Biochar mitigates the stimulatory effects of straw incorporation on N2O emission and N2O/(N2O + N2) ratio in upland soil.

Straw incorporation, a common agricultural strategy designed to enhance soil organic carbon (SOC), often leads to increased nitrous oxide (N2O) emission, potentially offsetting benefits of SOC sequestration. However, the mechanism and mitigation options for the enhanced N2O emission following straw incorporation remain unclear. Here, N2 and N2O emission rate, as well as N2O/(N2O + N2) ratio under four different fertilization treatments [i.e., non-fertilization (Control), conventional chemical fertilization (CF), conventional chemical fertilization plus straw incorporation (SWCF), and conventional chemical fertilization plus straw and biochar incorporation (SWBCF)] were investigated by a robotized sampling and analysis system. High-throughput sequencing was also employed to assess the variation of bacterial community across different treatments. The results showed CF, SWCF, and SWBCF fertilization treatments significantly increased N2O emission rate by 1.04, 2.01, and 1.29 folds, respectively, relative to Control treatment. Albeit no significant enhancements in N2 emission rate, the N2O/(N2O + N2) ratio significantly increased by 65.53%, 1.10 folds, and 69.49% in CF, SWCF, and SWBCF treatments, respectively. The partial least squares path modeling analysis further revealed that fertilization treatments slightly increased N2 emission rate by increasing DOC content and keystone OTUs abundance. While the enhanced N2O emission rate and N2O/(N2O + N2) ratio in the fertilization treatments was primarily determined by reducing DOC/NO3- ratio and specific bacteria module abundance dominated by Gaiellales, Solirubrobacterales, and Micrococcales. Furthermore, SWBCF treatment alleviated the increase in net global warming potential due to straw incorporation, as indicated by the higher SOC sequestration and lower N2O/(N2O + N2) ratio therein. Collectively, these findings suggest that simultaneous application of straw and biochar has the potential to mitigate the risk of increased N2O emission from straw incorporation. This study provides valuable insights for developing targeted strategies in C sequestration and greenhouse gas mitigation, tackling the challenge presented by global climate change.

First report of Neopestalotiopsis rosae causing leaf blight on Shatangju in southern China.

Shatangju (Citrus reticulata Blanco cv. Shatangju) belongs to genus Citrus and was cultivated extensively in southern China. In April 2022, a leaf blight-like symptom (firstly brown spots appeared on infecting leaves, then these brown spots extended, finally the whole leaves displayed blight-like symptom) was observed on 5%~10% of Shatangju seedlings (around five hundreds in total) in an orchard located in Wuhan city, Hubei, China. Diseased leaves from three seedlings were collected and cut into pieces (0.2 to 0.5 cm). These pieces were surface-sterilized using 75% ethanol for 3 min, rinsed with sterile distilled water for several times, then placed on potato dextrose agar (PDA) and incubated at 26°C with 12-h light/dark cycle. Over 20 pieces plated, wherein 30% were identified as Colletotrichum fructicola, 60% as Neopestalotiopsis spp., and 10% developed saprophytes. C. fructicola was a known pathogen on citrus, thus Neopestalotiopsis spp. was further investigated. Eight single-conidium colonies of the Neopestalotiopsis spp. were obtained, wherein STJ-8 was chosen as a representative for further study. The average growth rate of STJ-8 was 15.1±0.5 mm/day (n=5). Fungal colonies produced white cottony mycelium with abundant black acervuli distributed in concentric rings 6-8 days after planting, which ranged from 342.3 to 710.5 µm in diameter (n=100). Conidia were fusoid, five cells, four septa with average dimensions of 25.36×5.47 µm (n=100). Basal and apical cells were hyaline, wherein three middle cells were brown with darker septa. The apical cell was cylindrical with two to three transparent accessory filaments (13.7 to 30.5 µm in length, n=80). Basal cell was conic with an appendage (4.1 to 8.8 µm in length, n=40). Partial sequences of internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α), and ß-tubulin (TUB2) were amplified with reported primers (White et al. 1990; Lee et al. 2006; Maharachchikumbura et al. 2014), sequenced, and submitted to GenBank (accession nos. ITS: OP236541; TEF-1α: OP250124; TUB2:OP263094). BLASTn results showed 100% identity with the corresponding sequences of Neopestalotiopsis rosae. A multilocus phylogenetic analysis showed STJ-8 was closest to N. rosae. Thus, STJ-8 was identified as N. rosae. Pathogenicity tests were performed on one-year-old Shatangju seedlings and detached primary leaves by inoculating needle-wounded leaves with seven days old 5-mm mycelial plugs/acervuli (about 5000 spores) of STJ-8. Control seedlings/leaves were inoculated with 5-mm PDA plugs/sterile water drops. All inoculated detached leaves were cultured at same the place with STJ-8 cultured, while inoculated seedlings were put in a growth chamber at 26°C under a 16-h light/dark cycle (60% humidity). Symptoms developed on all inoculated leaves (except healthy control) 2 and 4 days post-inoculation by mycelial plugs and acervuli, respectively. N. rosae was re-isolated from the inoculated leaves, confirming Koch's postulates. N. rosae has been reported to cause diseases on various plants worldwide (Rebollar-Alviter et al. 2020; Xavier et al. 2021; Lawrence et al. 2022). In China, N. rosae has been reported to cause leaf spot/blight on pecan and strawberry (Wu et al. 2021; Gao et al. 2022), which caused great loss on these crops. To our knowledge, this is the first report of N. rosae causing leaf disease on citrus. Our study is important for developing control strategies against N. rosae in future.

Organocatalytic Enantioselective Synthesis of Axially Chiral N,N'-Bisindoles.

This study establishes the first organocatalytic enantioselective synthesis of axially chiral N,N'-bisindoles via chiral phosphoric acid-catalyzed formal (3+2) cycloadditions of indole-based enaminones as novel platform molecules with 2,3-diketoesters, where de novo indole-ring formation is involved. Using this new strategy, various axially chiral N,N'-bisindoles were synthesized in good yields and with excellent enantioselectivities (up to 87 % yield and 96 % ee). More importantly, this class of axially chiral N,N'-bisindoles exhibited some degree of cytotoxicity toward cancer cells and was derived into axially chiral phosphine ligands with high catalytic activity. This study provides a new strategy for enantioselective synthesis of axially chiral N,N'-bisindoles using asymmetric organocatalysis and is the first to realize the applications of such scaffolds in medicinal chemistry and asymmetric catalysis.

Characterization of pectin methylesterase gene family and its possible role in juice sac granulation in navel orange (Citrus sinensis Osbeck).

BACKGROUND: Citrus is one of the most important fresh fruit crops worldwide. Juice sac granulation is a physiological disorder, which leads to a reduction in soluble solid concentration, total sugar, and titratable acidity of citrus fruits. Pectin methylesterase (PME) catalyzes the de-methylesterification of homogalacturonans and plays crucial roles in cell wall modification during plant development and fruit ripening. Although PME family has been well investigated in various model plants, little is known regarding the evolutionary property and biological function of PME family genes in citrus. RESULTS: In this study, 53 non-redundant PME genes were identified from Citrus sinensis genome, and these PME genes were divided into four clades based on the phylogenetic relationship. Subsequently, bioinformatics analyses of gene structure, conserved domain, chromosome localization, gene duplication, and collinearity were performed on CsPME genes, providing important clues for further research on the functions of CsPME genes. The expression profiles of CsPME genes in response to juice sac granulation and low-temperature stress revealed that CsPME genes were involved in the low temperature-induced juice sac granulation in navel orange fruits. Subcellular localization analysis suggested that CsPME genes were localized on the apoplast, endoplasmic reticulum, plasma membrane, and vacuole membrane. Moreover, yeast one-hybrid screening and dual luciferase activity assay revealed that the transcription factor CsRVE1 directly bound to the promoter of CsPME3 and activated its activity. CONCLUSION: In summary, this study conducts a comprehensive analysis of the PME gene family in citrus, and provides a novel insight into the biological functions and regulation patterns of CsPME genes during juice sac granulation of citrus.

Solution structure of c-FLIP death effector domains.

The formation of death-inducing signaling complex (DISC) and death effector domain (DED) filament initiates extrinsic apoptosis. Recruitment and activation of procaspase-8 at the DISC are regulated by c-FLIP. The interaction between c-FLIP and procaspase-8 is mediated by their tandem DEDs (tDED). However, the structure of c-FLIPtDED and how c-FLIP interferes with procaspase-8 activation at the DISC remain elusive. Here, we solved the monomeric structure of c-FLIPtDED (F114G) at near physiological pH by solution nuclear magnetic resonance (NMR). Structural superimposition reveals c-FLIPtDED (F114G) adopts a structural topology similar to that of procaspase-8tDED. Our results provide a structural basis for understanding how c-FLIP interacts with procaspase-8 and the molecular mechanisms of c-FLIP in regulating cell death.

Metabolomics reveals the role of isopentenyl group in coumarins metabolism.

Coumarins are a group of natural compounds commonly found in the families of Rutaceae and Umbelliferae. 7-Isopentenyloxycoumarin (ISC), auraptene (AUR), and umbelliprenin (UM) belong to prenyloxycoumarins (PYCs), which link isopentenyl, geranyl, and farnesyl group at C7 position, respectively. The substituent of 7-ethoxycoumarin (ETC) is the ethyl group. In this study, UPLC-ESI-QTOF-MS (ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-MS)-based metabolomics was used to evaluate the in vivo and in vitro metabolism of PYCs. Results showed that ETC produced 10 known metabolites, and ISC was transformed into 17 metabolites in vivo and in vitro, which were undescribed compounds. A total of 35 AUR metabolites, including 34 undescribed metabolites were identified, and 21 metabolites were reported for the first time in UM. The results indicated that hydroxylation and N-acetylcysteine conjugation were the common metabolic reactions for PYCs. The metabolic rates of ETC, ISC, AUR and UM were 26%, 36%, 81%, and 38%, respectively, in human liver microsome, while they were 24%, 40%, 80%, and 37%, respectively, in mouse liver microsomes. In addition, recombinant cytochrome P450s (CYPs) screening showed that CYP1A1, 2C19, 3A4, and 3A5 were the major metabolic enzymes involved in the formation of hydroxylation metabolites. Together, these results suggest that the isopentenyl group plays an important role in the metabolism of PYCs.

First report of Fusarium oxysporum and Fusarium solani causing root rot on trifoliate orange rootstock in China.

Trifoliate orange (Poncirus trifoliata L) is a thorny tree of the Rue family, which is extensively used as citrus rootstock in China. In January 2021, several leaf yellowing, declining, and wilting citrus seedlings grafted on trifoliate orange rootstock with rotted main roots were observed in orchards located in Wuhan city, Hubei, China. In old orchards, the incidence of diseased roots was approximately 90%. Diseased roots from seven plants were collected and cut into small pieces (0.2 to 0.5 cm). These pieces were then surface-sterilized using 0.1% mercury bichloride for 3 min, 75% ethanol for 3 min, rinsed with sterile distilled water for several times, and then placed on potato dextrose agar (PDA) supplemented with 0.05% lactic acid (v/v), and incubated at at 25±2°C in dark. Fifty-threesingle-conidium isolates with morphological characteristics similar to Fusarium spp. were obtained (Leslie and Summerell 2006), which displayed two kinds of colony morphology. Thirty isolates showed white to orange-white abundant aerial mycelium in rings and acquired a yellow to orange pigmentation, tweenty-three isolates showed white to pink, fluffy aerial mycelium in rings and acquired an orange to red pigmentation. Isolate WG-1 and HrmY-9 from each group were used for future identification. The average colony growth rate of WG-1 and HrmY-9 on PDA was 0.95±0.06 and 0.69±0.11 mm/day, n=4, respectively. WG-1 produced numerous oval, unicellular microconidia without septa, 4.03-9.87×1.01-5.13 µm, n=80 and very few macroconidia with two to four septa, narrowed at both ends, 11.08-22.64×1.67-4.91 µm, n=30. HrmY-9 produced numerous curved macroconidia with three to four septa, 18.03-37.33×2.16-7.8 µm, n=80, microconidia were unicellular, oval, and 5.33-16.19×1.74-6.51 µm, n=50. Sequences of internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF-1α), and DNA-directed RNA polymerase largest subunit (RPB1) genes were amplified with the primers ITS1/ITS4, EF1a-F/EF1a-R, and RPB1-F5/RPB1-R8, respectively (White et al. 1990, O'Donnell et al. 1998, O'Donnell et al. 2010), sequenced and deposited in GenBank. Sequences of isolate WG-1 (GenBank accession No. ON045437, ON063232 and ON089664) and HrmY-9 (GenBank accession No. ON045438, ON063233 and ON089665) were 100% identical with the corresponding sequences of Fusarium oxysporum (OM876904, JF430180, and MT568959) and F. solani (MT605584, MK617767, and MT305110), respectively. Based on above results, WG-1 and HrmY-9 was identified as F. oxysporum and F. solani, respectively. Pathogenicity test were performed on healthy one-year-old trifoliate orange seedlings by dipping their injured roots into conidial suspension (50 ml, 1×106 conidia/mL) for 1 h and the rest of conidial suspension was added to the pot after replanting to make sure the inoculum was in contact with the roots. Roots of control plants were inoculated with sterilized water. All experiments were repeated twice. All plants were cultured at 26°C under a 16-h light/dark cycle. Typical symptoms developed on most of inoculated seedlings two months post inoculation. No disease symptoms appeared on control plants. Same colonies were reisolated from the inoculated roots, confirming Koch's postulates. To our knowledge, this is the first report of F. oxysporum and F. solani causing root rot on trifoliate orange rootstock in China. The identification of F. oxysporum and F. solani as the causal agents of the observed root rot on trifoliate orange rootstock is critical to the prevention and control of this disease in the future.

Two insulin receptors determine alternative wing morphs in planthoppers.

Wing polyphenism is an evolutionarily successful feature found in a wide range of insects. Long-winged morphs can fly, which allows them to escape adverse habitats and track changing resources, whereas short-winged morphs are flightless, but usually possess higher fecundity than the winged morphs. Studies on aphids, crickets and planthoppers have revealed that alternative wing morphs develop in response to various environmental cues, and that the response to these cues may be mediated by developmental hormones, although research in this area has yielded equivocal and conflicting results about exactly which hormones are involved. As it stands, the molecular mechanism underlying wing morph determination in insects has remained elusive. Here we show that two insulin receptors in the migratory brown planthopper Nilaparvata lugens, InR1 and InR2, have opposing roles in controlling long wing versus short wing development by regulating the activity of the forkhead transcription factor Foxo. InR1, acting via the phosphatidylinositol-3-OH kinase (PI(3)K)-protein kinase B (Akt) signalling cascade, leads to the long-winged morph if active and the short-winged morph if inactive. InR2, by contrast, functions as a negative regulator of the InR1-PI(3)K-Akt pathway: suppression of InR2 results in development of the long-winged morph. The brain-secreted ligand Ilp3 triggers development of long-winged morphs. Our findings provide the first evidence of a molecular basis for the regulation of wing polyphenism in insects, and they are also the first demonstration--to our knowledge--of binary control over alternative developmental outcomes, and thus deepen our understanding of the development and evolution of phenotypic plasticity.

Discovery and validation of quality markers of Fructus Aurantii against acetylcholinesterase using metabolomics and bioactivity assays.

Fructus Aurantii is a traditional medicated diet in East Asia. To determine the underlying chemical markers responsible for the quality and efficacy of Fructus Aurantii, a sensitive metabolomic method was applied to distinguish Fructus Aurantii in Jiangxi Province from other two geographical locations (Hunan Province and Chongqing City) in China. In the present study, multivariate analyses were adopted to compare chemical compositions in 21 batches of Fructus Aurantii samples. Among three geographical origins, 23 differential compounds were structurally identified. Serum pharmacochemistry exhibited that 22 components could be detected in rat serum. Six differential and absorbed components were selected as six potential markers. Statistical analysis revealed that the content of six markers varied widely in three origins of Fructus Aurantii. Six differential and absorbed components were evaluated further by biological activity. Neohesperidin, naringin, and meranzin showed inhibitory effect on acetylcholinesterase that regulates gastrointestinal motility in vitro and in silico, suggesting that these three components may be determined as the active biomarkers of Fructus Aurantii. These findings demonstrate the potential of biomarkers for identification and quality control of Fructus Aurantii.

Metabolic Profile of C-Prenyl Coumarins Using Mass Spectrometry-Based Metabolomics.

C-prenyl coumarins (C-PYCs) are compounds with similar structures and various bioactivities, which are widely distributed in medicinal plants. Until now, the metabolic characterizations of C-PYCs and the relationship between metabolism and bioactivities remain unclear. In this study, ultra-performance chromatography electrospray ionization quadrupole time-of-flight mass spectrometry-based metabolomics (UPLC-ESI-QTOF-MS) was firstly used to determine the metabolic characterizations of three C-PYCs, including meranzin hydrate (MH), isomeranzin (ISM), and meranzin (MER). In total, 52 metabolites were identified, and all of them were found to be novel metabolites. Among these metabolites, 10 were from MH, 22 were from ISM, and 20 were from MER. The major metabolic pathways of these C-PYCs were hydroxylation, dehydrogenation, demethylation, and conjugation with cysteine, N-acetylcysteine, and glucuronide. The metabolic rate of MH was much lower than ISM and MER, which was only 27.1% in MLM and 8.7% in HLM, respectively. Additionally, recombinant cytochrome P450 (CYP) screening showed that CYP1A1, 2B6, 3A4, and 3A5 were the major metabolic enzymes involved in the formation of metabolites. Further bioactivity assays indicated that all of these three C-PYCs exhibited anti-inflammatory activity, but the effects of ISM and MER were slightly higher than MH, accompanied by a significant decrease in inflammatory cytokines transcription induced by lipopolysaccharide (LPS) in macrophages RAW 264.7. Taken together, the metabolic characterizations of the three C-PYCs suggested that the side chain of the prenyl group may impact the metabolism and biological activity of C-PYCs.

Alterations in cellular RNA decapping dynamics affect tomato spotted wilt virus cap snatching and infection in Arabidopsis.

RNA processing and decay pathways have important impacts on RNA viruses, particularly animal-infecting bunyaviruses, which utilize a cap-snatching mechanism to translate their mRNAs. However, their effects on plant-infecting bunyaviruses have not been investigated. The roles of mRNA degradation and non-sense-mediated decay components, including DECAPPING 2 (DCP2), EXORIBONUCLEASE 4 (XRN4), ASYMMETRIC LEAVES2 (AS2) and UP-FRAMESHIFT 1 (UPF1) were investigated in infection of Arabidopsis thaliana by several RNA viruses, including the bunyavirus, tomato spotted wilt virus (TSWV). TSWV infection on mutants with decreased or increased RNA decapping ability resulted in increased and decreased susceptibility, respectively. By contrast, these mutations had the opposite, or no, effect on RNA viruses that use different mRNA capping strategies. Consistent with this, the RNA capping efficiency of TSWV mRNA was higher in a dcp2 mutant. Furthermore, the TSWV N protein partially colocalized with RNA processing body (PB) components and altering decapping activity by heat shock or coinfection with another virus resulted in corresponding changes in TSWV accumulation. The present results indicate that TSWV infection in plants depends on its ability to snatch caps from mRNAs destined for decapping in PBs and that genetic or environmental alteration of RNA processing dynamics can affect infection outcomes.

Functional analysis of apple stem pitting virus coat protein variants.

BACKGROUND: Although the canonical function of viral coat protein (CP) is to encapsidate the viral genome, they have come to be recognized as multifunctional proteins, involved in almost every stage of the viral infection cycle. However, CP functions of Apple stem pitting virus (ASPV) has not been comprehensively documented. This study aimed to characterize the functions of ASPV CP and any functional diversification caused by sequence diversity of six ASPV CP variants and studied their biological, serological, pathogenic and viral suppressor of RNA silencing (VSR) functions. METHODS: Six ASPV CP variants that have previously been shown to belong to different subgroups were selected here to study their diversity functions. Agrobacterium mediated infiltration (Agroinfiltration) was used to express YFP-ASPV-CPs in Nicotiana. benthamiana and infect Nicotiana. occidental with PVX-ASPV-CPs in. Confocal microscopy was used to detect YFP-ASPV-CPs florescence. CPs expressed in Escherichia coli BL21 (DE3) were induced by IPTG. RESULTS: In this study, we showed that recombinant CPs expressed in Escherichia coli BL21 (DE3) had different levels of serological reactivity to three anti-ASPV antibodies used to detect ASPV. Furthermore, fusion CPs with YFP (YFP-CPs) expressed in N. benthamiana cells differed in their ability to form aggregates. We also showed that ASPV isolates that harbour these CPs induced different biological symptoms on its herbaceous host N. occidentalis. At the same time, we found that all six CPs when expressed in PVX vector showed similar VSR activity and produced similar symptoms in N. occidentalis, despite their differences in amino acids. CONCLUSIONS: Different ASPV isolates induced different symptoms in N. occidentalis, however, ASPV CP variants expressed in PVX vector showed the same symptoms in N. occidentalis plants. Also, we showed that ASPV CP variants has the same level of VSR activity, but they have different abilities to aggregate in N. benthamiana.

Structural basis of the mechanism of ß-methyl epimerization by enzyme MarH.

Diverse derivatives of amino acids with different steric configurations are important biosynthetic building blocks. In biology, epimerization is an important way to generate steric diversity. MarH catalyzes the epimerization of the ß-position of (3R)-ß-methyl-indolepyruvate (MeInPy), forming (3S)-ß-MeInPy. Both compounds are derivatives of l-tryptophan (l-Trp) and are important precursors of bioactive natural products. Here, we report the crystal structures of MarH and the NMR structure of its complex with l-Trp, an analogue of its native substrate, (3R)-ß-MeInPy. Structural analysis and mutagenesis studies indicated that His25 acts as a base to remove Hß and generate a planar carbanion intermediate, which is then putatively reprotonated on the opposite face by a water molecule to form (3S)-ß-MeInPy in a stereospecific manner. The details of ß-site isomerization at the atomic level provide deeper insights into the epimerization mechanism of MarH and will facilitate further enzyme design to extend the substrate scope.

Site-specific peak intensity ratio (SPIR) from 1D 2H/1H NMR spectra for rapid distinction between natural and synthetic nicotine and detection of possible adulteration.

1H and 2H NMR spectra of 4 natural and synthetic nicotine samples were collected in a non-quantitative way and site-specific 2H/1H peak intensity ratio (SPIR) was calculated for 12 distinct sites of nicotine. Experimental results illustrated that the SPIRs at sites of 6, 2', and 5'ß of natural nicotine were significantly different from those of the synthetic nicotine, and could be used for nicotine authentication as the measured SPIRs were indicative of the site-specific natural isotope fractionation. We demonstrated that this method could be applied to detect adulteration of natural nicotine with as low as 20% synthetic nicotine, without the need to measure the site-specific δD values, which usually required time-consuming quantitative 2H NMR and additional IRMS for the overall 2H/1H isotopic ratio determination. The distinguishable 2H/1H SPIRs of nicotine, which can be quickly measured by NMR in non-quantitative way, can serve as an attractive alternative tool for tobacco authentication. Graphical abstract.

Electrochemiluminescence Platforms Based on Small Water-Insoluble Organic Molecules for Ultrasensitive Aqueous-Phase Detection.

Highly efficient detection in the aqueous phase for water-insoluble organic molecule probes is challenging. The bright aggregated-state electrochemiluminescence (ECL) of 1,1-disubstituted 2,3,4,5-tetraphenylsiloles by a co-reactant approach was discovered, and a heterogeneous aggregation-induced emission ECL (HAIE-ECL) was constructed at the electrode surface, showing very high ECL efficiency (37.8 %) and selective recognition for industrially important DNBP plasticizer with a low detection limit of 0.15 nm in the water phase. A mechanistic study indicates that ECL is mainly generated due to the high electron affinity of siloles and restriction of the intramolecular motions caused by their propeller-like noncoplanar structures. This system realizes the sensing of organic-based ECL in the water phase by solving the crucial problems of water insolubility and aggregation-caused quenching (ACQ), and demonstrates potential for further application because of its design and high efficiency.

Electrical pulse stimulation induces GLUT4 translocation in C2C12 myotubes that depends on Rab8A, Rab13, and Rab14.

The signals mobilizing GLUT4 to the plasma membrane in response to muscle contraction are less known than those elicited by insulin. This disparity is undoubtedly due to lack of suitable in vitro models to study skeletal muscle contraction. We generated C2C12 myotubes stably expressing HA-tagged GLUT4 (C2C12-GLUT4 HA) that contract in response to electrical pulse stimulation (EPS) and investigated molecular mechanisms regulating GLUT4 HA. EPS (60 min, 20 V, 1 Hz, 24-ms pulses at 976-ms intervals) elicited a gain in surface GLUT4 HA (GLUT4 translocation) comparably to insulin or 5-amino imidazole-4-carboxamide ribonucleotide (AICAR). A myosin II inhibitor prevented EPS-stimulated myotube contraction and reduced surface GLUT4 by 56%. EPS stimulated AMPK and CaMKII phosphorylation, and EPS-stimulated GLUT4 translocation was reduced in part by small interfering (si)RNA-mediated AMPKα1/α2 knockdown, compound C, siRNA-mediated Ca2+/calmodulin-dependent protein kinase (CaMKII)δ knockdown, or CaMKII inhibitor KN93. Key regulatory residues on the Rab-GAPs AS160 and TBC1D1 were phosphorylated in response to EPS. Stable expression of an activated form of the Rab-GAP AS160 (AS160-4A) diminished EPS- and insulin-stimulated GLUT4 translocation, suggesting regulation of GLUT4 vesicle traffic by Rab GTPases. Knockdown of each Rab8a, Rab13, or Rab14 reduced, in part, GLUT4 translocation induced by EPS, whereas only Rab8a, or Rab14 knockdown reduced the AICAR response. In conclusion, EPS involves Rab8a, Rab13, and Rab14 to elicit GLUT4 translocation but not Rab10; moreover, Rab10 and Rab13 are not engaged by AMPK activation alone. C2C12-GLUT4 HA cultures constitute a valuable in vitro model to investigate molecular mechanisms of contraction-stimulated GLUT4 translocation.

Bimodal Electrochemiluminescence of G-CNQDs in the Presence of Double Coreactants for Ascorbic Acid Detection.

How to improve the accuracy of target detection substance in low-content and complex of real sample, which is still a major challenge in the analysis field. There is no doubt that the internal standard method is the best choice in the analysis methods. The internal standard method of ECL strategy can furnish more accurate detection results in the changeable complex environment, and it can dispel the primary vaguest interference in the system through the self-calibration of two emission spectra. Herein, we effectually explored a strong and stable bimodal ECL system based on graphitic carbon nitride quantum dots (g-CNQDs) as single luminophore in the presence of double coreactants potassium persulfate (K2S2O8) and tetrabutylammonium bromide (TBAB) under the optimized conditions. ECL-1 at 2.82 V and ECL-2 at 1.73 V were observed when the potential was scanned between -3 and 3 V at the scan rate of 0.2 V·s-1. The ECL-1 was responding to the analyte, that is, ascorbic acid (AA) and the ECL-2 was not for a certain concentration of AA; hence, the developed bimodal ECL system was used as internal standard method for quantitative AA in human serum due to the different sensitivity of the double-peak ECL signals to the target analytes. The linear relationships were obtained based on the ln I (ECL-1/ECL-2) against the concentration of AA in the concentration range of 3.5 to 330 nM, with a detection limit of 110 pM (S/N = 3).

An adapted isotope dilution 1H-13C heteronuclear single-quantum correlation (ID-HSQC) for rapid and accurate quantification of endogenous and exogenous plasma glucose.

A wide variety of methods, such as enzymatic methods, LC-MS, and LC-MS/MS, are currently available for the concentration determination of plasma glucose in studies of diabetes, obesity, exercise, etc. However, these methods rarely discriminate endogenous and exogenous glucose in plasma. A novel NMR strategy for discriminative quantification of the endogenous and exogenous glucose in plasma has been developed using an adapted isotope dilution 1H-13C heteronuclear single-quantum correlation (ID-HSQC) with uniformly 13C-labeled glucose as a tracer of exogenous glucose. This method takes advantage of the distinct 1H-13C chemical shifts of the hemiacetal group of the α-D-glucopyranose and makes use of the 13C-13C one-bond J-coupling (1JCC) in uniformly 13C-labeled glucose to differentiate the 1H-13C HSQC signal of labeled glucose from that of its natural counterpart when data are acquired in high-resolution mode. The molar ratio between the endogenous and exogenous plasma glucose can then be calculated from the peak intensities of the natural and labeled glucose. The accuracy and precision of the method were evaluated using a series of standard mixtures of natural and uniformly 13C-labeled glucose with varied but known concentrations. Application of this method is demonstrated for the quantification of endogenous and exogenous glucose in plasma derived from healthy and diabetic cynomolgus monkeys. The results nicely agree with our previous LC-MS/MS results. Considering the natural abundance of 13C isotope at the level of 1.1% in endogenous glucose, comparable peak intensities of quantitatively measurable signals derived from natural and labeled glucose imply that the ID-HSQC can tolerate a significantly high ratio of isotope dilution, with labeled/natural glucose at ~ 1%. We expect that the ID-HSQC method can serve as an alternative approach to the biomedical or clinical studies of glucose metabolism.

Porphyrin aggregates decorated MWCNT film for solar light harvesting: influence of J- and H-aggregation on the charge recombination resistance, photocatalysis, and photoinduced charge transfer kinetics.

J- and H-aggregates of zinc tetraphenylporphyrin (ZnTPP) on carbon nanotube films (CNTFs) were prepared using the mixed solvent method. This resulted in completely different structures, such as the four-leaf clover and flower, on the CNTF, which were observed by recording SEM images. Characteristic changes in the electronic spectra of the ZnTPP monomer appeared when it underwent J- and H-aggregation. The measured photocurrent significantly varied for the same molecule when it was aggregated in two different ways on ITO and ITO/CNTF. The electron recombination resistance of the two aggregates, which was investigated using electrochemical impedance spectroscopy, was also different. The photocatalytic efficiency of the J- and H-aggregates was examined by performing methylene blue dye decoloration studies. In addition, a scanning electrochemical microscope was used to investigate the photoinduced charge transfer kinetics of the J- and H-aggregates at the electrode/electrolyte interface as a fresh attempt. The heterogeneous charge transfer constants for the J- and H-aggregates in the presence of light at varied intensities were calculated. Thereby, striking differences in the photophysical, photocatalytic, and photoelectrochemical properties of the J- and H-aggregates were visualized throughout our studies.

Antiviral Defense Involves AGO4 in an Arabidopsis-Potexvirus Interaction.

In plants, RNA silencing regulates gene expression through the action of Dicer-like (DCL) and Argonaute (AGO) proteins via micro RNAs and RNA-dependent DNA methylation (RdDM). In addition, RNA silencing functions as an antiviral defense mechanism by targeting virus-derived double-stranded RNA. Plants encode multiple AGO proteins with specialized functions, including AGO4-like proteins that affect RdDM and AGO2, AGO5, and AGO1, which have antiviral activities. Here, we show that AGO4 is also required for defense against the potexvirus Plantago asiatica mosaic virus (PlAMV), most likely independent of RdDM components such as DCL3, Pol IV, and Pol V. Transient assays showed that AGO4 has direct antiviral activity on PlAMV and, unlike RdDM, this activity does not require nuclear localization of AGO4. Furthermore, although PlAMV infection causes a decrease in AGO4 expression, PlAMV causes a change in AGO4 localization from a largely nuclear to a largely cytoplasmic distribution. These results indicate an important role for AGO4 in targeting plant RNA viruses as well as demonstrating novel mechanisms of regulation of and by AGO4, independent of its canonical role in regulating gene expression by RdDM.