Transcriptome Analysis of Intermittent Light Induced Early Bolting in Flowering Chinese Cabbage.

In flowering Chinese cabbage, early booting is one of the most important characteristics that is linked with quality and production. Through fixed light intensity (280 µmol·m-2·s-1) and fixed intermittent lighting in flowering Chinese cabbage, there was early bolting, bud emergence, and flowering. Moreover, the aboveground fresh weight, blade area, dry weight of blade, and quantification of the leaves in flowering Chinese cabbage were significantly reduced, while the thickness of tillers, tillers height, dry weight of tillers, and tillers weight were significantly increased. The chlorophyll contents and soil-plant analysis and development (SPAD) value decreased in the early stage and increased in the later stage. The nitrate content decreased, while the photosynthetic rate, vitamin C content, soluble sugar content, soluble protein content, phenolic content, and flavonoid content increased, and mineral elements also accumulated. In order to explore the mechanism of intermittent light promoting the early bolting and flowering of '49d' flowering Chinese cabbage, this study analyzed the transcriptional regulation from a global perspective using RNA sequencing. A total of 17,086 differentially expressed genes (DEGs) were obtained and 396 DEGs were selected that were closely related to early bolting. These DEGs were mainly involved in pollen wall assembly and plant circadian rhythm pathways, light action (34 DEGs), hormone biosynthesis and regulation (26 DEGs), development (21 DEGs), and carbohydrate synthesis and transport (6 DEGs). Three hub genes with the highest connectivity were identified through weighted gene co-expression network analysis (WGCNA): BrRVE, BrLHY, and BrRVE1. It is speculated that they may be involved in the intermittent light regulation of early bolting in flowering Chinese cabbage. In conclusion, intermittent light can be used as a useful tool to regulate plant growth structure, increase planting density, enhance photosynthesis, increase mineral accumulation, accelerate growth, and shorten the breeding cycle.

Extracellular vesicles released by cancer-associated fibroblast-induced myeloid-derived suppressor cells inhibit T-cell function.

Myeloid cells are known to play a crucial role in creating a tumor-promoting and immune suppressive microenvironment. Our previous study demonstrated that primary human monocytes can be polarized into immunosuppressive myeloid-derived suppressor cells (MDSCs) by cancer-associated fibroblasts (CAFs) in a 3D co-culture system. However, the molecular mechanisms underlying the immunosuppressive function of MDSCs, especially CAF-induced MDSCs, remain poorly understood. Using mass spectrometry-based proteomics, we compared cell surface protein changes among monocytes, in vitro differentiated CAF-induced MDSCs, M1/M2 macrophages, and dendritic cells, and identified an extracellular vesicle (EV)-mediated secretory phenotype of MDSCs. Functional assays using an MDSC/T-cell co-culture system revealed that blocking EV generation in CAF-induced MDSCs reversed their ability to suppress T-cell proliferation, while EVs isolated from CAF-induced MDSCs directly inhibited T-cell function. Furthermore, we identified fructose bisphosphatase 1 (FBP1) as a cargo protein that is highly enriched in EVs isolated from CAF-induced MDSCs, and pharmacological inhibition of FBP1 partially reversed the suppressive phenotype of MDSCs. Our findings provide valuable insights into the cell surface proteome of different monocyte-derived myeloid subsets and uncover a novel mechanism underlying the interplay between CAFs and myeloid cells in shaping a tumor-permissive microenvironment.

Study on the Function of Conveying, Kneading Block and Reversing Elements on the Mixing Efficiency and Dispersion Effect inside the Barrel of an Extruder with Numerical Simulation.

In order to better understand the extrusion process mechanism of plant protein inside a barrel, the parameter changes and flow characteristics of fluids under conveying, kneading block and reversing elements were investigated with numerical simulation. The results showed that the shear rate increased obviously with the increase in pitch; the shear rate value of the reversing element was larger, while that of the kneading block was the opposite. The screw combinations of conveying, kneading blocks and reversing elements all have a certain degree of mixing effect on the particles, and the reduction in pitch can effectively increase the mixing effect of the particles. The conveying element can provide a relatively constant acceleration for the particles, due to the pumping capability and pressure buildup as the pitch increases. The kneading block and the reversing element can increase the leakage flow between the discs and backflow, resulting in an extension of the residence time distribution that facilitates fluid interaction in the barrel and improves the dispersion of the particles. The restraint by the reversing element on the particles is obviously weaker than that of the kneading block and shows a higher particle mixing degree. Overall, the influence of different elements on the flow condition, mixing degree and residence time is significantly different, which improves the process controllability and provides references for potential applications to meet multiple demands.

Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster.

Males have finite resources to spend on reproduction. Thus, males rely on a 'time investment strategy' to maximize their reproductive success. For example, male Drosophila melanogaster extends their mating duration when surrounded by conditions enriched with rivals. Here we report a different form of behavioral plasticity whereby male fruit flies exhibit a shortened duration of mating when they are sexually experienced; we refer to this plasticity as 'shorter-mating-duration (SMD)'. SMD is a plastic behavior and requires sexually dimorphic taste neurons. We identified several neurons in the male foreleg and midleg that express specific sugar and pheromone receptors. Using a cost-benefit model and behavioral experiments, we further show that SMD behavior exhibits adaptive behavioral plasticity in male flies. Thus, our study delineates the molecular and cellular basis of the sensory inputs required for SMD; this represents a plastic interval timing behavior that could serve as a model system to study how multisensory inputs converge to modify interval timing behavior for improved adaptation.

Total Synthesis of (-)-Retigeranic Acid A: A Reductive Skeletal Rearrangement Strategy.

The asymmetric total synthesis of (-)-retigeranic acid A was described, which relies on a crucial reductive skeletal rearrangement cascade for the controllable assembly of diverse angular triquinane subunits. Taken together with an intramolecular Michael/aldol cyclization, an ODI-[5 + 2] cycloaddition/pinacol rearrangement cascade, a Wolff ring contraction and a stereoselective HAT reduction, our synthetic approach has enabled the access to (-)-retigeranic acid A in a concise and practical manner.

Conformational changes and physicochemical attributes of texturized pea protein isolate-konjac gum: With a new perspective of residence time during extrusion.

The present study aimed to investigate the effects of different extrusion temperatures (110, 130 and 150 °C) and konjac gum addition (0.1 %, 0.2 %, and 0.3 %) on the flow behavior, physicochemical properties and microstructure of extruded pea protein isolate (PPI). The results showed that the textured protein could be improved by enhancing the extrusion temperature and adding konjac gum during extrusion. The water/oil holding capacity of PPI decreased and the SH content increased after extrusion. With temperature and konjac gum content increased, the ß-sheet of extruded proteins transformed to other secondary structural components, and Trp residue transformed to a more polar environment, illustrating the changes in protein conformation. All extruded samples presented as yellow hue with little green and higher lightness, while excessive extrusion process reduced the brightness and promoted more formation of browning pigments. Extruded protein showed more associated layered with some air pores, and its hardness and chewiness increased with the increase of temperature and konjac gum concentration. Cluster analysis showed that the addition of konjac gum could effectively improve the quality characteristics of pea protein under low temperature extrusion, and the effect was similar to that of high temperature extrusion product. With the increase of konjac gum concentration, the flow pattern of protein extrusion gradually converted from plug flow to mixing flow, and the disorder degree of polysaccharide protein mixing system was enhanced. Moreover, Yeh-jaw model showed better fitting effect in F(θ) curves compared to Wolf-white.

Effect of Combined Infrared and Hot Air Drying Strategies on the Quality of Chrysanthemum (Chrysanthemum morifolium Ramat.) Cakes: Drying Behavior, Aroma Profiles and Phenolic Compounds.

Chrysanthemum (Chrysanthemum morifolium Ramat.) is a seasonal plant with high medicinal and aesthetic value, and drying is an effective practice to enhance its storability after harvesting. The effects of hot air drying (HAD), combined infrared and hot air drying (IR-HAD), and sequential IR-HAD and HAD (IR-HAD + HAD) on the drying behavior, color, shrinkage, aroma profiles, phenolic compounds, and microstructure of chrysanthemum cakes were studied. Results showed that the increasing temperature resulted in a decrease in drying time and an increase in drying rate and moisture diffusivity. The Logarithmic and Page models exhibited superior fit in describing the dehydration process. Among the three drying strategies, IR-HAD was more effective in reducing energy consumption, improving shrinkage, water holding capacity, water binding capacity and cellular microstructure, while IR-HAD + HAD showed better inhibitory effect on color deterioration. Furthermore, gas chromatography-mass spectrometry (GC-MS) analysis revealed that different drying strategies dramatically influenced the aroma profiles in samples, and IR-HAD obtained the highest concentration of volatiles. The results of ultra-performance liquid chromatography (UPLC) indicated that the introduction of infrared radiation contributed to increasing the contents of chlorogenic acid, luteolin, total phenolic and flavonoid. These suggested that IR-HAD was a promising technique for drying medicinal chrysanthemum.

Twin-Screw Extrusion of Oat: Evolutions of Rheological Behavior, Thermal Properties and Structures of Extruded Oat in Different Extrusion Zones.

Further investigation of material properties during the extrusion process is essential to achieve precise control of the quality of the extrudate. Whole oat flour was used to produce low moisture puffed samples by a twin-screw extruder. X-ray diffraction (XRD), Scanning electron microscopy (SEM), infrared spectroscopy (FTIR), thermal analysis, and rheological experiments were used to deeply characterize changes in the structure and cross-linking of oats in different extrusion zones. Results indicated that the melting region was the main region that changed oat starch, including the major transformation of oat starch crystal morphology and the significant decrease of enthalpy representing the starch pasting peak in the differential scanning calorimeter (DSC) pattern (p < 0.05). Moreover, the unstable structure of the protein increased in the barrel and then decreased significantly (p < 0.05) after being extruded through the die head. The viscosity of oats increased in the cooking zone but decreased after the melting zone. A transformation occurred from elastic-dominant behavior to viscoelastic-dominant behavior for oats in the melting zone and after being extruded. This study provides further theoretical support for the research of the change of materials during extrusion and the development of oat-based food.

Establishing and Validating Cellular Functional Target Engagement Assay for Selective IRAK4 Inhibitor Discovery.

One of the main reasons for the lack of drug efficacy in late-stage clinical trials is the lack of specific and selective target engagement. To increase the likelihood of success of new therapeutics, one approach is to conduct proximal target engagement testing during the early phases of preclinical drug discovery. To identify and optimize selective IRAK4 inhibitors, a kinase that has been implicated in multiple inflammatory and autoimmune diseases, we established an electrochemiluminescence (ECL)-based cellular endogenous IRAK1 activation assay as the most proximal functional evaluation of IRAK4 engagement to support structure-activity relationship (SAR) studies. Since IRAK1 activation is dependent on both the IRAK4 scaffolding function in Myddosome formation and IRAK4 kinase activity for signal transduction, this assay potentially captures inhibitors with different mechanisms of action. Data from this IRAK1 assay with compounds representing different structural classes showed statistically significant correlations when compared with results from both IRAK4 biochemical kinase activity and functional peripheral blood mononuclear cell (PBMC)-derived tumor necrosis factor α (TNFα) secretion assays, validating the biological relevancy of the IRAK1 target engagement as a biomarker of the IRAK4 activity. Plate uniformity and potency reproducibility evaluations demonstrated that this assay is amenable to high throughput. Using Bland-Altman assay agreement analysis, we demonstrated that incorporating such proximal pharmacological assessment of cellular target engagement to an in vitro screening funnel for SAR studies can prevent compound optimization toward off-target activity.

Effects of sodium arsenite and dimethyl arsenic acid on Liaoning cashmere goat skin fibroblasts.

The morphology and oxidation state of arsenic in its compounds affects the skin cell toxicity. Accordingly, the present study was conducted to explore the effects of two different arsenic compounds on the proliferation and survival of Liaoning cashmere goat skin fibroblasts. Based on MTT assay results, at 24 h, the proliferation concentration, critical concentration, and half inhibitory concentration (IC50) of sodium arsenite were 0.50, 5.00, and 45.66 µmol/L, respectively. The corresponding values for dimethyl arsenic acid were 0.85, 1.00, and 38.68 mmol/L. Immunofluorescence, transmission electron microscopy, and mitochondria membrane potential (MMP) assays showed that sodium arsenite promotes microtubule polymerization and increases MMP, while cells treated with dimethyl arsenic acid exhibited cytoskeletal collapse and decreased MMP. In the IC50 groups for both arsenic agents, the cytoskeletons collapsed, microtubules were gathered into bundles, and MMP was significantly decreased. Dimethyl arsenic acid had a stronger effect on MMP than sodium arsenite. Flow cytometry revealed a slightly lower occurrence of apoptosis in the sodium arsenite proliferation group, while it was slightly increased in the dimethyl arsenic acid proliferation group. Apoptosis was increased more significantly in the sodium arsenite IC50 group than in the dimethyl arsenic acid IC50 group. These results indicate that the differences in cell proliferation and cytotoxicity induced by inorganic and organic arsenic are related to their effects on cellular structures.

Effect of high pressure homogenization treatment on the rheological properties of citrus peel fiber/corn oil emulsion.

BACKGROUND: Citrus fiber is a main component in the peel of citrus and contains natural dietary fiber. It is often used as a functional additive to improve the texture or nutritional property of food. It is also widely used to reduce the content of absorbable fat in sausages and other meat products, and to improve food stability as an emulsifier. In this research, the dynamic rheological properties (linear and non-linear) of citrus peel fiber/corn oil (CF/CO) emulsion system under high pressure homogenization (HPH) treatment was investigated. RESULT: Rheological results illustrated HPH treatment significantly increased the apparent viscosity of the emulsion, reduced the activation energy of the emulsion and distinctly improved the viscoelasticity of the emulsion. Meanwhile, HPH treatment increased the linear viscoelastic region of the sample, and the behavior of the emulsion converted from strain thinning (without HPH treatment) to weak strain overshoot (with HPH treatment). Lissajous curves indicated the viscosity of the sample increased first and then decreased with strain increasing and the third harmonic contributed much more to the first harmonic compared with the fifth harmonic. Chebyshev stress decomposition revealed that, as strain increased, the samples with HPH treatment showed internal-cycle strain hardening behavior first, then turned to internal-cycle softening behavior. CONCLUSION: HPH treatment can significantly improve the processing performance of CF/CO emulsion as well as the stability against large periodic oscillations in food processing. © 2020 Society of Chemical Industry.

Cancer-Associated Fibroblasts Promote Immunosuppression by Inducing ROS-Generating Monocytic MDSCs in Lung Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8+ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.

Total Synthesis of (+)-Jatrophalactam.

The first asymmetric total synthesis of (+)-jatrophalactam was reported, which unambiguously determined the absolute configuration of the titled natural product. The key features entail a conformationally controlled cyclopropanation, a Meldrum's acid adduct-engaged macrolactam formation, and a Pd(II)-mediated oxidative cyclization.

Spongian diterpenes from Chinese marine sponge Spongia officinalis.

3-Nor-spongiolide A (1), belonging to the extremely rare 3-nor-spongian carbon skeleton, and spongiolides A (2) and B (3), having γ-butenolide instead of furan ring as usual for ring D, together with six related known metabolites were isolated from South China Sea sponge Spongia officinalis as its metabolic components. Their structures were elucidated on the basis of extensive spectroscopic analysis. The absolute configurations of three new compounds 1-3 were determined by ECD calculations.

Asymmetric Total Synthesis of Distaminolyne A and Revision of Its Absolute Configuration.

The first total synthesis of a marine derived polyacetylene, distaminolyne A, and its enantiomer were achieved from the commercially available undec-10-en-1-ol. A key proline-catalyzed asymmetric α-aminooxylation of an aldehyde intermediate was used to introduce the chiral center en route to the enantiomerically pure 1,2-amino alcohols. The absolute configuration of both synthesized enantiomers of distaminolyne A was confirmed by using chiral derivatizing agents, leading to revision of the natural product absolute configuration from 2S to 2R. Antibacterial, pancreatic lipase (PL) inhibitory, and protein-tyrosine phosphatase 1B (PTP1B) inhibitory activities were evaluated.

Discovery of 1-(1H-Pyrazolo[4,3-c]pyridin-6-yl)urea Inhibitors of Extracellular Signal-Regulated Kinase (ERK) for the Treatment of Cancers.

The ERK/MAPK pathway plays a central role in the regulation of critical cellular processes and is activated in more than 30% of human cancers. Specific BRAF and MEK inhibitors have shown clinical efficacy in patients for the treatment of BRAF-mutant melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the ERK signal pathway. Acquired resistance to these agents has led to greater interest in ERK, a downstream target of the MAPK pathway. De novo design efforts of a novel scaffold derived from SCH772984 by employing hydrogen bond interactions specific for ERK in the binding pocket identified 1-(1H-pyrazolo[4,3-c]pyridin-6-yl)ureas as a viable lead series. Sequential SAR studies led to the identification of highly potent and selective ERK inhibitors with low molecular weight and high LE. Compound 21 exhibited potent target engagement and strong tumor regression in the BRAF(V600E) xenograft model.

Efficient identification of novel leads by dynamic focused screening: PDK1 case stud.

A dynamic, focused screening strategy that utilized a limited but diversified set of target-specific compounds was explored as an efficient means for the identification of inhibitors of the protein kinase PDK1. Approximately 21,500 compounds, including a 19,000 molecule kinase-focused compound collection (KFCC), were screened at two concentrations to identify initial leads. The KFCC included several empirically-derived, general kinase libraries and molecules chosen by PDK1-specific virtual screens. As was expected, this initial screen mostly identified potent leads with limited novelty. In order to overcome this limitation, the data from the screen were used to drive several rounds of a customized iterative focused screening (IFS) campaign. A machine-learning technique was used to build a predictive model to identify compounds to be screened in subsequent rounds. Molecules deemed not to be novel were removed from the training set for the next round, which allowed this campaign to progressively walk away from the chemical space covered by the KFCC. This resulted in the identification of PDK1 inhibitors which are uniquely different from publicly known chemotypes after just three rounds of screenings. A retrospective analysis of this IFS approach against an ultra-high throughput screen (uHTS) indicated that while uHTS is still the most prolific paradigm for lead identification, this dynamic, focused screening approach was successful in discovering novel scaffolds for a medicinal chemistry effort. Finally, a theoretical optimization suggested the dynamic, focused screening approaches could provide either a complementary or alternative approach to uHTS for the efficient and rapid lead identification.

Development of high-throughput TR-FRET and AlphaScreen assays for identification of potent inhibitors of PDK1.

The PI3K/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a Ser/Thr protein kinase, which catalyzes the phosphorylation of a conserved residue in the activation loop of a number of AGC kinases, including proto-oncogenes Akt, p70S6K, and RSK kinases. To find new small-molecule inhibitors of this important regulator kinase, the authors have developed PDK1-specific high-throughput enzymatic assays in time-resolved fluorescence resonance energy transfer (TR-FRET) and AlphaScreen formats, monitoring phosphorylation of a biotinylated peptide substrate derived from the activation loop of Akt. Development of homogeneous assays enabled screening of a focused kinase library of approximately 21,500 compounds in 1536-well TR-FRET format in duplicate. Upon validation of hits in an alternative 384-well AlphaScreen assay, several classes of structurally diverse PDK1 inhibitors, including tetracyclics, tricyclics, azaindoles, indazoles, and indenylpyrazoles, were identified, thus confirming the utility and sensitivity of the developed assays. Further testing in PC3 prostate cancer cells confirmed that representatives of the tetracyclic series showed intracellular modulation of the PDK1 activity, as evident from decreased phosphorylation levels of AKT, RSK, and S6-ribosomal protein.

SM16, an orally active TGF-beta type I receptor inhibitor prevents myofibroblast induction and vascular fibrosis in the rat carotid injury model.

OBJECTIVE: TGF-beta plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis. METHODS AND RESULTS: The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF-beta and activin-induced Smad2/3 phosphorylation and TGF-beta-induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle alpha-actin-positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells. CONCLUSIONS: These results are the first to demonstrate the efficacy of an orally active, small-molecule ALK5/ALK4 inhibitor in a vascular fibrosis model and suggest the potential therapeutic application of these inhibitors in vascular fibrosis.

Quality assessment and analysis of Biogen Idec compound library.

A subset of the compound repository for lead identification at Biogen Idec was characterized for its chemical stability over a 3-year period. Compounds were stored at 4 degrees C as 10 mM DMSO stocks, and a small subset of compounds was stored as lyophilized dry films. Compound integrity of 470 discrete compounds (Compound Set I) and 1917 combinatorial chemistry-derived compounds (Compound Set II) was evaluated by liquid chromatography/mass spectrometry from the time of acquisition into the library collection and after 3 years of storage. Loss of compound integrity over the 3 years of storage was observed across the 2 subsets tested. Of Compound Set I, 63% of samples retained > 80% purity, whereas 57% of samples from Compound Set II had purity greater than 60%. The stability of the lyophilized samples was superior to the samples stored as DMSO solution. Although storage at 4 degrees C as DMSO solution was adequate for the majority of compounds, the authors observed and quantified the level of degradation within the compound collection. Their study provides general insight into compound storage and selection of library subsets for future lead identification activities.