Macrophages exposed to the Th2 cytokines interleukin (IL) IL-4 and IL-13 exhibit a distinct transcriptional response, commonly referred to as M2 polarization. Recently, IL-4-induced polarization of murine bone marrow-derived macrophages (BMDMs) has been linked to acetyl-CoA levels through the activity of the cytosolic acetyl-CoA-generating enzyme ATP-citrate lyase (ACLY). Here, we studied how ACLY regulated IL-4-stimulated gene expression in human monocyte-derived macrophages (MDMs). Although multiple ACLY inhibitors attenuated IL-4-induced target gene expression, this effect could not be recapitulated by silencing ACLY expression. Furthermore, ACLY inhibition failed to alter cellular acetyl-CoA levels and histone acetylation. We generated ACLY knockout human THP-1 macrophages using CRISPR/Cas9 technology. While these cells exhibited reduced histone acetylation levels, IL-4-induced gene expression remained intact. Strikingly, ACLY inhibitors still suppressed induction of target genes by IL-4 in ACLY knockout cells, suggesting off-target effects of these drugs. Our findings suggest that ACLY may not be the major regulator of nucleocytoplasmic acetyl-CoA and IL-4-induced polarization in human macrophages. Furthermore, caution should be warranted in interpreting the impact of pharmacological inhibition of ACLY on gene expression. Macrophages respond to changes in their environment, such as bacterial or viral infection, hormones, cytokines, or nutrients, with remodeling their transcriptome. Consequently, they alter their phenotype, a response known as macrophage polarization . Historide novo lipogenesis and thus, cell proliferation  . IL-4 trSince we previously noticed considerable differences in metabolic requirements of human vs. murine macrophages toward IL-4-induced polarization , we quesHuman peripheral blood mononuclear cells were isolated from commercially obtained buffy coats  using Ficoll  density centrifugation. Monocytes were separated from lymphocytes by adherence to plastic after 1 h-incubation in serum-free medium, and differentiated into macrophages by culture in RPMI-1640 medium  containing 3% heat-inactivated AB-positive human serum for 7–10 days. THP-1 cells were purchased from ATCC (cat. no. TIB-202) and maintained in RPMI-1640 medium containing 10% fetal bovine serum, penicillin and streptomycin. THP-1 cells were differentiated to macrophages by 24 h-treatment with 50 nM phorbol myristate acetate  followed by overnight culture in serum-free medium. Unless indicated otherwise, cells were treated with 5 μM BMS-303141 (cat. no. 4609), 25 μM SB 204990 (cat. no. 4962) (both Tocris), 100 μM MEDICA 16 (cat. no. M5693), 20 mM hydroxycitrate (cat. no. 59847), 5 mM sodium acetate (cat. no. S2889), 5 mM sodium octanoate (cat. no. C5038), 1,2,3-benzene-tricarboxylic acid (cat. no. 51520), CTP inhibitor (cat. no. SML0068) , 5 μg/mL TOFA (cat. no. 10005263) (Cayman), 20 ng/mL IL-4 (cat. no. 200-04), or IL-13 (cat. no. 200-13) (Peprotech).−(Ct(target)−Ct(reference))] using expression of β2-microglobulin or GAPDH as reference genes.Total RNA from macrophages was isolated using PeqGold RNAPure kit  followed by reverse transcription using cDNA Synthesis kit . Quantitative real time PCR (Q-PCR) was carried out on a CFX96 system from Bio-Rad using iQ SYBR green . Primer sequences are listed in Supplementary Table Total cell lysates were prepared by scraping the cells into Laemmli buffer  containing protease inhibitors  followed by sonification. For histone extraction, cell pellets were lyzed in PBS containing 0.5% Triton X-100 , 5 mM sodium butyrate , and protease inhibitors for 10 min on ice. Lysates were centrifuged 0.5 min at 16,000 g, 4°C, and pellets containing nuclei were incubated with 0.2 M HCl  overnight. After centrifugation of insoluble material for 10 min at 500 g, 4°C, supernatants were neutralized with NaOH , mixed with 5x Laemmli buffer and heated 5 min at 95°C.Protein lysates from macrophages were run on 7.5–15% polyacrylamide gels and blotted on nitrocellulose membranes. Following primary antibodies were used: pSTAT6 (Y641) (cat. no. #9361), STAT6 (clone D3H4) (cat. no. #5397), pSTAT3 (Y705) (cat. no. #9131), STAT3 (clone 124H6) (cat. no. #9139), pACLY (S454) (cat. no. #4331), histone H3 acK14 (clone D4B9) (cat. no. #7627), histone H3 acK23 (clone D6Y7M) (cat. no. #14932), DDK tag (clone 9A3) (cat. no. #8146) , ACLY , histone H3 acK9 (clone Y28) , histone H3 acK27 , histone H3 , nucleolin . Membranes were incubated with IRDye 700/800-coupled secondary antibodies, scanned and quantified using Odyssey imaging system (Licor).Silencing of ACLY was performed using siGENOME SMARTpool  at 50 nM and Hyperfect transfection reagent . Cells were treated 96 h post-transfection.Human MDMs were transfected with 1 μg DDK-tagged ACLY  or GFP-encoding plasmids  using Viromer Red transfection reagent  according to manufacturer's instructions. Twenty-four hours after transfection, cells were stimulated with 20 ng/mL IL-4 for 24 h.13C2-acetyl-CoA . Acetyl-CoA concentration was determined by reference to the standard. Analyst 1.6.2 and MultiQuant 3.0 (both Sciex), were used for data acquisition and analysis, respectively. Acetyl-CoA amounts in the sample were normalized to sample DNA concentration measured following incubation with Höchst 33342 fluorescent DNA dye  on a Tecan fluorescence plate reader.Cells were rapidly washed with saline, and metabolism was quenched by putting the dishes into liquid nitrogen. Cells were scraped into methanol:water (5:3) mix on ice/dry ice followed by addition of cold chloroform , and vortexing for 10 min at 4°C. Aqueous phase was separated, evaporated, and re-suspended in 10% methanol . Acetyl-CoA concentration was determined with a Sciex QTrap5500 mass spectrometer operating in multiple reaction monitoring mode in positive electrospray ionization mode. Chromatographic separation was performed on an Agilent 1290 Infinity LC system (Agilent) using an Acquity HSS T3 column. The mobile phase consisted of (A) water, 10 mM ammonium formate , 0.01% ammonia, and (B) methanol, 10 mM ammonium formate, 0.01% ammonia. Elution of analytes was carried out under gradient conditions at a flow rate of 0.3 mL/min going from 2% B to 70% B in 5 min, increasing to 95% B in 1 min, hold 95% B for 0.5 min, and equilibrate at 2% B for 2.5 min. Calibration curve was performed with an authentic standard. All samples and dilutions of the standards were spiked with heavy isotope labeled internal standard containing pLentiCRISPRv2 (Addgene: cat. no. #52961) vectors harboring different sgRNAs designed using the benchling software package Table . Cell-frpost-hoc means comparison using GraphPad Prism. Differences were considered statistically significant at p < 0.05.Data are presented as means ± S.E. of at least three independent experiments. Data were analyzed by one-way analysis of variance (ANOVA) with Bonferroni Investigations were conducted in accordance with the ethical standards and according to the Declaration of Helsinki and to the national and international guidelines and have been approved by the authors' institutional review board. The ethics committee of Goethe-University waived the necessity of written informed consent when using the buffy coats from anonymized blood donors.To investigate the role of ACLY in IL-4-stimulated human macrophage polarization we initially analyzed mRNA expression of arachidonate 15-lipoxygenase (ALOX15) in MDMs in the presence of pharmacological ACLY inhibitors BMS 303141 , SB 2049A previous study showed that ACLY was necessary for IL-4-induced expression of a subset of IL-4 target genes in murine macrophages . To asseNext, we assessed the effect of silencing ACLY mRNA expression on IL-4-induced MDM polarization. For this, we treated MDMs for 96 h with control or ACLY siRNAs prior to 24 h-treatment with IL-4. Surprisingly, a knockdown of ACLY failed to reproduce the effect of ACLY inhibitors on IL-4-stimulated gene expression Figures , despiteConsidering these discrepancies, we decided to investigate the impact of ACLY inhibitors on human macrophage metabolism in more detail. Surprisingly, we did not find any differences in the levels of acetyl-CoA in cells treated with BMS 303141 or SB 204990 for 24 h Figure . AccordiWe then investigated the influence of ACLY inhibitors on initial steps of IL-4-induced signal transduction by pre-incubating MDMs with inhibitors for 1 h followed by 0.5 h-treatment with IL-4. ACLY inhibitors did not affect IL-4-triggered tyrosine phosphorylation of STAT6 with the exception of BMS 303141 and hydroxycitrate Figure . We notiMurine BMDMs responded to IL-4-stimulation with an Akt-dependent increase of ACLY phosphorylation at Ser454, which exhibited a delayed kinetics as compared with IL-4-induced Akt phosphorylation . We follde novo lipogenesis . Thus, the role of ACLY in metabolism and epigenetic regulation of terminally differentiated human macrophages remains unclear and warrants further research. Of note, ACLY may have greater impact during human macrophage differentiation, since this process is characterized by a temporary rise of de novo lipogenesis . ACSS2 contributes to histone acetylation in cancer cells, especially under hypoxia or glucose deprivation , 38, andOur results also highlight the notorious proneness of pharmacological inhibitors to off-target effects, which is in our case particularly remarkable, since we used structurally dissimilar substances . Off-target effects of ACLY inhibitors on IL-4-stimulated gene transcription could only be revealed using ACLY knockout cell line. Our findings also point to the limitation of studying human primary cells, since ACLY knockdown leaves substantial residual activity left whereas pharmacological inhibitors are unsuitable due to off-target activities. Whether induced pluripotent stem cell—derived macrophages, where creation of knockout models is possible , will beDN designed the study, performed the experiments, and wrote the manuscript. SZ and IF contributed to acetyl-CoA measurements. FS, NK, and DF contributed to CRISPR/Cas9 THP-1 knockout cell line creation. BB contributed to study design and edited the final manuscript. All authors contributed to manuscript revision, read, 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.


Coastal regions experiencing declining dissolved oxygen are increasing in number and severity around the world. However, despite the importance of microbial metabolism in coastal hypoxia, few metagenomic surveys exist. Coastal regions experiencing declining dissolved oxygen are increasing in number and severity around the world. However, despite the importance of microbial metabolism in coastal hypoxia, few metagenomic surveys exist. Our data set from within the second largest human-caused hypoxic region provides opportunities to more deeply explore the microbiology of these systems. The northern Gulf of Mexico experiences seasonal bottom water hypoxia that can exceed 22,000 km2 (http://www.noaa.gov/media-release/gulf-of-mexico-dead-zone-is-largest-ever-measured), impacting fisheries and other coastal industries (Marine systems suffering from declines in dissolved oxygen (DO) are becoming more numerous across the globe . Many codustries ; howeverActinobacteria (n = 12), Alphaproteobacteria (n = 5), Bacteroidetes (n = 6), Gammaproteobacteria (n = 3), Gemmatimonidetes (n = 1), Ignavibacteriae (n = 2), Nitrospina (n = 5), Planctomycetes (n = 7), Proteobacteria (n = 1), Synechococcus (n = 3), Verrucomicrobia (n = 3), and unclassified (n = 2). Twenty-four of the MAGs were estimated at >50% complete, with 16 estimated at >75% complete based on CheckM (https://doi.org/10.6084/m9.figshare.6911729.v1). All but 2 MAGs had estimated contamination of <9%, with 40 having estimated contamination of <3%. While several of our Actinobacteria MAGs grouped with sequences near the important OM1 clade of marine Actinobacteria (8https://doi.org/10.6084/m9.figshare.6911729.v1), but make useful references for future studies of the group.Site selection, marine chemistry metadata, and extraction, sequencing, and assembly methods were previously described , 4. Brieetes n = , Proteobacteria 810, theseeria n = , Gemmatihttps://doi.org/10.6084/m9.figshare.6911729.v1). Nitrospina MAGs were the only ones with predicted nitrite-oxidizing metabolism, matching observations from 2012 , including dissimilatory nitrate reduction to ammonium, and a few had predicted capacity for sulfur lithotrophy and possible autotrophy  (see Table S1 at https://doi.org/10.6084/m9.figshare.6911729.v1). Future comparisons of these data with those from other low-DO systems will illuminate common functional features associated with hypoxia and also provide information about biogeographic distinctions among taxa associated with these regimes.Metabolic reconstruction and carbohydrate-active enzyme (CAZyme) prediction were also completed as described  database under the accession numbers tandards . The ann


Serratia marcescens strains cause serious nosocomial infections in humans. Here, we present the annotated genome sequence of S. marcescens podophage Pila. Similar to its closest relative, Enterobacteria phage T7, Pila has a 38,678-bp genome, predicted to encode 51 protein-coding genes, and contains 148-bp direct terminal repeats.Multidrug-resistant  Serratia marcescens strains cause serious nosocomial infections in humans. Here, we present the annotated genome sequence of S. marcescens podophage Pila. Similar to its closest relative, Enterobacteria phage T7, Pila has a 38,678-bp genome, predicted to encode 51 protein-coding genes, and contains 148-bp direct terminal repeats.Multidrug-resistant  Serratia marcescens, a facultative, Gram-negative bacterium of the family Enterobacteriaceae, causes nosocomial infections in severely immunocompromised or critically ill patients  aerobically at 30°C and 37°C in LB broth and agar (BD). This phage was plaque purified, via the soft-agar overlay method (www.bioinformatics.babraham.ac.uk/projects/fastqc) was used for quality control of the 4,413 total reads in the phage-containing index, and reads were trimmed with the FastX Toolkit v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit). SPAdes v3.5.0 was used with default parameters to assemble a single contig at 14.9-fold coverage , using default parameters unless otherwise stated . PhageTerm analysis predicted the genomic termini to be 148-bp direct terminal repeats, typical for T7-like phages .The 38,678-bp double-stranded DNA genome of podophage Pila has a G+C content of 48.8%. The 51 predicted protein-coding genes correspond to a coding density of 92.5%. Notably, Pila shares 83.7% nucleotide identity with e phages , which iMN098329, BioProject accession no. PRJNA222858, SRA accession no. SRR8892142, and BioSample accession no. SAMN11408657.The genome sequence and associated data for phage Pila were deposited under GenBank accession no.


Its’ LOD is more than 74 times lower than the traditional Cu@Ni CSNPs modified working electrode. It also has higher sensitivity and wider linear range. This indicates the great potential of applying this kind of nano composites in electrode modification.In present work, a highly sensitive biosensor with high selectivity for glucose monitoring is developed based on novel nano-composites of nitrogen doped graphene quantum dots (N-GQDs) and a novel bimetallic Cu/Ni core-shell nanoparticles (CSNPs) (Cu@Ni CSNPs/N-GQDs NCs). With the tuned electronic properties, N-GQDs helped bimetallic core-shell structure nanomaterials from aggregation, and separate the charges generated at the interface. This novel nano-composites also have the good electrical conductivity of N-GQDs, catalyst property of Cu/Ni bimetallic nano composite, Cu@Ni core-shell structure and the synergistic effect of the interaction between bimetallic nano composite and N-GQDs. While modified the electrode with this novel nano-composites, the sensor’ linear range is 0.09 ~ 1 mM, and the limit of detection (LOD) is 1.5 μM (S/N = 3) with a high sensitivity of 660 μA mM With the rapid development of nanotechnology, a lot of carbon materials have emerged in recent years, such as fullerenes, multi-wall carbon nanotubes, carbon nanofibers, graphene, graphenOwing to these properties, GQDs have gained wide attention for their enormous potential in varies applications. For instance they are widely applied in photovoltaics, organic light emitting diodes, fuel cells, photocatalysis, bioimaging, biosensing, biomedicine, environmental monitoring, thermal interface materials etc–8. Biose2 NTs[core-shellnanostructure of Ni/Cu bimetallic have attracted researchers’ attention for glucose sensing[On the other hand, various nano-structured metals, alloys and metal oxides have been extensively applied in non-enzymatic electrode modification, due to their increased specific surface area, rapid mass transport, significant catalytic activity and so on–22. Amon2 NTs, Ni/Cu/M2 NTs are all 2 NTs–39. On te sensing.-1 cm-2. Comparing with the sensor based on Cu@Ni CSNPs modified working electrode, the electrode modified with Cu@Ni CSNPs/ N-GQDs NCs makes the sensor’s LOD more than 74 times lower, higher sensitivity and wider linear range. These indicate that well designed N-GQDs’ composites have great application prospects in improving electron migration rate and electrocatalytic performance of electrode surface.In this work, the novel bimetallic Cu/Ni and N-GQDs nano-composites (Cu@Ni CSNPs/N-GQDs NCs) have been synthesized by hydrothermal method and a one-pot solvothermal method. With our gentle synthesis method, the size of N-GQDs can be uniform with the nitrogen content about 12.93%. The size of Cu @ni CSNPs/N-GQDs NCs is 30–80 nm, and N-GQDs is uniformly coated on its surface, with bimetallic Cu/Ni co-catalytic performance and good electrical conductivity. A series of non-enzymatic glucose sensors are constructed with Cu@Ni CSNPs/N-GQDs NCs modified glassy carbon electrode (Cu@Ni CSNPs/N-GQDs/GCE), Cu@Ni CSNPs/GCE and Cu@Ni CSNPs/N-GQDs/GCE. Their electrochemical properties and electrocatalytic activities are compared. It is excited that Cu@Ni CSNPs/N-GQDs/GCE shows the best electrocatalytic performance for glucose oxidation, and displays the lowest detection limit(LOD) 1.5 μM (S/N = 3), a wider linear range from 0.09 mM to 1 mM and high sensitivity 660 μA mM2·2H2O, 99.0%), Nickel (II) chloride hexahydrate , NaOH (96.0%), AA (≥99.7%), sucrose, glucose and NaCl (≥99.5%) were purchased from XiLong Scientific. Citric acid (99.5%) and DA (98%) were purchased from Aladdin. 5wt% of Nafion was purchased from Sigma Aldrich. Ethylene glycol , maltose, fructose, D-galactose and UA (99%) were purchased from Sinopharm Chemical Reagent Co., Ltd. Ultrapure water  was used throughout the experiments, and all reagents which were not mentioned above were of analytical grade.Urea (≥99.0%), N, N-dimethyl formamide , Copper (II) chloride dihydrate (CuCl2 20), ultraviolet-visible spectra , Fourier transform infrared spectra  and X-ray diffraction measurement . All electrochemical experiments were performed on the electrochemical workstation . The working electrodes were modified glassy carbon electrode , the reference electrode was saturated calomel electrode (SCE), and a platinum wire electrode was usedas the auxiliary electrode.The samples were characterized by X-ray photoelectron spectra , transmission electron microscopy (TEM) and high resolution transmission electron microscope , Ipc = 6.1 ×10−3 v + 0.45125 (R2 = 0.99109), as shown in Ipa = -5.12 ×10−3 v—0.0932 (R2 = 0.99914), Ipc = 2.56 ×10−3 v + 0.06168 (R2 = 0.99952), as shown in 3+ and Ni2+ species[The modified Cu@Ni CSNPs/Nafion/GCE and Cu@Ni CSNPs/N-GQDs/Nafion/GCE electrodes were placed in cell containing 0.1 M NaOH solution to be tested at different scan rates. As shown in + species,42.-1. In Ipa = -0.00127 c—2.12802 (R2 = 0.95372), Ipc = 2.34641 ×10−5 c + 0.59356 (R2 = 0.63127). In Ipa = - 8.9 ×10−4 c—0.9307 (R2 = 0.99045), Ipc = -1.8 ×10−4 c + 0.3803 (R2 = 0.99435). Compared with the above two sets of experiments, Cu@Ni CSNPs/N-GQDs/Nafion/GCE has better linearity and better determination of glucose concentration.CVs was used to study the relationship between current and glucose concentration. The CVs was obtained by placing modified electrodes in solutions containing different glucose concentrations at scan rate of 100 mV si-t curve test.The CVs of modified Cu@Ni CSNPs/N-GQDs/Nafion/GCE electrode at different scan rates in 0.1 M NaOH containing 10 μM glucose is shown in i-t curve of Cu@Ni CSNPs/Nafion/GCE at different glucose concentrations. The linear relationship between current and glucose concentration is shown in i = 0.0853 c—0.00018, R = 0.99973). The detection limit of glucose using Cu@Ni CSNPs/Nafion/GCE is found to be 111.4 μM (S/N = 3) with the sensitivity of 85.3 μA mM-1 cm-2. As shown in i-t curve of Cu@Ni CSNPs/N-GQDs/Nafion/GCE at different glucose concentrations. The linear relationship between current and glucose concentration is shown in i = 0.66 c + 0.125, R = 0.99952). The detection limit of glucose using Cu@Ni CSNPs/N-GQDs/Nafion/GCE is found to be 1.5 μM (S/N = 3) with the sensitivity of 660 μA mM-1 cm-2. From this, it can be clearly concluded that Cu@Ni CSNPs/N-GQDs/Nafion/GCE is more sensitive to glucose determination. This is because GQDs itself is conductive, and N-GQDs has more hole electron pairs due to nitrogen atoms have been successfully doped, which greatly improve the electrical conductivity. Glucose is catalyzed by Cu and Ni in the composites, and N-GQDs are coated on the surface of Cu@Ni CSNPs, which improves the electron mobility between the electrode and the electrolyte, so that the working electrode to detect glucose within a very short time, which greatly improves the sensitivity of the electrode and reduces the detection limit, providing conditions for real-time, rapid and accurate determination of glucose concentration.Under the optimal conditions, +0.6 V is selected as the constant potential in the range of anodic potential of +0.5 ~ 0.7 V. In The anti-interference property and selectivity for glucose determination are crucial in the development of glucose biosensors. Chemical species such as uric acid (UA), dopamine (DA), ascorbic acid (AA), and NaCl that easily oxidize are always present with glucose in human blood. In this study, interference experiments were detected by adding 0.1 mM interference component to a 0.1 M NaOH solution containing 0.5 mM glucose. As shown in -1 cm-2, rapid response time (3 s). Comparing with the sensor based on Cu@Ni CSNPs modified working electrode, this novel nano-composite of Cu@Ni CSNPs/ N-GQDs NCs makes the sensor’s LOD more than 74 times lower, also has higher sensitivity and wider linear range. Furthermore, the interference components show insignificant interference in determination of glucose, and Cu@Ni CSNPs/N-GQDs/Nafion/GCE has high selectivity for glucose determination. The results indicate that the biosensor based on Cu@Ni CSNPs/N-GQDs/Nafion/GCE has potential application prospect in the determination of glucose, the application of GQDs in biosensor has a great prospect. It also indicates the great potential to apply this kind of nano composites in electrode modification and high sensitivity biomolecule detection.In summary, Cu@Ni CSNPs and Cu@Ni CSNPs/N-GQDs NCs were successfully synthesized by one-pot solvothermal method. Our experiments show that the electrochemical response of Cu@Ni CSNPs/N-GQDs/Nafion/GCE to glucose determination was the highest. Cu@Ni CSNPs/N-GQDs/Nafion/GCE has the advantages of low cost, high sensitivity and good selectivity. One more advantage of Cu@Ni CSNPs/N-GQDs/Nafion/GCE is its’ wide linear range is 0.09 ~ 1 mM. And the detection limit is 1.5 μM (S/N = 3), high sensitivity of 660 μA mMS1 Table(TIF)Click here for additional data file.S1 FigA) full spectrum, (B) C1s spectrum, (C) N1s spectrum, (D) O1s spectrum.((TIF)Click here for additional data file.S2 FigA) C1s spectrum, (B) Cu2p spectrum, (C) Ni2p spectrum.((TIF)Click here for additional data file.S3 FigMapping of Cu@Ni CSNPs/N-GQDs NCs (a), mapping-Cu (b), mapping-Ni (c).(TIF)Click here for additional data file.S4 FigA) and the histogram (B) of the Cu@Ni CSNPs/N-GQDs/GCE with successive addition of 0.5 mM glucose, 0.1 mM DA, 0.1 mM AA, 0.1 mM UA, 0.1 mM NaCl and 0.5 mM glucose in 0.1 M NaOH solution at +0.6 V, respectively.Amperometric response ((TIF)Click here for additional data file.S5 FigA) and the histogram (B) of the Cu@Ni CSNPs/N-GQDs/GCE with successive addition of 0.5 mM glucose, 0.1 mM maltose, 0.1 mM sucrose, 0.1 mM fructose, 0.1 mM D-galactose, 0.5 mM glucose in 0.1 M NaOH solution at +0.6 V, respectively.Amperometric response ((TIF)Click here for additional data file.


Testing sampling effort and relative abundance descriptors of belowground ectomycorrhizal fungi in a UK planted scots pine woodland. Mycology. Agerer R, Ammirati J, Blanz P, Courtecuisse R, Desjardin DE, Gams W, Hallenberg N, Halling R, Hawksworth DL, Horak E, Korf RP, Mueller GM, Oberwinkler F, Rambold, G, Summerbell RC, Tribel D and Watling R. 2000. Open letter to the scientific community of mycologists. Canadian Journal of Botany. 78:981–983Agerer R. 1991. Characterization of ectomycorrhiza. In: Norris JR, Read DJ, Varma AK, editors. Techniques for the study of mycorrhiza. Methods in microbiology. 23:25–73.Palmer MW. 1991. Estimating species richness: the second order jackknife reconsidered. Ecology. 72:1512–1513.Erland S and Taylor AFS. 2002. Diversity of ecto-mycorrhizal fungal communities in relation to the abiotic environment. In: van der Heiden MGA and Sanders IR, editors. Mycorrhizal ecology. Ecological Studies. Berlin: Springer; p. 163–200.When the above article was first published online, the following references were omitted by mistake:The caption for Figure 2 incorrectly read:“(a) Illustration of transect line (blue dashed line) and replicated sampling points (red dots). (b) Field sampling procedure in stand 8. (1) transect line, (2) soil samples in a bucket, (3) soil corer. (c) Detail of soil sample (1) in the soil corer (2) (3) Ruler 15 cm.’’This has now been corrected to:“(a) Illustration of transect line (dashed line) and replicated sampling points (dots). (b) Field sampling procedure in stand 8. (1) transect line, (2) soil samples in a bucket, (3) split soil core sampler. (c) Detail of soil sample (1) in the split soil core sampler (2) (3) Ruler 15 cm.’’The caption for figure 5 incorrectly read:“Result of regression analysis: r2 = 0.676**, p < 0.01**.’’This has now been corrected to:“Result of regression analysis: r2 = 0.676, p < 0.01.’’In Table 1 the reference to Agerer, mistakenly listed the year as 2006 in three places, this has now been corrected to Agerer 1987–2002.These items have now been corrected in both the print and online versions of the journal.Taylor and Francis apologises for these errors.


This article has been corrected: During the assembly of Figure 2B, the same image was inadvertently used for both H1299 (pEGFP-C3) and H1299 (pEGFP-4.1N) groups at 0h time point. The proper Figure 2 B specific for H1299 (pEGFP-C3) and H1299 (pEGFP-4.1N) groups are shown below. The authors declare that these corrections do not change the results or conclusions of this paper.509-523. https://doi.org/10.18632/oncotarget.6312Original article: Oncotarget. 2016; 7:509–523.


Butia ordorata) and fishtail palm (Caryota uren), using liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF/MS) and assess their antioxidant potential. The total phenolic content (TPC), total tannins content (TTC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant assay and 2,2′-azinobis-(3-ethylbenzo-thiazoline-6-sulfonic acid) (ABTS) scavenging abilities and ferric reducing antioxidant power (FRAP) were higher in the jelly palm fruit while total flavonoid contents (TFC) were higher in the fishtail palm. The LC-ESI-QTOF/MS tentatively identified a total of 86 phenolic compounds in both jelly and fishtail palm fruits. Although both palm fruits exhibited different phenolic profiles, hydroxycinnamic acids and flavonols were the most common in both. In high performance liquid chromatography photodiode array (HPLC-PDA) quantification, 4-hydroxybenzoic acid (317.46 ± 4.68 µg/g) and catechin (4724.00 ± 32.39 µg/g) were the most abundant phenolic acid and flavonoid quantified in the jelly palm fruit, respectively. Quercetin (557.28 ± 7.81 µg/g) and kaempferol 3-O-glucoside (220.99 ± 2.06 µg/g) were the most abundant flavonoids quantified in the fishtail palm. Our study indicates that palm fruit is a good source of polyphenols and has strong antioxidant potential for health promotion. Furthermore, this study provides the scientific basis for an exploitation of jelly and fishtail palm fruits in the food, pharmaceutical and nutraceutical industries. Palm fruits have gained growing attention for their nutrition values and health promotion perspectives. They have a diverse range of bioactive compounds including carotenoids, vitamins, dietary fibres and especially polyphenolic compounds. These polyphenolic compounds contribute to the putative health benefits of palm fruits. Nevertheless, the detailed information about these polyphenols in palm fruits is limited. The present work was conducted to comprehensively characterize polyphenols in two palm fruits, jelly palm ( Palm fruits are mostly grown in tropical regions and are rich sources of bioactive compounds such as polyphenols, vitamin E, carotenoids and unsaturated fatty acids . BioactiButia ordorata) is a tropical palm species whose fruits and leaves can be processed into different food products +m/z 339.1081) and 3-O-methylrosmarinic acid ([M + H]+m/z 375.1069) were tentatively characterized in fishtail palm. Hoffman et al. [p-coumaroylquinic acid (p-coumaric acid) in the methanolic extract of jelly palm pulp and nectar.Compound 2 and 5 (retention time (RT) = 19.734 and 37.691 min) with [M − H]ectively , while c355.1017 . There wion mode , while flm fruit . Sinapinly palms . The ferly palms . The prely palms ,29,30. C+ at m/z 139.0394 and 4-hydroxybenzoic acid 4-O-glucoside with RT = 7.456 min and [M − H]− at m/z 299.0793 in extracts of jelly and fishtail palm, respectively (m/z 137.0242) and hydroxybenzoic hexose (m/z 299.0070) have already been characterized in jelly palm by Hoffmann, Carvalho, Barbieri, Rombaldi and Chaves + at m/z 167.0687 and 227.0916 were assigned as 3-hydroxyphenylpropionic acid and dihydrosinapic acid, while 3-hydroxy-3-(3-hydroxyphenyl) propionic acid was tentatively identified by a molecular ion at m/z 181.0513 in negative ionization mode. The hydroxyphenylpentanoic acid compounds conjugated with valerolactone were proposed for both palm fruits  and 5--γ-valerolactone ([M − H]−m/z 207.0667) were detected  and 23  detected in ethanolic extract of fishtail palm fruits were tentatively identified as kaempferol 3-O-glucosyl-rhamnosyl-galactoside and kaempferol 3-O-glucoside, respectively, in negative ionization mode (O-glucoside (14) tentatively identified by the parent ion at m/z 449.1072 in ESI+ mode , quercetin 3-O-rutinoside (16), and quercetin (17) exhibiting precursor ion at m/z 465.1008, 611.1579 and 303.0486 in positive ionization mode were proposed for jelly palm fruit (O-glucoside (RT = 37.133 min) giving precursor ion at m/z 463.0878 in ESI− mode . The detection of aglycone quercetin in the jelly palm was reported previously d Chaves  also cha studies . Compounm/z 595.1634 and 595.163, respectively, was tentatively characterized in extracts of jelly and fishtail palm fruits. Compound 21  in the jelly palm and compound 32  in the fishtail palm were tentatively identified as apigenin 7-O-apiosyl-glucoside and apigenin 6-C-glucoside, respectively. The aglycone apigenin has been identified in different solvent extracts of jelly palm [− ion at m/z 461.1093 and [M + H]+ ion at m/z 549.1220, were proposed as chrysoeriol 7-O-glucoside and chrysoeriol 7-O-, respectively  and fishtail palm fruit , respectively. The aglycone hesperetin displaying [M − H]− ion at m/z 301.0707 has been proposed for jelly palm fruits in the study of Hoffmann, Carvalho, Barbieri, Rombaldi and Chaves + ion at m/z 481.1340 was proposed as 3’-O-methyl-(-)-epicatechin 7-O-glucuronide. Catechin has also been detected in ethanolic extracts of jelly palm in a study reported by Beskow, Hoffmann, Teixeira, Fachinello, Chaves and Rombaldi Rombaldi . ProcyanO-sambubioside-5-O-glucoside (RT = 25.648 min) and peonidin 3-O-diglucoside-5-O-glucoside (RT = 26.857 min) exhibiting precursor ions at m/z 756.2092 and 786.2195 were detected in jelly palm (O-sophoroside (RT = 44.148 min) with parent ions at m/z 624.1664 was tentatively characterized in fishtail palm (m/z 772.2033 and 564.1457 were assigned as cyanidin 3-O-diglucoside-5-O-glucoside (29) and pelargonidin 3-O-sambubioside (32), respectively. In fishtail palm fruit, compound 40 exhibiting precursor ion at m/z 520.1225 was proposed as petunidin 3-O-(6’’-acetyl-glucoside). Previously, two cyanidin derivative anthocyanins were characterized in jelly palm fruit + ion at m/z 151.1108 was detected in fishtail palm. Pyrogallol was the only compound belonging to other polyphenols subclass detected in both palm fruits in the positive mode of ionization (Compound 54 (RT = 33.745 min) and 55 RT = 79.779 min) tentatively characterized in the extract of fishtail palm fruit with [M − H]opoletin . 3-Methynization . Previou.779 min The screening and characterization of polyphenolic compounds showed that some of the polyphenols presented in two palm fruits have strong antioxidant potential. Hydroxycinnamic acid derivatives, hydroxybenzoic acids and their derivatives, protocatechuic acid, chlorogenic acid, catechin, matairesinol, hydroxytyrosol, quercetin and kaempferol derivatives are regarded as potential compounds showing considerable free radical scavenging capacity ,38,39. TO-glucoside, catechin, quercetin and kaempferol 3-O-glucoside) based on the LC-ESI-QTOF/MS characterization and previously reported antioxidant activities. The quantification of individual polyphenols was computed considering the UV–Vis absorption and calibration curves of external standards.Seven polyphenols were targeted to quantify through HPLC-PDA including three phenolic acids  and four flavonoids (quercetin 3-fw) was higher than fishtail palm fruit (140.23 ± 1.78 µg/g fw). Boeing et al. [fw) was only detected in jelly palm fruit, while protocatechuic acid  was only found in fishtail palm. Beskow, Hoffmann, Teixeira, Fachinello, Chaves and Rombaldi [The polyphenols quantified through HPLC-PDA in the jelly palm were higher than in fishtail palm. These HPLC results support our TPC values determined using the Folin–Ciocalteu method. Among the three selected phenolic acids, chlorogenic acid was the only phenolic acid detected in both palm fruits . The cong et al.  already fw in jelly palm and 557.28 ± 7.81 µg/g fw in fishtail palm fruit. Beskow, Hoffmann, Teixeira, Fachinello, Chaves and Rombaldi [fw), which might be involved in higher antioxidant activity.Flavonols were found to be higher in fishtail palm fruit as compared to jelly palm fruits. Quercetin was the major compound in this group, with a concentration of 360.19 ± 4.53 µg/g It has been established in this work that LC-ESI-QTOF/MS is an effective and powerful analytical tool to characterize most of the polyphenols present in jelly and fishtail palm fruits. The TPC, TTC, DPPH, FRAP and ABTS scavenging activity was higher in jelly palm compared to fishtail palm. LC-ESI-QTOF/MS characterizes a total of 42 and 60 phenolic compounds in jelly and fishtail palm fruits, respectively. Hydroxycinnamic acids and flavonols were the most common polyphenols reported in both palm fruits. The HPLC-PDA enabled the quantification of some targeted phenolic compounds and found that catechin and quercetin were the most abundant polyphenols in jelly and fishtail palm, respectively. In short, both palm fruits are a good source of polyphenols and could be utilized in food, feed and pharmaceutical industries.


P < 0.001, RFS; P < 0.001), cytoplasmic expression of FAM83H , nuclear expression of PANX2 , cytoplasmic expression of PANX2 , co-expression pattern of nuclear FAM83H and nuclear PANX2 . In multivariate analysis, nuclear expression of FAM83H  and the co-expression pattern of nuclear FAM83H and PANX2  were independent indicators of shorter survival of CCRCC patients. Cytoplasmic expression of FAM83H was associated with shorter RFS (P = 0.030) in multivariate analysis. In Caki-1 and Caki-2 CCRCC cells, knock-down of FAM83H decreased PANX2 expression and cell proliferation, and overexpression of FAM83H increased PANX2 expression and cell proliferation. These results suggest that FAM83H and PANX2 might be involved in the progression of CCRCC in a co-operative manner, and their expression might be used as novel prognostic indicators for CCRCC patients.FAM83H is primarily known for its role in amelogenesis; however, recent reports suggest FAM83H might be involved in tumorigenesis. Although the studies of FAM83H in kidney cancer are limited, a search of the public database shows a significant association between FAM83H and pannexin-2 (PANX2) in clear cell renal cell carcinomas (CCRCCs). Therefore, we evaluated the clinicopathological significance of the immunohistochemical expression of FAM83H and PANX2 in 199 CCRCC patients. The expression of FAM83H and PANX2 were significantly associated with each other. In univariate analysis, individual, and co-expression pattern of FAM83H and PANX2 was significantly associated with shorter overall survival (OS) and relapse-free survival (RFS) of CCRCC patients: nuclear expression of FAM83H (OS;  Family with sequence similarity 83 H (FAM83H) is one of the eight FAM83 family members, from FAM83A to FAM83H , 2. Accohttp://www.oncolnc.org). In addition, the cBioPortal database  indicated pannexin-2 (PANX2) as the gene most significantly associated with FAM83H expression in CCRCCs   is the most common type of kidney cancer . Most CCn CCRCCs , 12. Altst 2018) , 12. PANst 2018) . Howeverst 2018) . TherefoThis study evaluated CCRCC patients who underwent surgical resection between July 1998 and August 2011. Among them, 199 CCRCCs with complete medical records were available with original H&E slides, and paraffin-embedded tissue blocks. Clinical information was obtained by reviewing the medical records. The histopathologic information was retrospectively reviewed according to the 2016 World Health Organization classification of the renal tumors , and staIn this study, tissue microarrays were used for immunohistochemical staining. The tissue microarrays have 3.0 mm cores, and one core was arrayed per case from the areas composed mainly of tumor cells with no degenerative change or necrosis. The histologic sections from the tissue microarray tissue blocks were deparaffinized and boiled for 20 min in pH 6.0 antigen retrieval solution  in a microwave oven. Primary antibodies for FAM83H  and PANX2  was used. The slides were visualized with the enzyme substrate 3-amino-9-ethylcarbazole and counterstained with hematoxylin. The scoring for the immunohistochemical staining slides was performed without clinical information by two pathologists (KYJ and KMK) with consensus under a multi-viewing microscope. The immunohistochemical positivity for FAM83H and PANX2 was separately scored according to their expression patterns in the cytoplasm and nuclei. The immunohistochemical scores were obtained by adding their staining intensity score ranging from zero to three  and staining area score ranging from zero to five  , 19. The2 and 37°C.In this study, we used two human CCRCC cell lines. Caki-1 and Caki-2 cells were purchased from the Korean Cell Line Bank . The Caki-1 and Caki-2 cells were cultured in DMEM medium with 10% fetal bovine serum  and streptomycin and penicillin (100 U/ml) in 5% COThe vector of shRNA for FAM83H was purchased from GenePharma . The sense and antisense sequences of the FAM83H shRNA were the 5′-CAC CGC TCA TCT TCA GCA CGT CAC ATT CAA GAG ATG TGA CGT GCT GAA GAT GAG CTT TTT TG-3′ and 5′-GAT CCA AAA AAG CTC ATC TTC AGC ACG TCA CAT CTC TTG AAT GTG ACG TGC TGA AGA TGA GC-3′, respectively. The vector for FAM83H overexpression  was purchased from GeneCopoeia . JetPRIME transfection reagent  was used for transfection.To isolate protein, the cells were lysed with PRO-PREP Protein Extraction Solution  with 1x phosphatase inhibitor cocktails 2, 3 (Sigma-Aldrich). The primary antibodies for FAM83H , PANX2 , and actin (Sigma-Aldrich) were used for western blots.RNA was isolated with an RNeasy Mini Kit . The isolated RNA was reverse transcribed by probing 1.5 μg RNA with Reverse Transcription kits  to generate cDNA that was used for quantitative polymerase chain reaction using Applied Biosystems Prism 7900HT sequence Detection System and SYBR Green Master PCR Mix (Applied Biosystems). The values were normalized to the expression of the glyceraldehyde-3-phosphate dehydrogenase reference housekeeping gene. All experiments were performed in triplicate. The sequences of the primers used in quantitative reverse-transcription polymerase chain reaction are listed in Table 5) and Caki-2 (2 × 105) cells were seeded in 24-well plates and the number of viable cells was counted by using hemocytometer. For the MTT assay, Caki-1 (3 × 103) and Caki-2 (3 × 103) cells were seeded in 96-well culture plates, and absorbance was measured at 560 nm using a microtiter plate reader .The proliferation of cells was evaluated by counting the number of cells and performing a 3--2,5-diphenyltetrazonium bromide (MTT)  assay and a colony-forming assay. To compare the number of cells in control, FAM83H knock-down, and FAM83H overexpression groups, Caki-1  was used throughout, and P < 0.05 were considered statistically significant.The cut-off points of immunohistochemical staining scores for FAM83H and PANX2 were determined by receiver operating characteristic curve analysis , 19. SurP < 0.001), Cy-FAM83H (P < 0.001), Nu-PANX2 (P < 0.001), and Cy-PANX2 (P = 0.002) was significantly associated with death of patients from CCRCC  and higher tumor stage (P = 0.001) (Table P = 0.004), larger tumor size (P < 0.001), higher tumor stage (P < 0.001), and higher histologic grade (P = 0.037) (Table P = 0.009), tumor size (P = 0.001), tumor stage (P < 0.001), and tumor necrosis (P = 0.003) (Table P = 0.032), tumor size (P < 0.001), tumor stage (P < 0.001), lymph node metastasis (P = 0.018), nuclear grade (P = 0.006), and tumor necrosis (P = 0.011) , tumor size (P < 0.001), tumor stage (P < 0.001), tumor necrosis (P = 0.006), Nu-FAM83H (P < 0.001), Cy-FAM83H (P < 0.001), Nu-PANX2 (P < 0.001), and Cy-PANX2 (P < 0.001) (Table P = 0.033), age (P = 0.022), tumor size (P < 0.001), tumor stage (P < 0.001), lymph node metastasis (P = 0.010), nuclear grade (P = 0.006), Nu-FAM83H (P < 0.001), Cy-FAM83H (P < 0.001), Nu-PANX2 (P < 0.001), and Cy-PANX2 (P < 0.001) , Nu-FAM83H , Cy-FAM83H , Nu-PANX2 , and Cy-PANX2 positivity  are presented in Figure Nuclear positivity of FAM83H expression had a 6.205-fold  greater risk of death and a 4.949-fold  greater risk of relapse or death of CCRCC patients. Cy-FAM83H positivity showed a 4.617-fold  greater risk of death and a 5.369-fold  greater risk of relapse or death of CCRCC patients (Table P = 0.047), tumor stage (P = 0.018), tumor necrosis (P = 0.031), and Nu-FAM83H positivity (P < 0.001) (Table P < 0.001), Nu-FAM83H positivity (P = 0.003), and Cy-FAM83H positivity (P = 0.030) were the factors significantly associated with RFS of CCRCC patients in multivariate analysis , intermediate (Nu-FAM83H+/Nu-PANX2−), and poor prognostic (Nu-FAM83H+/Nu-PANX2+) groups that were significantly associated with OS  and RFS  in univariate analysis  greater risk of OS and 4.903-fold  greater risk of RFS compared with the Nu-FAM83H−/Nu-PANX2− and Nu-FAM83H−/Nu-PANX2+ subgroups  greater risk of lower OS and a 160%  greater risk of lower RFS compared with the Nu-FAM83H−/Nu-PANX2− and Nu-FAM83H−/Nu-PANX2+ subgroups , higher expression of FAM83H RNA was associated with shorter OS of CCRCC   predicted shorter survival of hepatocellular carcinoma patients . In a reConcerning the mechanism how FAM83H is involved in tumorigenesis, the role of FAM83H in the proliferation of cancer cells has been suggested in prostatic cancer cells . In addihttps://www.proteinatlas.org) also reported FAM83H expression to be in the cytoplasmic membrane and cytoplasm. In colorectal cancer cells, FAM83H expression was localized in the cytoplasm in association with keratin cytoskeleton structures   , 12, theP = 0.004) and RFS (P = 0.015). The patients with Nu-PANX2-positive CCRCC had a 3.186-fold  greater risk of cancer-related death and a 2.302-fold  greater risk of relapse or cancer-related death compared with the patients with Nu-PANX2-negative CCRCC. Therefore, despite limited reports on the role of PANX2 in human cancers, our results suggest PANX2 as a potential biologic marker of CCRCC. In addition, when we searched the OncoLnc database (http://www.oncolnc.org), higher expression of PANX2 RNA was associated with shorter OS of CCRCC patients  greater risk of death compared to CCRCC patients with low expression of PANX2 RNA. However, in contrast, PANX2 inhibited proliferation and tumor formation of C6 glioma cells in in vitro and in vivo   , 12. Morn; 0.36) . In addiIn conclusion, this study presents the roles for FAM83H and PANX2 in CCRCCs and suggests that FAM83H and PANX2 are closely associated and involved in the progression of CCRCCs. Especially, the expression patterns of FAM83H and PANX2 were significantly associated with shorter survival of CCRCC patients. In addition, the co-expression patterns of FAM83H and PANX2 were also significantly associated with the survival of CCRCC patients. Therefore, individual and co-expression patterns of FAM83H and PANX2 might be useful prognostic indicators for CCRCC patients. Furthermore, understanding for the role of FAM83H in conjunction with PANX2 might be helpful in establishing a new therapeutic strategy for CCRCCs.KMK, UH, JB, S-HP, KSK, SH, HP, HL, MC, WM, MK, and KJ participated in the study design. KMK, UH, and JB performed the experiment. KMK, UH, JB, S-HP, KSK, SH, HP, HL, MC, WM, MK, and KJ were involved in data collection and data interpretation. KMK, UH, JB, S-HP, KSK, SH, HP, MC, WM, and KJ participated in the statistical analyses. KMK, UH, JB, S-HP, KSK, SH, HP, HL, MC, WM, MK, and KJ wrote the manuscript. All authors read and approved the final manuscript.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.


The correct citation is: Bispo PC, Balzter H, Malhi Y, Slik JWF, Santos JR, Rennó CD, et al. (2017) Drivers of metacommunity structure diverge for common and rare Amazonian tree species. PLoS ONE 12(11): e0188300. In the Funding section, the author Fernando D. Espírito-Santo’s initials are listed incorrectly. The sentence should read: FDES was supported by Natural Environment Research Council (NERC) grants (BIO-RED NE/N012542/1 and AFIRE NE/P004512/1) and Newton Fund (The UK Academies/FAPESP Proc. N°: 2015/50392-8 Fellowship and Research Mobility).


The family environment is often overlooked in caregiver research and assessment, despite having implications for caregiver health and well-being . The purpose of the present study was to examine differences on two types of family conflict (beliefs and support) among a diverse sample of caregivers. The present sample consisted of help-seeking (n = 375) and non-help-seeking (n = 415) caregivers . Caregivers filled out the Caregiver Reaction Scale , a multidimensional assessment of the caregiver experience. Results of a 2  x 2  ANOVA indicated that help-seeking caregivers reported significantly more conflict over family beliefs than did non-help-seeking caregivers , F = 21.10 p < .001. Adult children caregivers reported significantly greater conflict over family beliefs (M = 1.91) than did spouse caregivers (M = 1.60), F = 10.66, p < .001. Adult children caregivers also reported significantly greater conflict over family support (M = 1.87) than did spouse caregivers (M = 1.57), F = 16.23, p < .001. Results highlight that certain caregiving contexts  potentially increase family conflict, which has implications for caregiver burden. Family conflict over beliefs is also related to help-seeking in caregivers. Findings inform appropriate assessment and intervention regarding the family environment in caregiving.