using the Fit Model platform of JMP ), and the percent retention of each compound after 8 weeks of storage was calculated using the fit model equation. The effect of storage temperature on phenolic compounds stability was not evaluated in this study due to the length of time (4 months) the WBB powder was stored between processing the products in Experiment 1 (products stored at 21 °C) and Experiment 2 (products stored at 4.4 °C). During this four-month storage time, the powder stored at 15.5 °C presumably absorbed moisture evident by powder clumping, resulting in different amounts of polyphenolics in the products immediately after processing. Principal component analysis (PCA) was performed with the total and individual anthocyanins variables, using the Multivariate platform in JMP, on the mean value (m/z) values and elution orders with previous studies [m/z of 303, but the molecular ion m/z value was ambiguous. Many of the anthocyanins were present in acylated form. Two of the cyanidin glycosides  were acylated with malonic acid, whereas delphinidin, cyanidin, and malvidin galactosides as well as petunidin, peonidin, and malvidin glucosides were acylated with acetic acid moieties.The WBB powder used to prepare the products contained at least 22 anthocyanins , which w studies ,5,34. Blm/z) values and elution orders with previous studies [m/z of 300, but the molecular ion m/z of 623 was ambiguous.The WBB powder used to prepare the products contained at least 12 flavonols , which w studies ,32. The p = 0.0056), but the percent retention remained high with 93% of total anthocyanins retained in the product after eight weeks. Consistent with our results, total anthocyanin content of frozen blueberries was stable over three months of storage at −20 °C [The total anthocyanin content of the ice pop over eight weeks of storage at −20 °C is shown in t −20 °C . Changesp = 0.0008) and 21 °C (p = 0.0008). The mean total anthocyanin content of both storage temperatures over eight weeks of storage is shown in The total anthocyanin content of the oatmeal bar decreased with storage time at 4.4 °C (p = 0.0003) and 21 °C (p < 0.0001). The mean total anthocyanin content of both storage temperatures over eight weeks of storage is shown in The total anthocyanin content of the graham cracker cookie decreased with storage time at 4.4 °C (p < 0.0001). The total anthocyanin content of juice stored at 4.4 °C and 21 °C is shown in The total anthocyanin content of the juice decreased with storage time for each storage temperature (p < 0.0001). The total anthocyanin content of the gummy product stored at 4.4 °C and 21 °C is shown in The total anthocyanin content of the gummy product decreased with storage time for each storage temperature .The distribution of the products according to their individual anthocyanin profile as affected by storage time  can be visualized on a PCA scores plot . The firChanges in percent polymeric color (% PC) values in oatmeal bar, graham cracker cookie, juice, and gummy product stored at 4.4 °C and 21 °C over eight weeks of storage are shown in p = 0.0012) and 21 °C (p = 0.0173). For samples stored at 4.4 °C, % PC values increased from 7% at day 0 to 9.6% at eight weeks, and from 5.7% at day 0 to 9.6% at eight weeks in samples stored at 21 °C. Percent polymeric color values showed significant inverse correlations to levels of total anthocyanins at 4.4 °C  and at 21 °C  during storage.Percent polymeric color values of oatmeal bar samples increased over storage at 4.4 °C (p < 0.0001), but not at 21 °C (p = 0.2075). For samples stored at 4.4 °C, % PC values increased from 2.5% at day 0 to 6.3% at eight weeks, but when stored at 21 °C, % PC values remained at 6.2% on average during eight weeks of storage. Percent polymeric color values showed significant inverse correlations to levels of total anthocyanins at 4.4 °C  and 21 °C  during storage.Percent polymeric color values of graham cracker cookie samples significantly increased over storage at 4.4 °C (p = 0.0008) and 21 °C (p < 0.0001). For samples stored at 4.4 °C, % PC values increased from 13.6% at day 0 to 20.8% at eight weeks, and the values increased from 13.8% at day 0 to 23.5% at eight weeks when stored at 21 °C. Percent polymeric color values showed significant inverse correlations to levels of total anthocyanins at 4.4 °C , and more strongly at 21 °C  during storage.Percent polymeric color values of juice samples significantly increased over storage at 4.4 °C (p = 0.0229) and 21 °C (p < 0.0001). For samples stored at 4.4 °C, % PC values increased from 6.8% at day 0 to 12.7% at eight weeks, and in samples stored at 21 °C, % PC values increased from 6.9% at day 0 to 23.8% at eight weeks of storage. Percent polymeric color values showed a moderate inverse correlation to levels of total anthocyanins at 4.4 °C , but a much greater inverse correlation at 21 °C  during storage.Percent polymeric color values of gummy samples significantly increased over storage at 4.4 °C . At 4.4 °C, the chlorogenic acid content decreased from 4.3 to 3.6 mg/g WBB powder in the juice and from 3.0 to 2.6 mg/g WBB powder in the oatmeal bar. At 21 °C, chlorogenic acid in the juice showed a slight increasing trend; however, this change was not statistically significant (p = 0.6496). Chlorogenic acid was also stable in the ice pop over eight weeks of storage at −20 °C (p = 0.8332), with an average value of 6.5 mg/g WBB powder over storage . Flavonols in the juice stored at 21°C were also stable over time despite an upward trend, but the increase was not significant (p = 0.1935). Consistent with our findings, total flavonol concentrations were found to be relatively stable (<15% losses) in blueberry jam, juice, puree, and canned berries over six months of storage at 25 °C [The stability of total flavonols in the five blueberry products is shown in at 25 °C ,42.p = 0.0085) and graham cracker cookie (p = 0.0237) stored at 21 °C. Total flavonol levels declined by 45.7% and 28.5%, respectively, in these products over eight weeks of storage. In the gummy product, the most marked loss occurred from six to eight weeks of storage. Moisture loss during late storage presumably led to hardening of the gummies, resulting in incomplete extraction of the flavonols.However, total flavonol content significantly decreased over storage in the gummy product (p = 0.038). Quercetin-3-rutinoside (p = 0.0225), quercetin-3-galactoside (p = 0.0195), quercetin-3-glucoside (p = 0.037), quercetin-3-rhamnoside (p = 0.0058), quercetin-3-(6″-acetyl) galactoside (p = 0.0195), and syringetin-3-galactoside/glucoside (p = 0.0237) were all retained at levels from 61.1% to 70.1%. Four individual flavonols significantly decreased in the gummy product stored at 4.4 °C. The percent retention of quercetin-3-glucuronide (p = 0.0012) and quercetin-3-rhamnoside (p = 0.0016) were 33% and 38.5%, respectively. In the gummy product stored at 21 °C, only two flavonols (myricetin-3-glucoside and the syringetin derivative) showed no significant decrease over eight weeks of storage. All other flavonols in the gummy product stored at 21 °C were adversely affected by storage time with retentions ranging between 45.2 and 60.1% after eight weeks.Individual flavonols in oatmeal bar and juice were all stable for both storage temperature as well as in graham cracker cookie stored at 4.4 °C (data not shown). In the graham cracker cookie stored at 21 °C, myricetin-3-galactoside was the most unstable flavonol showing 57.8% retention after eight weeks . The other food items may also be used for these applications, but the relative retention of bioactive polyphenolics as outlined herein should be taken into account when dose and delivery are designed. The gummy product showed relatively poor retention of anthocyanins and flavonols after eight weeks of storage, which may be due to extraction issues rather than true losses. In summary, incorporating WBB powder into food products in which key molecules remain intact during storage can improve the consumption of blueberry phytochemicals. The effect of the product matrix on bioavailability of retained polyphenolic compounds needs further investigation.


Picea glauca, a boreal species at risk under global warming trends. Although moderate drought treatment reduced the photosynthetic rate by over 70%, metabolic flux through the MEP pathway was reduced by only 37%. The activity of the putative rate-limiting step, 1-deoxy-D-xylulose-5-phosphate synthase (DXS), was also reduced by about 50%, supporting the key role of this enzyme in regulating pathway metabolic flux. However, under severe drought, as flux declined below detectable levels, DXS activity showed no significant decrease, indicating a much-reduced role in controlling flux under these conditions. Both MEP pathway intermediates and the MEP pathway product isoprene incorporate administered 13CO2 to high levels (75–85%) under well-watered control conditions indicating a close connection to photosynthesis. However, this incorporation declined precipitously under drought, demonstrating exploitation of alternative carbon sources. Despite the reductions in MEP pathway flux and intermediate pools, there was no detectable decline in most major MEP pathway products under drought (except for violaxanthin under moderate and severe stress and isoprene under severe stress) suggesting that the pathway is somehow buffered against this stress. The resilience of the MEP pathway under drought may be a consequence of the importance of the metabolites formed under these conditions.The methylerythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis produces chlorophyll side chains and compounds that function in resistance to abiotic stresses, including carotenoids, and isoprene. Thus we investigated the effects of moderate and severe drought on MEP pathway function in the conifer  These intermediates arise from two different pathways in plants, the mevalonate (MVA) pathway located in the cytosol, ER and peroxisomes, and the more recently identified methylerythritol 4-phosphate (MEP) pathway localized in plastids   . However (MEcDP) . Among it phases . Meanwhit phases , but theD-xylulose-5-phosphate synthase (DXS), has been generally assumed to be rate limiting based on studies in which overexpression of the corresponding gene led to increases in MEP pathway products  Voss], 3 years old, were purchased from a local nursery in Jena, Germany and grown outdoors at the Max Planck Institute of Chemical Ecology under natural conditions. At the end of May 2015, the trees were transferred inside a greenhouse with supplemental lighting. Relative humidity was maintained between 50 and 60%, light period was 14 h, and the temperature was 23°C:19°C, day: night. The experiment was performed in August after needles had fully expanded. Drought was applied by withholding water. At the beginning of the experiment, trees were irrigated and excess of water was allowed to drain for 2–3 h. Then the initial pot weight was measured using a digital balance to a precision of 1 g . Subsequently, pot weights were recorded daily. Each pot was enclosed with a bag to avoid soil evaporation. Therefore, loss of water was attributed to plant transpiration only. The experiment lasted 20 days. The availability of water under the different stress regimes, was described by the fraction of transpirable soil water (FTSW). The FTSW was calculated by using the daily pot weight according to the formula  was achieved . Samplinendpoint .13CO2 labeling for 50 min (see details below), twigs were harvested, frozen immediately in liquid nitrogen, and transferred to a −80°C freezer. Only needles were used for biochemical analysis. After grinding, the total fresh weight was measured. Analysis of chlorophylls, carotenoids, β-cyclocitral, monoterpenes, and the DXS assay was carried out with fresh tissue, while analyses of MEP intermediate metabolites, sugars, and abscisic acid were carried out after freeze-drying. For each tree, 100 mg of fresh tissue were weighed before and after freeze-drying to determine fresh-to-dry weight conversion factors. Due to the differences in leaf water content between stressed and control trees all measurements were reported on a dry weight basis. Rates of photosynthesis and isoprene emission were also referenced to the dry weight of the needles.After –2 s–1). Leaf temperature was set at 30°C, and the relative humidity in the cuvette ranged between 45 and 50%. After harvesting the measured tissue, A, the rate of carbon fixation, was calculated on a dry weight basis according to the LI-COR manual pages 16–51.Photosynthesis was measured with a portable gas exchange system  using a chamber for measuring conifer needles supplied with the instrument. Measurements were performed between 10:00 and 14:00 under conditions of photosynthetic photon flux density (PPFD)  as an internal standard. The solution was incubated at 20°C for 30 min in a heating block shaking at 1000 rpm. After centrifugation at 18,000 × g at 4°C for 20 min, the supernatant was analyzed by using an Agilent 1260 Infinity high-performance liquid chromatography (HPLC) system  coupled to an API 5000 tandem mass spectrometer . A Zorbax Eclipse XDB-C18 column  was used for the chromatographic separation with a formic acid (0.05% in water)/acetonitrile gradient . ABA was detected via multiple reaction monitoring and quantified relative to the peak area of the standard.A 10 mg quantity of freeze-dried, ground leaf material was extracted with 1 ml methanol containing 40 ng mlg and 4°C for 5 min. The extracts were further diluted 1:10 with water before analysis on an Agilent 1260 Infinity HPLC system connected to an API 5000 triple quadrupole mass spectrometer. An external standard curve made with authentic standards (Sigma-Aldrich) at concentrations ranging between 1.25 and 20.0 μg ml–1 was used for quantification. Samples were analyzed directly by LC-MS/MS after a 1:20 (v/v) dilution in water. Separation was made via hydrophilic interaction chromatography on an apHera NH2 polymer column . Water and acetonitrile were used as mobile phases A and B, respectively. The elution profile was: 0–0.5 min, 80% B in A; 0.5–13 min, 80–55% B in A; 13–14 min, 55–80% B in A; and 14–18 min, 80% B in A. The ion spray voltage was maintained at −4500 eV. The turbo gas temperature was set at 600°C. Nebulizing gas was set at 50 psi, curtain gas at 20 psi, heating gas at 60 psi, and collision gas at 5 psi. Multiple reaction monitoring (MRM) was used to monitor isotopic composition of glucose and fructose by the following precursor ion → product ion reactions: m/z 178.8 → 58.7, m/z 179.8 → 58.7, m/z 179.8 → 59.7, m/z 180.8 → 58.7 m/z 180.8 → 59.7, and m/z 180.8 → 60.70. For sucrose, we used m/z 340.9 → 58.8, m/z 341.9 → 58.8, m/z 341.9 → 59.8, m/z 342.9 → 58.8 m/z 342.9 → 59.8, m/z 342.9 → 60.8, m/z 343.9 → 58.8, m/z 343.9 → 59.8, m/z 343.9 → 60.8, m/z 344.9 → 58.8, m/z 344 → 59.8, m/z 344.9 → 60.8, m/z 345.9 → 58.8, m/z 345.9 → 59.8, m/z 345.9 → 60.8, m/z 346.9 → 58.8, m/z 346.9 → 59.8, m/z 346.9 → 60.8, m/z 347.9 → 58.8, m/z 347.9 → 59.8, m/z 347.9 → 60.8, m/z 348.9 → 58.8, m/z 348.9 → 59.8, m/z 348.9 → 60.8, m/z 349.9 → 58.8, m/z 349.9 → 59.8, m/z 349.9 → 60.8, m/z 350.9 → 58.8, m/z 350.9 → 59.8, m/z 350.9 → 60.8, m/z 351.9 → 59.8, m/z 351.9 → 60.8, and m/z 352.9 → 60.8. The percentage of 13C labeling in each metabolite after 50 min was calculated by summing all 13C atoms incorporated in the sugar isotopes, and dividing this number by the overall sum of unlabeled and labeled C atoms.Freeze dried needles (5 mg) were extracted with 1 ml 80% MeOH. The solution was vortexed for 10 min at ambient temperature and centrifuged at 13,000 × D-xylulose 5-phosphate (DXP), 2-C-methyl-D-erythritol 4-phosphate (MEP), 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME), 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP), isopentenyl diphosphate and dimethylallyl diphosphate (IDP + DMADP), samples of 5 mg dry weight were extracted twice with 250 μl of 50% acetonitrile containing 10 mM ammonium acetate, pH 9.0. After vortexing and centrifugation in a micro-centrifuge at 20,000 × g for 5 min, 200 μl of the supernatant from both extracts were combined, transferred into a new 1.5 ml tube and dried under a stream of nitrogen gas at 40°C. The residue was dissolved in 100 μl of 10 mM ammonium acetate, pH 9.0, and after vortexing, 100 μl of chloroform was added. The upper aqueous phase, separated by centrifugation at 20,000 × g, was transferred into a new tube and diluted with 1 volume of acetonitrile. To remove any precipitate, the supernatant was transferred to an HPLC vial after centrifugation at 20,000 × g.To quantify the MEP pathway metabolites, 1-deoxy-TM high pressure pre-column filter  using two solvents: 20 mM ammonium bicarbonate adjusted to pH 10.5 with LC-MS grade ammonium hydroxide (solvent A) and 80% acetonitrile containing 20 mM ammonium bicarbonate, pH 10.5 (solvent B). The solvent gradient profile started with 100% of solvent B, which decreased to 60% in the first 15 min, followed by an isocratic elution with 100% solvent B. Separation was performed at 25°C with a flow rate of 500 μl min–1.Methylerythritol 4-phosphate pathway metabolites were analyzed on an Agilent 1260 Infinity HPLC system connected to an API 5000 triple quadrupole mass spectrometer. A 5 μl quantity of the extract was injected and the metabolites were separated on a hydrophobic interaction liquid chromatography (HILIC) XBridge Amide columns  with a HILIC guard column containing the same sorbent  and a SSIm/z 212.9 → 96.9, m/z 213.9 → 96.9, m/z 214.9 → 96.9, m/z 215.9 → 96.9, m/z 216.9 → 96.9, and m/z 217.9 → 96.9 . MEcDP and its isotope distribution were monitored by the following precursor ion → product ion reactions: m/z 277.0 → 78.9, m/z 278.0 → 78.9, m/z 279.0 → 78.9, m/z 280.0 → 78.9, m/z 281.0 →7 8.9, and m/z 282.0 → 78.9 . IDP/DMADP and their isotope distributions were monitored by the following precursor ion → product ion reactions: m/z 244.9 → 78.9, m/z 245.9 → 78.9, m/z 246.9 → 78.9, m/z 247.9 → 78.9, m/z 248.9 → 78.9, and m/z 249.9 → 78.9 . Analyst 1.6 software (Applied Biosystems) was used for data acquisition and processing.The mass spectrometer was used in the negative ionization mode with the following instrument settings: ion spray voltage −4500 V, turbo gas temperature 700°C, nebulizer gas 70 psi, heating gas 30 psi, curtain gas 30 psi, and collision gas 10 psi. DXP and its isotope distribution were monitored by the following precursor ion → product ion reactions: 13C abundance. Normalization to added unlabeled standards was accomplished by analyzing each sample twice, once with and once without the addition of 25 ng of DXP, 55 ng of MEcDP, and 24 ng of DMADP + IDP standards dissolved in 10 ml of water . Isoprene was monitored with the mass signal m/z 69. The raw count-rate (cps) of the isoprene signal was normalized (ncps) to the sum of the primary ion and water cluster, and to the drift tube pressure. The average of the normalized signal during the steady state period was used to calculate the emission rate, after subtracting the background (empty chamber without needles). In vivo labeling was accomplished by replacing the 12CO2 in the air entering the cuvette with 13CO2  at the same ambient concentration (380 μmol mol–1). Labeling was performed for 50 min. The appearance of protonated masses of isoprene was followed in the PTR MS by monitoring m/z 70 (13C12C4H9), m/z 71 (13C212C3H9), m/z 72 (13C312C2H9), m/z 73 (13C412CH9), and m/z 74 (13C5H9). The percentage of 13C labeling was calculated by summing all 13C atoms incorporated in the isoprene isotopes, and dividing this number by the overall sum of unlabeled and labeled carbon atoms of isoprene   was emplisoprene . Isopren2, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% (v/v) glycerol, 1% (w/v) polyvinylpyrrolidone , 4% (w/v) polyvinylpolypyrrolidone (PVPP), 4% (w/v) Amberlite XAD-4, and 0.1% (v/v) Tween 20. Extraction was carried out in an 1.5 ml Eppendorf tube shaken in an Eppendorf Thermoshaker at 4°C and 1400 rpm. After centrifugation for 20 min at 4°C and 20,000 × g, the supernatant was passed through a 2 ml Zeba Spin desalting column with a 7 kDa molecular weight cut-off  to exchange the buffer to 50 mM Tris–HCl, pH 8.0, with 10% (v/v) glycerol and 10 mM MgCl2. The total protein concentration was estimated by measuring absorbance at 280 nm using a NanoDrop 1000 UV-Vis spectrophotometer (Thermo Scientific).Freshly ground needles (200 mg) were immersed in 1 ml of extraction buffer containing 250 mM MOPSO (pH 6.8), 5 mM ascorbic acid, 5 mM sodium bisulfite, 5 mM dithiothreitol (DTT), 10 mM MgCl2, 2.5 mM dithiothreitol, 1 mM thiamine pyrophosphate, 2 mM imidazole, 1 mM sodium fluoride, 1.15 mM molybdate, 1% (v/v) protease inhibitor cocktail, and 10 mM each of the substrates pyruvate and glyceraldehyde-3-phosphate. The final volume of 100 μl was incubated in a water bath at 25°C for 2 h. As a control for non-enzymatic conversion and the presence of assay product in the original plant extract, 30 μl of the enzyme extract were heated to 90°C for 10 min to deactivate the enzyme, and then combined with 70 μl of mixture assay and incubated for 2 h as well. The enzyme reaction was stopped by vigorously vortexing for 5 s with 100 μl of chloroform. After centrifugation in a microcentrifuge to complete phase separation, the upper aqueous phase, was transferred into a new tube and diluted with 1 volume of acetonitrile. As an internal standard, 25 ng of [13C5] DXP dissolved in water was added to the final solution dissolved in water.The DXS enzyme assay was carried out in accord with a previous protocol . Briefly–1 with a column temperature of 25°C. The solvent profile started with a linear gradient from 0 to 16% A over 5 min and followed with an isocratic separation for 10 min. After a linear gradient from 16 to 40% A over 5 min, solvent A was returned to 0% over 15 min of equilibration. The mass spectrometer was used in the negative ionization mode with the following instrument settings: ion spray voltage −4500 V, turbo gas temperature 700°C, nebulizer gas 70 psi, heating gas 30 psi, curtain gas 30 psi, and collision gas 10 psi. DXP was monitored using the following precursor ion → product ion transition m/z 212.95 → 78.9 and DXP. The DXP produced by the DXS enzyme reaction was normalized to the [13C5] DXP internal standard monitored with the transition m/z 217.94 → 96.9.The enzymatic end product DXP was quantified on an Agilent 1260 Infinity HPLC system connected to an API 5000 triple quadrupole mass spectrometer. DXP was separated via hydrophilic interaction liquid chromatography with the column and solvent system mentioned above for the analysis of MEP pathway metabolites. The flow rate was 1 ml min13C-label incorporation to that of IDP + DMADP, and using these fractions to determine plastidial content. As isoprene labeling could be followed on-line instantaneously with the PTR-MS without the need for individual sampling, these measurements were taken as the instantaneous labeling state of the IDP + DMADP pool. This assumption was justified since isoprene is produced from DMADP in a single step, and the volatile isoprene gas escapes from the leaf. Moreover the assumption was verified experimentally by ensuring that the final label incorporation after 50 min in the isoprene and IDP + DMADP pools was identical . Thus, the plastidial concentrations of DXP and MEcDP were estimated by calculating the ratio of their final dentical . Followif(t) is the fractional labeling of isoprene  as a function of time, m is the maximal fractional labeling at the end of the run, A, B, and C are the pool sizes of DXP, MEcDP, and IDP + DMADP, respectively, J is the pathway flux and t is time. Equation (1) assumes that the pools of the other MEP pathway intermediates  are too small to significantly delay the label incorporation into downstream metabolites; these intermediates were below the limit of detection in the HPLC-MS analysis. To calculate the flux, Eq. (1) was fitted to isoprene labeling time-courses obtained from the PTR-MS [f(t)], with the plastidial pool sizes of DXP, MEcDP and IDP + DMADP entered as parameters A, B, and C, respectively  by using a gradient of acetone (solvent A) and 1 mM NaHCO3 in water (solvent B). The flow rate was 1.5 ml min–1. The initial mobile phase consisted of 65/35% (v/v) solvent A/solvent B. Then, solvent A was linearly increased up to 90% in 12 min and to 100% in 8 min. 100% solvent A was kept for 2 min and then decreased to 65% in 3 min. Quantification was done using external standard curves. Authentic standards of chlorophylls and β-carotene (Santa Cruz Biotechnology) were analyzed in a range from 0.1 to 0.00625 mg ml–1. Lutein, neoxanthin, and violaxanthin were considered to have the same response factors as β-carotene.After grinding the needles in liquid nitrogen, 50 mg were extracted in light-protected tubes with 1 ml of acetone by shaking for 6 h at 4°C in the dark. After centrifugation for 5 min at 2300 × g and 4°C. The supernatant was centrifuged as above for 10 min, and 200 μl of the new supernatant were taken for the analysis using an Agilent 1260 Infinity high-performance liquid chromatography (HPLC) system coupled to an API 5000 tandem mass spectrometer. β-Cyclocitral was separated on an Zorbax Eclipse XDB-C18 column  Chromatographic separation was performed by using a gradient of formic acid 0.05% in water, (solvent A) and acetonitrile (solvent B). The flow rate was set at 0.5 ml min–1. The initial mobile phase consisted of 95/5% (v/v) solvent A/solvent B. Then, solvent A was decreased to 50% in 2 min and held for 3 min. After solvent A was decreased to 0% in 9 min and held for 11 min, it was raised again to 95%. β-Cyclocitral was monitored by following the precursor ion → product ion reaction: m/z 153.191 → 109.0. Quantification was done using an external standard curve made with an authentic standard of β-cyclocitral (Sigma-Aldrich).A 100 mg quantity of fresh tissue was extracted with 1 ml of methanol. The suspension was mixed by vortexing for 5 min and centrifuged for 20 min at 20, 000 × –1 isobutylbenzene as internal standard, and shaken for 14 h at room temperature. The ethereal extract was transferred to a fresh vial and washed with 0.3 ml of 0.1 M (NH4)2CO3 (pH 8.0) in order to purify the extracted terpenes from other organic acids. The sample was filtered through a Pasteur pipette column filled with silica gel (Sigma 60 Å) and anhydrous MgSO4. Monoterpene analysis was performed by GC-MS with a Hewlett-Packard 6890 system, using a DB-WAX column . Split injection was carried out at 220°C. Helium was used as carrier gas at a constant flow of 1 ml min–1. The GC was programmed with an initial oven temperature of 40°C (3-minute hold), a ramp of 5°C min–1 until 80°C, then a ramp of 5°C min–1 until 200°C, followed by a final ramp of 60°C min–1 until 280°C (4-minute hold). For GC-FID analysis, the flame ionization detector operated at 300°C. GC-FID- and GC-MS-generated peaks were integrated using Hewlett-Packard Chemstation software. Identification of terpenes was based on comparison of retention times and mass spectra with those of authentic standards or with mass spectra in the Wiley library. In order to calculate needle monoterpene concentrations on a ng mg–1 dry weight basis, the residue of 100 mg extracted material of each trees was dried and weighed as described by According to the procedure of post hoc test at P < 0.05. Normality was tested by the Shapiro-Wilk test.The effect of drought treatment was tested using one-way ANOVA. When test results were significant, the means were compared using Tukey’s Picea glauca (white spruce) trees. In the moderate stress treatment, transpiration rate was reduced to 50% of that in well-watered trees, which occurred when the fraction of transpirable soil water (FTSW) was 12%. Under severe stress transpiration rate was reduced to 20% of that in well-watered trees, which occurred when the FTSW was 3%  and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) declined significantly under moderate and severe drought stress in white spruce  was the most abundant at all time points after 20 min  under both moderate and severe drought treatments, but no significant difference was observed between the two drought levels . The activity of DXS measured t levels . This det levels .20 side chain of chlorophylls a and b is synthesized by the MEP pathway. However, the sum of chlorophyll a and chlorophyll b did not show any significant reduction with drought despite a declining trend (ANOVA one-way P = 0.109) (P = 0.110) and lutein (ANOVA one way P = 0.449) also did not decline significantly, although declining trends were evident (The decrease in metabolic flux through the MEP pathway might be expected to impact the levels of carotenoid and chlorophyll pigments since carotenoids are wholly derived from the MEP pathway while the C= 0.109) , even th= 0.109) . Among t evident . By cont evident  while ne evident . An oxid evident  increase evident .P. glauca and other conifers, and are also produced by the MEP pathway. The pool size of stored monoterpenes in needles was not influenced by drought treatments, and the amounts of individual monoterpenes were also unaltered . Similar 13CO2 measurements of the illuminated rosettes of Arabidopsis thaliana conducted by 13CO2 in illuminated A. thaliana rosettes. The MVA pathway is also known from other studies to be less active during the day due to the negative effect of light on the transcription of pathway genes  pathway of isoprenoid biosynthesis, localized in the cytosol, ER and peroxisomes, also produces DMADP and IDP. However, our data give no support to a role for products of the MVA pathway in supplying the MEP pathway under stress. Import of DMADP or IDP from outside the plastid would decrease increase . Thus thay genes .Arabidopsis thaliana based on metabolic control analysis   , 4-hydrose (DXR)  and 4-hyDespite the substantial decline in MEP pathway flux under drought, we found no commensurate reduction in the major products of the MEP pathway produced in photosynthetic cells, the chlorophylls, lutein and β-carotene. Depending on the turnover rate of these pigments, it is possible that the drought treatment was not long enough to observe any net depletion. Some depletion of β-carotene is indicated by the sharp increase in β-cyclocitral, a β-carotene oxidation product . HoweverPicea abies, the size of the stored pool of monoterpenes in current year needles did not change after the first 2 months of growth in July, and did not change in older needles at all over the entire growing season (Picea abies to a low atmospheric CO2 concentration (50 ppm) also did not change the amount of monoterpenes stored in current year needles (Under severe drought (but not under moderate drought), there was a very significant reduction (>50%) in the MEP pathway product isoprene. This may have diverted enough MEP pathway flux to chlorophyll and carotenoid formation to keep the pools of these pigments stable. However, transgenic silencing of isoprene formation in poplar led to only slight increases in the levels of chlorophylls and carotenoids , but theg season . Our exp needles .13CO2 incorporation, the percentage of isotopic label in isoprene was virtually identical to the percentage of labeling in the DMADP + IDP pool under all conditions  was not one of these alternative sources. More investigations are needed to determine how alternative carbon sources are recruited to the MEP pathway under stress and how this is regulated. The relative importance of the MEP pathway under drought may be a consequence of the number of pathway products shown to help protect against drought-associated oxidative stresses, including carotenoids, tocopherols and isoprene . ControlAll datasets presented in this study are included in the article/EP and LW designed the experiments. EP performed the experiments, analyzed the data, and wrote the manuscript. JR performed the calculation of the metabolic flux. JR, DG-C, FL, AS, JG, and LW supervised the study and complemented writing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Their structures differ in conformation at the glycine moiety, and the monoclinic form contains additional, disordered water molecules. Despite the high stability of Schiff bases, these newly obtained compounds hydrolyze in aqueous media, the process being accelerated by metal cations. These studies, accompanied by mechanistic considerations and solid-state moisture and thermal analysis, clarify the structure and behavior of this amino acid Schiff base and shed new light on the role of water in its stability.Salens, as chelating, double Schiff base ligands, are an important group utilized in transition metal catalysis. They have been used to build interesting functional metal-organic frameworks (MOFs). However, salens interacting with amino acids have also found applications in receptors. Here, we intended to form a “green” glycine-derived salen fragment, but the available literature data were contradictory. Therefore, we optimized the synthetic conditions and obtained the desired product as two different crystallographic polymorphs (orthorhombic Pcca and monoclinic P Schiff bases, due to their conjugation with the aromatic system, constitute an important group of stabilized imines. Salens, which consist of a disalicylic moiety and an aliphatic diamine, have been extensively studied over the past 80 years, resulting in myriad publications . CatalysN,O-ligands led us to literature references suggesting a simple condensation of salicylaldehyde with glycine in ethanol. The synthetic procedures for the desired imine described in the literature differed at several points, and a survey of these procedures is presented in 2 atmosphere. Contrary to Zhu, NaOH was used as the base. After 2 h, the solid product was formed and recrystallized with methanol-ether (1:1) to give, after filtration, the ligand with a 58% yield. Panchal was the first to publish a synthetic method that avoided water [Our quest for ed water . He carred water  performe1—potassium N-[(2-hydroxyphenyl)methylene]glycinate. It is worth noting that we identified the factor determining success. Initial efforts focused on obtaining the non-salt product. Chohan’s conditions glycinate (1) could be successfully synthesized with high yields in basic conditions in methanol. The spectroscopic data agree with the structure proven by X-ray analysis. The overall analysis reveals the presence of two crystallographic forms differing in color (yellow and orange), crystallographic system, and degree of hydration. Traces of water present in the reactants  are beneficial for obtaining the product. However, in alcoholic solution, one equivalent of water with respect to compound 1 leads to its hydrolysis. This process is additionally accelerated by the presence of Lewis acids such as Fe3+ or Cu2+. On the other hand, Cu complexes with ligand 1 could be obtained in non-aqueous media.Based on the solid-state studies, the behavior of the Schiff base  and one equiv. of KOH  were dissolved in methanol (50 mL). A solution of salicylaldehyde  in methanol (50 mL) was added. The color of the solution rapidly turned yellow with the formation of an orange precipitate. The mixture was stirred for 1 h at room temperature. The resulting solid product was filtered and air-dried. During drying, the color of the product changed from orange to yellow. Yield 74%,  m.p. = 219–221 °C.® software (license No. 2013781467484).NMR spectra were recorded on an Agilent 400 MHz spectrometer at room temperature. Aromatic signals were assigned by simulations using ChemDraw1H-NMR  δ 8.34 , 7.30 , 7.27 , 6.79 , 6.73 , 4.23 . 13C-NMR  δ 175.80 (COO), 168.00 (CH=N), 167.92 (CAr-OH), 134.85 (p-CAr-H), 133.61 (o-CAr-H), 119.78 (m-CAr-H), 119.17 (CAr-CH=N), 117.88 (CAr-H-CAr-OH), 60.89 (=N-CH2). MS for C9H9NO3 [M+H]+: calculated = 180.1; found: 180.2.HPLC analyses were carried out using a Dionex UltiMate 3000 HPLC system equipped with an autosampler. Chromatographic separation was achieved with Thermo Scientific Hypersil GOLD HPLC  column, using water  and methanol . Sample injection volumes were 10 μL, the flow rate was 1 mL/min, and the column temperature was maintained using an oven at T = 23 °C.1 was mixed with 10 mL of a 1 mM solution of metal sulfate and stirred for 1 h. 20 μL was then taken and diluted with distilled water to 2 mL, filtered through a 0.22 μm pore size syringe filter, and analyzed by HPLC.In a 50 mL round bottom flask, 10 mL of a 1 mM solution of ligand 1 is described in the The synthesis of the Cu complex with 1 was dissolved in 10 mL of water and stirred for 40 h. After 1, 2, 16, 24, and 40 h, a 20 μL solution aliquot was diluted with distilled water to 2 mL, filtered through a 0.22 μm pore size syringe filter, and analyzed by HPLC.In a 25 mL round bottom flask, 1 mmol of ligand 1 in methanol (HPLC grade) was prepared, and then one equivalent or 10 equivalents of water was added, respectively. These solutions were transferred sequentially to the cuvette, capped, and measured for at least 6 h. 10 mL of a 1 mM solution of ligand 1 in methanol (HPLC grade) was then measured as a blank, followed by the addition of 0.1 and 1 equivalent FeCl3 or CuCl2, respectively, which were measured for at least 3 h. Additionally, the following solutions were prepared and measured: 1 mmol solution of salicylaldehyde + 1 equiv. KOH in methanol followed by the addition of 1 equiv. of FeCl3; and 1 mmol solution of glycine + 1 equiv. KOH + 1 equiv. FeCl3. All UV-Vis spectra are attached to the UV-Vis analyses were carried out using a Hitachi U-2910 in the wavelength range 190–900 nm. 10 mm quartz cuvettes were used. Sample preparation: 10 mL of a 1 mM solution of ligand 1a and using a SuperNova diffractometer with Cu Kα radiation (λ = 1.54184 Å) at 120 K for crystal 1b. Data were integrated with CrysAlisPro software  [2 using SHELXL-2018/3 [X-ray diffraction data were collected using an Xcalibur diffractometer with Mo Kα radiation (λ = 0.71073 Å) at room temperature for crystal  for 1b) . The pha for 1b) . The modL-2018/3 . All proL-2018/3 .1a represents a pure compound without any additional solvent molecules. Compound 1b is a hydrate and is also a pure compound. The presence of the additional atoms results from the disordering of the carboxyl group and the K+ ions. The number of water molecules can be defined based on an analysis of the occupancies of the oxygen atoms in the water molecules. The O1W atom from the first water molecule has an occupancy equal to 0.5. This atom is placed in the special position , and thus the real occupancy is 1. The O2W atom from the second water molecule has an occupancy equal to 0.46(1). In the final refinement, it was fixed at 0.5. The hydrogen atoms that are present at the O2W atom were found on the difference Fourier map. In order to preserve the correct geometry of the water molecules, the hydrogen atoms were placed using a suitable HFIX. In the case of the O1W atoms, the hydrogen atoms were not visible on the difference Fourier map.http://www.ccdc.cam.ac.uk/conts/retrieving.html .CCDC 1957836-1957837 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via 1a : orthorhombic, space group Pcca (no. 54), a = 13.9484(12) Å, b = 18.5280(16) Å, c = 7.0215(8) Å, V = 1814.6(3) Å3, Z = 8, μ(MoKα) = 0.562 mm−1, Dcalc = 1.591 g/cm3, 12,574 reflections measured (6.8° ≤ 2Θ ≤ 52.6°), 1842 unique  which were used in all calculations. The final R1 was 0.0387 (I > 2σ(I)), and wR2 was 0.0900 .Crystal Data for 1b : monoclinic, space group P21/c (no. 14), a = 18.879(3) Å, b = 6.2395(6) Å, c = 8.7555(9) Å, β = 99.765(12)°, V = 1016.4(2) Å3, Z = 4, μ(CuKα) = 4.567 mm−1, Dcalc = 1.531 g/cm3, 3619 reflections measured (9.5° ≤ 2Θ ≤ 117.6°), 1350 unique ) and wR2 was 0.1854 .Crystal Data for TGA curves were recorded using a LINSEIS STA PT1600 at a heating rate of 10 °C/min under an argon atmosphere over a 25 °C to 800 °C temperature range. Moisture analysis was performed on a Radwag MA 50/1.R.WH.Mass spectrometry employed an API 4000 QTRAP tandem mass spectrometer detector equipped with an electrospray ionization (ESI) source .


Candida species that has clinical importance. Aqueous extraction of DAV samples was subjected to Liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF-MS) profiling. Reverse phase (RP) separation was used due to the process extraction using water as a solvent to separate polar compound. The data was interpreted using Profile Analysis 2.1V. The DAV samples were also tested for the effect on the biofilm formation of seven Candida species in a 96 well plate Deer antler velvet (DAV) has been traditionally used in Chinese medicine, including treatment on toothache  Value of the Data•Cervus timorensis) antler velvet.First report on the LC-MS profile of Malayan deer (•Candida biofilm enables to understand the potential of DAV to improve oral health by inhibiting Candida infection.The data on DAV showing reduction of •Data will enable understanding the importance of chemical composition of DAV in bone formation. Both Positive and negative ionization mode was analyzed.Raw OD values represents microbial biofilm density of different 22.1Male Malayan deer antler was collected from local company; D'Paradise Deer Valley and Exotic Farm Sdn. Bhd. Dried samples was powdered using variable speed laboratory blender . 10 g of powdered sample was soaked in 450 ml double distilled deionized water with periodic shaking for 30 min intervals for 8 h; then the mixture will be allowed to settle for 16 h. After filtration, the residue was washed with a small portion of water and followed by freeze-drying .2.2C. glabrata ATCC 90030, C. tropicalis ATCC 13803, C. krusei ATCC 14243, C. parasilopsis ATCC 22019, C. lusitaniae ATCC 64125, C. dubliniensis ATCC MYA-2975, C. albicans ATCC MYA-4901) were procured from American Type Culture Collection (ATCC). While ALC2 (isolate from HIV positive patient) and ALC3  representing Candida albicans isolates from Melbourne dental school, University of Melbourne, Australia were tested for biofilm formation with or without DAV extract of known concentration in RPMI-1640 media following the previously established procedure A total of 7 Candida species  at the rate of 0.5 ml/min. The mobile phase was prepared using the binary solvent manager with solvent A and B.  Briefly, the gradients elution were 99% A and 1% B (0.00 min) 99% A and 1% B (0.00–0.50 min), 65% A and 35% B (0.50–16.00 min), 0% A and 100% B (16.00–18.00 min), 99% A and 1% B (18.00–20.00 min), with the flow rate of 0.6 mL/min. The solvent A is made of 0.1% formic acid  plus water . However, solvent B was acetonitrile at a seal wash time and highest-pressure limit of 5 min and 1800 psi, respectively. Positive and negative ionization mode was performed at the following setting: capillary voltage: 2.50 kV, nebulizer pressure: 1.2 bar, drying gas: 8 l/min at 200 °C, mass range: 50–1000 m/z This research work does not require ethical approval.Arzmi M.H: Conceptualization, Methodolog;, John B.A.: Data interpretation, Writing - manuscript; Rismayuddin N.A.R, Kenali N.M: Laboratory analysis; Arzmi M.H: Supervision; Darnis D.S: Validation; Arzmi M.H., John B.A., Rismayuddin N.A.R.: Writing - Reviewing and Editing. All authors equally contributed to this manuscript.Authors have no competing interest.


L1) and urea (L2) have been synthesized and studied for a variety of anions by UV-Vis and colorimetric techniques in DMSO. Results from the binding studies suggest that both L1 and L2 bind halides showing the order: fluoride > chloride > bromide > iodide; and oxoanions showing the order: dihydrogen phosphate > hydrogen sulfate > nitrate > perchlorate. Each receptor has been shown to form a 1:1 complex with an anion via hydrogen bonding interactions, displaying distinct color change for fluoride and dihydrogen phosphate in solution. As compared to the urea-based receptor L2, the thiourea-based receptor L1 exhibits stronger affinity for anions due the presence of more acidic thiourea functional groups.Two rationally designed 4-nitrophenyl-based molecular clefts functionalized with thiourea ( Because of the fundamental role played by anions in biological, industrial, and environmental processes, selective binding and sensing of anions by synthetic receptors has become one of the most extensively studied areas in supramolecular chemistry . As anioosteosarcoma  and shows donor-acceptor properties similar to those of the water molecule. Hence, the recognition and detection of these key inorganic anions by efficient artificial sensors is much anticipated. In a recent report, it was shown that C3v-symmetric receptors possessing both H-bond donor and acceptor exhibited high selectivity for H2PO4− over other common inorganic anions  receptors exhibiting interactions with oxoanions, while both bisurea and thiourea were shown to form tris-chelate complexes with phosphate ion (PO43−) in the solid state. C3v-symmetric tripodal receptors with various flexible and rigid frameworks have been shown to exhibit for the chromogenic and fluorogenic sensing of anions through the formation of hydrogen bonded host-guest complex followed by the deprotonation of amide-NH protons  and other containing urea (L2) functionalities . Elemental analysis was carried out using an ECS 4010 Analytical Platform (Costech Instrument) at Jackson State University.All reagents and solvents were purchased from commercial suppliers and used without further purification. Nuclear magnetic resonance (NMR) spectra were recorded at 25 °C on a Varian Unity INOVA 500 FT-NMR instrument and the chemical shifts were recorded in parts per million (ppm) using tetramethylsilane as a reference. All NMR data were processed and analyzed with MestReNova software. The following abbreviations in m-xylylenediamine  with 4-nitrophenyl isothiocyanate  for L1 or 4-nitrophenyl isocyanate  for and L2 in dry acetonitrile under constant stirring at room temperature. After overnight stirring, the yellowish precipitate was filtered off and washed with acetonitrile and THF. The residues were dried under vacuum to give L1 (88% yield) and L2 (92% yield).The receptors were successfully synthesized  by the rL1: 1H NMR  δ (ppm): 4.77 , 5.76 , 7.26–7.27 , 7.33–7.36 , 7.82–7.84 , 8.15 , 8.71 , 10.26 .13C NMR  δ (ppm): 47.5 (xylyl–CH2), 120.0 (Ar–C), 124.9 (Ar–C), 126.9 (Ar–C), 128.9 (Ar–C), 138.9 (Ar–C), 180.7 (C=S). IR : 3,334, 3,190, 2,930, 1,594, 1,533, 1,506, 1,326, 1,303, 1,245. Anal. Calcd. for C22H20N6O4S2: C, 53.21; H, 4.06; N, 16.92. Found: C, 53.25; H, 4.07; N, 16.88.L2: 1H NMR  δ (ppm): 4.774 , 5.760 , 7.259–7.272 , 7.335–7.356 , 7.825–7.838 , 8.149–8.162 , 8.717 , 10.270 .13C NMR  δ (ppm): 44.5 (xylyl–CH2), 118.2.0 (Ar–C), 125.69 (Ar–C), 128.0 (Ar–C), 141.8 (Ar–C), 146.2 (Ar–C) 152.2 (C=S). IR : 3360, 3340, 2,940, 1734, 1,594, 1,495, 1,250, 1,207, 1,177. Anal. Calcd. for C22H20N6O6: C, 56.89; H, 4.34; N, 18.10. Found: C, 56.85; H, 4.37; N, 18.08.−1 M) and receptors (5 × 10−3 M) were prepared separately in DMSO. In order to obtain an optimal absorption, the solutions of L1 and L2 were diluted to 2.5 × 10−5 M. In the UV-Vis screening experiment, the samples were prepared by mixing 10 equivalents of the stock solutions of different anions into a quartz optical cell of 1 cm path length filled with 2.0 ml of individual receptor solution. In a typical titration experiment, UV abortion spectra were recorded from the gradual addition  of a respective anionic solution (2.5 × 10−3 M) to L1 or L2. The receptor-anion binding constant values (log K) were determined from UV-Visible titration studies using a 1:1 binding model  including F−, Cl−, Br−, I−, HSO4−, H2PO4−, NO3− and ClO4− as their salts of [n-Bu4N]+ to the probes, we observed remarkable spectral changes of L1 and L2 in terms of both intensity and wavelength with fluoride and dihydrogen phosphate, as compared to other anions included in our study. This observation suggests that these two anions (fluoride and dihydrogen phosphate) are capable to induce a colorimetric response in DMSO due to the formation of a strong anion complex. The corresponding colorimetric experiments, as performed with the addition of 10 equivalents of the respective anions to L1 and L2 (2 × 10−3 M) are shown in As shown in L1 with fluoride, as shown in max = 6–7 nm), yielding an isosbestic point at around 395 nm. A new peak appears in the visible range at around 478 nm, showing an increase in intensity (hyperchromic shift) with the concentration of fluoride, suggesting the formation of a receptor-fluoride complex. This spectral change is consistent with the visual color change  due to the gradual addition of fluoride provided the best fit to a 1:1 binding mode, yielding a binding constant of 3.70 , while the peak was shifted to about λmax at 363 nm at the end of the titration  may be because of more converging dipodal arms . Notably, no shifting of λmax was found (− anion (log K = 3.70). Under the same condition, it binds other anions  weakly, agreeing with the reported binding trend for other neutral receptors  at 357 nm followed by a red shift (Δλmax = 10–12 nm), while a new peak appeared at around 480 nm was shown to increase with the incremental addition of fluoride to the receptor. With dihydrogen phosphate, it showed a red shift (Δλmax = 15 nm) of the absorption band (λmax) at 357 nm at the end of the titration  and dihydrogen phosphate (from light yellow to red) in solution. In particular, each of the receptors exhibited strong affinities for fluoride with the binding trend for halides in the order: fluoride > chloride > bromide > iodide; and for oxoanions in the order: dihydrogen phosphate > hydrogen sulfate > nitrate > perchlorate, forming 1:1 complexes with anions by virtue of hydrogen bonding interactions. The thiourea-based receptor as compared to its urea analogue, exhibits an increased affinity for anions due the presence of enhanced acidity of thiourea units. This study could serve as an insight for the rational design of new functional chemosensors for selective sensing of anions in an application oriented field.In summary, we have synthesized two simple dipodal urea/thiourea clefts; each appended on a The original contributions presented in the study are included in the article/MH conceived the project idea. UM, BP, and MN synthesized and studied the compounds. TE assisted in titration studies. UM prepared the initial manuscript. All authors approved the manuscript.The project described was supported by the US Department of Defense (Grant No. W911NF-19-1-0006). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.