Leishmania braziliensis, responsible for cutaneous leishmaniasis, is the most prevalent species in Brazil. Superoxide dismutase (SOD) belongs to the antioxidant pathway of the parasites and human host. Despite the differences between SOD of Leishmania braziliensis and human make this enzyme a promising target for drug development efforts. No medicinal chemistry effort has been made to identify LbSOD inhibitors. Herein, we show that thermal shift assays (TSA) and fluorescent protein-labeled assays (FPLA) can be employed as primary and secondary screens to achieve this goal. Moreover, we show that thiazole derivatives bind to LbSOD with micromolar affinity.Leishmaniasis is considered as one of the major neglected tropical diseases due to its magnitude and wide geographic distribution.  Besides, MORENO-VIGURI et al. (2016) have reported that arylaminoketone derivatives inhibit FeSOD from trypanosomatids but have little effect over human CuZnSOD.The superoxide dismutase from L. braziliensis is a promising target for drug design efforts. However, no medicinal chemistry effort to target this protein has been carried out before. Aiming at overcoming this knowledge-gap, this work describes how thermal shift assays (TSA) and fluorescent protein-labeled assays (FPLA) can be employed as primary and secondary HTS-friendly alternatives to indirect-kinetic assays that measure SOD catalytic activity. TSA relies on environment-sensitive fluorophores that reversibly binds to exposed hydrophobic regions when protein unfolds due to temperature increaseL. braziliensis FeSOD.These results suggest FeSOD from F254) with visualization by UV light 254 nm in the appropriated system for each compound. The Infrared spectra  were recorded on a Bruker IFS66 spectrophotometer , the wave numbers were given in cm−1 and are uncorrected. The 1H NMR and 13 C NMR spectra were recorded on a VARIAN VNMRS 400-MR , 400 MHz for 1H and 75.4 MHz for 13 C. The 1H spectra were recorded in DMSO-d6 while 13 C spectra were recorded in CDCl3 and DMSO using tetramethylsilane (TMS) as the internal standard. The peak and abbreviations were used to indicate multiplicity: s (singlet), d (doublet), dd (double doublet), ddd , t (triplet), and m (multiplet). The chemical shifts were reported in δ units and the coupling constants (J) were reported in Hertz. Mass spectra were recorded on a Varian MAT 711 spectrometer  70 eV electron impact.Chemical reagents and solvents were purchased from Sigma-Aldrich  with analytical grade purity. Melting points were determined in open capillaries on a Buchi apparatus and are uncorrected. Thin-layer chromatography (TLC) was carried out on aluminium-supported silica gel plates (Merck 60 −3 mol) of thiourea dissolved in 15 ml of methanol followed by the addition of 0.513 g (2.3 × 10−3 mol) of substituted acetophenone halide dissolved in 20 ml methanol, to form a white suspension. The reaction was left for 15 min at room temperature under magnetic stirring. After that, the mixture was dissolved in 0.6 ml of HCl (dropwise). The reaction was heated to 90 °C in an oil bath, the pH adjusted between 4 and 5. The reaction time was 2 h. The reaction was monitored by thin layer chromatography. The product was ice-cooled and filtered. The compound was purified by crystallization from methanol.The 2-aminothiazole derivatives were obtained from the mixture of equimolar amounts 0.175 g  MP 216–217 °C. Infrared: 3390–3317 (NH2), 3110 (C–H) 1620 (C = N), 1570 (C = C Ar),1185 (C–N), 1058(C–S–C), 815 (Ar) 739 (Ar–Br). 1HNMR: 6.39 (s 2H); 6,99 (s 1H); 7.77–7.74 (d 2H) J = 8.50 Hz; 7.76–7.71 (d 2H) J = 8.50 Hz. 13HNMR: 121.5, 129.5, 135.6, 129.5, 121.5, 123.4, 136.6, 124.8, 115.0, 125.6. +HRMS, calculated: 252.9561, found: 252.9558.Chemical formula: C9H7BrN2S, molecular weight: 255.1343, yield 92%, Rf = 0.47 . MP 202–204 °C. Infrared 3389–3520 (NH2), 3116 (C–H) 1614 (C = N), 1575 (C = C Ar),1180 (C–N), 1067 (C–S–C), 817 (Ar) 740 (Ar–Br). 1HNMR: 8.33 ; 8.10 ; 8.12 (s 1H CH); 7.76 (d 1H Ar J = 8.1); 7.63 ; 7.66 . 13HNMR: 148; 124; 126; 140; 178; 161; 130; 132; 129; 137; 163. +HRMS, calculated: 255.9493, found: 253.9513.Chemical formula: C−3 mol of 4–-2-aminothiazole and 2.3 × 1.0 −3 mol of substituted benzaldehydes. The substituted benzaldehydes were dissolved in 15 ml of MeOH under stirring at room temperature. After that, 0.08 ml of HCl (conc.) dissolved in MeOH was added dropwise. After 5 min. under cold stirring, the starting material -2-aminothiazole previously dissolved in 15 ml of MeOH was added dropwise. The pH was then adjusted between 4 and 5 and the reaction was warmed to 75 °C and refluxed in an oil bath for 2–24 h. The progress of the reaction was monitored by TLC. At the end, the solvent was reduced to one-half volume and ice-cooled. The precipitate was filtered off and recrystallized from the appropriate solvent.The 4–(4-bromophenyl)-thiazol-2-imine-arylidene-substituted and 4–(3 or 4-nitrophenyl)-thiazol-2-imine-arylidene-substituted derivatives were synthesized from the mixture of equimolar amounts 2.3 × 1.016H9Cl2N3O2S, MW 378.2326, Rdt 55%, Rf = 0.45 . MP 218.6–220.0 °C. Infrared: 3109 (C–H), 1610 (C = N), 1575 (C = C Ar),1189 (C–N), 1055(C–S–C), 810 (Ar), 730 (Ar–Cl). 1HNMR: 8.35 ; 8.10 , 8.11 (s 1H CH–S); 8,28 (s 1H CH = N); 8.27 (s 1H Ar); 7.63 ; 7.67  13CNMR: 124, 130, 135, 129, 121, 124, 111, 152, 173, 179, 133, 131, 137, 136, 130, 129. +HRMS, calculated: 376.9793, found: 378.9633.Chemical Formula: C16H10BrN3O2S, MW 388.2385, Rdt 45%, Rf = 0.54 : MP 262 °C. Infrared 3112 (C–H), 1613 (C = N), 1575 (C = C Ar), 1179 (C–N), 1061(C–S–C), 815 (Ar), 737 (Ar–Br). 1HNMR: 8.33 ; 8.10 , 8.11 (s 1H CH); 7.74 (d 1H Ar J = 8.1) 7.63 ; 7.67  13CNMR: 148; 126; 127; 140; 175; 3. 161; 129; 133; 129; 137; 162. +HRMS, calculated: 386.9677, found: 388.9557.Chemical Formula: C16H9Br2N3O2S, MW 467.1346, Rdt 70%, Rf = 0.53 : Melting Point = 273 °C. Infrared 3115 (C–H), 1615 (C = N), 1571 (C = C Ar), 1182 (C–N), 1064 (C–S–C), 818 (Ar), 739 (Ar–Br). 1HNMR: 8.33 ; 8.10 ; 8.11 (s 1H CH); 7.74 (d 1H Ar J = 8.1); 7.63 ; 7.67 . 13CNMR: 148; 126; 127; 140; 175; 161; 129; 133; 129; 137; 162. +HRMS, calculated: 464.8782, found: 466.8657.Chemical Formula: C17H13BrN2OS, Molecular Weight: 373.2669. Rdt 70%, Rf = 0.53 : MP 273–274 °C. Infrared: 3114 (C–H), 1615 (C = N), 1576 (C = C Ar), 1180 (C–N), 1063 (C–S–C), 815 (Ar), 740 (Ar–Br). 1HNMR: 8.34 ; 8.10 ; 8.10 (s 1H CH); 7.74 (d 1H Ar J = 8.1); 7.64 ; 7.64. 13CNMR: 147; 124; 126; 141; 175; 160; 1 133; 129; 138;162. +HRMS, calculated: 371.9933, found: 371.9630.Chemical Formula: C16H10BrN3O2S, Molecular Weight: 388.2385, yield 53%, Rf = 0.531 . MP 233–234 °C. Infrared 3115 (C–H), 1615 (C = N), 1575 (C = C Ar), 1181 (C–N), 1066 (C–S–C), 817 (Ar), 740 (Ar–Br). 1HNMR: 8.34 ; 8.11 ; 8.11 (s 1H CH); 7.74 (d 1H Ar J = 8.1); 7.63 ; 7.66 . 13CNMR: 147; 124; 127; 141; 177; 160; 130; 133; 129; 138;163. +HRMS, calculated: 385.9725., found: 386.9678.Chemical Formula: C16H10BrN3O2S, Molecular Weight: 388.2385, yield 55%, Rf = 0.55 . MP 155–156 °C. Infrared 3116 (C–H), 1614 (C = N), 1575 (C = C Ar), 1180 (C–N), 1067 (C–S–C), 817 (Ar), 740 (Ar–Br). 1HNMR: 8.33 ; 8.10 ; 8.12 (s 1H CH); 7.76 (d 1H Ar J = 8.1); 7.63 ; 7.66 . 13CNMR: 148; 124; 126; 140; 178; 161; 130; 132; 129; 137; 163. +HRMS, calculated: 387.9725, found: 385.9688.Chemical Formula: C16H10FN3O2S, Molecular Weight: 327.3329, yield 80%, Rf = 0.6 . MP 182–183 °C. Infrared: 3116 (C–H), 1614 (C = N), 1575 (C = C Ar), 1230 (Ar–F), 1180 (C–N), 1068 (C–S–C), 818 (Ar). 1HNMR: 8.29 ; 8.12 ; 8.11 (s 1H CH); 7.76 ; 7.64 ; 7.67 . 13CNMR: 149; 121; 124; 138; 179; 163; 132; 131; 130; 138; 164. +HRMS, calculated: 326.0525, found: 325.9786.Chemical Formula: C16H10FN3O2S, Molecular Weight: 327.3329, yield 63%, Rf = 0.53 . MP 118.4–119.5 °C. Infrared: 3117 (C–H), 1612 (C = N), 1571 (C = C Ar), 1241 (Ar–F), 1183 (C–N), 1069 (C–S–C), 820 (Ar). 1HNMR: 8.31 ; 8.11 ; 8.09 (s 1H CH); 7.73 (d 1H Ar J = 8.11); 7.64 ; 7.67 . 13CNMR: 149; 121; 124; 137; 179; 168; 133; 131; 129; 138; 162. +HRMS, calculated: 326.3448, found: 325.9581.Chemical Formula: C16H11N3O3S, Molecular Weight: 325.3418, yield 63%, Rf = 0.45 . MP 118.4–119.5 °C. Infrared: 3340 (O–H), 3118 (C–H), 1612 (C = N), 1572 (C = C Ar), 1182 (C–N), 1070 (C–S–C), 820 (Ar). 1HNMR: 8.30 ; 8.09 ; 8.08 (s 1H CH); 7.72 (d 1H Ar J = 8.11); 7.65 ; 7.68 . 13CNMR: 149; 120; 123; 137; 179; 166; 133; 132; 130; 138; 161. +HRMS, calculated: 325.0519, found: 326.0537.Chemical Formula: C16H11N3O3S, Molecular Weight: 325.3418, yield 59%, Rf = 0.46 (Hex/AcOEt 0.5:0.5). MP 187.5 °C. Infrared: 3338 (O–H), 3120 (C–H) 1613 (C = N), 1574 (C = C Ar), 1181 (C–N), 1072 (C–S–C), 821 (Ar). 1HNMR: 8.31 ; 8.08 ; 8.08 (s 1H CH); 7.72 (d 1H Ar J = 8.11); 7.65 ; 7.68 . 13CNMR: 149; 121; 123; 137; 180; 166; 131; 132; 130; 136; 160. +HRMS, calculated: 324.0569, found: 325.9581.Chemical Formula: C17H13N3O2S, Molecular Weight: 323.369, yield 61%, Rf = 0.56 (CH2Cl2/MeOH 0.98:0.02). MP 213–214 °C. Infrared: 3118 (C–H), 1613 (C = N), 1572 (C = C Ar), 1181 (C–N), 1070 (C–S–C), 821 (Ar). 1HNMR: 8.29 ; 8.11 ; 8.08 (s 1H CH); 7.71 (d 1H Ar J = 8.11); 7.64 ; 7.67 . 13CNMR: 149; 121; 123; 137; 179; 167; 133; 132; 131; 138; 160. +HRMS, calculated: 322.3810, found: 324.9580.Chemical Formula: CE. coli BL21 (DE3) cell carrying the pETM11-LbSOD plasmid were cultured at 37 °C/180 rpm in Luria Bertani medium supplemented with 30 μg/mL kanamycin until OD600 reached 0.6–0.9. At this moment, 1 mM  isopropyl-β-D-thiogalactoside (IPTG) was added to the culture and the temperature was reduced to 20 °C. After 16 h, the cells were harvested by centrifugation  and resuspended in lysis buffer  supplemented with 1 mM phenylmethanesulphonylfluoride (PMSF). Next, cells were incubated with Lysozyme 0.5 mg/mL, for 30 min and then disrupted by sonication . These steps were carried out in ice-bath. The soluble fraction was clarified by centrifugation  and then loaded onto a HisTrap HP column , pre-equilibrated with PBS 50 mM, NaCl 100 mM, 20 mM Imidazole pH 7.0.Amicon Ultra centrifugal filters , 4000 rpm at 4 °C.The column was washed with 20 column volumes of PBS 50 mM, 100 mM NaCl, 20 mM Imidazole pH 7.0 and then steps of increasing concentration of imidazole (50–500 mM) were used. Purification was monitored by UV absorbance measurement at 280 nm and the level of protein purity was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 12%. The protein fraction was dialyzed in PBS 50 mM, NaCl 100 mM pH 7 with − 1 cm−1 at 280 nm calculated using ExPASy ((http://web.expasy.org/protparam/).Protein concentrations were determined spectrophotometrically using a theoretical extinction coefficient of 55775 M Tobacco Etch Virus) protease digestion (1 mg per 20 mg LbSOD), 4 °C overnight. After the proteolysis step, LbSOD was loaded again onto His-Trap column to separate cleaved and uncleaved His-tag LbSOD from TEV protease. The column was pre-equilibrated with PBS 50 mM, NaCl 100 mM pH 7 and then the elution of cleaved LbSOD was performed with 10 column volumes of this same buffer. The purification fractions were analyzed by UV measurement at 280 nm and gel electrophoresis (SDS-PAGE 12%) to confirm purification of the protein. The cleaved His-tag LbSOD was concentrated  to 10 mg/mL and stored in 30% glycerol at −80 °C.The enzyme was subjected to TEV , sealed with transparent capping strips . The plates were centrifuged for 2 min, 2000 rpm, 25 °C. After the plates were heated from 25 to 85 °C in increments of 1 °C per minute and fluorescence signal was monitored with SYPRO Orange® dye, with 492 (excitation) and 610 nm (emission) wavelengths.The assays were carried out in the RT-PCR Applied Biosystems 7500 , in triplicate, using 96-well PCR plate (PCR plates 96 well BioRadftp://ftp.sgc.ox.ac.uk/pub/biophysics).The results obtained by Applied Biosystems 7500 proprietary software (v2.0) were submitted to processing and analysis in Excel 2007 worksheet . Comparisons between Tm of different conditions (ΔTm) were considered as significantly different when p < .05, according to the Kruskal–Wallis test followed by Dunn's post-test for multiple comparisons.The melting temperature (Tm) values were calculated by non-linear fitting of the melting curves to a Boltzmann sigmoidal function, using GraphPad Prism version 5.0 for Windows  in the presence of SYPRO Orange®  (1:100 dilution) in ultrapure water qsp 20 µL. Then, the buffers  and pHs (4–9) were evaluated at a final concentration of 50 mM for each buffer, SYPRO Orange® (1: 100 dilution) and LbSOD. Finally, the influence of DMSO  on the thermal stability of the protein was investigated.Conditions were optimized to LbSOD was evaluated at a single concentration  and an equivalent volume of DMSO was employed as a control (ΔTm = 0.0). Briefly, LbSOD (5 μM) was diluted in 50 mM sodium phosphate buffer (pH 7.0) supplemented with 100 mM NaCl and SYPRO Orange® (1:100 dilution). The 96 well plates were heated from 25 to 85 °C in increments of 1 °C per minute. The results were analyzed as described in the previous section.The effect of the thiazoles derivatives over the melting temperature of LbSOD (2 mg/mL) was incubated with fluorescein-5-isothiocyanate (FITC) dye (10 mg/mL) for 2 h, at 25 °C, with continuous stirring. Then, the solution was loaded on a Hi-Trap HP desalting column , previously equilibrated with 50 mM sodium phosphate buffer (pH 8) and 1.5 column volumes of the buffer were injected. The absorbance of the collected fractions was monitored at 280 nm and 490 nm, so correct to the contribution of dye to Absorbance280nm could be calculated according to Aprotein stands for the protein absorbance after correction; A280 is the absorbance at 280 nm of the purified sample; Amax: maximal dye absorption at 280 nm or correction factor (CF) corresponding to 0.3 to fluorescein.LbSOD was diluted to 5 µM and the influence of putative inhibitors (50 µM) over its fluorescence was measured at 25 °C for 10 min on an Applied Biosystems 7500 RT-PCR machine. The experiments were carried out in triplicate using 96-well PCR plates (PCR plates 96 well BioRad®). Fluorescence data was recorded on the Applied Biosystems 7500 Software v2.0 and then analyzed on GraphPad Prism V5.0 software .Next, LbSOD (20 µM) diluted in sodium phosphate buffer 0.05 M pH 7 with DMSO (5%v/v). The injection syringe was loaded with ligand 400 µM (previously dissolved in the same solution as the protein). Control experiments were carried out with sodium phosphate buffer 0.05 M pH 7 and DMSO (5%v/v) in the sample cell to determine the dilution heat. Injections were started after baseline stability had been achieved. The ligands were titrated in the sample cell through 21 injections of 13 µL and the first injection of 10 µL. The solutions of the sample cell were stirred during the experiment at 200 rpm to ensure mixing.ITC experiments were carried out in a Microcal VP-ITC Model, at 298 K. All the solutions were thoroughly degassed prior to the titrations to avoid the formation of bubbles during the experiment. The reference cell contained distilled water. The sample cell was filled with The heat variation, following each injection of the ligand in the sample cell, were employed to build the titration isotherms, as available in Origin package supplied with the calorimeter.The thermodynamic parameters ΔG (Gibbs free energy), ΔS (entropy), and ΔH  were calculated using the L. infantum FeSOD than human CuZnSODLbSOD inhibitorsPrevious studies have shown that 1,3-thiazole derivatives have leishmanicidal activity1HNMR and 13CNMR absorption of CH = N between 4.92–4.04 and 171.76–168.25 ppm, respectively. The stereochemistry of CH = N bond is assumed to be E, as reported in literature for thiazole Schiff bases derivativesThe starting material 2-aminothiazole-4-substituted Ju-436 and Ju-533 were prepared, as described previously,E. coli Bl21 (DE3) cells were transformed with plasmid LbSOD-pET-M11 and LbSOD expression was performed with IPTG 1 mM at 20 °C for 16 h.6xhistidine tag, which allows the expression of the His-tag fused LbSOD and its purification by affinity chromatography. The purification was based on the interaction of histidine residues with nickel ions immobilized on the chromatographic column (Ni-sepharose) and LbSOD was eluted in a higher concentration of imidazole (500 mM)   . The oveLbSOD was obtained, the next step consisted the identification of potential inhibitors (LbSOD due to the absorption of light at the same wavelength of the assay (320 nm) (data not shown), the thermal shift assays (TSA) and fluorescent protein-labeled assays (FPLA) were employed to evaluate the potential LbSOD inhibitors.Once the pure hibitors . Conside,,LbSOD, buffer, pH, and organic solvent (DMSO), as described next.In general, TSA is used to identify the conditions and molecules that influence protein stabilityLbSOD concentrations  in this range  showed that Propionibacterium shermanha Fe-SOD activity decreases at pH <5 Plasmodium vickey also decreases at pH <6.0,Comparison of is range . Either LbSOD was also investigated. The assessment of different DMSO concentrations suggests that up to 5% (v/v) of DMSO does not significantly affect the thermal stability of LbSOD (p > .05)   and, forLbSOD hit identification campaigns. In order to increase the number of true-positives, it is common practice to employ a counter-screening method, whose end-point is different from the first screening assay. Accordingly, the first screening method described in this work relies on fluorescein-5-isothiocyanate (FITC) covalent binding to the target proteinLbSOD was labeled with FITC, any change in the protein conformation would lead to a change in the fluorescence. In case thiazole derivatives cause a conformational shift that exposes FITC-labeled residues, an increased fluorescence signal would be observed. On the other hand, if ligand binding hides the FITC-labeled residues, a decreased signal is expected. Then, the conformational modification due to thiazole derivatives binding to LbSOD was evaluated at a single concentration. This approach suggests that Ju-450 and Ju-480 are the most promising hits , the change in the fluorescence signal might be low and promising compounds would be discarded. An even more troublesome scenario would be a covalent modification of a residue that is crucial for binding. Considering all these shortcoming of protein covalent labeling, we resorted to thermal shift assays as a secondary counter-screening approach. Although this strategy also depends on a fluorescent label (Sypro-orange®), covalent binding is not an issue anymore.When protein labeling occurs far from the LbSOD (ΔTm ≅ +1.0 °C). However, only Ju-450, Ju-480 have a significant effect over LbSOD thermal stability (p < .05). Taking both results into consideration, Ju-450 and Ju-480 would be considered a true hit.TSA shows that Ju-514, Ju-517, and Ju-546 stabilize p-nitrophenyl moiety at the 4-position of the thiazole ring and increase the fluorescence signal in FLPA. On the other hand, Ju-514 has a p-bromophenyl moiety at the equivalent position and reduces the fluorescence signal in the FLPA. Given the overall similarity between these compounds, we decided to include Ju-514 in subsequent assays to make sure no true ligand was excluded from our study . Hence, structure-activity relationships based on those compounds would be of little help to design potent LbSOD inhibitors. Instead, we decided to focus our efforts on Ju-480, that shows low-micromolar affinity to the macromolecular target (Kd= 4.8 ± 12.5 μM) and employ a label-free method to study its thermodynamic signatureAlthough all evaluated compounds exhibited dose-response profile that are typical of true ligands, Ju-450 and Ju-514 have low affinity to ΔH= −4.22 ± 1.6970 kcal/mol) and entropic  contributions to affinity (LbSOD surface, as was observed in the optimization of HIV-1 protease inhibitors,Once ITC measures the heat released or absorbed upon the ligand-macromolecule interaction, it is possible to directly determine thermodynamic parameters ΔH and ΔSaffinity . The entLbSOD ligands. On the other hand, the combined use of TSA and FPLA assays lead to the identification of Ju-480, a thiazole derivative that has a low-micromolar affinity to L. braziliensis Superoxide dismutase. Label-free methods confirm that Ju-480 binds to LbSOD and suggests that structure rigidification might increase the ligand’s affinity to its macromolecular target.Although TSA is commonly employed for screening putative ligands of promising targets, the results presented in this work highlight that its use alone might lead to the selection of false-positive


The results of preliminary bioassays showed that the target compounds possessed good activities against Tetranychus cinnabarinus, Plutella xylostella, and Aphis craccivora. Most of the target compounds exhibited moderate to good acaricidal activity against Tetranychus cinnabarinus at a concentration of 400 µg/mL, and some showed moderate activity at a concentration of 200 µg/mL; in particular, compounds 8m and 8p exhibited 70.0% mortality. In addition, some of the target compounds exhibited good insecticidal activities against Plutella xylostella at a concentration of 200 µg/mL, especially compounds 8i and 8o, which achieved 100.0% mortality at a concentration of 100 µg/mL. Interestingly, some of the target compounds exhibited potent anti-aphid activity against Aphis craccivora at a concentration of 200 µg/mL; furthermore, compounds 8p and 8q demonstrated 100.0% anti-aphid activity at a concentration of 50 µg/mL. The preliminary analyses of the structure–activity relationships (SAR) indicated that the acaricidal and insecticidal activities varied significantly depending on the type of substituent and substitution pattern, which provides guidance for the further investigation of such structural modifications.New 1,3,5-trimethylpyrazole-containing malonamide derivatives based on pyflubumide were designed, synthesized, and characterized using  Amide bonds are an important functional group in many of the active ingredients of agrochemicals, such as the representative amide herbicides acetochlor and metolachlor ,2 and thA . However, it showed low activity against other pests, including hemiptera and lepidoptera (LC50 > 500 µg/mL). Furthermore, it is harmless to non-target arthropods, beneficial insects, and natural enemies +), 511.1939; found, 511.1931.1-(2-methyl-4-(perfluoropropan-2-yl)phenyl)-N3-malonamide2-Isobutyl-N (8b): White solid; m.p. 204–206 °C; yield 56.5%; 1H-NMR : δ 9.48 , 8.41 , 8.11 , 7.43-7.41 , 3.69 , 3.63 , 2.35 , 2.10 , 2.01-1.87 , 1.80-1.71 2), 1.00 2). 13C-NMR : δ 171.19 (C=O), 169.13 (C=O), 142.64 , 137.89 , 134.42 , 128.81 , 127.34 , 123.79 , 122.28 , 121.43 , 120.22 , 113.60 , 90.97 2), 53.53 (O=C-C-C=O), 42.50 (O=C-CH-CH2), 35.89 , 26.17 (CH(CH3)2), 22.11 (CH(CH3)2), 21.69 (CH(CH3)2), 17.66 (Ar-CH3), 10.88 (pyrazole-CH3), 9.09 (pyrazole-CH3). HRMS calcd. for C23H27F7N4O2 ([M+H]+), 525.2095; found, 525.2086.1-(2-methyl-4-(perfluoropropan-2-yl)phenyl)-N3-malonamide2-Isopentyl-N (8c): Light yellow solid; m.p. 200–202 °C; yield 48.4%; 1H-NMR : δ 9.57 , 8.20 , 8.16 , 7.44–7.42 , 3.70 , 3.43 , 2.36 , 2.12 , 2.09-2.03 , 1.66–1.57 2), 1.41–1.33 2), 0.89 2). 13C-NMR : δ 171.29 (C=O), 168.90 (C=O), 142.66 , 137.93 , 134.48 , 128.70 , 127.33 , 123.82 , 122.21 , 121.34 , 120.22 , 113.55 , 90.98 2), 55.15 (O=C-C-C=O), 36.31 , 35.91 (CH2-CH(CH3)2), 31.64 (CH(CH3)2), 27.39 (O=C-CH-CH2), 21.97 (CH(CH3)2), 21.89 (CH(CH3)2), 17.72 (Ar-CH3), 10.93 (pyrazole-CH3), 9.13 (pyrazole-CH3). HRMS calcd. for C24H29F7N4O2 ([M+H]+), 539.2252; found, 539.2244.1-(2-Methyl-4-(perfluoropropan-2-yl)phenyl)-2-(prop-2-yn-1-yl)-N3-malonamideN (8d): White solid; m.p. 83–85 °C; yield 50.2%; 1H-NMR : δ 9.70 , 9.25 , 7.83 , 7.52-7.49 , 3.80 , 3.62 , 2.94 , 2.84–2.80 , 2.33 , 2.05 , 1.96 . 13C-NMR : δ 167.39 (C=O), 166.93 (C=O), 141.82 , 139.32 , 134.23 , 132.07 , 127.24 , 124.42 , 123.52 , 122.23  120.42 , 114.80 , 91.24 2), 81.57 (C≡CH), 72.83 (C≡CH), 52.45 (O=C-C-C=O), 36.08 , 19.33 (CH2-C≡CH), 17.90 (Ar-CH3), 11.22 (pyrazole-CH3), 9.10 (pyrazole-CH3). HRMS calcd. for C22H21F7N4O2 ([M+H]+), 507.1625; found, 507.1619.1-(2-methyl-4-(perfluoropropan-2-yl)phenyl)-N3-malonamide2-Allyl-N (8e): Light yellow solid; m.p. 155–157 °C; yield 61.7%; 1H-NMR : δ 9.67 , 8.38 , 8.13 , 7.43–7.41 , 5.96–5.78 , 5.27–5.12 , 3.68 , 3.54 , 3.42–3.33 , 2.83 , 2.35 , 2.09 , 2.04–1.99 . 13C-NMR : δ 170.56 (C=O), 168.14 (C=O), 142.68 , 137.83 , 134.58 , 132.98 (CH=CH2), 128.94 , 127.33 , 123.78 , 122.33 , 121.53 , 115.71 , 118.36 (CH=CH2), 113.51 , 90.96 2), 54.39 (O=C-C-C=O), 37.41 , 35.86(O=C-CH-CH2), 17.71 (Ar-CH3), 10.90 (pyrazole-CH3), 9.08 (pyrazole-CH3). HRMS calcd. for C22H23F7N4O2 ([M+H]+), 509.1782; found, 509.1777.1--2-methylphenyl)-2-isopropyl-N3-malonamideN (8f): Light yellow solid; m.p. 192–193 °C; yield 58.4%; 1H-NMR : δ 9.50 , 8.59 , 8.09 , 7.40–7.37 , 3.68 , 3.47 , 3.20 , 2.47–2.40 2), 2.35 , 2.11 , 1.13 2). 13C-NMR : δ 170.71 (C=O), 168.39 (C=O), 142.68 , 137.20 , 134.48 , 129.81 (Ar-C2), 128.54 (Ar-C3), 126.30 (Ar-C5), 123.36 (Ar-C4), 122.06 , 121.26 (Ar-C6), 113.73 , 82.38 2), 62.65 (O=C-C-C=O), 53.83 (OCH3), 35.89 , 32.70 (CH(CH3)2), 20.52 (CH(CH3)2), 20.27 (CH(CH3)2), 17.78 (Ar-CH3), 10.99 (pyrazole-CH3), 9.15 (pyrazole-CH3). HRMS calcd. for C23H28F6N4O3 ([M+H]+), 523.2138; found, 523.2132.1--2-methylphenyl)-2-isobutyl-N3-malonamideN (8g): White solid; m.p. 204–205 °C; yield 51.5%; 1H-NMR : δ 9.43 , 8.47 , 8.06 , 7.40–7.38 , 3.69 , 3.64 , 3.47 , 2.35 , 2.10 , 2.03–1.88 , 1.82–1.69 2), 1.00 2). 13C-NMR : δ 171.20 (C=O), 169.09 (C=O), 142.65 , 137.13 , 134.44 , 129.80 (Ar-C2), 128.63 (Ar-C3), 126.30 (Ar-C5), 123.49 (Ar-C4), 122.06 , 121.39 (Ar-C6), 113.63 , 82.38 2), 53.84 (OCH3), 53.53 (O=C-C-C=O), 42.51 (O=C-CH-CH2), 35.90 , 26.18 (CH(CH3)2), 22.12 (CH(CH3)2), 21.73 (CH(CH3)2), 17.75 (Ar-CH3), 10.89 (pyrazole-CH3), 9.11 (pyrazole-CH3). HRMS calcd. for C24H30F6N4O3 ([M+H]+), 537.2295; found, 537.2288.1--2-methylphenyl)-2-isopentyl-N3-malonamideN (8h): White solid; m.p. 186–187 °C; yield 56.1%; 1H-NMR : δ 9.55 , 8.40, 8.10 , 7.41–7.38 , 3.69 , 3.47 , 3.45 , 2.35 , 2.12 , 2.10–2.03 , 1.65–1.56 2), 1.41–1.33 2), 0.88 2). 13C-NMR : δ 171.30 (C=O), 169.01 (C=O), 142.63 , 137.17 , 134.45 , 129.81 (Ar-C2), 128.64 (Ar-C3), 126.28 (Ar-C5), 123.45 (Ar-C4), 122.06 , 121.38 (Ar-C6), 113.68 , 82.38 2), 55.06 (O=C-C-C=O), 53.81 (OCH3), 36.30 , 35.86 (CH2-CH(CH3)2), 31.62 (CH(CH3)2), 27.37 (O=C-CH-CH2), 21.96 (CH(CH3)2), 21.88 (CH(CH3)2), 17.78 (Ar-CH3), 10.92 (pyrazole-CH3), 9.10 (pyrazole-CH3). HRMS calcd. for C25H32F6N4O3 ([M+H]+), 551.2451; found, 551.2446.1--2-methylphenyl)-2-(prop-2-yn-1-yl)-N3-malonamideN (8i): Light yellow solid; m.p. 54–55 °C; yield 46.7%; 1H-NMR : δ 9.61 , 8.48 , 8.05 , 7.40-7.37 , 3.69-3.64 , 3.46 , 2.97 , 2.33 , 2.15 , 2.11 . 13C-NMR : δ 169.54 (C=O), 166.75 (C=O), 142.79 , 136.94 , 134.78 , 129.80 (Ar-C2), 129.09 (Ar-C3), 126.27 (Ar-C5), 123.77 (Ar-C4), 122.04 , 121.79 (Ar-C6), 113.47 , 82.36 2), 79.41 (C≡CH), 71.64 (C≡CH), 53.83 (O=C-C-C=O), 52.84 (OCH3), 35.83 , 22.06 (CH2-C≡CH), 17.78 (Ar-CH3), 10.86 (pyrazole-CH3), 9.03 (pyrazole-CH3). HRMS calcd. for C23H24F6N4O3 ([M+H]+), 519.1825; found, 519.1818.1--2-methylphenyl)-N3-malonamide2-Allyl-N (8j): White solid; m.p. 146–148 °C; yield 62.5%; 1H-NMR : δ 9.56 , 8.30 , 8.07 , 7.40–7.38 , 5.93–5.79 , 5.28–5.12 , 3.68 , 3.55 , 3.47 , 2.82 , 2.34 , 2.09 . 13C-NMR : δ 170.56 (C=O), 168.07 (C=O), 142.71 , 137.08 , 134.61 , 133.04 (CH=CH2), 129.80 (Ar-C2), 128.73 (Ar-C3), 126.31 (Ar-C5), 123.54 (Ar-C4), 122.05 , 121.49 (Ar-C6), 118.35 (CH=CH2), 113.50 , 82.38 2), 54.41 (O=C-C-C=O), 53.84 (OCH3), 37.40 , 35.88 (O=C-CH-CH2), 17.81 (Ar-CH3), 10.90 (pyrazole-CH3), 9.11 (pyrazole-CH3). HRMS calcd. for C23H26F6N4O3 ([M + H]+), 521.1982; found, 521.1976.1--2-methoxyphenyl)-2-isopropyl-N3-malonamideN (8k): Light yellow solid; m.p. 74–76 °C; yield 58.3%; 1H-NMR : δ 9.39 , 8.42 , 8.37 , 7.16 , 7.05 , 3.91 , 3.68 , 3.49 , 3.09 , 2.51–2.40 2), 2.11 , 1.11 2). 13C-NMR : δ 169.60 (C=O), 168.48 (C=O), 148.25 (Ar-C2), 142.72 , 134.46 , 128.74 , 122.90 (Ar-C4), 122.05 , 120.76 (Ar-C5), 119.24 (Ar-C6), 113.84 , 109.51 (Ar-C3), 82.45 2), 63.58 (O=C-C-C=O), 55.64 (Ar-OCH3), 53.90 (OCH3), 35.87 , 32.35 (CH(CH3)2), 20.45 (CH(CH3)2), 20.24 (CH(CH3)2), 10.95 (pyrazole-CH3), 9.19 (pyrazole-CH3). HRMS calcd. for C23H28F6N4O4 ([M+H]+), 539.2088; found, 539.2081.1--2-methoxyphenyl)-2-isobutyl-N3-malonamideN (8l): White solid; m.p. 193–195 °C; yield 51.8%; 1H-NMR : δ 9.32 , 8.41 , 8.29 , 7.16 , 7.05 , 3.92 , 3.68 , 3.55 , 3.49 , 2.10 , 2.04–1.84 , 1.80-1.67 2), 0.99 2). 13C-NMR : δ 170.02 (C=O), 169.05 (C=O), 148.18 (Ar-C2), 142.72 , 134.47 , 128.70 , 122.98 (Ar-C4), 120.78 (Ar-C5), 122.04 , 119.27 (Ar-C6), 113.72 , 109.46 (Ar-C3), 82.45 2), 55.64 (Ar-OCH3), 54.35 (OCH3), 53.92 (O=C-C-C=O), 41.75 (O=C-CH-CH2), 35.90 , 26.16 (CH(CH3)2), 22.11 (CH(CH3)2), 21.78 (CH(CH3)2), 10.85 (pyrazole-CH3), 9.15 (pyrazole-CH3). HRMS calcd. for C24H30F6N4O4 ([M+H]+), 553.2244; found, 553.2236.1--2-methoxyphenyl)-2-isopentyl-N3-malonamideN (8m): Light yellow solid; m.p. 70–72 °C; yield 47.2%; 1H-NMR : δ 9.32 , 8.43 , 8.17 , 7.17 , 7.06 , 3.92 , 3.69 , 3.49 , 3.36 , 2.12 , 2.09-2.03 , 1.67–1.54 2), 1.40–1.31 2), 0.88 2). 13C-NMR : δ 170.05 (C=O), 169.01 (C=O), 148.23 (Ar-C2), 142.71 , 134.47 , 128.71 , 122.98 (Ar-C4), 120.78 (Ar-C5), 122.04 , 119.34 (Ar-C6), 113.74 , 109.48 (Ar-C3), 82.45 2), 56.01 (Ar-OCH3), 55.63 (O=C-C-C=O), 53.90 (OCH3), 36.27 , 35.88 (CH2-CH(CH3)2), 31.01 (CH(CH3)2), 27.43 (O=C-CH-CH2), 22.01 (CH(CH3)2), 21.92 (CH(CH3)2), 10.88 (pyrazole-CH3), 9.16 (pyrazole-CH3). HRMS calcd. for C25H32F6N4O4 ([M+H]+), 567.2401; found, 567.2391.1--2-methoxyphenyl)-2-(prop-2-yn-1-yl)-N3-malonamideN (8n): Light yellow solid; m.p. 178–180 °C; yield 60.2%; 1H-NMR : δ 9.64 , 9.37 , 8.31 , 7.17 , 7.08 , 3.92 , 3.86 , 3.62 , 3.48 , 2.93 , 2.78–2.74 , 2.05 , 1.96 . 13C-NMR : δ 167.45 (C=O), 166.67 (C=O), 148.93 (Ar-C2), 141.74 , 134.18 , 129.47 , 122.35 , 121.93 (Ar-C4), 120.64 (Ar-C5), 120.51 (Ar-C6), 114.70 , 110.17 (Ar-C3), 82.47 2), 81.48 (C≡CH), 72.89 (C≡CH), 56.28 (Ar-OCH3), 54.48 (O=C-C-C=O), 52.50 (OCH3), 36.09 , 19.32 (CH2-C≡CH), 11.15 (pyrazole-CH3), 9.05 (pyrazole-CH3). HRMS calcd. for C23H24F6N4O4 ([M+H]+), 535.1775; found, 535.1768.1--2-methoxyphenyl)-N3-malonamide2-Allyl-N (8o): White solid; m.p. 190–191 °C; yield 55.2%; 1H-NMR : δ 9.45 , 8.40 , 8.34 , 7.15 , 7.05 , 5.91-5.77 , 5.25-5.08 , 3.90 , 3.66 , 3.50 , 3.48 , 2.84-2.76 , 2.09 . 13C-NMR : δ 169.48 (C=O), 168.11 (C=O), 148.29 (Ar-C2), 142.71 , 134.55 , 133.23 (CH=CH2), 128.67 , 123.00 (Ar-C4), 122.03 , 120.74 (Ar-C5), 119.40 (Ar-C6), 118.05 (CH=CH2), 113.72 , 109.50 (Ar-C3), 82.44 2), 55.61 (Ar-OCH3), 55.14 (O=C-C-C=O), 53.88 (OCH3), 36.74 , 35.83 (O=C-CH-CH2), 10.85 (pyrazole-CH3), 9.10 (pyrazole-CH3). HRMS calcd. for C23H26F6N4O4 ([M+H]+), 537.1931; found, 537.1922.1--3-isopropylphenyl)-2-isopropyl-N3-malonamideN (8p): White solid; m.p. 86–88 °C; yield 51.7%; 1H-NMR : δ 9.46 , 8.39 , 8.06 , 7.41 , 7.08 , 3.68 , 3.56 , 2.97 , 2.95–2.86 2), 2.53–2.38 2), 2.09 , 1.26 2), 1.12 2). 13C-NMR : δ 168.66 (C=O), 168.48 (C=O), 151.92 (Ar-C3), 142.66 , 137.24 , 134.50 , 129.37 (Ar-C4), 122.40 (Ar-C5), 122.04 , 121.35 (Ar-C6), 113.80 , 112.11 (Ar-C2), 84.49 2), 65.18 (O=C-C-C=O), 54.28 (OCH3), 35.89 , 33.50 (Ar-CH(CH3)2), 31.66 (CH(CH3)2), 23.08 (Ar-CH(CH3)2), 20.36 (CH(CH3)2), 20.28 (CH(CH3)2), 10.80 (pyrazole-CH3), 9.12 (pyrazole-CH3). HRMS calcd. for C25H32F6N4O3 ([M+H]+), 551.2451; found, 551.2443.1--3-isopropylphenyl)-2-isobutyl-N3-malonamideN (8q): White solid; m.p. 67–69 °C; yield 48.8%; 1H-NMR : δ 9.41 , 8.33 , 8.02 , 7.39 , 7.06 , 3.66 , 3.54 , 3.45 , 2.94–2.85 2), 2.06 , 2.00–1.83 , 1.77–1.68 2), 1.24 2), 0.99 2). 13C-NMR : δ 169.08 (C=O), 169.02 (C=O), 151.90 (Ar-C3), 142.67 , 137.17 , 134.54 , 129.36 (Ar-C4), 122.46 (Ar-C5), 122.09 , 121.57 (Ar-C6), 113.79 , 112.21 (Ar-C2), 84.49 2), 55.20 (OCH3), 54.27 (O=C-C-C=O), 40.72 (O=C-CH-CH2), 35.87 , 33.50 (Ar-CH(CH3)2), 26.04 (CH(CH3)2), 23.07 (Ar-CH(CH3)2), 22.10 (CH(CH3)2), 21.81 (CH(CH3)2), 10.70 (pyrazole-CH3), 9.05 (pyrazole-CH3). HRMS calcd. for C26H34F6N4O3 ([M+H]+), 565.2608; found, 565.2601.1--3-isopropylphenyl)-2-isopentyl-N3-malonamideN (8r): White solid; m.p. 63–64 °C; yield 54.8%; 1H-NMR : δ 9.40 , 8.33 , 7.97 , 7.39 , 7.07 , 3.66 , 3.54 , 3.27 , 2.98–2.84 2), 2.07 , 2.05–1.97 , 1.68–1.55 2), 1.37–1.29 2), 1.25 2), 0.90 2). 13C-NMR : δ 169.04 (C=O), 168.96 (C=O), 151.92 (Ar-C3), 142.68 , 137.15 , 134.56 , 129.36 (Ar-C4), 122.49 (Ar-C5), 122.09 , 121.66 (Ar-C6), 113.77 , 112.25 (Ar-C2), 84.49 2), 57.03 (O=C-C-C=O), 54.27 (OCH3), 36.24 , 35.88 (CH2-CH(CH3)2), 33.50 (Ar-CH(CH3)2), 29.94 (CH2-CH(CH3)2), 27.47 (O=C-CH-CH2), 23.08 (Ar-CH(CH3)2), 22.06 (CH2-CH(CH3)2), 21.88 (CH2-CH(CH3)2), 10.74 (pyrazole-CH3), 9.07 (pyrazole-CH3). HRMS calcd. for C27H36F6N4O3 ([M+H]+), 579.2764; found, 579.2756.1--3-isopropylphenyl)-2-(prop-2-yn-1-yl)-N3-malonamideN (8s): White solid; m.p. 69–71 °C; yield 56.5%; 1H-NMR : δ 9.44 , 8.27 , 7.93 , 7.39 , 7.08 , 3.66 , 3.56–3.51 , 2.98–2.93 , 2.93–2.86 2), 2.12 , 2.08 , 1.25 2). 13C-NMR : δ 167.52 (C=O), 166.65 (C=O), 151.97 (Ar-C3), 142.79 , 136.79 , 134.83 , 129.37 (Ar-C4), 122.78 (Ar-C5), 122.14 (Ar-C6), 122.09 , 113.54 , 112.65 (Ar-C2), 84.43 2), 79.52 (C≡CH), 71.27 (C≡CH), 54.86 (O=C-C-C=O), 54.30 (OCH3), 35.87 , 33.47 (Ar-CH(CH3)2), 23.07 (Ar-CH(CH3)2), 20.88 (CH2-C≡CH), 10.71 (pyrazole-CH3), 9.05 (pyrazole-CH3). HRMS calcd. for C25H28F6N4O3 ([M+H]+), 547.2138; found, 547.2131.1--3-isopropylphenyl)-N3-malonamide2-Allyl-N (8t): White solid; m.p. 58–60 °C; yield 56.8%; 1H-NMR : δ 9.39 , 8.28 , 7.90 , 7.39 , 7.08 , 5.92-5.78 , 5.29–5.10 , 3.66 , 3.54 , 3.38 , 2.96-2.87 2), 2.80 , 2.06 , 1.25 2). 13C-NMR : δ 168.30 (C=O), 168.14 (C=O), 151.95 (Ar-C3), 142.71 , 136.97 , 134.66 , 133.18 (CH=CH2), 129.37 (Ar-C4), 122.09 , 122.66 (Ar-C5), 121.87 (Ar-C6), 118.12 (CH=CH2), 113.60 , 112.42 (Ar-C2), 84.47 2), 56.32 (O=C-C-C=O), 54.28 (OCH3), 35.89 , 35.86 (O=C-CH-CH2), 33.48 (Ar-CH(CH3)2), 23.09 (Ar-CH(CH3)2), 10.72 (pyrazole-CH3), 9.08 (pyrazole-CH3). HRMS calcd. for C25H30F6N4O3 ([M+H]+), 549.2295; found, 549.2287.All of the biological assays were performed on representative test organisms reared in the laboratory. Each bioassay was repeated at 25 ± 1 °C according to statistical requirements. All compounds were dissolved in dimethyl sulfoxide and diluted with distilled water containing Triton X-100 to obtain a series of concentrations. Control check (CK) was treated as the same without compounds. Assessments were based on a dead/alive scale, and mortality rates were rectified using Abbott’s formula . EvaluatTetranychus cinnabarinus were evaluated using the reported procedure [Phaseolus vulgaris) with primary leaves expanded to 10 cm in size were selected and cut back to one plant per pot. A small piece was cut from a leaf taken from the main colony and placed on each leaf of the test plants. This was done approximately 2 h before treatment to allow the mites to move to the test plant and lay eggs. The size of the leaf piece from the main colony was varied to obtain approximately 30 mites per leaf. At the time of the treatment, the piece of leaf used to transfer the mites was removed and discarded. The mite-infested plants were dipped in the test solution for 3 s with agitation and set in a fume hood to dry. Plants were kept for 48 h before the numbers of live and dead adults were counted. Each treatment was repeated with triplicate experiments, and each replicate involved 30 adult mites.The acaricidal activities of the target compounds and fenpyroximate against rocedure . Sieva bPlutella xylostella were evaluated using the reported procedure [Plutella xylostella larvae. Mortality was assessed 48 h after treatment. Each treatment was repeated with triplicate experiments and each replicate involved 10 third-instar Plutella xylostella larvae.The insecticidal activities of the target compounds and flubendiamide against rocedure . Leaf diAphis craccivora were evaluated using the reported procedure [The insecticidal activities of the target compounds and imidacloprid against rocedure . Leaves Tetranychus cinnabarinus, Plutella xylostella, and Aphis craccivora. Preliminary bioassay results indicated that compounds 8m and 8p were found to have moderate activities against Tetranychus cinnabarinus. Compounds 8i and 8o exhibited good insecticidal activities against Plutella xylostella. In addition, compounds 8p and 8q possessed potent insecticidal activities against Aphis craccivora. Although the target compounds did not exhibit excellent acaricidal activities against Tetranychus cinnabarinus, as we expected, some of the target compounds showed good insecticidal activities against Plutella xylostella and Aphis craccivora, respectively. The preliminary analyses of structure–activity relationships (SAR) indicated that the methoxy-substituted hexafluoroisopropyl group and variations of Y groups in the position of phenyl ring markedly affected the acaricidal and insecticidal activities. In addition, different types of substituents (R2) have a great influence on the biological activity of different pest species. Notably, these findings demonstrated that the synthesis of 1,3,5-trimethylpyrazole-containing malonamide derivatives could be considered as a new template for pesticide development. Further structural optimization and bioactivities are in progress.In summary, twenty novel 1,3,5-trimethylpyrazole-containing malonamide derivatives were designed and synthesized. The target compounds were evaluated for acaricidal and insecticidal activities against


Hyperglycemia is the main characteristic of diabetes and is the result of an absolute or partial deficit in the production or action of insulin. Recent evidence suggests that hyperglycemia increases the risk of frailty. This issue is of great importance for the Mexican population given the high prevalence of diabetes, particularly in older adults. Our objective was to analyze the association between hyperglycemia and the incidence of frailty in a cohort of rural older adults in Mexico. Prospective cohort study with 600 rural older adults, with measurements made in 2009, 2013 and 2018. Fragility was defined using the proposal of Fried and colleagues. The determination of glycosylated hemoglobin was performed through the A1CNow® device, with capillary blood; hyperglycemia was defined considering the recommendation of the American Diabetes Association; where values greater than 6.5% (140 mg / dL) of glycosylated hemoglobin were considered hyperglycemia. We used an ordinal logistic regression model to analyze the relationship between hyperglycemia and incidence of frailty. In the baseline measurement (2009), 8.6% of older adults presented frailty. The incidence of frailty was 6.9%. After adjusting for health and sociodemographic characteristics, hyperglycemia was significantly associated with the incidence of frailty (RR = 2.24 P = 0.018). These findings allow us to determine that hyperglycemia is a prognostic factor of the incidence of frailty. Because frailty is preventable, future interventions for the prevention of frailty should consider the presence of hyperglycemia.


Several binding targets have previously been proposed for Cltx but none effectively explain its pleiotropic effects; its true target remains ambiguous and is the focus of this study.Chlorotoxin (Cltx) isolated from scorpion venom is an established tumor targeting and antiangiogenic peptide. Radiolabeled Cltx therapeutic (A peptide-drug conjugate (ER-472) composed of Cltx linked to cryptophycin as warhead was developed as a tool to probe the molecular target and mechanism of action of Cltx, using multiple xenograft models.Neuropilin-1 (NRP1), an endocytic receptor on tumor and endothelial cells, was identified as a novel Cltx target, and NRP1 binding by Cltx increased drug uptake into tumor. Metabolism of Cltx to peptide bearing free C-terminal arginine, a prerequisite for NRP1 binding, took place in the tumor microenvironment, while native scorpion Cltx with amidated C-terminal arginine did not bind NRP1, and instead acts as a cryptic peptide. Antitumor activity of ER-472 in xenografts correlated to tumor NRP1 expression. Potency was significantly reduced by treatment with NRP1 blocking antibodies or knockout in tumor cells, confirming a role for NRP1-binding in ER-472 activity. Higher cryptophycin metabolite levels were measured in NRP1-expressing tumors, evidence of NRP1-mediated enhanced drug uptake and presumably responsible for the superior antitumor efficacy.NRP1 was identified as a novel Cltx target which enhances tumor drug uptake. This finding should facilitate tumor selection for chlorotoxin-based therapeutics and diagnostics.The online version of this article (10.1186/s12964-019-0368-9) contains supplementary material, which is available to authorized users. Leiurus quinquestriatus and named for its ability to block chloride channels and cause neurotoxicity [131I-TM601, TM601 refers to chemically synthesized Cltx) as a clinical candidate for glioblastoma [131I-TM601 also demonstrated tumor-specific uptake beyond glioma in multiple diverse cancer indications in preliminary clinical investigations [https://clinicaltrials.gov/).Chlorotoxin (Cltx) is a small basic peptide first isolated from the venom of the scorpion toxicity . Injectitoxicity . The tumtoxicity . They reigations . Additioigations , https:/vβ3 was identified as a receptor for Cltx and proposed to play a role in the observed anti-invasive activity [Tumor-specific binding by Cltx, first described in glioma, was more broadly observed in a variety of diverse tumor types, in contrast to normal tissue which consistently remained refractory to Cltx binding . Cltx’s activity . Treatmeactivity . Nonetheactivity .Further investigation revealed that Cltx will bind and undergo internalization by proliferating endothelial cells, the first reported interaction of the peptide with a normal, untransformed cell type. These findings led to the identification of annexinA2 as an additional target of Cltx; annexinA2 is expressed on the surface of many tumor cells as well as vascular endothelial cells , 12. TheA more complete understanding of its mechanism of action is essential to fully realize the broad potential of Cltx as a therapeutic, a diagnostic, or a tumor targeting drug delivery vehicle. Toward this objective, we describe the novel peptide drug conjugate (PDC) ER-472, comprised of Cltx linked to a novel cryptophycin analog as cytotoxic warhead. ER-472 was used as a tool to comprehensively investigate the true molecular target and mechanism of action of Cltx. In studies described herein the endocytic receptor neuropilin-1 (NRP1) was identified as a novel Cltx binding partner. A C-terminal arginine residue proved essential for binding of peptides to NRP1. Native Cltx with amidated arginine at its C-terminus did not bind NRP1 but acted as a cryptic peptide; its metabolism to peptides bearing a free C-terminal arginine and the capacity to bind NRP1 took place in the tumor microenvironment. Cltx-NRP1 binding enhanced tumor uptake of ER-472 active metabolite that was blocked by anti-NRP1 antibody treatment or NRP1 knockdown and resulted in diminished ER-472 antitumor activity. This newly identified targeting mechanism of Cltx via NRP1 binding appeared to be separate and distinct from the neurotoxic effects of Cltx and will be useful in selection of tumors for Cltx application.2) was synthesized at CPC Scientific  by routine methods and stored dry at -20 °C. Recombinant Cltx expressed in E.coli  has a carboxylated C-terminal arginine residue (Cltx-COOH). Cltx peptide was dissolved in PBS for routine studies. The PDC ER-472 and compounds ER-271 and S-methyl cryptophycin were synthesized at Eisai; details provided in [Native Cltx (Cltx-CONHvided in . For in BxPC-3 cells (BW125058) were from Perkin Elmer , MIA PaCa-2  and PC-3  were from ATCC. All cell lines were maintained according to their instructions. Cells were used within a short time of receipt, or cell line authenticity was verified by STR typing  and all cells were certified mycoplasma-free prior to culture .2)/2 = mm3, where l and w refer to the larger and smaller perpendicular dimensions collected at each measurement. Tumor dimensions were recorded twice per week starting when tumors reached an approximate size of 100 to 150 mm3. For xenograft models MIA PaCa-2, BxPC-3 and PC-3, cells were injected subcutaneously near the right axillary area, into female Nu/Nu mice (Charles River Labs), approximately 6–8 weeks old. CRISPR engineering of the PC-3 cells resulted in isogenic cell lines that were less tumorigenic than parental cells and therefore these xenograft models were performed in NOD SCID mice. For efficacy studies treatments were initiated when the average tumor size was approximately 200 mm3. ER-472 was administered at ¼, ½ or full MTD dose  once every 4 days, 3 times (Q4Dx3).All studies were performed according to IACUC approved protocols. The general health of mice was monitored daily. Tumor volume was determined by caliper measurements (mm), using the formula . Tumors were cryofractured using the Cryoprep System , lysed 3:1 (v:w) in PBS containing 1x Halt™ Protease Inhibitor Cocktail , and were extracted 3:1 with methanol:acetonitrile 1:1 (v:v) containing 0.1% acetic acid. ER-472 and S-methyl metabolite were then quantified using liquid chromatography coupled with tandem mass spectrometry analysis (LC-MS/MS).For studies to detect tumor levels of active metabolite, a single dose of drug was administered when tumors volumes reached ~ 300 to 500 mmTo detect Cltx and derived peptides in PC-3 tumor lysate, Cltx peptide was administered at 150 mg/kg and tumors were harvested 1 h later. Following cryofracture, tumors were lysed in Super B buffer  containing protease inhibitors, centrifuged, dried and re-constituted in acetonitrile, ammonium bicarbonate, pH 8. Proteins and peptides were reduced with dithiothreitol and the cysteine residues were capped with iodoacetamide prior to LC-MS/MS analysis. BiopharmaLynx™ version 1.33 software (Waters) was used to analyze and identify Cltx-related peptides.Cell proliferation assays were performed in 96 well plates with 3000 cells seeded per well; ER-472 was added at various concentrations and plates were incubated for various time points. Cell growth was assessed using the CellTiter-Glo® luminescent cell viability assay .n = 4) using the RNeasy Mini Kit . Total RNA (2 μg) was converted to cDNA using SuperScript VILO Master Mix  and amplified using Applied Biosystems TaqMan expression assays using RNA-specific primers for NRP1 (Hs00826128_m1), GUSB (Hs99999908_m1), and HPRT1 (Hs00000009_m1). QPCR was performed with TaqMan Fast Advanced Master Mix  on the Applied Biosystems 7900 instrument and relative gene expression was calculated after normalization against reference genes GUSB and HPRT1 using the using the 2 − ΔΔCT method in the Applied Biosystems SDS 2.4 software.Tumors were harvested from various models and flash frozen prior to lysis and analysis of thiol, protein or mRNA levels. For thiol and western studies, total protein was measured in tumor lysates following cryofracture and resuspension in lysis buffer, using the Pierce™ BCA protein assay kit (Thermo Fisher Scientific Cat# 23225). Antibodies used in westerns were NRP1 , CLC-3 (D8Y5Q) , MMP2 (D204T) , AnnexinA2 (D11G2)  and tubulin . In certain blots recombinant human proteins were included as positive controls: NRP1, Phe22-Lys644 , CLC-3 , MMP2  and AnnexinA2  using an Odyssey Infrared Imaging System (LI-COR Biosciences). RNA was extracted from excised tumors  (~ 40 μg/mL) was added to Cltx biotinylated at K27 or N-terminus (10 mg/mL) and incubated for 24 h at 37 °C, pH 8.5. The resulting peptides were separated by preparative HPLC and peptide fragments were characterized by MALDI. Small linear Cltx-derived peptides were also synthesized by routine methods and were biotinylated at the N-terminus. In some cases, peptides were reduced with TCEP at pH 8 and the cysteine residues were capped using iodoacetamide.D) was determined by measuring binding over the range of NRP1 concentrations . For competition studies, binding of biotinylated VEGF165  to NRP1 (390 nmol/L) was measured in the presence of Cltx-CONH2 or Cltx-COOH at concentrations from 50 to 800 μmol/L.Assessment of binding between NRP1and Cltx was performed by Bio-Layer Interferometry (BLI) using the Octet Red96 System . Cltx and derived fragments were captured on streptavidin biosensors (Cat# 18–5020) under approximately equivalent loading conditions; sensors were then quenched with EZ-Link Biocytin and washed in assay buffer . Binding to NRP1  at concentrations from 0 to 1750 nmol/L was determined by association then dissociation (1 min each) in assay buffer. Binding constant  using an SOP generated by TransMolecular Inc. Cltx peptide (20 μg) in sterile water was injected through the ventral surface of the thorax in the region of the sub-esophageal ganglion of crayfish which were then subjected to physical challenge defined as multiple, continuous, gentle prods in random parts of the body from head to tail. The animal’s response is based on avoidance, ability to right itself, and clasping with chelae in a defensive manner. Time from injection to total incapacitation was recorded; total incapacitation is defined as the time when the tested crayfish is not responsive when physically challenged.The 36 amino acid sequence of Cltx is shown in Fig. 50 values and kinetics of cell growth inhibition  revealed differential sensitivity. At the MTD dose, ER-472 treatment resulted in significant antitumor activity in all 3 models, but at ½ MTD no antitumor activity was observed in MIA PaCa-2 or BxPC-3 xenografts  did not bind to NRP1 , lysine, leucine or glutamic acid. The binding affinity of NRP1 for Cltx-derived peptides ranged from 100 to 400 nM as compared to ~ 50 nM for the native substrate VEGF  did not bind to NRP1  of ~ 240 nM . Furthermore, in competition experiments, Cltx-COOH but not Cltx-CONH2 competed with biotinylated VEGF for binding, suggesting that Cltx and VEGF interact with NRP1 at a similar binding site ; moreover, NRP1 levels appeared to further increase upon tumor recurrence  in a bioassay to investigate neurotoxicity. Cltx peptides were injected into crayfish which were immediately assessed for onset of paralysis. The 2 versions of Cltx were indistinguishable in this assay; both peptides induced paralysis in crayfish with a very rapid and almost identical timeframe for total incapacitation  does not bind NRP1. Deamidation occurs in tumor and the resultant Cltx-COOH and several derived peptides bind to NRP1. Thus, systemically, Cltx acts as a cryptic peptide, with NRP1 binding ability subsequently revealed upon reaching the tumor.The tumor specificity of Cltx was previously attributed to the distinct targets; CLC-3, MMP2 and annexinA2, and while all 3 are likely involved in aspects of Cltx function, none fully accounts for its multiple diverse activities. ER-472, a PDC of Cltx and the potent tubulin agent cryptophycin was developed to further probe the mechanism of action of Cltx. Treatment of xenografts with ER-472 resulted in a wide range of therapeutic activity which did not correlate with tumor expression of the previously described Cltx targets. In this study, we identified the cell surface endocytic receptor NRP1 as a novel target of Cltx. A role was demonstrated for NRP1 in tumor-specific uptake of Cltx and, notably, expression of NRP1 on tumors positively correlated with the antitumor activity of ER-472. A key finding is that NRP1 binding only occurs when the C-terminal arginine of Cltx is deamidated while native Cltx from scorpion venom . Since these enzymes have not yet been identified in eukaryotes, deamidation in the case of Cltx is likely the result of a spontaneous, non-catalyzed reaction. Studies of the anti-apoptotic protein Bcl-xoperties , illustrNRP1 is a multifunctional protein essential for development of both CNS and vasculature . It funcIn the case of Cltx and derived peptides, the previously reported RXXR motif was not required for NRP1 binding; peptides bound to NRP1 when they terminated with arginine regardless of the preceding sequence. However, the C-terminal arginine residue itself was essential since no NRP1 binding occurred when Cltx peptides terminated with any other amino acid, including lysine. This absolute requirement for arginine contrasts with reports of NRP1 binding by peptides with C-terminal lysine .Binding of deamidated Cltx and derived peptides increased uptake of cryptophycin metabolite into tumors; these findings are in accord with reports that NRP1 binding increases tumor uptake of a wide variety of anticancer drugs from small molecules to larger biologicals . Uptake In most cases NRP1 acts with a co-receptor, transporter, etc. and often a multi-protein complex is involved in mediating cellular uptake. While identification of additional co-receptors and a detailed examination of the endocytosis mechanism upon Cltx-NRP1 binding were beyond the scope of this study, it is of interest to note that in early Cltx studies, cells treated with Cltx were reported to have reduced surface expression of both MMP2 and CLC-3 . These pDuring embryogenesis, NRP1 binds VEGF to mediate angiogenesis and this natural ligand (VEGF-165) bears a C-terminal arginine residue which appears essential for NRP1 binding. Loss of NRP1 results in vascular remodeling and branching defects and thus, blocking NRP1 is an attractive approach for targeting angiogenesis as an anticancer therapy. Indeed, Genentech developed an anti-NRP1 antibody which was assessed in clinical studies alone and in combination with Avastin® , 30. In Finally, our studies evaluating paralysis induction in crayfish demonstrate, for the first time, that the neurotoxic and tumor targeting properties of Cltx can be definitively separated. The NRP1 binding, tumor targeting capability of Cltx appears irrelevant and unnecessary for its neurotoxicity.Collectively, our studies provide an improved understanding of Cltx targeting to tumors. Binding of Cltx to the endocytic receptor NRP1 results in an increase in drug uptake, which translates to enhanced antitumor activity in vivo. The identification of NRP1 as a Cltx binding partner also contributes mechanistic insight into its antiangiogenic properties. The revised and improved knowledge of Cltx SAR strongly supports using NRP1 as a stratification and selection marker for any Cltx-containing diagnostics and therapies and should help to significantly advance research in the Cltx field.Additional file 1:Figure S1. Structure of ER-472 PDC. Figure S2. Metabolism of ER-472 PDC in vivo. Figure S3. CLC-3, MMP2 and annexinA2 expression in MIA PaCa-2, BxPC-3 and PC-3 tumor lysates: lack of correlation with therapeutic activity. Figure S4. Knockout of NRP1 by gene editing in PC-3 tumor cells. Figure S5. NRP1 expression in human tumors, including glioblastoma. Figure S6. Neurotoxicity of Cltx peptides in crayfish bioactivity assay. Table S1. Cell growth inhibition by ER-472 in MIA PaCa-2, BxPC-3 and PC-3 cell lines in vitro. Table S2. Basal thiol levels in MIA PaCa-2, BxPC-3 and PC-3 tumor lysates. (DOCX 1260 kb)


Endometrioid endometrial carcinoma (EEC) is the most common gynaecologic malignancy worldwide. Long non‐coding RNAs have previously been demonstrated to play important roles in regulating human diseases, particularly cancer. However, the biological functions and molecular mechanisms of long non‐coding RNAs in EEC have not been extensively studied. Here, we describe the discovery of Lnc‐NA from the promoter of the transcription factor nuclear receptor subfamily 4 group A member 1 (NR4A1) gene. The role and function of Lnc‐NA in EEC remain unknown. In this study, we used quantitative real‐time polymerase chain reactions to confirm that Lnc‐NA expression was down‐regulated in 30 EEC cases (90%) and in EEC cell lines compared with that in the paired adjacent tissues and normal endometrial cells. In vitro experiments further demonstrated that overexpressing Lnc‐NA decreased EEC cell proliferation, migration and invasion and promoted apoptosis via inactivation of the apoptosis signalling pathway. Moreover, the results show that Lnc‐NA expression was positively correlated with NR4A1. Furthermore, Lnc‐NA regulated NR4A1 expression and activated the apoptosis signalling pathway to inhibit tumour progression. In summary, our results demonstrate that the Lnc‐NA‐NR4A1 axis could be a useful tumour suppressor and a promising therapeutic target for EEC. Lnc‐NA is a lncRNA‐transcribed antisense adjacent to nuclear receptor subfamily 4 group A member 1 (NR4A1) on chromosome 12q13.13. Several studies have reported NR4A1 as a tumour suppressor. For example, NR4A1 and Nor1 double knockout mice develop acute myeloid leukaemia.22.1This study was carried out according to the guidelines approved by the review board of the ethics committee of the Hospital of Shandong Provincial, Shandong University. Written informed consent was obtained from all the subjects in our research (In Supporting information). Pairs of EEC samples and adjacent normal endometrial tissues were collected from 30 patients who underwent a hysterectomy for EEC at the Department of Provincial Hospital affiliated with Shandong University. All adenocarcinoma samples were diagnosed and evaluated according to the International Federation of Gynecology Oncology criteria. The diagnoses of these EEC samples were verified by pathologists. Detailed pathologic and clinical data were collected for all samples.2.22. Cell transfections were performed using Lipofectamine 3000  according to the manufacturer's instructions.The following cell lines were obtained from the China Type Culture Collection  and the Be Na Culture Collection : KLE, RL95‐2, Ishikawa, HEC‐1‐b and ESC. Cells were grown in RPMI 1640 medium  supplemented with 10% FBS  at 37°C in a humidified atmosphere of 5% CO2.3The Lnc‐NA gene expression plasmid, the gene expression inhibitor plasmid and the respective empty vector plasmids were purchased from Shanghai GeneChem Co., Ltd. . Neomycin was used as the stable cell line selection marker.2.4−∆∆Ct), and human GAPDH was used as an internal control. The sequences of the primers used in the study are shown in Table Total RNA was extracted using TRIzol reagent  according to the manufacturer's protocol. RNA quantity was measured with a NanoDrop ND‐2000 spectrophotometer , and RNA integrity was assessed using standard denaturing agarose gel electrophoresis. Lnc‐NA expression in tissue samples and cultured cells was quantified using SYBR Green I Master  and a LightCycler 480  according to the manufacturer's instructions. Fold changes were calculated using relative quantification  supplemented with protease and phosphatase inhibitors . Total protein samples were separated by electrophoresis with 10% SDS‐polyacrylamide gels and transferred onto PVDF membranes . The membranes were blocked in 5% skim milk and incubated with primary antibodies Table  overnigh2.63 cells per well. CCK‐8 solution was added to each well. After 1 h, the absorbance of each well at 450 nm was determined with a microplate reader . For cell apoptosis analysis, cells were washed with PBS and resuspended in Annexin V binding buffer. Then, 5 μL of PE Annexin V and 5 μL of 7‐amino‐actinomycin (7‐AAD)  were added to each sample and incubated for 15 minutes at room temperature in the dark according to the manufacturer's protocol. Immediately afterward, cell apoptosis was analysed with FACSCalibur flow cytometry .2‐(2‐Methoxy‐4‐nitrophenyl)‐3‐(4‐nitrophenyl)‐5‐‐2H‐tetrazolium salt  assays were conducted according to the manufacturer's instructions to evaluate cell proliferation rates. Briefly, log‐phase cells were trypsinized into a single‐cell suspension and plated into 96‐well plates at a density of 2 × 102.75 cells were seeded on the upper insert in serum‐free medium. After incubation for 24 hours, the insert was fixed, stained, counted, and analysed using an inverted light microscope (Nikon). Each experiment was performed in triplicate. For the cell invasion assay, the procedure was similar to that of the cell migration assay, except the transwell membranes were coated with Matrigel (Corning).For the cell migration assays, migration chambers (8‐μm pores)  placed into a 24‐well plate were used. In the lower chamber, medium with 10% FBS was added as a chemoattractant. A total of 2 × 102.8TM Dual Luminescence Assay Kits (GenecopoeiaTM). The results were obtained from three independent experiments performed in duplicate.The NR4A1 promoter was PCR‐amplified from human genomic DNA and cloned into the pEZX‐LvPG04 Luciferase Reporter Vector (Genecopoeia). 293T cells were transfected with NR4A1 promoter plasmids or the control plasmid and Lnc‐NA plasmid. Forty‐eight hours following transfection, luciferase activity was analysed using the Secrete‐Pair2.9t test to compare data. P < 0.05 was considered statistically.Data are presented as the means ± SD. All statistical analyses were performed using ANOVA or a two‐tailed Student's 33.1P = 0.0014, Figure P = 0.0002, Figure r = 0.454, P = 0.0191) . However, compared with control siRNA transfection, sh‐Lnc‐NA transfection reduced Lnc‐NA expression levels (P < 0.001).To elucidate the underlying mechanisms of Lnc‐NA in EEC development, we performed in vitro assays to examine the effects of Lnc‐NA on cell proliferation and apoptosis in EEC cells. Thus, we selected and transfected two recipient EEC cell lines: Ishikawa cells were transfected with Lnc‐NA or Lnc‐NA‐control, and KLE cells were transfected with Lnc‐NA‐inhibitor or Lnc‐NA‐inhibitor‐control. Cell lines with Lnc‐NA overexpression and knockdown were established and designated Ishikawa‐Lnc‐NA and KLE‐sh‐Lnc‐NA. As shown in Figure P < 0.001). In contrast, KLE cells transfected with sh‐Lnc‐NA had higher growth index values at 72 and 96 hours post‐transfection than those transfected with sh‐Lnc‐NA‐control . The effects of Lnc‐NA on cell apoptosis in EEC cells were determined by flow cytometry. As shown in Figure P < 0.01), whereas Lnc‐NA knockdown in KLE cells inhibited cell apoptosis (P < 0.05).The effects of Lnc‐NA on EEC cell growth were determined by CCK‐8 assays. Ishikawa cells transfected with Lnc‐NA had lower growth index values  at 48, 72, and 96 hours post‐transfection than those transfected with Lnc‐NA‐control . Studies have shown that Bcl2/Bax are involved in apoptosis in tumours.P < 0.001). However, Lnc‐NA knockdown inhibited Bax expression and promoted Bcl2 expression. Our results suggest that Lnc‐NA inhibits cell progression and promotes cell apoptosis and that Lnc‐NA and NR4A1 expression levels are consistent in EEC cells.To further analyse the relationship between Lnc‐NA and NR4A1, we examined NR4A1 expression after Lnc‐NA up‐regulation and down‐regulation. Our results showed that compared with the controls, Lnc‐NA overexpression increased NR4A1 expression levels, and Lnc‐NA knockdown decreased NR4A1 expression levels . Ishikawa cells also exhibited significant impairments in invasion ability after transfection with Lnc‐NA for 48 hours . In addition, we performed migration and invasion assays to examine the effects of Lnc‐NA knockdown on cell migration and invasion ability in KLE cells. Compared to the controls, Lnc‐NA knockdown caused a significant increase in the number of migrated and invaded cells .After verifying the inhibitory role of Lnc‐NA in EEC cell viability, we further investigated whether this lncRNA participates in cell migration and invasion. Compared with Ishikawa cells transfected with the negative vector, cells transfected with Lnc‐NA for 48 hours showed significantly decreased migration ability . In contrast, MMP2/9 expression was up‐regulated in Lnc‐NA knockdown KLE cells compared with that in control cells.Matrix metalloproteinases play an important role in tumour invasion and migration.Collectively, these data suggest that Lnc‐NA decreased cell invasion and migration in EEC cells.3.4P < 0.001). Next, we performed flow cytometry analyses to determine the role of NR4A1 in cell apoptosis. As shown in Figure P < 0.01). In addition, the migration and invasion assay results showed that cell numbers were higher in NR4A1 knockdown cells than in Ishikawa‐Lnc‐NA cells . As a control, we examined the role of NR4A1 overexpression in Ishikawa cells, and the results showed that overexpression of NR4A1 inhibited cell proliferation, migration, and invasion while promoting cell apoptosis . Meanwhile, to demonstrate that Lnc‐NA exerts its effects by targeting NR4A1, we measured NR4A1, Bcl2/Bax, and MMP2/9 expression levels by Western blot. Consistent with the cell function experiment results, Bcl2 expression levels were increased, and Bax expression levels were decreased upon NR4A1 knockdown. NR4A1 knockdown increased MMP2 and MMP9 protein expression levels compared with those of the control group .To determine the importance of NR4A1 in Lnc‐NA‐mediated proliferation, apoptosis, migration, and invasion in EEC cells, we silenced NR4A1 expression in the Ishikawa‐Lnc‐NA cell line. First, CCK‐8 assay results showed that compared with the Ishikawa‐Lnc‐NA group, NR4A1 knockdown significantly increased cell proliferation . These data suggested that NR4A1 is a target of Lnc‐NA, which inhibits the progression of EEC by promoting the expression of NR4A1.Furthermore, we examined the effect of down‐regulating Lnc‐NA in cell lines overexpressing NR4A1. We found that Lnc‐NA did not reverse the proliferation, apoptosis, invasion, and migration of cells caused by overexpression of NR4A1. At the same time, the results of Western blotting indicate that down‐regulation of Lnc‐NA did not alter the associated protein expression , NR4A2 (Nurr1), and NR4A3 (NOR‐1) are nuclear orphan receptors of the Nur77 family and are widely expressed in different types of tissues, such as skeletal muscle, adipose tissue, heart, kidney, T cells, liver, and brain. As recently described, these receptors are also involved in tumorigenesis.Some studies suggest that upstream antisense RNAs may exert some biological functions by regulating corresponding genes.Although NR4A1 target genes are poorly characterized, the proapoptotic effects of NR4A1 may be achieved by its transactivation function of acting as a transcription factor (TF) for apoptosis‐inducing genes.Several genome‐wide studies have suggested that promoters of protein‐coding genes are the origins of LncRNA transcription.In conclusion, we discovered that lncRNA Lnc‐NA is frequently down‐regulated in EECs. Lnc‐NA overexpression decreased proliferation, migration, and invasion and increased apoptosis in EEC cells by up‐regulating NR4A1. Furthermore, our findings indicate that the Lnc‐NA‐NR4A1 axis plays a tumour suppressing role in EEC progression by activating apoptosis signalling. Therefore, the Lnc‐NA‐NR4A1 axis could serve as a candidate prognostic marker for EEC.The authors declare no conflicts of interest for this article.LY Sun and YR Zhao designed the experiments; LY Sun carried out all of the experiments and data collection; RF Zhou, S Liu, LC Wang, PJ He, and XW Liu helped with the collection of clinical samples. LY Sun wrote the manuscript and prepared the figures; J Dong, SN Hu, YL Jiao, and XB Zhao provided technical support; LY Sun, YR Zhao, and GS Jiang revised the manuscript. All authors approve and agree to be responsible for all aspects of this work. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Surveying the landscape of the educational experience of medical physicists, there are many relatively recent accomplishments, and our educational organizations have been active promoting some necessary improvements. Maintenance of Certification became a reality in the early years of this century. The requirement to complete a residency program to sit for the American Board of Radiology (ABR) certification examinations is a reality as of 2014. The Commission on Accreditation of Medical Physics Education Programs (CAMPEP) has accredited over 50 academic programs, over 100 residency programs in therapy physics and over 25 residency programs in imaging physics. The majority of these programs were first accredited in this decade. Additionally, the American Association of Physicists in Medicine (AAPM) Education Council and the Society of Directors of Academic Medical Physics Programs have been active promoting medical physics education, encouraging quality, providing for coordination between education programs and establishing best practices in medical physics education. The AAPM has hundreds of Councils, Committees, Subcommittees, Task Groups and Working groups, all involved in some aspects of the evolving nature of technology, clinical practice, and the knowledge base of our profession.Even with all of this activity, however, keeping up with changes in the field and providing for contemporary education is a daunting task and challenging to do well. Continuing education is absolutely necessary in this profession. If we fail to keep up, our practice skills can become obsolete in a few years. So as a profession, we face two challenges in education: how do we bring students up to a baseline level of practice in our academic and residency programs, and how do we maintain a level of practice proficiency? This editorial discusses one aspect of the first challenge.JACMP and Medical Physics. An argument easy to make is that an advanced structured course in both radiation oncology and imaging physics of one semester each would be not only desirable, but perhaps essential for the training of the next generation of medical physicists. But what would such a course look like?Respecting CAMPEP academic programs, there is only one full structured course required in radiation oncology physics (at the level of the Khan or Hendee text) and one required in imaging physics (at the level of Bushberg). These courses, usually one semester each, can do little more than provide the most basic of introduction to the science of the field. Thus, a growing knowledge gap exists between the material in these courses and the information published each month in our scholarly journals, the We envision the second course in therapy physics to be site/disease based and would focus on integrating the technology with the clinical challenges we face each day. As an example, consider prostate cancer. Three or four lectures would include the cancer biology and progression, staging and scoring, treatment approaches, planning and optimization algorithm details, Image Guided Radiation Therapy (IGRT), outcomes, and avenues for research. Other disease sites, including Central Nervous System (CNS), Gynocology (GYN), brain, lung, head and neck, and others would follow similarly. An advanced course in imaging physics would deal with specific sites , using the latest insights into use of advanced techniques such as molecular imaging, dual‐energy imaging, image registration, artificial intelligence, computer‐aided diagnosis and other emerging technologies.The justification for such courses is that this is the type of work therapy and imaging physicists face every day. It is our calling to solve imaging problems for specific disease sites and to assure knowledgeable and precise clinical practice respecting therapy interventions. Yet so often, entry‐level medical physicists are less prepared than they could be to undertake these challenges.JACMP would be an ideal place to publish such articles since the journal is open access and the author retains ownership of the copyright. There would therefore be no restriction on the use in an educational setting as the articles are free to access and use. These review articles would be peer reviewed and would form the basis for the second advanced course in therapy or imaging physics.The basis for these advanced courses would include, among other references, a series of review articles published in a public forum. The JACMP has an established category to publish Education Articles, we are issuing a call for the community to volunteer its time to create and publish Education Review Articles as described. To avoid duplication of effort, please email the Editor and volunteer your article for a therapy or imaging site along with a timetable for submission. We believe these articles will be very valuable to the community and essential to prepare medical physics students and residents for clinical practice.Since the


Eutonina indicans  medusa swimming in a predominantly linear path. Bell contractions produced pairs of vortex rings with opposite rotational sense. Analyses of the momentum flux in these wake structures demonstrated that vortex dynamics related directly to variations in the medusa swimming speed. Furthermore, a bulk of the momentum flux in the wake was concentrated spatially at the interfaces between oppositely rotating vortices rings. Similar thrust-producing wake structures have been described in models of fish swimming, which posit vortex rings as vehicles for energy transport from locations of body bending to regions where interacting pairs of opposite-sign vortex rings accelerate the flow into linear propulsive jets. These findings support efforts toward soft robotic biomimetic propulsion.Swimming bell kinematics and hydrodynamic wake structures were documented during multiple pulsation cycles of a  Medusae present an important opportunity for understanding animal propulsion because they were likely the earliest animal phyla to develop muscular swimming. Cnidarians are one of the most ancient animal lineages ,2 and meThe early focus of medusan swimming mechanics centered on jet-propelled species. Early descriptions of jet propulsion depended upon contraction of muscular fibers encircling the swimming bell to eject fluid that is accelerated into a thrust-generating jet as it passes through a narrow aperture, the velum. A model of this process , involviEutonina indicans is such a species which, in combination with digital particle imaging velocimetry methods [One obstacle to documenting flows of swimming medusae is that flow initiation occurs within the subumbrellar cavity, a region often surrounded by body tissue with limited transparency. Measurements of these flows are hindered when the medusa’s body wall obscures the ability to observe flow patterns. This limitation can be at least partially overcome by using target species possessing a highly transparent body that permits imaging through the body wall and into the enclosed subumbrellar fluids. The hydromedusa  methods ,15, prov methods .Eutonina indicans is a jetting medusa. However, unlike some other hydromedusae, it does not possess a pronounced velum at the oral end of its body. This reduced velar component facilitates the aforementioned optical access to the flow in the subumbrellar cavity. Moreover, the oblate fineness ratio  of its body relative to other jetters made it a compelling scientific focus for this study, as its body plan is intermediate between many jetters and rowers [d rowers . Thus, tEutonina indicans medusae (0.7–1.3 cm relaxed bell diameter) were obtained from the New England Aquarium and maintained in 20 L aquaria at 20 C. Swimming kinematics and fluid interactions during swimming were measured for individuals placed into a glass filming vessel seeded with neutrally buoyant, hollow glass spheres (10 μm). Only recordings of animals swimming upwards were used in the analysis to eliminate the possibly of gravitational force from aiding forward motion of the animal between pulses. One swimming sequence with multiple pulsation cycles (n = 5) was selected for detailed study of kinematic and fluid dynamic data.−1 using a high-speed digital video camera  placed perpendicular to the laser sheet. The laser sheet illuminated a two-dimensional sheet of the glass spheres around the medusae and the cross-section through the center of the medusan bell. Velocities of particles illuminated in the sheet were determined using digital particle image velocimetry (DPIV) software  that analyzed sequential video frames using a cross-correlation algorithm. Image pairs separated in time by 6 ms were analyzed with shifting, 50% overlapping interrogation windows of decreasing size . Prior testing with these interrogation window settings and interval duration demonstrated >2 pixel average particle motion between image pairs, as well as the most consistent velocity and vorticity estimates at the experimental seeding density and magnification. This analysis generated a 128 × 128 gird of velocity vectors around the swimming medusae. Medusae were illuminated using a 680-nm wavelength laser sheet and recorded at 1000 frames sf, whereU, as:x and y are the coordinates of the apex of the bell in successive images. Pixel discretization errors were filtered by neglecting bell position changes of less than 33 μm. This was <20% of average position changes occurring during periods of bell contraction and forward swimming.Bell kinematics, such as subumbrellar volume and velar aperture diameter, were quantified from the cross-sectional images of the bell and were analyzed using Image J software . The bell and swimming kinematics were quantified using previously reported methods . Changesfluxp) of fluid across transects positioned across the velar diameter at the fluid exit from the subumbrellar cavity. The video frames were re-oriented so that the trajectory of swimming medusae was parallel to the y-axis . With this orientation, the velocity of the jet of fluid emerging from the subumbrellar cavity had a predominant y-component (yu). The momentum flux [fluxp) using the fluid velocity vector (u), the direction perpendicular to the linear transection (n), and the y-component of the jet (yu): ρ is the density of seawater and l is the length of the horizontal transect. Estimation of total momentum flux through the velar aperture assumed a circular velar aperture area with a diameter of the transect line. Analyses of wake structures were performed from DPIV velocity vector fields. The contribution of different fluid regions to the starting vortex rings during bell contraction was measured as the momentum flux  within starting vortex rings on either side of the bell margin was quantified as: Eutonina indicans medusae generated a characteristic series of opposite-sign vortex rings during bell pulsation. Starting from rest  of Eutonina relative to prior studies [The relatively rapid return to zero swimming velocity B likely  studies , which iVortex interactions dominated momentum transfer from the swimming medusa to the fluid in the medusa’s wake. Bell contraction forced fluid with stopping vortex circulation out of the bell cavity and through the expanded velar aperture. As this fluid was ejected, it was forced into contact with fluid characterized by opposite-sense rotation that was part of the developing starting vortex formed at the bell margin A. The twEutonina indicans medusa.The velocity magnitudes at VIA regions, and consequently momentum flux patterns, were directly related to circulation magnitudes of the interacting vortices. Starting vortex ring circulation magnitudes were approximately equal on either side of the bell  and maxip = 0.67) but they were lower than the central axis of the animal . The lower VIA angles . Peak vswimming . Althoug−1 for predicted and observed values, respectively, E. indicans medusa can be predicted based on analysis of the momentum flux in the symmetry plane of its wake, despite the complex vortex interactions that occur downstream. The similarities between thrust force estimates based on wake momentum flux and those of an alternative, kinematically-based hydrodynamic model , indicating that inertial forces strongly influence the wake dynamics and jet formation. DPIV imaging allowed us to link the kinematics of bell motion to generation of rotational flows (One important contribution of this study is the documentation of vortex interactions that provide the fluid mechanical basis of thrust production by y Daniel  used kiny Daniel . Resultsal flows , jet proal flows , momentual flows , and, ulal flows B,C. The al flows  but, beiWake structure patterns demonstrate that momentum transfer from animal to surrounding fluids does not occur evenly through the wake. Instead, momentum in the wake is concentrated at interfaces between opposite-rotation vortices generated by the medusa . While tDirect measurement of momentum flux away from the swimmer’s body permits evaluation of both thrust force magnitude and direction. Because the VIA jets are strongly linearly directed, their orientation relative to the medusan central axis provides information about the resulting directionality of body motion, as well as its magnitude. For the swimming sequence used within this study, the VIA jet directions at either bell margin were directed slightly away from the medusa’s central axis, generating primarily straight swimming with a small deviation of body motion in the opposite direction of VIA jets . The abi