Nanidorus spp. was pathogenic to seashore paspalum  turfgrass as its population increased from 100 to 2,080 nematodes per pot 180 days after inoculation under greenhouse conditions. Morphological measurements of adult females were similar to those described for N. minor. Molecular analysis also confirmed the morphological identification by targeting three different regions of the genomic DNA. Three primer pairs targeting 18S rDNA (360F/932R), 28S rDNA (D2A/D3B) and ITS1 rDNA (BL18/5818) were used in singleplex PCR. Forward and reverse sequences of each individual primer set were then subjected to multiple alignment and the complimentary sequences were assembled into a consensus sequence. Upon nucleotide blast on the NCBI website, they were all confirmed to be N. minor. A one-step multiplex PCR method using specific primers and a fragment size of 190 bp also confirmed the identity of N. minor. To the best of our knowledge, this is the first report of N. minor infecting seashore paspalum turfgrass in Georgia.We found that  Nanidorus spp., are one of the most common plant-parasitic nematodes found associated with different species of turfgrasses in Georgia . In March 2019, five soil samples were submitted to the Nematode Diagnostic Laboratory, University of Georgia, Athens for diagnosis of plant-parasitic nematodes associated with seashore paspalum  grown on a turf farm located in South Georgia. While processing these samples, 112 stubby-root nematodes per 100 cm3 of soil were found to be associated with this turfgrass, which is currently grown in many sport fields and golf courses in Georgia and Florida. This prompted us to first study the pathogenicity of Nanidorus spp. to seashore paspalum turfgrass and second to determine the species identity using both molecular and morphological techniques. Accurate species identification of the nematodes can be helpful to develop effective control strategies. For the pathogenicity study, nematode-free seashore paspalum plants were grown from sprigs in five clay pots containing 500 g sandy-loam soil and allowed to establish for two weeks. After two weeks, three pots were inoculated with 100 handpicked, mixed-stages of juveniles and females of Nanidorus spp. per pot extracted from original soil samples using the decanting and flotation technique  included body length 664.5 (562.1-768.9) μm; body width 31.9 (27.2-36.4) μm; onchiostyle 31.6 (31.0-33.4) μm; anterior end to esophagus-intestinal valve 115.0 (99.6-126.1) μm; vagina length 8.4 (7.2-10.3) μm; a 21.1 (15.9-27.3) μm; b 5.0 (4.6-6.0) μm; and V 51.4% (47.5-54.1%) μm. Measurements of morphological characters of adult females were similar to N. minor (Colbran) Siddiqi  using Extract-N-Amp™ Tissue PCR Kit  following kit protocol with modifications. Individual nematodes were transferred into a sterile petri dish in 5 µL water and cut into 2 to 3 pieces using pipette tip under an inverted microscope. Subsequently, each individual nematode mix was transferred into a sterile 1.5 mL microcentrifuge tube containing 20 μL of extraction buffer with 5 µL of Tissue Preparation Solution from the kit. After careful vortexing, the mixture was incubated at room temperature (23 ± 2°C) for 10 min and then at 95°C for 3 min. Finally, 20 µL of neutralization solution was added to the tube and vortexed briefly. This DNA extract was stored at −20°C and used as DNA template for PCR reactions in 2 µL aliquots. Three primer pairs targeting 18S rDNA (360F/932R), 28S rDNA (D2A/D3B) and ITS1 rDNA (BL18/5818) (N. minor. The top pairwise identity with N. minor references was 100% for 360F/932R (accession no. MT36688) and BL18/5818 (MT372094), and 99.73% for D2A/D3B (MT371258). In contrast, the highest identity to other members of the Nanidorus sp. was in the range of 96% or lower. A one-step multiplex PCR method was also performed using specific primers as described by N. minor, confirming the identity of this species. To the best of our knowledge, this is the first report of N. minor infecting seashore paspalum turfgrass in Georgia. This nematode species was reported to cause damage on St. Augustine grass, switchgrass, Vidalia onion and field and sweet corn in Georgia   were use18/5818) . The con Georgia .


H-pyrrolobis benzothiazine (TRPZ). TRPZ is a heteroacene with a rigid, pi-conjugated structure, multiple reactive sites, and unique spectroscopic properties. Here we coupled TRPZ to a tert-butyl carbamate (BOC) protected 2,2-bis(hydroxymethyl)propanoic acid (bisMPA) dendron via azide-alkyne Huisgen cycloaddition. Deprotection of the protected amine groups on the dendron afforded a cationic terminated amphiphile, TRPZ-bisMPA. TRPZ-bisMPA was nanoprecipitated into water to obtain nanoparticles (NPs) with a hydrodynamic radius that was <150 nm. For comparison, TRPZ-PG was encapsulated in pluronic-F127 (Mw = 12 kD), a polymer surfactant to afford NPs almost twice as large as those formed by TRPZ-bisMPA. Size and stability studies confirm the suitability of the TRPZ-bisMPA NPs for biomedical applications. The photophysical properties of the TRPZ-bisMPA NPs show a quantum yield of 49%, a Stokes shift of 201 nm (0.72 eV) and a lifetime of 6.3 ns in water. Further evidence was provided by cell viability and cellular uptake studies confirming the low cytotoxicity of TRPZ-bisMPA NPs and their potential in bioimaging.The challenges faced with current fluorescence imaging agents have motivated us to study two nanostructures based on a hydrophobic dye, 6 Bioimaging techniques are crucial to understanding biological processes of living systems. Among many imaging techniques, fluorescence imaging  is a powerful, noninvasive method for diagnostics. It can provide excellent spatiotemporal resolution that affords the investigation of biological systems in real-time . In lighInorganic hybrids such as single-wall carbon nanotubes (SWCNTs)  and inorH-pyrrolobisbenzothiazine (TRPZ). The structures of interest are shown in Current research efforts in bioimaging have taken a more supramolecular approach in which nanoparticles (NPs) are beginning to dominate . These sTRPZ is similar to pentacene and pyrene in that it possesses a conjugated structure and unique spectroscopic properties. Its structure readily forms pi-aggregates/excimers at high concentrations due to the stacking interactions of the pi-conjugated backbone resulting in a red-shift of the fluorescence and a bright green-yellow emission. Additionally, it possesses multiple reactive sites with the central pyrrole nitrogen atom offering a diverse library of derivatives with potentially unique functionalities.TRPZ-PG) and coupled it to a dendritic bisMPA alanine possessing protected amine termini via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Deprotection of tert-butyl carbamates (BOC) protected amine groups with trifluoroacetic acid (TFA) afforded a cationic terminated TRPZ-bisMPA amphiphile. In aqueous media, TRPZ-bisMPA is capable of self-assembling into nanoparticles (NPs) where TRPZ makes up the core and bisMPA is the corona or exterior. In parallel, TRPZ-PG was encapsulated in pluronic-F127 (Mw = 12 kD), a polymer surfactant, to make a second NP system, TRPZ-127 NPs. We report the size and photophysical properties of the two resulting NP systems. Based on these results, TRPZ-bisMPA was then assessed via biological studies to demonstrate TRPZ as a promising platform for designing new versatile bioimaging probes.Here we propargylated the central nitrogen on TRPZ .Synthesis of methanol (2): The 2-(hydroxymethyl)-2-methylpropane-1,3-diol  was stirred in 50 ml of acetone, 2,2-dimethoxypropane (DMP)  and PTSA  were added under room temperature. After the completion of the addition, the reaction mixture was stirred for 4 h. Then it was filtered through an amberlyst column, and the solvent was evaporated, and the residue was put under 60°C and full vacuum for 2 h. Then it was put under vacuum overnight to give (2) as a colorless liquid with 96% yield. 1H NMR  δ 3.67 , 3.60 , 2.55 , 1.44 , 1.40 , 0.83 .Synthesis of methyl 4-methylbenzenesulfonate (3): Compound (2)  was dissolved in 34 ml of pyridine, and it was added dropwise to the stirred solution of p-toluenesulfonyl chloride  in 48 ml of pyridine at 0°C under nitrogen. After the complete addition, the reaction mixture was stirred 48 h at room temperature. Then the reaction mixture was added dropwise to 100 ml of 40% ammonium chloride solution at 0°C. After complete addition, it was allowed to stir at room temperature for 2 h. Then it was filtered and washed with DI water until the pyridine smell was gone. Then the residue was dissolved in 25 ml of DCM and extracted with half saturated ammonium chloride and saturated NaCl solution. Yellow DCM solution was dried with anhydrous sodium sulfate. Then the solvent was evaporated, and the residue was placed under full vacuum for 12 h to give (3) as a yellow solid with 89% yield. 1H NMR  δ 7.86–7.79 , 7.38 , 4.11 , 3.58 , 2.47 , 1.39 , 1.25 , 0.84 .Synthesis of 5-(azidomethyl)-2,2,5-trimethyl-1,3-dioxane (4): Compound (3) , NaN3 , water (10 ml), and DMF (80 ml) were stirred at 110°C for 48 h under reflux. The mixture was poured into 150 ml water and extracted four times with Et2O (4 × 200 ml). The organic phase was dried over anhydrous MgSO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography with silica gel (100 g) and ethyl acetate/n-hexane (1:4) to give 7.48 g of a colorless liquid with an 87% yield. 1H NMR  δ 3.58 , 3.51 , 1.40 , 0.81 .Synthesis of 2-(azidomethyl)-2-methylpropane-1,3-diol (5): Compound (4)  was dissolved in 35 ml of methanol. 7.00 g of a Dowex, acid resin was added, and the reaction mixture was stirred for 12 h at 50°C. When the reaction was complete, the Dowex was filtered off in a vacuum filter under a low vacuum and carefully washed with methanol. The methanol was evaporated to give 5.41 g of white crystals with a 93% yield. 1H NMR  δ 3.73–3.58 , 3.56–3.43 , 2.19 , 0.89 .Synthesis of A-MPA-4-AC (6): 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid was prepared using similar method mentioned in compound (2) synthesis. 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid , 1,1′-carbonyldiimidazole (CDI)  were dissolved in 30 ml of ethyl acetate and it was stirred 1 h at 50°C. CsF , Compound (5)  were dissolved in 10 ml of ethyl acetate separately, and it was slowly added to the reaction mixture under nitrogen at 50°C. It was stirred for 12 h. When the reaction was complete 200 ml DI water was added and allowed to stir for 2 h at room temperature. Then it was extracted with 1 M HCl (200 ml × 3), 1 M NaHSO4 (200 ml × 3), 10% Na2CO3, saturated NaCl (200 ml), and it was dried under anhydrous MgSO4. Ethyl acetate was evaporated to give 5.22 g of colorless oil liquid with a 92% yield. 1H NMR  δ 4.21 , 4.13–4.09 , 3.68 , 3.42–3.39 , 1.43 , 1.18 , 1.08 .Synthesis of A-MPA-4-OH (7): Compound (6)  was dissolved in 20 ml of methanol. 5.00 g of a Dowex, acid resin was added, and the reaction mixture was stirred for 12 h at 50°C. When the reaction was complete the Dowex, acid resin was filtered off in a vacuum filter under a low vacuum and carefully washed with methanol. The methanol was evaporated to give 3.96 g of colorless liquid with a 96% yield. 1H NMR  δ 4.09 , 3.88 , 3.78–3.67 , 3.43 , 3.38 , 1.12–1.09 , 1.09–1.07 .Synthesis of A-MPA-4-ala (8):N-boc-alanine , 1,1′-carbonyldiimidazole (CDI)  were dissolved in 30 ml of dry ethyl acetate, and it was stirred for 1 h at 50°C. CsF , compound (7)  were dissolved in 5 ml of dry ethyl acetate and it was slowly added to the reaction mixture under nitrogen at 50°C. It was stirred for 12 h. When the reaction was complete 250 ml DI water was added and allowed to stir for 2 h at room temperature. Then it was extracted with 1 M HCl (200 ml × 3), 1 M NaHSO4 (200 ml × 3), 10% Na2CO3, saturated NaCl (200 ml), and it was dried under anhydrous MgSO4. Ethyl acetate was evaporated to give 3.15 g with a 91% yield. 1H NMR  δ 5.19 , 4.31–4.19 , 4.02 , 3.38 , 3.33 , 2.55 , 1.27 , 1.02 .Synthesis of 3,4-dichloro-1-(prop-2-yn-1-yl)-1H-pyrrole-2,5-dione (9): A total of 0.40 g of dichloromaleimide (2.40 mmol) and 2-ethylhexylbromide  and DMF (10 ml) in a 2-neck round bottom flask under nitrogen. The mixture was vigorously stirred at 140°C for 24 h. After that, the solution was quenched with 0.1 M HCl and extracted with diethyl ether. The organic layer was dried over Na2SO4 and concentrated under reduced pressure, and purification by silica gel column chromatography (30% DCM/Hexane) resulted in a cream-colored powder of N-(propargyl) dichloromaleimide . 1H NMR  δ 4.31–4.30(d), 2.23(s).Synthesis of TRPZ-Propagyl (10): A mixture of 2-aminothiophenol  and N-(propargyl) dichloromaleimide  was dissolved in 24 ml of acetic acid. The reaction mixture was refluxed under a nitrogen atmosphere for 24 h. After being cooled to room temperature, the mixture was quenched with brine and extracted with ethyl acetate. The organic layer was separated, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The product was purified via flash column chromatography (30% chloroform/hexane) and obtained as orange solid . 1H NMR  1H NMR  δ 7.72 , 7.34–7.26 , 7.18 , 5.20 , 2.29 . 13C NMR  δ 148.79, 136.97, 130.48, 128.26, 127.11, 126.57, 111.17, 77.23, 73.36, 33.08. 1H–1H correlation spectroscopy (COSY) spectrum is provided in the SI. HRMS (ESI/Q-TOF) m/z: [M + H]+ Calcd for C19H11N3S2 345.0394; Found 345.0365 with an isotope pattern similar to the predicted pattern.Synthesis of TRPZ-bisMPA-amine-BOC (11): This procedure was modified from previously reported procedures   was added and allowed to stir under nitrogen at 55°C for 48 h. The reaction mixture was precipitated to 200 ml diethyl ether. After settling, the diethyl ether layer was decanted, and the remaining product was air-dried. This precipitation, decanting, and the air-drying process was repeated twice more. The crude product was dissolved in 100 ml of DCM. It was extracted with 0.1 M EDTA solution. It was further purified using size exclusion chromatography (Sephadex LH-20) to give 0.59 g of TRPZ-bisMPA-amine-Boc with 74% yield. 1H NMR  δ 8.03 , 7.51 , 7.44 , 7.32 , 7.22 , 6.77 , 5.32 , 4.41 , 4.16 , 3.94 , 3.12 , 2.41 , 1.34 , 1.17 , 0.84 . HRMS (ESI/Q-TOF) m/z: [M + H]+ Calcd for C66H90N10O20S2 1407.5852; Found 1407.5813 with an isotope pattern similar to the predicted pattern.ocedures . To the Synthesis of TRPZ-bisMPA (12): TRPZ-bisMPA-amine-Boc was dissolved in 5 ml of chloroform and 2 ml of trifluoroacetic acid (TFA) was slowly added and stirred for 45 min. The reaction mixture was air-dried and dissolved in 10 ml of chloroform. It was added dropwise to 500 ml of diethyl ether and stirred for 2 h. It was filtered, and the precipitation procedure was repeated three times. Finally, the resulting solid was put under the vacuum for 24 h to give 0.39 g of the pure product with 93% yield. The formation of the TRPZ-bisMPA was confirmed via the disappearance of BOC groups (1.34 ppm). 1H NMR  δ 8.07 , 7.53 , 7.44 , 7.33 , 7.24 , 5.33 , 4.42 , 4.22 , 3.94 , 3.01 , 2.64 , 1.17 , 0.85 .TRPZ-bisMPA separate glass vial. The solution was added dropwise to a separate vial of Milli-Q water at pH 7.0 (2 ml) while gently stirring to obtain a 1 mg/ml final concentration. THF was allowed to evaporate under a stream of nitrogen. NP solutions were allowed to equilibrate for 12 h before further study.For the nanoprecipitation method , 100 μL TRPZ-127 NPs was prepared and modified according to the previously reported procedure (TRPZ-PG (2 mg) was dissolved under rapid sonication in THF (2 ml). Then, a THF solution (1 ml) containing TRPZ-PG (1 mg/ml) and Pluronic F-127 (5 mg/ml) was used to prepare TRPZ-127 NPs by rapidly injecting the solution into deionized water (1 ml) under continuous sonication using a bath sonicator. After sonication for an additional 1 min, THF was evaporated under a nitrogen atmosphere.rocedure . First, o at 25°C. The size measurements were performed in triplicate for each sample at 0.5 mg/ml concentration to ensure consistency. The morphological study of the aggregates formed from the NPs was carried out by TEM using a JEOL 1230 TEM operated at 100 kV to collect the TEM images using a Gatan Orius 831 bottom mounted CCD camera.Aggregate sizes and ζ-potentials measurements were carried out on a Malvern Instrument Zetasizer Nano ZS using a He–Ne laser with a 633 nm wavelength, a detector angle of 173–5 M) and excited close to their maximum absorption. The spectroscopic energy gap . While the fluorescence studies were performed on Horiba Quantamaster fluorimeter with a xenon lamp and PMT detector using glass cuvettes. Fluorescence quantum yields were measured with samples of low sample concentration . Nanoparticles were added to tissue culture media and allowed 24 h incubation period in cytotoxicity studies. Cytotoxicity of the nanoparticles was evaluated with a CyQUANT LDH Cytotoxicity Assay Kit (Invitrogen) using a microplate reader (BioTek Synergy H1). Following manufacturer protocols, both negative and positive controls are used in the assay. Experimental values are transformed based on two values: zero-cytotoxicity value (background) and 100% cytotoxicity value (cells treated with lysis buffer based on manufacturer protocol). Each experiment is represented by relative values based on the control values. Imaging of particle distribution in cells was done 30 min after the addition of 10 μg/ml NPs to culture media. TRPZ fluorescence in HEK cells was observed with a Leica Stellaris STED confocal microscope using both conventional and STED modes.Human embryonic kidney (HEK293) cells were used for the assay. HEK cells were grown under standard conditions , TRPZ-127 NPs. Although frequently employed, the encapsulation strategy has significant drawbacks  that limit its application. Often the nature of the molecule being encapsulated has varying physiochemical properties that contrast to the solubilizing block of the amphiphilic polymer being employed. This, of course, leads to lower encapsulation efficiencies and can destabilize the resulting nanostructure  and DMF to obtain compound 4. The following step was carried out to deprotect dimethoxy moiety using an anion exchange resin-Dowex to obtain compound 5. Before proceeding to the next step, 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid was synthesized using a similar method to compound 2. Then, 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid was employed in a Malkoch esterification , is shown in fication  with comTRPZ-bisMPA (12) consists of four synthetic steps (TRPZ-PG was synthesized via a condensation method between 2-aminothiophenol and N-(propargyl) dichloromaleimide (compound 10). Then TRPZ-PG (10) and A-MPA-4-ala (8) were reacted using click-chemistry to obtain TRPZ-bisMPA-amine-Boc (compound 11). Finally, acid was used for deprotection of the tert-butyl carbamates (BOC) protected amine groups to afford a cationic terminated TRPZ-bisMPA amphiphile (compound 12).The synthesis of ic steps . First, TRPZ-bisMPA was done by nanoprecipitation. The hydrodynamic diameter was analyzed via dynamic light scattering (DLS) .The particle formation for ng (DLS) . SupplemTRPZ-PG was encapsulated with Pluronic F-127 (Mw = 12.6 kDa). It is not readily soluble in water unless incorporated within a water-soluble surfactant. Pluronic F-127 is known to self-assembly independently with sizes that are much smaller than those observed in this study (<50 nm) (TRPZ-bisMPA NPs. TRPZ-127 NPs exhibit a hydrodynamic diameter of 323.5 nm (±97 nm)   . The DLS(±97 nm)  and PDI nm (±66) . The encvia ζ-potential values aid in supporting the sizes obtained from DLS and TEM. The ζ-potential, which depends on the surface charge, is essential for the stability of NPs in suspension. Additionally, it is a critical factor in regards to the function and toxicity of NPs specifically for biological application. TRPZ-bisMPA NPs exhibit ζ-potentials of 17.2 mV. The positive charge results from the outer surface consisting of cationic amines. In contrast, TRPZ-127 NPs show -0.5 mV of average surface charge, which is close to neutral due to the lack of charge on the polymer surfactant used. The positive surface charge on TRPZ-bisMPA NPs corresponds to small and more stable aggregates . The emission maximum of the fluorophore is at 678 nm with a Stokes shift of 0.76 eV (200 nm). Note that the propargyl group does not contribute to the observed photophysical properties via pi orbital overlap.The normalized absorption and emission spectra of TRPZ and its NPs are shown in m in THF . The banTRPZ-127 NPs display three absorbance peaks at 452 nm, 480 nm, and 515 nm in water, as shown in normalized absorbance spectra  in the spectra suggest enhanced intermolecular interactions due to aggregation in the NP core.  spectra . CompareTRPZ. TRPZ-bisMPA NPs show absorbance peaks at 460 and 494 nm in water (TRPZ-bisMPA NPs exhibit a strong neon green fluorescence with an emission band centered at 695 nm and a Stokes shift of 0.72 eV (201 nm). The optical profile for TRPZ-bisMPA NPs resembles that of “free” TRPZ-PG in THF, with a 20 nm red shift for the latter. This behavior is presumably due to an aggregation behavior that is reminiscent of a monomeric form  of the dye with extended electron delocalization. The weaker emission of TRPZ127 NPs relative to TRPZ-bisMPA NPs is supported by a negative solvatochromism observed for TRPZ-PG  that are not dominant with the amphiphilic derivative.Interestingly there is a contrast in optical properties between the amphiphile and encapsulated in water . Upon ex TRPZ-PG . These rTRPZ-bisMPA support this notion where HOMO orbitals showed proaromatic molecular orbitals (MOs) and an aromatic excited state (LUMO) predominately at the pyrrole ring , , low reoole ring . Electroole ring  were empole ring . Using tole ring .TRPZ-bisMPA NPs possess a high quantum yield (Φ) in water, Φ = 49%. Typically, the Φ for conventional bioimaging dyes are much lower in water due to unfavorable solvent-solute interactions, which induce non-radiative pathways and quench emission (TRPZ-PG) . However, due to its poor solubility, comparison in aqueous media was not achievable.Along with a large Stokes shift, emission . In thisTRPZ-PG) . TRPZ-12TRPZ-PG in THF shows a signal exponential component of 5.3 ns and TRPZ-bisMPA NPs exhibits a lifetime of 6.3 ns. In contrast, TRPZ-127 NPs shows a dual exponential lifetime: t1 of 0.8 ns and t2 of 5.7 ns. The two exponentials are due to both the aggregated and monomeric  form being present inside the NP. When compared with TRPZ-PG and the absence of a lower (t1) value, the lifetime of 0.8 ns can be assigned to high-energy aggregates, which in this case is the dominant form (66.9%).Experimental lifetime plots are shown in SI . FluoresIn vivo testing will be needed to further assess the effect of the NPs on physiology; however, these results suggest our NPs will be well-tolerated in biological environments.The impact of the NPs on cell viability was tested by LDH assay . Even atTRPZ-bisMPA NPs and TRPZ-127 NPs accumulate most significantly in endosomes (TRPZ-bisMPA NPs leads to apparent cellular uptake via electrostatic interactions with anionic cell membranes. Similar results have been observed with other dendrimer-based NPs modified with anime groups (Confocal microscopy found that both ndosomes . The cate groups . Likely e groups .TRPZ-127 NPs show not only accumulation in the lysosome but also labeling of various cellular membranes, including nuclear membranes (TRPZ-127 NPs and TRPZ-bisMPA NPs aid in comparing cellular uptake where fluorescence labeling is localized to the membrane of the organelle and not the interior, as observed with TRPZ-bisMPA NPs (TRPZ-127 is much more aggressively loading into cells; however, this is not the case. We speculate that the encapsulated dye may have escaped its NPs and, due to its hydrophobic nature, became embedded into membrane interiors. The unloaded dye being hydrophobic actively segregates into cells while TRPZ-bisMPA NPs remain intact and localized in the lysosome.embranes . StaininsMPA NPs . It appeIn summary, a comparative analysis of two NP systems was conducted. Evaluation via spectroscopic, light scattering and microscopic techniques confirm colloidal stability and provided insight on photophysical properties of the molecular scaffold as a potential bioimaging agent. Cell viability studies indicate low cytotoxicity of the materials and their suitability for biological application. However, cellular distribution of TRPZ originating from the two sets of NPs suggests a fundamentally different interaction with cells. Such results suggest that a variation in formulation could be used for different fundamental applications. Overall the approach described here opens up avenues towards developing fluorescent NPs from a simple yet appealing scaffold to afford materials with tuneable properties for bioimaging applications.


This study aimed to validate the Sick, Control, One stone, Fat, and Food (SCOFF) questionnaire in relation to the Eating Disorders Examination Questionnaire (EDE-Q) and to examine the appropriateness of a question concerning weight loss among Japanese university students. The psychometric properties of the two Japanese versions were determined among 649 Japanese college students. The original version (SCOFF-O) employed the original item 3, whereas the revised version (SCOFF-2.5) modified the item to “Have you recently lost more than 2.5 kg within three months?” Validity was tested relative to EDE-Q.r = 0.53 and r = 0.56. The sensitivity and specificity of SCOFF-O were 65.2 and 89.7, and those of SCOFF-2.5 were 69.5 and 86.5, respectively. There were significant correlations between the question concerning losing 2.5 kg and the EDE-Q subscales. The Japanese version of SCOFF-2.5 is an appropriate tool for the early screening of eating disorders among Japanese university students.The test–retest reliabilities of SCOFF-O and SCOFF-2.5 were 0.52 and 0.57, while the correlations of SCOFF-O and SCOFF-2.5 with EDE-Q were  The emergence of eating disorders (EDs) in Asia over the past three decades is multifactorial and includes causes related to pathological and cultural transformation . A studyTherefore, the early detection of EDs in adolescents and undergraduate college students is of concern. A feasible assessment tool is needed to screen large populations for EDs or those at risk of developing EDs. Given the time constraints facing school health care, the screening tool needs to be brief, validated, and easy to remember.The Eating Attitude Test-26 (EAT-26)  is most 2) of young Japanese people. The percentage of women and men aged 20–29 years who were (at the time of data collection) underweight (BMI < 18.5 kg/m2) was 21.7% and 9.0%, respectively [The Sick, Control, One stone, Fat, and Food (SCOFF) questionnaire, consisting of five questions, was developed as a simple screening instrument for EDs . Leung eectively , 11.2 [2. In the present study, item 3 was modified to, “Have you recently lost more than 2.5 kg within three months?” This item 3b was subsequently included in the Japanese version of the SCOFF questionnaire in addition to the original question (item 3a). For early detection among young people, losing 2.5 kg would be a useful measure among the average 20-year-old woman, where it would result in an average BMI score of 19.7 kg/m2 (from 20.8 kg/m2), whereas for men of the same age, losing 2.5 kg would result in a decrease in average BMI from 21.8 kg/m2 to 20.9 kg/m2. We hypothesize that the questionnaire asking about losing 2.5 kg (SCOFF-2.5) would be more sensitive than the SCOFF-O .In the original SCOFF questionnaire, Question 3 refers to a weight loss of one stone over three months. In the current study, the Japanese version of the SCOFF questionnaire uses “6.35 kg” instead of “one stone.” The average BMI of 20-year-old Japanese women is 20.8 kg/m2  and the The first aim of this study was to verify the reliability and validity of both the Japanese SCOFF-O and the SCOFF-2.5 questionnaires relative to the Japanese version of EDE-Q among university students. The second aim of this study was to examine the appropriateness of the question concerning body weight loss in the SCOFF questionnaire.An online survey of Chiba University students was administered between 2016 and 2017 as part of an online mental health checkup. Participants were asked to complete the Japanese version of the SCOFF and EDE-Q questionnaires and to provide demographic variables, including body height and weight.SD = 3.28) participated in this study. An additional 82 students  with a mean age of 22.22 years (SD = 2.67) took part as a measure of the test–retest reliability of SCOFF-O and SCOFF-2.5. All participants could speak and understand Japanese fluently.A total of 649 college students  with a mean age of 22.37 years (1. Do you make yourself sick (vomit) because you feel uncomfortably full?2. Do you worry that you have lost control over how much you eat?3a. Have you recently lost more than 6.35 kg within three months?3b. Have you recently lost more than 2.5 kg within three months?4. Do you believe yourself to be fat when others say you are too thin?5. Would you say that food dominates your life?The original SCOFF questionnaire was translated into Japanese by four experts in EDs, back-translated into English, and subsequently approved by the original author.  The itemThe total score of the SCOFF questionnaire  is referred to as the original questionnaire (SCOFF-O). The total score of items 1, 2, 3b, 4, and 5 are referred to as SCOFF-2.5.The EDE-Q was developed to screen and evaluate EDs severity . The JapThe study was approved by the Ethics Committee of Chiba University. After reading about the purpose of the study, participants gave their informed consent. It took approximately 15 min to complete the questionnaire package. To examine test–retest reliability, SCOFF and EDE-Q were re-administered at two- to four-week intervals.Data were analyzed using SPSS statistics version 24.0 software . The reliability of the SCOFF questionnaire was tested with interclass correlation coefficients and internal consistency measures using Cronbach’s alpha. Concurrent validity was analyzed by its correlations with the EDE-Q. .SD = 2.28) completed the questionnaires twice. The average weight, height, and BMI scores of the women were 50.96 kg (SD = 5.94), 158.86 cm (SD = 5.40), and 20.18 kg/m2 (SD = 2.07), and those for men were 65.23 kg (SD = 8.89), 171.94 cm (SD = 5.36), and 22.00 kg/m2 (SD = 2.15). The test–retest reliability of the SCOFF-O and SCOFF-2.5 questionnaires and the EDE-Q were 0.52  and 0.56 . The internal consistencies of the SCOFF-O, SCOFF-2.5, and EDE-Q subscales ranged from 0.41. to 0.42, 0.40 to 0.46, and 0.58 to 0.93, respectively.To examine test–retest reliability, the Japanese SCOFF questionnaire and EDE-Q were tested at two- to four-week intervals. Fifty-eight out of 82 students  with a mean age of 22.03 years (n = 377), and 41.9% were women (n = 272). The mean age of the students was 22.37 (SD = 3.28). The average weight, height, and BMI of the women were 51.54 kg (SD = 6.34), 158.02 cm (SD = 5.30), and 20.63 kg/m2 (SD = 2.24), and that for the men was 63.55 kg (SD = 12.13), 171.56 cm (SD = 6.13), and 21.55 kg/m2 (SD = 3.72), respectively; 12.3% of participants were underweight , while 9.5%  were overweight (> 25 kg/m2). The average weight and height at each age in this study were close to those reported in Statistics of Japan . Of the participants, 12.4%  were SCOFF-O-positive (scoring two or more), while 15.4%  were SCOFF-2.5-positive. Table Complete data regarding the SCOFF-O and SCOFF-2.5 questionnaires were obtained from 649 students, of which 58.0% were men . Conversely, there were no significant correlations between the question concerning “losing 6.35 kg” and either the subscale or global scores of the EDE-Q (Table The answer “yes” for item 3b losing 2.5 kg) was observed among 10.6% of respondents, which is more compatible than the 0.77% “yes” response rate for item 3a (losing 6.35 kg)  and Sweden , 26.9% and 11.1% of secondary school students were SCOFF-positive  because 15 The significant correlation between item 3b and EDE-Q subscales and global scores suggests that, as a screening tool for EDs, the inclusion of the question concerning 2.5 kg weight loss in the SCOFF may be more appropriate for early detection among Japanese university students.To detect Japanese adolescents at risk for EDs, a new question  was included. The answer “yes” for item 3b (losing 2.5 kg) was registered among 10.6% of participants, which is more compatible than the 0.77% observed response rate for item 3a (losing 6.35 kg), with the rate (6.1%) of the Swedish version of SCOFF.Both the original SCOFF and the revised SCOFF 2.5 questionnaires indicate a significant correlation with their global EDE-Q scores. Specifically, the question concerning losing 2.5 kg showed a significant correlation with their global scores of the EDE-Q.SCOFF-O and SCOFF-2.5 showed good levels of specificity, while SCOFF 2.5 showed better levels of sensitivity than SCOFF-O. In particular, the version with the question concerning the 2.5 kg bodyweight loss is an appropriate tool for early screening of EDs among Japanese university students.The participants were not diagnosed using structured interviews. The present study modified item 3 based on the average weight and height of adolescents in Japan. Future studies should test the SCOFF questionnaire among other generations with younger participants.


PPE) was encapsulated in hydroxypropyl beta-cyclodextrin (HP-β-CD) by a spray-drying technique and the formation of an inclusion complex was confirmed by microscopy and infrared spectroscopy. The phenolic profile of the complex was analyzed by LC-ESI-LTQ-Orbitrap-MS and the encapsulation efficiency of the phenolic compounds was determined. A total of 42 compounds were identified, including stilbenes, flavonoids, and phenolic acids, and a complex of (epi)catechin with β-CD was detected, confirming the interaction between polyphenols and cyclodextrin. The encapsulation efficiency for the total extract was 80.5 ± 1.1%, with restrytisol showing the highest value (97.0 ± 0.6%) and (E)-resveratrol (32.7 ± 2.8%) the lowest value. The antioxidant capacity of the inclusion complex, determined by ORAC-FL, was 5300 ± 472 µmol TE/g DW, which was similar to the value obtained for the unencapsulated extract. This formulation might be used to improve the stability, solubility, and bioavailability of phenolic compounds of the GCPPE for water-soluble food and pharmaceutical applications.Grape canes, the main byproducts of the viticulture industry, contain high-value bioactive phenolic compounds, whose application is limited by their instability and poorly solubility in water. Encapsulation in cyclodextrins allows these drawbacks to be overcome. In this work, a grape cane pilot-plant extract (GC The generation of food and agricultural waste is a growing problem, with negative impacts on the economy, environment, and human health. Therefore, the integral valorization of these wastes by conversion into bioenergy or recovery of chemical compounds for biobased products is a technological challenge for achieving a circular economy. Alternative ways of disposing of food waste include the valorization of byproducts as a source of phenolic compounds used to fortify high-consumption foods or to formulate new functional foods .V. vinifera L. produced during pruning are the main byproduct of viticulture, and millions of tons are generated worldwide every year —  were detected and identified by comparison with the mass spectra reported by Żyżelewicz et al. [According to the supplier, the HP-z et al. . The detPPE is a complex mixture of various compounds that have different physical and chemical properties and abilities to form interactions and bind within the HP-β-CD cavity. As shown earlier, the phenolic compounds in the microencapsulated extract are phenolic acids and derivatives, flavonoids, and stilbenes. The individual EE (%) for twenty of these compounds was calculated and presented in GCPPE+HP-β-CD+MD).The average EE for all the analyzed compounds was 80.5 ± 1.1%. Several authors have studied the encapsulation of phenolic compounds from wine byproducts using different encapsulation materials. Davidov-Pardo et al.  reportedO-hexoside 1 (54.3 ± 2.4%) and the highest for hydroxybenzaldehyde (95.8 ± 0.6%). The EE for protocatechuic acid was 66.8 ± 9.8%, higher than the value reported by Taofiq et al. [2) to promote alginate gelation. The EE for hydroxybenzaldehyde obtained here is higher than the value (46.50%) reported for p-hydroxybenzaldehyde in a β-CD inclusion complex [The average EE for the phenolic acids and derivatives was 81.5 ± 0.7%, the lowest value being obtained for protocatechuic acid-xoside 1 .3 ± 2.4% complex .β-CD and the lyophilization method. However, Olga et al. [β-CD, a result that was reduced to 77.34% when the gallic acid was co-encapsulated with trans-ferulic acid. The authors suggested a possible antagonistic relationship between the two phenols in the HP-β-CD cavity.The EE for gallic acid, 83.4 ± 0.6%, was slightly higher than the value reported by da Rosa et al. , who deta et al.  obtainedρ-coumaric acid (42 ± 1%) [β-CD nanosponges [The EE of 87.6 ± 0.5% observed for ellagic acid pentoside was considerably higher than the 55.2% reported for ellagic acid (aglycone) encapsulated with polyvinyl alcohol . The EE 42 ± 1%)  and chloosponges , respectThe average EE for flavonoids was 85.2 ± 2.4%. The values for astilbin isomer (2) and astilbin (1) were particularly high, 92.0 ± 1.1% and 82.7 ± 2.3%, respectively. Zheng and Zhang  reportedO-glucuronide (88.0 ± 3.1%) and quercetin-O-glucoside (81.8 ± 7.0) also had high EE values. Tchabo et al. [O-glucoside in a spray-dried mulberry leaf extract prepared with MD, yet the EE was higher (91.71–93.95%) when sodium carboxymethyl cellulose was used. To the best of our knowledge, no EE values have been previously reported for quercetin-3-O-glucuronide.Quercetin-3-o et al.  obtainedThe EE for eriodictyol was 74.2 ± 4.7%, similar to the almost 70% reported for naringenin (another flavanone) in polymer PLGA nanoparticles prepared by an emulsion-diffusion-evaporation method .E)-resveratrol (32.7 ± 2.8%). Nevertheless, previous studies have shown that the cyclodextrin encapsulation of resveratrol increases its solubility, stability, and bioactivity (antioxidant and anticarcinogenic properties) [β-CD complex exhibits a strong H-bonding interaction with molecules such as oxyresveratrol [β-CD.The stilbene group had the lowest mean EE (78.6 ± 1.9%), mainly because of the low value obtained for (perties) . Furtherveratrol . In our E)-ε-viniferin (76.8 ± 3.5%), and an undefined resveratrol dimer (94.1 ± 2.1%). Previously, ε-viniferin, a resveratrol dimer, was encapsulated in phospholipid-based multi-lamellar liposomes called spherulites or onions. This formulation gave a lower EE (58 ± 3%) than in our study, but increased the water solubility of this stilbene more than five-fold and provided protection against its UV-induced isomerization [A good EE was obtained for stilbene dimers such as pallidol (74.6 ± 3.8%),  by ORAC-FL was 5300 ± 472 µmol TE/g DW, similar to the 4612 ± 155 µmol TE/g DW reported in our previous GCPPE study [PPE microencapsulated with HP-β-CD retains its antioxidant capacity and its formulation may improve stability, solubility, and bioavailability for applications in the food, cosmetic and pharmaceutical industries.The antioxidant capacity of the IC (GCPPE+HP-β-CD+MD). The microencapsulated extract was rich in stilbenes, especially oligomers, flavonoids, and phenolic acids. A complex of (epi)catechin and β-CD was detected by mass spectrometry, which confirmed the interaction between polyphenols and cyclodextrin. The formation of the inclusion complex was also supported by FTIR-ATR and SEM analyses. HP-β-CD provided a high EE for phenolic compounds, with a mean of 80.5 ± 1.1%, the highest values being obtained for restrytisol (97.0 ± 0.6%), stilbenoid heterodimer (1) (96.8 ± 0.4%) and hydroxybenzaldehyde (95.8 ± 0.6%), and the lowest for (E)-resveratrol (32.7 ± 2.8%). The antioxidant capacity of the inclusion complex was similar to the unencapsulated extract. Considering the protection afforded the phenolic compounds by the inclusion complex, it is expected that the formulation may improve their stability, solubility, and bioavailability in water-soluble applications for the food and pharmaceutical industries.Phenolic compounds from a grape cane pilot-plant extract were successfully encapsulated in an inclusion complex (GCV. vinifera and the application of HP-β-CD to form the IC and the use of MD as a coating agent are described in the Patent [The preparation of grape canes before extraction in the pilot plant followed the procedure described in the Patent . The fore Patent .


Wai-Tai-Mi-Yao, is a potentially effective treatment for AD. We aimed to clarify the anti-inflammatory and anti-pruritus mechanisms of HLJDT in AD treatment. We performed immunohistochemistry, Western blotting, reverse transcriptase-polymerase chain reaction, Luminex-based direct multiplex immunoassay, enzyme-linked immunosorbent assays, and flow cytometry to address the abovementioned aims. HLJDT significantly reduced clinical symptoms and ear swelling in AD-like mice by inhibiting the production of cytokines , substance P (SP), transient receptor potential cation channel subfamily V member 1 (TRPV-1), and gastrin-releasing peptide (GRP). Additionally, HLJDT significantly suppressed the protein expression levels and positive cell percentage of CD28, CD80, CD86, CD207, CD326, MHCII, and OX40 in the lymphoid nodes. Moreover, HLJDT significantly suppressed mRNA and protein expression of tyrosine–protein kinase (JAK1), histamine H4 receptor, and IL-4Rα, as well as the protein expression of GRP, SP, and TRPV-1 in the root ganglion. Our findings indicate that HLJDT can treat AD by regulating the antigen presentation function of dendritic cells, weakening T-lymphocyte activation, and subsequently exerting anti-inflammatory and anti-pruritus effects.Atopic dermatitis (AD) is a common chronic skin disease driven by a T-cell-mediated immune response, with inflammation and pruritus being its main clinical manifestations. Huanglian Jiedu decoction (HLJDT), which is an ancient Chinese medicine herbal formula derived from  Atopic dermatitis (AD) is a common, highly heritable chronic skin disease that is usually mediated by IgE and is clinically characterized by erythema, itch, eczema skin lesions, skin thickening, and inflammation . WorldwiAD involves an aberrant immune response involving several crosslinks, including antigen recognition, processing, and presentation in Langerhans cells (LCs) and dendritic cells (DCs); Th2-dominant abnormal immune responses; compromised function of regulatory T cells; IgE overexpression; and increased eosinophil levels . AbnormaWai-Tai-Mi-Yao. HLJDT is known to possess therapeutic effects of clearing heat and detoxification in Chinese medicine practice and is widely used in skin diseases, such as eczema and AD , Scutellaria baicalensis Georgi , Phellodendron amurense Rupr. , and Gardenia jasminoide J. Ellis , at a ratio of 3:2:2:3. It exerts therapeutic effects of heat clearing and detoxification against the fire-toxicity syndrome (Huanglian Jiedu decoction (HLJDT) is an ancient Chinese medicine herbal formula derived from a and AD . HLJDT isyndrome . Modern syndrome . Althougsyndrome , the antThis study aimed to determine whether AD treatment by HLJDT mainly involved anti-inflammatory and anti-pruritus activities, as well as to systematically explore these anti-inflammatory and anti-pruritic mechanisms.Coptis chinensis Franch, Scutellaria baicalensis Georgi, Phellodendron amurense Rupr, and Gardenia jasminoides J. Ellis were purchased from the Hangzhou Mintai (Bozhou) Chinese Herbal Medicine Co. Ltd.  and identified by professor Rui-Ping Huang  following the Chinese Pharmacopoeia (2015 Edition). Voucher specimens of the aforementioned herbs were deposited in the Department of Pharmacy of Taizhou Central Hospital , Taizhou, Zhejiang, with reference no. Xu1-4. We purchased 2,4-dinitrofluorobenzene (DNFB) from the Shanghai McLean Biochemical Technology Co., Ltd. . All the other chemicals used were of analytical grade.Coptis chinensis Franch, Scutellaria baicalensis Georgi, Phellodendron amurense Rupr., and Gardenia jasminoides J. Ellis, with a ratio of 3:2:2:3) was measured in proportion. Water was used as the solvent to extract twice for 1 h each; the filtrate was collected with a treatment of hot filtration and evaporated to obtain the appropriate concentrated soup. Subsequently, the quality of HLJDT was evaluated through high liquid chromatography, and the contents of berberine and baicalin in HLJDT were determined to be 4.51 and 7.92 mg/g, respectively; the HPLC fingerprinting was constructed as quality control for HLJDT as previously described . In brief, 600 g of the mixed Chinese medicine (escribed . Furtherescribed . SupplemWe purchased specific pathogen-free (SPF) male C57BL/6 mice  from the Shrek Jinda Laboratory Animals Co., Ltd. . The animals were kept under SPF laboratory conditions; moreover, all animal experimental procedures were approved by the ethics committee of the institution (ID number: SHDSYY-2020-9001).n = 8) as follows: the control group, model group, and three HLJDT groups . DNFB was dissolved in acetone/olive oil (4:1) to prepare the proper solutions before administration to the mice. The procedure was performed as previously described, with slight modifications (2) of the mice on the first day. Next, 200 μl of 1% DNFB was used to sensitize the abdominal skin on days 1, 4, and 7 (once a day). Subsequently, the model and HLJDT groups were administered with 200 μl of 0.5% DNFB on the back skin and right ear on days 14, 17, 19, 22, 24, 27, and 29 (once a day), with the control group being smeared with an equal volume of DNFB matrix solution at the same time points. Next, the HLJDT groups were intragastrically administrated with 3.2, 6.4, and 12.8 g/kg HLJDT once a day for 16 days, while the control and model groups received an equal saline volume at the same time points. After the last administration, the mice were anesthetized using pentobarbital sodium followed by blood sample collection to obtain serum. Finally, the animals were sacrificed, followed by the collection of the dorsal root ganglion and lymphoid nodes. The obtained tissues and serum were placed in a refrigerator at −80°C for subsequent analyses. Some of the tissues were cut and immersed in 4% paraformaldehyde for immunohistochemical analysis.Based on our preliminary findings, C57BL/6 mice were randomly divided into five groups , gastrin-releasing peptide (GRP), substance P (SP), and transient receptor potential cation channel subfamily V member 1 (TRPV-1) were determined in the serum of mice.Mouse Custom ProcartaPlex 8-plex kit  was used to detect IL-3, IL-4, IL-5, IL-13, IL-17A, IL-31, and IL-33 levels using the Luminex-based direct immunoassay platform , following the instructions of the manufacturer. Moreover, the data were analyzed using ProcartaPlex Analyst 1.0 software. Enzyme-linked immunosorbent assays (ELISA) kits were purchased from Cloud-Clone Corp  for measuring the histamine, IgE, GRP, SP, and TRPV-1 levels.® reagent . Subsequently, we conducted reverse transcription and produced cDNAs using the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) , which were subjected to RT-PCR analysis using 2× SYBR Green PCR Master Mix  on CFX Connect™ PCR instrument  according to the protocol of the manufacturer. The primers were as follows: HRH4 specific primer , IL-4Rα specific primer , JAK1 specific primer , GRP specific primer , SP specific primer , TRPV-1 specific primer , and β-actin specific primer . After normalization to the β-actin level, we calculated the expression levels of target mRNAs.Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was used to detect HRH4, IL-4Rα, JAK1, GRP, SP, TRPV-1, and β-actin mRNA expression. Total RNA was isolated from the dorsal root ganglion tissues using TRIzolWestern blot analysis was performed as previously described, with slight modifications . BrieflyNext, equal protein amounts of the different samples were separated and electroblotted through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polyvinylidene difluoride (PVDF) membranes. After blocking using 3% nonfat milk overnight, the PVDF membranes were incubated with the primary antibody at 4°C for 3 h, followed by rinsing with TBST thrice for 10 min each. Next, the PVDF membranes were incubated with a secondary antibody solution for 2 h at room temperature. Subsequently, they were washed and visualized using an ECL  method through a high-sensitivity chemiluminescence imaging system . Antibodies for Western blot analysis included anti-H4R (Omnimabs # OM209489), anti-JAK1 , anti-p-JAK1 (Affinity # AF 2012), anti-IL-4Rα (Affinity # DF8567), and anti-β-actin (Abcam # ab8226) antibodies.Immunohistochemical staining was performed as previously described, with slight modifications . Briefly7 times/ml; subsequently, we added 200 μl of cell suspension with 5 μl of PE-CD28/CD80/CD207 fluid antibody, followed by incubation for 30 min in a water bath at a constant temperature of 37°C. After incubation, the cells were resuspended in 200 μl of phosphate-buffered saline and incubated with 5 μl of FITC-OX40/CD86/MHC-II/CD326 fluid antibody at the same condition. All the antibodies were purchased from BD Pharmingen. Phycoerythrin and fluorescein isothiocyanate signals were analyzed using CytoFlex . DC populations were selected according to the forward- and side-scatter.Lymph node tissues were ground and disrupted using a glass homogenizer in an ice bath, with a single-cell suspension being prepared using a 200-mesh filter. The cell concentration was adjusted to 1 × 10p < 0.05.Data were presented as mean ± standard deviation. Statistical analyses were performed by one-way analysis of variance using the statistical software SPSS 20.0 . Statistical significance was set as p < 0.01), which was significantly reduced by 6.4 and 12.8 g/kg of HLJDT (p < 0.01). Compared with the control group, the model group showed significantly higher between-ear thickness differences (p < 0.01); furthermore, 6.4 g/kg and 12.8 g/kg HLJDT significantly inhibited the DNFB-induced ear swelling (p < 0.01)  .p < 0.01), which was indicative of abnormal cytokine secretion mainly related to inflammation and pruritus in the model group. Notably, 12.8 g/kg HLJDT inhibited the abnormal secretion of the aforementioned cytokines (p < 0.01) except for IgE (p > 0.05) ; furthermore, 6.4 g/kg HLJDT only significantly decreased IL-4 and IL-17A levels (p < 0.05), IL-3, IL-5, IL-13, and IL-33 (p < 0.01) compared with the levels in the model group. Finally, 3.2 g/kg HLJDT had no effect in the model group  from the dorsal root ganglion. The mRNA expression levels of all pruritus-related markers, except JAK1, were significantly upregulated in the model group, which were downregulated by 12.8 g/kg HLJDT. Additionally, 6.4 g/kg HLJDT downregulated HRH4 and GRP mRNA expressions, while 3.2 g/kg HLJDT only downregulated HRH4 mRNA expression. .The model group showed significantly upregulated protein expressions of p-JAK1, HRH4, and IL-4αR, which were significantly downregulated by 12.8 g/kg HLJDT. Furthermore, 6.4 g/kg of HLJDT could downregulate HRH4 protein expression, while 3.2 g/kg HLJDT had no effect  The ori.p < 0.01). Compared with the model group, the 6.4- and 12.8-g/kg HLJDT groups showed weakly positive expression of p-JAK1, HRH4, IL-4αR, GRP, SP, and TRPV-1 proteins; moreover, there was a significant decrease in the number of positive cells (p < 0.05 or p < 0.01). Although this result was consistent with the RT-PCR and Western blot, 12.8 g/kg HLJDT had a more stable effect on AD than the 6.4 g/kg of HLJDT.As shown in p < 0.01), which were significantly downregulated by 12.8 g/kg HLJDT (p < 0.01). Additionally, 3.2 g/kg of HLJDT downregulated CD28 and CD80 expressions, while 6.4 g/kg of HLJDT downregulated CD28, CD86, CD207, MHCII, and OX40 expressions compared with the expression levels in the model group (p < 0.05 or p < 0.01). Taken together, 12.8 g/kg HLJDT had the strongest anti-AD effect.As shown in p < 0.01), which were significantly reduced by 3.2, 6.4, and 12.8 g/kg of HLJDT (p < 0.01). Furthermore, 12.8 g/kg HLJDT was the most efficient.As shown in HLJDT has good therapeutic effects on AD , which iepidermis, with different functions in different skin regions  during their migration to draining lymph nodes after antigen uptake, which induces AD by stimulating T-lymphocyte activation . MHC-Ⅱ iule CD28 . In addiule CD28 . We founAD is characterized by abnormalities in innate and adaptive immunity. After antigen presentation by DCs, T cells are activated to secrete various cytokines and chemokines that play an immunomodulatory role to maintain and expand the overall inflammatory response . We founPruritus is a main characteristic of AD. GRP and SP are crucially involved in histamine-dependent itch neurotransmission; furthermore, GRP upregulation has been presented in both chronic dermatitis and pruritus mice, which suggests that GRP-expressing nerves have a vital role in mediating pruritus . MoreoveScutellaria baicalensis and Phellodendron amurense). For anti-AD mechanism of pharmacodynamic material basis from HLJDT, some blockers or agonists will be used as positive controls for further research, which also helps in the development of yellow detoxification soup and lay the foundation for clinical application.In this experiment, the positive control was not used for AD owing to there is no AD-specific drugs, and mostly, AD patients use hormone drugs. So far, early clinical and basic research had already demonstrated the effectiveness of HLJDT for AD, and the research of ConclusionsIn conclusion, HLJDT could exert anti-inflammatory and anti-pruritic effects by regulating the antigen presentation function of DCs, weakening T lymphocytes, and downregulating the expression of indices in the dorsal root ganglion. Future studies should investigate the specific components of HLJDT involved in AD treatment. Our findings provide evidence regarding the theoretical and scientific basis for the clinical treatment of AD using HLJDT.


This paper reports HIV prevalence, incidence, progress towards the UNAIDS (90-90-90) targets, and HIV drug resistance among adolescents living with HIV in South Africa. We conducted secondary analyses using data extracted from the South African national HIV prevalence surveys (2005–2017). Analyses were stratified by sex and age (10–14 and 15–19-years), presenting weighted descriptive statistics, and realised totals.HIV prevalence increased from 3.0% in 2012 to 3.7% in 2017, translating to 360 582 (95% CI 302 021-419 144) HIV positive adolescents in 2017. Female adolescents bear a disproportionate HIV burden of 5.6% prevalence versus 0.7% for males. HIV incidence remained relatively stable. For the UNAIDS 90-90-90 targets, approximately 62.3% of adolescents knew their HIV status, 65.4% of whom were on antiretroviral therapy, and of these 78.1% on antiretroviral therapy had attained viral load suppression. There are knowledge gaps pertaining to the magnitude of perinatal infections and postnatal infections, and socio-behavioural risk factors for HIV transmission among adolescents in South Africa. There is still a need for focussed interventions targeting adolescent (1) gender disparities in HIV risk (2) screening for HIV, (3) sustained access and adherence to antiretroviral therapy and (3) retention in care to maintain viral load suppression. In 2020 approximately 1.7 million children under 15 years were living with HIV (ALHIV) globally; majority of whom reside in sub-Saharan Africa . AdolescImproved understanding of the circumstances of ALHIV is critical in designing adolescent-friendly health services to facilitate their successful transition into adult care. Compared to adults, adolescents have lower rates of HIV testing, disclosure , treatmeIn South Africa, routine data on ALHIV’s health are also not readily available . PertineWe used data from four South African National HIV Prevalence, Incidence, Behaviour and Communication Surveys (known as “SABSSM”) from 2005 to 2017. These are population-based household surveys, using a multi-stage stratified cluster random sampling design, described in detail in the survey reports –20. SociThe survey reports describe HIV testing assays for each SABSSM survey , 18–20. Non VLS (VL ≥ 1000 copies/ml) samples were evaluated for HIVDR using Next Generation Sequencing , 24, 25.Awareness of HIV status was used to estimate.the 90-90-90 UNAIDS’ targets, which stipulates by 2020, 90% of PLHIV know their HIV status, 90% of those diagnosed with HIV receive ART, and 90% of all people receiving ART achieved VLS. The gaps in achieving the UNAIDS’ targets were calculated based on the 90%-81%-73% cascade .We used cross-sectional benchmarked weights per each survey wave and did not pool these for analyses. We calculated descriptive statistics for the sample characteristics by each indicator variable, using Pearson’s Chi-square tests to detect differences among categorical variables, reporting 95% confidence intervals (CIs). Where CIs did not overlap, this was used to conclude statistical significance. Significance at p ≤ 0.05 are reported. All analyses except incidence estimates were performed in Stata version 15.0 , accounting for the complex survey design. The computational tools for the incidence estimates were developed by the South African Centre for Epidemiological Modelling and Analysis .Among 8 762 adolescents tested for HIV in 2017, the majority were Black African (95.4%), attending school (79.1%) and unemployed (99.3%)  in 2005 to 3.0% (95% CI 2.5–3.7) in 2012, then increased to 3.7% in 2017 (95% CI 3.2–4.3) , comprising 136,913 aged 10–14 years and 223,669 aged 15–19 years. Of these, 202,923 were female, and 157,659 were male. In 2017, HIV prevalence among female adolescents aged 15–19 years remained unchanged at 5.6% (95% CI 4.5–6.9) whilst for their male counterparts, it increased to 4.2% (95% CI 3.0–5.8) from 0.7% in 2012.Among ALHIV who knew their status, more females, , said they had disclosed their HIV status to a main sexual partner compared to males , although there was a small sample for males (n = 9).HIV incidence among adolescents aged 10–19 years was 0.50% (95% CI 0.44–0.56) in 2012, declining to 0.39% (95% CI 0.35–0.43) in 2017. HIV incidence in adolescents aged 15–19 years was 0.87% (95% CI 0.82–0.92) in 2012 and remained relatively stable at 0.82% (95% CI 0.74–0.90) in 2017. There were limited data to estimate HIV incidence in adolescents aged 10–14 years [HIV viral load results were available for 398 10–19 years ALHIV. Among these, 48.9% (95% CI 41.5–56.3) had VLS (VL < 1000 copies/ml). A higher proportion  of ALHIV aged 10–14 years had VLS compared to their older counterparts , but these differences were non significant.Of n = 182 ALHIV samples not virally suppressed, n = 106 were successfully amplified and sequenced and of these, 33.7%  had HIVDR. Of the 33 samples, 64.5%  had non-nucleoside reverse transcriptase inhibitor (NNRTI) mono resistance, while 30.4%  had dual NNRTI and nucleoside reverse transcriptase inhibitor resistance. We found no mutations to protease inhibitor drugs. Of the samples successfully amplified and sequenced for DRMs and analysed for ART exposure (n = 82), 12% (95% CI 6.0–22.5) were exposed to ART).Approximately 62.3% (95% CI 54.7–69.2) of ALHIV aged 10–19 years knew their HIV status and of these 65.4% (95% CI 54.3–75.0) were on ART. Among ALHIV on treatment 78.1% (95% CI 69.0–85.1) were virally suppressed.Overall awareness of their HIV status was significantly lower (p < 0.001) for ALHIV aged 10–14 years at 44.5% (95% CI 33.1–56.5) compared to 73.1% for those aged 15–19 years (95% CI 64.2–80.5). Among ALHIV who knew their status, ART uptake was significantly higher (p = 0.001) among 10–14-year-old adolescents  compared to their 15–19 -year-old counterparts . For ALHIV who were on treatment, 75.2% (95% CI 55.2–88.1) of those aged 10–14 years had VLS, and a similar proportion of 15–19-year-old ALHIV had VLS .Similar proportions of male and female ALHIV aged 10–19 years knew their HIV status (1st 90) i.e., 60.1% (95% CI 49.6–69.7) for males and 63.9% for females (95% CI 53.4–73.3). However, ART uptake was lower among females at 62.1% (95% CI 50.1–72.8) than males  (2nd 90). Among ALHIV on ART, more females had VLS at 82.8% (95% CI 71.3–90.3) than their male counterparts  (3rd 90). Overall, the gap in attaining VLS was 43% among male ALHIV and 40% among female ALHIV (Fig. There were approximately 360 600 ALHIV aged 10–19 years in South Africa in 2017. Female adolescents bear a disproportionate burden of HIV compared to their male counterparts. The gender differential is consistent with HIV incidence among adolescents living in sub-Saharan Africa  and can Progress towards the UNAIDS target of the first 90 at 62.3% being aware of their HIV status, is substantially lower compared to the general population, where 85% of PLHIV aged 15–64 years knew their status .We found low levels of ART use and low levels of VLS among ALHIV. Like other African region-based studies, ART uptake was lower for females than males , 33. TheThe high HIV prevalence among female adolescents and steady incidence highlight the need to prevent new infections among adolescents. There remains a need for improved access to quality sexual and reproductive health services that are youth-friendly and gender sensitive.This study is limited by the small sample size, particularly for those aged 10–14 years. Collection of blood specimens for determination of HIV status in this group are influenced by parents’ or caregivers’ reluctance to give consent as mentioned by other researchers in this area . The sam


Current issues in this field include a poor catalytic efficiency and a low substrate loading.Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block Bacillus badius to create an enhanced biocatalyst for efficient l-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic efficiency. Seven superior mutants were acquired, and the optimal triple-site mutant, V309G/L306V/V144G, showed a 12.7-fold higher kcat value, and accordingly a 12.9-fold higher kcat/Km value, than that of the wild type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to l-HPA in 210 min, and the specific space–time conversion was 30.9 mmol g−1 L−1 h−1. The substrate loading and specific space–time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones.In this study, we report a structure-guided steric hindrance engineering of PheDH from l-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of The online version contains supplementary material available at 10.1186/s13068-021-02055-0. The 2-OPBA retention time was 9.04 min. The ee value of l-HPA was determined using a Develosil® ODS-UG-5 column  (Phenomenex) after derivatization with 1-fluoro-2,4-dinitrophenyl-5-l-alanineamide (FDAA). A 5-μL reaction sample, 4 μL of 1 M NaHCO3, and 20 μL of 1% (w/v) FDAA in acetone were mixed and heated at 40 °C for 60 min. Then 4 μL of 1 M HCl and 467 μL of 40% (v/v) aqueous acetonitrile were added to the mixture. The latter was then passed through a 0.22-μm filter and subjected to HPLC. The conditions for determining the ee value of l-HPA were as follows: injection volume, 20 μL; mobile phase B (5% (v/v) acetonitrile, 0.05% (v/v) trifluoroacetic acid, and 1% (v/v) methanol); mobile phase C (60% (v/v) acetonitrile, 0.05% (v/v) trifluoroacetic acid, and 1% (v/v) methanol); linear gradient from 0 to 100%; mobile phase C, 45 min at 30 °C; flow rate, 1.0 mL min−1; and UV detection at 340 nm. The l-HPA retention time was 33.65 min.The conversion of 2-OPBA and the ee value of BbPheDH and its mutants were constructed using the SWISS-MODEL server (https://swissmodel.expasy.org/) [Rhodococcus sp. M4 (PDB: 1C1D) [https://pubchem.ncbi.nlm.nih.gov/) [http://biophysics.cs.vt.edu/H++) [BbPheDH and its mutants and was set as the search space for suitable substrate-binding poses. Energies of the interactions between the ligands and the receptors were calculated with MGLTools v. 1.5.4. The lowest energy docking pose was selected for the subsequent molecular dynamics simulations.Structural homology models of sy.org/)  based onB: 1C1D) . The 3D ih.gov/) . Ligand ih.gov/) . AutoDocih.gov/)  was usededu/H++)  to mimicBbPheDH and its mutant M3–2 were calculated with the CAVER v. 3.0 plugin of PyMOL [BbPheDH and its mutants were calculated with the ProteinsPlus tool (https://proteins.plus/) [The tunnels of of PyMOL . The pros.plus/) . The PDBAdditional file 1: Table S1. Primers used for site-directed mutagenesis.Table S2. Enzyme thermostabilities of BbPheDH and its superior mutants. Table S3. Effects of substrate concentration on the asymmetric reductive amination of 2-OPBA catalyzed by mutant M3–2. Table S4. Comparison between M3–2 and other reported PheDHs for the synthesis of l-HPA from 2-OPBA. Table S5. Calculated substrate-binding pocket volume and area of BbPheDH and its superior mutants. Table S6. Kinetic parameters of BbPheDH and its superior mutants toward NADH.Additional file 2: Figure S1. Time course of asymmetric reductive amination of 2-OPBA catalyzed by BbPheDH and GkPheDH. Figure S2. Amino acid sequence alignment of the PheDHs from different sources.Figure S3. Relative activity of the single-site mutants constructed in the first round of steric hindrance engineering. Figure S4. Relative activity of the double-site mutants constructed in the second round of steric hindrance engineering. Figure S5. Relative activity of the triple-site mutants constructed in the third round of steric hindrance engineering. Figure S6. Relative activity of the quadruple-site mutants constructed in the fourth round of steric hindrance engineering. Figure S7. Relative activity of BbPheDH and GluDH in different concentrations of NH4OH/HCOONH4 buffer (pH 8.5). Figure S8. Binding poses of native substrate PPA (a) and bulky substrate 2-OPBA (b) with BbPheDH. Figure S9. SDS-PAGE analysis of BbPheDH and its superior mutants.


Rho family mechano-signaling through the actin cytoskeleton positively regulates physiological TEAD/YAP transcription, while the evolutionarily conserved Hippo tumor suppressor pathway antagonizes this transcription through YAP cytoplasmic localization/degradation. The mechanisms responsible for oncogenic dysregulation of these pathways, their prevalence in tumors, as well as how such dysregulation can be therapeutically targeted are not resolved. We demonstrate that p53 DNA contact mutants in human tumors, indirectly hyperactivate RhoA/ROCK1/actomyosin signaling, which is both necessary and sufficient to drive oncogenic TEAD/YAP transcription. Moreover, we demonstrate that recurrent lesions in the Hippo pathway depend on physiological levels of ROCK1/actomyosin signaling for oncogenic TEAD/YAP transcription. Finally, we show that ROCK inhibitors selectively antagonize proliferation and motility of human tumors with either mechanism. Thus, we identify a cancer driver paradigm and a precision medicine approach for selective targeting of human malignancies driven by TEAD/YAP transcription through mechanisms that either upregulate or depend on homeostatic RhoA mechano-signaling. Crosstalk between the p53 and Hippo pathway has been reported in different physiological contexts. Here, the authors show that p53 DNA contact mutations upregulate TEAD/YAP transcription indirectly and transform cells via hyperactivation of RhoA/ROCK/actomyosin signaling. The great majority involve driver mutations that directly activate oncogenic signaling within the same pathway2. There remains a critical need to better understand oncogenic mechanisms that have not yet proven amenable to biologically targeted therapies. One such mechanism involves the evolutionarily conserved Hippo pathway, which plays an important role in organ size control and normal tissue homeostasis3 through the actions of its core kinases, MST1/2 and LATS1/2, which negatively regulate TEA domain (TEAD) transcription through phosphorylation and cytosolic retention/degradation of TEAD co-activators, YAP and TAZ (hereafter designated as YAP)4. Recurrent tumor mutations in the Hippo pathway through loss of function (LOF) of LATS or NF2 or YAP amplification/over expression result in constitutive upregulation of TEAD/YAP transcription and acquisition of the transformed phenotype in culture5 and in mouse genetic models in vivo6. While these findings have strongly implicated this pathway in human malignancy7, there are as yet no targeted approaches available for Hippo pathway dysregulated tumors10.The ability to selectively target oncogene or tumor suppressor pathways that afflict particular tumors or tumor types has led to significant increases in patient survival over the past two decades11 and has a critical role in normal cellular homeostasis12. There is also evidence of crosstalk between p53 and Hippo pathways in various physiological contexts13. More than 50% of human tumors exhibit p53 LOF mutations, and around three-quarters of these are missense mutations14, which have been reported to also confer a gain of function (GOF)15. Over the past 2 decades, numerous GOF mechanisms have been set forth with most implicating mutant p53 interactions with transcription factors ranging from p53 related family members to NF-Y, Sp1, Ets-1, SREBPs, VDR, or YAP18. Among these, Freed Pastor et al. reported that p53 missense mutants upregulated the activity of SREBPs, master transcriptional regulators of the mevalonate (MVA) pathway19. Geranylgeranyl pyrophosphate, an MVA pathway product, is known to increase membrane localization and activation of the Rho family20, and a p53 missense mutant that increased SREBP activity was subsequently reported to upregulate TEAD/YAP transcription through a RhoA dependent mechanism21. However, activating mutations analogous to Ras oncogenic mutations in members of the Rho family of GTPases22, are observed very infrequently in human tumors23.The p53 tumor suppressor gene is similarly highly conserved in evolution24, to selectively antagonize the growth and motility of a substantial fraction of human tumors harboring previously untargeted p53 missense or Hippo pathway mutations.In the present study, we investigated the GOF mechanism through which certain p53 missense mutants induce the transformed phenotype and identify a paradigm for cancer driver mutations that act indirectly to hyperactivate wild-type RhoA mechano-signaling to levels required for oncogenic TEAD/YAP transcription. We also investigated the role of physiological RhoA/ROCK mechano-signaling in upregulating TEAD/YAP transcription and the proliferation of tumors with Hippo pathway genetic aberrations. Our findings provide proof of concept for the application of ROCK inhibitors, which have been clinically tested in humans for other indications21, was shared by other p53 GOF mutants. To do so, we screened different p53 missense mutants exogenously expressed in MCF10A immortalized human breast epithelial cells, which are wild-type for components of the Hippo pathway and p53, for the ability to activate a TEAD transcriptional reporter25. Those with hot spot as well as less common mutations of the class that specifically impairs DNA contact with the promoters of p53 wild type (WT) target genes in 10-15% of tumors21, increased TEAD reporter activity comparably to overexpressed YAP  and fatty acid biosynthesis pathways29. We observed no detectable p53 protein interactions with YAP by co-IP in tumor cells endogenously expressing a p53 DNA contact mutant , at levels comparable to those in RhoA exogenously expressing MCF10A cells  are among several RhoA targetslls Fig. . Moreovells Fig. . Of notells Fig. .42. We observed that the ROCK1/2 dual inhibitor, H-1152, markedly antagonized TEAD reporter activity in p53 R273H MCF10A cells in a concentration dependent manner . Moreover, the ROCK inhibitor markedly antagonized TEAD reporter activity , YAP amplification (SF268 glioma) or both Hippo pathway and p53 DNA contact mutations (MDA-MB-231 breast carcinoma). In each case, these tumors were dependent on TEAD/YAP transcriptional activity for proliferation 2. In some cases, recurrent driver mutations can either activate or cause loss of function of cancer genes within a common pathway as is the case with Wnt or p53 signaling54. The present studies establish a paradigm in which a cancer driver mutation in a given pathway indirectly causes an imbalance in normal homeostasis in a different pathway to drive oncogenesis. We showed that this was the case for p53 DNA contact mutations, which through a gain of function that upregulates the MVA pathway, indirectly hyperactivate RhoA/ROCK1/actomyosin mechano-signaling to drive oncogenic TEAD/YAP transcription. p53 knockdown antagonized upregulated TEAD/YAP transcription, cell proliferation and motility in human tumors with this class of endogenous p53 missense mutations. Moreover, dnTEAD4 or pharmacological inhibitors of RhoA/ROCK1 mechano-signaling antagonized both upregulated TEAD/YAP transcription and the transformed phenotype in human tumors harboring these p53 missense mutations or in MCF10A cells exogenously expressing such mutants. While hot spot p53 conformational mutants also induced the transformed phenotype, and knockdown of their endogenous expression antagonized tumor cell proliferation, these missense mutants did not activate TEAD/YAP transcription in tumors or when exogenously expressed in MCF10A cells and thus, must utilize a different GOF mechanism(s). Our findings that a specific class of p53 missense mutations drives oncogenic TEAD/YAP transcription provides a PCR based molecular approach for their detection in tumor tissues or liquid biopsy.Cancer driver genes including those encoding autocrine growth factors, their receptors, and critical downstream effectors such as Ras and Raf share a common signaling pathway to confer a growth advantage23, but such mutations are very rare in human tumors55. Recurrent RhoA mutations at other sites have been identified at high frequency in a subset of human gastric carcinomas56. However, these mutations have recently been reported to exert biological effects primarily through activation of FAK signaling57. Thus, hyperactivation of wild type RhoA mechano-signaling and upregulation of TEAD/YAP transcription by p53 DNA contact mutations appears to be the most frequent mechanism activating RhoA oncogenic function in human malignancies.Mutations analogous to those that activate Ras oncogenes confer transforming properties to Rho family members in culture3, altered this homeostatic balance in the MCF10A model without detectably altering physiological RhoA mechano-signaling. Yet, the imbalance created by Hippo pathway aberrations resulted in increased TEAD/YAP transcription to levels sufficient to induce the transformed phenotype  was used as a control in all experiments in which chemicals were solubilized in DMSO. Latrunculin B  and Blebbistatin . ROCK inhibitors were as follows:HEK293T, MDA-MB-231, MDA-MB-468, MDA-MB-157, U373MG, U251MG, SK-LMS-1, U138MG, LN229, M059J, M059K, and BT-549 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 50 units/ml of penicillin/streptomycin. H2052, H2373, MSTO-211H, Snu387, PC-9, HCC193, SF295, SK-BR-3, HCC1395, HCC1954, H1299, HCC1937 and HCC1806 cells were grown in RPMI-1640  medium supplemented with 10% FBS and 50 units/ml of penicillin/streptomycin. SK-MEL-2 cells were grown in Eagle’s Minimum Essential Medium (MEM) (Sigma-Aldrich) supplemented with 1% non-essential amino acids (NEAA), 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich), 50 units/ml of penicillin/streptomycin. MCF10A cells were grown in DMEM/F12 medium supplemented with, 5% horse serum, 10 μg/ml insulin, 100 ng/ml cholera toxin, 0.5 mg/ml hydrocortisone, 20 ng/ml EGF, and 50 units/ml of penicillin/streptomycin. All cells were cultured at 37 °C and 90% humidity in a 5% COSAR407899  Y-27632 , GSK429286A , CAY10622  and KD025 . H-1152  was dissolved in water. All the other inhibitors were dissolved in DMSO to yield 10 mM stock, and treatments were as indicated.oC. The resulting mixture was stirred overnight at room temperature. The progress of the reaction was monitored by TLC. After removal of the solvent, the residue was purified by flash chromatography on silica gel with eluent  to yield desired the sulfonamide as white solid. To a stirring solution of the above solid in methanol (20 mL) was added hydrogen chloride in dioxane  at 0 °C and the resulting mixture was stirred overnight at room temperature. The mixture was concentrated and the resulting crystals were harvested in diethyl ether to provide the desired product H-1152 di-HCl salt as white solid . Purity: >95% (determined by 1H NMR).To a stirring solution of tert-butyl (S)-3-methyl-1,4-diazepane-1-carboxylate  and potassium carbonate  in acetonitrile (30 mL) was added 4-methylisoquinoline-5-sulfonyl chloride  at 01H NMR  δ 9.79 , 8.74 , 8.67 , 8.58 , 8.13 , 4.63–4.60 , 3.86–3.81 , 3.67 , 3.53 , 3.43–3.38 , 3.27 , 2.30–2.28 , 2.18–2.15 , 1.34 .1H NMR  δ 9.17 , 8.57 , 8.20 , 8.08 , 7.63 , 4.39 , 3.87 , 3.68 , 3.55 , 3.48 , 3.33 , 3.03 , 2.44–2.40 , 1.51 .1H NMR  δ 9.55 , 8.65 , 8.57 , 8.26 , 7.93 , 4.46 , 4.25 , 3.79–3.72 , 3.64–3.61 , 3.56–3.50 , 3.39–3.22 , 3.04 , 2.11–1.99 , 1.24 .13C NMR  δ 147.89, 138.70, 136.96, 136.22, 133.81, 133.56, 130.91, 129.70, 127.93, 51.85, 50.04, 45.48, 43.97, 39.79, 39.66, 39.52, 39.38, 39.24, 39.10, 38.96, 38.60, 27.04, 21.19, 16.64.m/z) for C16H22N3O2S+ [M + H]+ calculated 320.1427; found: 320.1428.HRMS  motif cloned at ClaI and NheI restriction sites within NV-luciferase vector25. Shp53.1 and shp53.2 were cloned in the pLKO.1 backbone with sequences as followed: shp53.1: GTCCAGATGAAGCTCCCAGAA; shp53.2 CACCATCCACTACAACTACAT; shScr CCTAAGGTTAAGTCGCCCTCG. All the other shRNAs were obtained from Sigma (MISSION® shRNA library). Mutant p53 constructs containing a substitution of single amino acids, were generated by PCR site-directed mutagenesis using the QuickChange® Lightning Site-Directed Mutagenesis Kit  with WT-p53 cDNA as template as previously described74. Primers were as follows: R175H (g524a):Forward: ATGGTGGGGGCAGTGCCTCACAACCTC, Reverse GAGGTTGTGAGGCACTGCCCCCACCAT; G245S (g733a): Forward CCTCCGGTTCATGCTGCCCATGCAGGAAC, Reverse GTTCCTGCATGGGCAGCATGAACCGGAGG; R248Q (g743a): Forward GAGGATGGGCCTCTGGTTCATGCCGCC, Reverse GGCGGCATGAACCAGAGGCCCATCCTC; R273H (g818a): Forward AGGACAGGCACAAACATGCACCTCAAAGCTGTTC, Reverse GAACAGCTTTGAGGTGCATGTTTGTGCCTGTCCTG; R273C (c817t) Forward 5′-GGACAGGCACAAACACACACCTCAAAGCTGTTC-3′, Reverse 5′-GAACAGCTTTGAGGTGTGTGTTTGTGCCTGTCC-3′; R248Q (g743a) Forward: 5′-GAGGATGGGCCTCTGGTTCATGCCGCC-3′, Reverse: 5′-GGCGGCATGAACCAGAGGCCCATCCTC-3′; S241F (c722t) Forward: 5′-CCGCCCATGCAGAAACTGTTACACATGTAGTTGTA-3′, Reverse: 5′-TACAACTACATGTGTAACAGTTTCTGCATGGGCGG-3′. All constructs were verified by DNA sequencing. To generate retroviral stocks, HEK293T cells were co-transfected with the appropriate retroviral expression vector and pCL-ampho packaging plasmid. For lentiviral stocks, HEK293T cells were co-transfected with the appropriate lentiviral expression vector, pCMVΔR8.74 packaging vector and pMD2 VSVG envelope vector. Titers for each virus stock were determined by colony formation following marker selection of a common assay cell, HT1080, making it possible to compare results using similar amounts of virus in different experiments. Retroviral and lentiviral infections were carried out on all cell lines in the presence of 8 μg/ml polybrene (Sigma). Cells were selected for antibiotic resistance by treatment with 2 μg/ml puromycin or 100 μg/ml hygromycin) and expanded to mass populations.pQCXIH-Myc-YAP was purchased from Addgene (#33091) and pQCXIH vector control was generated by re-ligating backbone after removal of YAP. dnTEAD4 was cloned from pSPORT6 vector  into NSPI-CMV-MCS lentiviral vector using Nhe1 and BamH1 restriction site containing primers: Forward: TAAGCAGCTAGCGCCACCTTGGAGGGCACGGCCGGCAC and Reverse: ACTATGGGATCCTCA TTCTTTCACCAGCCTGTGGATGTGGTGCTGAGC. Site directed mutagenesis was used to generate Y429H (TAC— > CAC) dominant negative (dn) mutation in TEAD4 gene4 cells/well in 24 well plates and treated or genetically manipulated as described. Firefly and renilla luciferase activities were assayed with the dual luciferase assay system , as directed, and firefly luciferase activity was normalized to renilla luciferase activity. Firefly and renilla luminescence were measured with a TD-20e Luminometer (Turner).Cells were plated at 2 × 103 MCF10A cells in 1 ml of growth media containing 0.3% agar (BD 214050) on top of 1 ml of 0.48% agar in 35-mm dishes. Cells were fed every 4 days for 3 weeks with 0.2 mL of growth medium. Colonies were stained with 1% crystal violet in ethanol and experiments were conducted in triplicate with representative results shown.Growth in soft agar was determined by seeding 2.5 × 10Total RNA was extracted from cells using QIAshredder  and RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. First-Strand cDNA synthesis was performed using Superscript II reverse transcriptase  according to the manufacturer’s instructions.Quantitative PCR was carried out using the ViiA™ 7 Real-Time PCR System (Life Technologies) and the FastStart SYBR Green Master mix . PCR was performed in 384 well plates with 10 µl under the following conditions: 95 °C for 15 min, followed by 40 cycles of 94 °C for 15 s, 61 °C for 30 s, and 72 °C for 30 s. Specificity was verified by a dissociation curve. Results were analyzed with ViiA7 RUO software (Life Technologies). Gene expression levels were normalized to GAPDH expression. Primer sequences are provided in Supplementary Table 3 cells in 6-well plates. Cells were treated or genetically manipulated as described. After 10 to 14 days, colonies were stained with 1% crystal violet in ethanol and photographed.For clonogenic assays, cells were plated in triplicate at 1 × 10Cells were harvested in EBC lysis buffer , supplemented with Complete Mini Protease and Phosphatase Inhibitor Cocktails . Cells were lysed and 30–80 µg protein subjected to SDS-PAGE followed by transfer onto an Immobilon-FL PVDF membrane  and incubation with the indicated antibodies. Detection was carried out with an Odyssey Infrared Imaging System  with IR dye-tagged secondary antibodies . The following antibodies were utilized: p53 , YAP, CTGF, MYC, MLC2, RhoA, pan-Rac, Cdc42, ROCK1, ROCK2, GAPDH , p-YAP, p-Cofilin, p-MLC2, Cofilin, pPAK1/2, pPAK4/5, PAK2, PAK4, , TEAD4, , mouse anti-α-tubulin , mouse anti-β-actin .Cells were harvested in RIPA lysis buffer  supplemented with Complete Mini Protease and Phosphotase Inhibitor Cocktails. 800 μg proteins were incubated with 10 μg of antibody overnight at 4 °C. Anti-mouse or anti-rabbit IgG  was used as a negative control. The following antibodies were utilized: p53 , YAP  TEAD4 and pan-TEAD . Immunoprecipitated complexes were captured by 2 h incubation at 4 °C with Dynabeads Protein A/G B , followed by three washes in lysis buffer. Immunoprecipitated complexes were eluted by boiling for 5 min with Laemmli buffer  with 10% of the total lysates run on the same gel for comparative immunoblot analysis.6 cells were collected by centrifugation, crosslinked for 10 min at room temperature with 1% formaldehyde in PBS and neutralized by the addition of 125 mM glycine for 5 min. Cells were washed twice in PBS and lysed with 0.6 ml SDS lysis buffer , 50 mM Tris (pH 8.0)) supplemented with protease inhibitors . Samples were then sonicated to generate an average of 500 bp DNA fragments using Bioruptor  on high setting (30 s on and off), and insoluble material was removed by centrifugation. A total of 200 μg of cleared samples were diluted 10-fold in ChIP dilution buffer (16.7 mM Tris (pH 8.0), 1.2 mM EDTA (pH 8.0), 167 mM NaCl, 1.1% Triton X-100) and incubated with 10 μg of p53 (DO-1) monoclonal antibody  or 10 μg of normal IgG  overnight at 4 °C. In all, 20 μg of samples without primary antibody were saved as 10% input controls. Immunoprecipitated complexes were captured by 2 h incubation at 4 °C with Dynabeads Protein A , followed by 5 min washes: once with low salt wash buffer , 5 mM EDTA (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), once with high salt wash buffer , 5 mM EDTA (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), four times with LiCl wash buffer , 1% NP-40, 0.5% sodium deoxycholate) and twice with TE buffer (100 mM Tris (pH 8.0), 100 mM EDTA (pH 8.0)). Immunoprecipitated complexes and 10% input controls were eluted in two sequential 10 min incubations with 75 μl fresh elution buffer (50 mM Tris (pH 8.0), 10 mM EDTA (pH 8.0), 1% SDS) at 65 °C. DNA-protein crosslinks were reversed by the addition of 200 mM NaCl and incubation at 65 °C overnight. DNA was purified with QIAquick PCR Purification Kit , resuspended in 50 μl H2O and quantified by real-time PCR with FastStartSYBR Green Master  on the Stratagene MxPro3005 system, using 2 μl DNA in a 15 μl reaction mixture. All reactions were performed in duplicate with primers described in Supplementary Table adjusted input − Ctenriched], where input Ct is adjusted from 10 to 100% by: Ctadjusted input = Ct10% input − log2 10.Around 5 × 10g to remove cell debris. The cell lysate (100 μl) was centrifuged at 100,000 × g for 1 h at 37 °C to pellet F-actin with G-actin remaining in the supernatant. F-actin in the pellet was resuspended in 100 μl of F-actin destabilizing buffer on ice for 1 h with frequent pipetting. Equal volumes of G-actin and F-actin fractions were mixed with 5× SDS sample buffer and run on SDS-PAGE. Western blot was performed using anti-actin antibody provided in the kit and F-/G-actin ratios were determined by densitometry using Image StudioTM Lite Software (Li-Cor). For all F-/G-actin fractionation experiments, 1 μM Latrunculin B treatment for 24 h served as positive control for F-actin dissociation and 10 μM Phalloidin treatment for 30 min as a positive control for F-actin stabilization39.G-actin and F-actin were isolated using the G-actin/F-actin in vivo assay kit  according to the manufacturer’s instructions. Briefly, cells were lysed in pre-warmed lysis/F-actin stabilizing buffer supplemented with protease inhibitor and ATP. The cell lysate was centrifuged for 5 min. at 350 × 50,000 MCF10A cells were plated on sterile coverslips and allowed to attach overnight. Cells were fixed using paraformaldehyde for 10 min followed by permeabilization with methanol for 5 min. After an hour of incubation in 5% BSA in PBS and 0.01% triton-x100, cells were incubated with YAP primary antibody  overnight at 4 degree C. Excess antibody was washed off with PBS followed by secondary antibody incubation at room temperature for an hour. After washing off secondary antibody with PBS, the cells were mounted in DAPI vector shield. Imaging was performed using a Leica TCS SP8 microscope. Signal intensity was quantified for 100 cells per experimental condition using Image J software and plotted as a ratio of nuclear versus cytoplasmic signal intensity.6 MDA-MB-231, MDA-MB-468 or HCC1935 cells were inoculated orthotopically into the fat pads of the fifth mammary glands of 6-week-old immunocompromised female SCID mice. The tumor volume was measured with a caliper twice per week, using the formula volume = length × width2/2. When tumors reached 100 mm3, mice were split into two groups (5 mice each group) and either treated with vehicle control (PBS) or 25 mg/kg H-1152 twice daily for 31 days. All mouse experiments were approved by the Icahn School of Medicine at Mount Sinai Animal Care and Use Committee (Protocol no. LA12-00037). Maximal tumor burden permitted by Mount Sinai’s IACUC is 10% of mouse body weight or tumor diameter of 10 mm in any dimension. We confirm that tumors did not exceed maximum approved tumor size or burden.For in vivo experiments, 5 × 10Paraffin-embedded tissue sections were subjected to HE staining and immunodetection of different proteins using antibodies as indicated. For immunohistochemistry, sections were incubated with primary antibodies Caspase3 or Ki67  overnight at 4 °C, followed by Dako EnVision+System-HRP Labelled Polymer anti rabbit (Dako) for 1 h at room temperature. Sections were counterstained with hematoxylin, and visualized by light microscopy.t, as determined by the processing software. Wound confluence is expressed as a percentage of the scratch wound that is filled with cells at any given time t, when compared with the initial scratch. Wound closure was monitored at 60-min intervals for at least 48 h. Videomicroscopy was performed with a ×10 objective. Experiments were performed in triplicate at 37 °C.Cells were plated on 96-well Image-lock plates (Essen Bioscience) and analyzed for confluency as a monolayer via light microscopy before initiation of a scratch wound. Scratches were made using a 96-pin tool (Woundmaker) as per the protocol. Data processing and analysis of migration were performed using the IncuCyte 96-well Kinetic Cell Migration and Invasion Assay software module. Data were then exported to Excel for further analysis. Wound width is defined as the area of the wound at any time 30,000 cells (control empty vector and shP53 MDA-MB-468 cells) were processed for FACS 24 h after plating. Cell pellets were washed with phosphate-buffered saline and fixed/permeabilized with 70% ice-cold ethanol. Pellets were washed with phosphate-buffered saline, resuspended in 50 μg/ml ribonuclease A and 62.5 μg/ml propidium iodide and run on BD FACSCanto™ II Cell Analyzer. Data analysis was performed with FloJo 10.4.2 software. Using the FSC/SSC gating, debris was removed by gating on the main cell population  (https://www.gsea-msigdb.org/gsea/index.jsp) was used to assess whether the TEAD/YAP conserved gene signature52 was enriched. The samples are separated into 2 groups based on the mutation status of p53 for TNBC and the following genes: NF2, LATS1, LATS2, YAP1 and WWTR1 for Mesothelioma. The gene expression boxplots use the DESeq2 VST normalized read counts.The public data sets TCGA-Breast Invasive Carcinoma and TCGA-Mesothelioman = 3 biologically independent replicates using two-tailed t-tests, and statistical significance was set at p = 0.05 . The actual calculated p values for all data are provided in the source data file. All western blots were performed with n = 2 biologically independent replicates and representative data are shown.All data analysis was performed using GraphPad Prism version 6 for Macintosh OS X . All p-values were determined from Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary