The organocatalytic transformation of resorcinols is extremely rare. In this article, we report a highly enantioselective, organocatalytic intramolecular cyclization of these systems by a Friedel–Crafts‐type 1,4‐addition using a Jørgensen‐Hayashi‐like organocatalyst with a large silyl protecting group, and show that heat improves reaction yield with virtually no detriment to enantioselectivity. A variety of bicyclic resorcinols were obtained with excellent enantioselectivities (up to 94%). To show the utility of these constructs, and as part of a wider project involving the synthesis of cannabinoid‐like compounds, the resorcinol formed was used to generate both ‘normal’ and ‘abnormal’ cannabidiol (CBD) derivatives which were shown to have anticonvulsant activity.  A variety of catalysts were screened (Table I (Entry 1), a bifunctional thiourea II (Entry 2), – a system with which we have much experience, a selection of chiral phosphoric acids III (Entry 3) and a range of secondary amines IV–X (Entries 4–10). Interestingly, the cinchona alkaloid derived primary amine I, used by Takao and co‐workers in the only other successful organocatalytic resorcinol derived Friedel−Crafts process known to date,VI and VIII did not work at all (Entry 3). In contrast, the MacMillan catalysts IX and X gave better yields but poorer enantioselectivities.In order to test our hypothesis, we accessed . This was confirmed insomuch that a screen of additives including acid co‐catalysts and bases did not significantly improve the reaction (see supporting information for complete study), leading us to conclude that such additives might be catalyzing the non‐enantioselective background process.In view of this, and because of its superior selectivity, we decided our best option was to optimize the yield of the conditions described in entry 10. To begin with, we performed a solvent screen Table  which shN‐substituted systems. All gave excellent enantioselectivities, including 8 c and 8 d, which can open the door to constrained δ‐amino acids which are of potential use within foldamer systems.8 k whilst suffering from poorer conversion gave a reasonable enantioselectivity. Substrates containing electron‐withdrawing substituents, unfortunately, failed to convert (see supporting information). However, post‐cyclization modification to introduce these is possible as demonstrated in compound 14 discussed vide infra.To expand the scope of this reaction, we explored the use of various substrates Table . Compoun8 a – see supporting information), we propose the transition state shown in figure E‐iminium ion blocks the approach of the resorcinol nucleophile from the re‐face. Cyclization via SNAr thus leads to the (S)‐tetrahydronaphthalene system reported.Based on the observed stereochemical outcome , and further confirmed the previously assigned absolute stereochemistry. Additionally, not only did this system allow us to obtain more crystalline material for XRD, but it also has two further clear advantages – first of course, that only the ‘normal’ regioisomer is obtained, and second that the aryl bromide can be derivatized to further systems via aryl coupling methodologies.In our first synthesis, removal of the primary alcohol of In conclusion, we have developed a highly enantioselective organocatalytic method for the synthesis of bicyclic resorcinols. Such transformations are rare, but we have exploited the nucleophilic character of the resorcinol towards an organocatalytic intramolecular Friedel−Crafts type 1,4‐addition. The key features of this reaction are that it requires a catalyst with a bulky silyl protecting group and can tolerate high temperatures. Furthermore, we have applied our reaction to the synthesis of cannabidiol analogues, a class of compound our group has had a long‐term interest in owing to their biological character and with the aim of developing new therapies towards the treatment of epilepsy and other conditions. The cannabinoid compounds within this report have been shown to have promising anticonvulsant activity and this will be the subject of a future communication elsewhere.General procedure for the organocatalytic cyclization of resorcinols. To a solution of (R)‐α,α‐Bis‐2‐pyrrolidinemethanol tert‐butyldimethylsilyl ether (0.2 equiv.) in CHCl3 (0.5 M) in a sealed tube was added the α,β‐unsaturated aldehyde (1 equiv.). The mixture was heated to 50 °C and stirred until consumption of starting which was monitored by NMR. Once the reaction was complete, the solution was cooled to 0 °C and reduced using NaBH4 (1.5 equiv.) and MeOH (0.2 M). After 20 min the reaction was quenched with saturated NH4Cl and distilled water. The solution was diluted with EtOAc and extracted three times. The combined organic layers were washed with brine and dried over MgSO4. The crude mixture was purified using silica gel on column chromatography.1H NMR  δ 6.30 , 6.24 , 3.81 , 3.77 , 3.73–3.62 , 3.11–3.08 , 2.80–2.66 , 1.88–1.63 ; 13C NMR  δ 158.45 (Ar), 158.00 (Ar), 138.75 (Ar), 122.28 (Ar), 104.88 (Ar), 96.33 (Ar), 61.81 (CH2OH), 55.52 (OMe), 55.38 (OMe), 38.61 (CH2CH2OH), 29.70 (Cy), 27.53 (Cy), 27.40 (Cy), 18.14 (Cy). [α]D20+28.0  IR (diamond) υ 3357, 3285, 2992, 2939, 2916, 2865, 2837, 1605, 1591,1067, 822 cm−1; HRMS (EI) Exact mass calculated for C14H20O3 [M+H]+ 237.1491; Found 237.1490; HPLC analysis: Daicel Chiralpak OD, hexane/iso‐propanol=99:1, flow rate=0.8 mL/min, λ=210 nmYield: 1.19 g, 68% (yellow oil). Purified using column conditions: Hex/EtOAc, 3:1. 1H NMR  δ 6.06 , 6.03 , 4.27–4.22 , 4.17–4.11 , 3.80 , 3.75–3.66 , 3.10–3.05 , 2.04–1.86 , 1.80 , 1.75–1.67 , 1.60  ppm; 13C NMR  δ 159.60 (Ar), 158.46 (Ar), 155.55 (Ar), 107.15 (Ar), 93.77 (Ar), 91.42 (Ar), 62.29 (Cy), 61.11 (CH2OH), 55.71 (OMe), 55.42 (OMe), 38.90 (CH2CH2OH), 26.72 (Cy), 24.30 (Cy). [α]D20+50.5 . m.p: 82.1–83.1 °C. HRMS (ES‐Tof) Exact mass calculated for C13H18O4 [M+H]+ 239.1283, Found 239.1272 IR (Diamond) υ 3387, 3303, 2982, 2958, 1269, 1612, 1588, 1051, 818, 810, 789 cm−1; HRMS (ES‐Tof) Exact mass calculated for C13H18O4 [M+H]+ 239.1283, Found 239.1272 HPLC analysis: Daicel Chiralpak OD, hexane/iso‐propanol=99:1, flow rate=1.0 mL/min, λ=210 nmYield: 0.073 g, 51% (orange solid). Purified using column conditions: Hex/EtOAc, 65:35. 1H NMR  δ 7.01 , 6.23 , 3.81 , 3.79 , 3.73–3.60 , 3.53–3.47 , 3.32–3.30 , 2.32 , 1.95–1.89 , 1.87–1.78 , 1.73–1.67 , 1.52  ppm; 13C NMR  δ 158.37 (Ar), 156.87 (Ar), 154.10 (C=O), 139.35 (Ar), 114.65 (Ar), 101.59 (Ar), 94.25 (Ar), 80.97 (C(CH3)3), 61.17 (Cy), 55.86 (OMe), 55.53 (OMe), 42.17 (CH2OH), 36.13 (Cy), 28.58 (CH3), 28.48 (CH2CH2OH), 26.02 (Cy). [α]D20−24.0  HRMS (ES‐Tof) IR (Diamond) υ 3512, 2938, 1689, 1609, 1204, 825, 744 cm−1 Exact mass calculated for C18H27NO5 [M+H]+ 338.1967, Found 338.1967 HPLC analysis: Daicel Chiralpak OD, hexane/iso‐propanol=96.5:3.5 flow rate=1.0 mL/min, λ=210 nmYield: 0.17 g, 77% . Purified using column conditions: Hex/EtOAc, 75:25. 1H NMR  δ 7.77 , 7.55 , 7.40 , 7.32–7.28 , 7.01 , 6.27 , 4.61–4.51 , 4.28 , 3.83 , 3.74 , 3.70–3.61 , 3.52–3.46 , 3.36–3.30 , 2.27 , 1.93–1.82 , 1.72–1.59 . 13C NMR  δ 158.62 (Ar), 156.95 (Ar), 154.93 (8‐C), 144.02 and 143.97 (2×rotamer Ar), 141.53 (Ar), 138.78 (Ar), 127.89 and 127.88 (2×rotamer Ar), 127.24 and 127.22 (2×rotamer Ar) 125.15 (2×rotamer Ar), 120.15 (Ar), 115.01 (Ar), 101.54 (Ar), 94.85 (Ar), 67.64 (OCH2CH), 61.13 (CH2OH), 55.54 (OMe), 55.55 (OMe), 47.45 (CH(Fmoc)), 42.25 (Cy), 36.11 (CH2CH2OH), 28.37 (Cy), 25.92 (Cy). [α]D20+6.5  m.p: 50.5–51.0 °C. IR (Diamond) υ 3457, 3001, 2968, 2943, 2866, 2827, 1700, 1696, 1608, 1492, 1002, 726 cm−1 HRMS (EI) Exact mass calculated for C28H29NO5 [M+H]+ 460.2124, Found 460.2118 HPLC analysis: Daicel Chiralpak AD‐H, hexane/iso‐propanol=92.5:7.5, flow rate=1.0 mL/min, λ=210 nmYield: 0.28 g, 69% (white solid). Purified using column conditions: Hex/EtOAc, 55:45. 1H NMR  δ 7.41–7.30 , 7.01 , 6.24 , 5.29 , 5.20 , 3.85–3.72 , 3.68 , 3.65–3.57 , 3.53–3.46 , 3.36–3.30 , 2.32–3.27 , 1.98–1.92 , 1.90–1.80 , 1.76–1.66  ppm; 13C NMR  δ 158.50 (Ar), 156.90 (Ar), 154.88 (C=O), 138.71 (Ar), 136.40 (Ar), 128.70 (Ar), 128.32 (Ar), 128.24 (Ar), 114.79 (Ar),101.16 (Ar), 94.82 (Ar), 67.72 (CH2Ph), 61.10 (CH2OH), 55.89 (OMe), 55.42 (OMe), 42.36 (Cy), 36.01 (CH2CH2OH), 28.27 (Cy), 25.91 (Cy). [α]D20 −21.0 . IR (Diamond) υ 3417, 2943, 2844, 1696, 1607, 1587, 1206, 826, 697 cm−1 HRMS (ES‐Tof) Exact mass calculated for C21H25NO5 [M+H]+ 372.1811, Found 372.1816 HPLC analysis: Daicel Chiralpak OD, hexane/iso‐propanol=94:6, flow rate=1.0 mL/min, λ=210 nmYield: 0.26 g, 78% . Purified using column conditions: Hex/EtOAc, 7:3. 1H NMR  δ 7.41–7.31 , 7.20 , 5.32 , 5.18  3.89 , 3.82–3.72 , 3.65–3.61 , 3.50–3.44 , 3.31–3.26 , 2.57 , 2.02–1.96 , 1.91–1.82 , 1.74 , 1.66–1.58  ppm; 13C NMR  δ 154.90 (C=O), 151.43 (Ar), 150.02 (Ar), 138.02 (Ar), 136.37 (Ar), 132.96 (Ar), 128.73 (Ar),128.39 (Ar), 128.27 (Ar), 119.51 (Ar), 104.44 (Ar), 67.75 (CH2Ph), 61.45 (OMe), 61.04 (OMe), 60.88 (CH2OH), 55.96 (OMe), 42.25 (Cy), 35.91 (CH2CH2OH), 28.31 (Cy), 26.78 (Cy). [α]D20 −14.5 . [M+H]+ 402.1917, Found 402.1908 IR (Diamond) υ 3480, 3011, 2941, 2866, 2827, 1696, 1685, 1207, 833 cm−1 HRMS (EI) Exact mass calculated for C22H27NO6 HPLC analysis: Daicel Chiralpak OD, hexane/iso‐propanol=96:4, flow rate=1.0 mL/min, λ=210 nmYield: 0.16 g, 46% (dark yellow oil). Purified using column conditions: Hex/EtOAc, 55:45. 2O, 9:1. 1H NMR  δ 7.49 , 7.19 , 6.28 , 3.94–3.89 , 3.83 , 3.78 , 3.54 , 3.25–3.12 , 3.08–3.02 , 2.42 , 2.07 , 1.75–1.60 , 1.27–1.22 , 0.80  ppm; 13C NMR  δ 158.75 (Ar), 157.27 (Ar), 143.88 (Ar), 137.69 (Ar), 135.58 (Ar), 129.62 (Ar), 127.27 (Ar), 114.82 (Ar), 101.20 (Ar), 95.61 (Ar), 60.72 (CH2OH), 55.87 (OMe), 55.65 (OMe), 43.44 (Cy), 36.61 (CH2CH2OH), 27.22 (Cy), 25.71 (Cy), 21.62 (ArCH3). [α]D20 +66.0 , IR (Diamond) υ 3557, 3386, 2941, 2882, 1607, 1585, 1127, 1120, 669 cm−1. HRMS (ES‐Tof) Exact mass calculated for C20H25 NO5S [M+H]+ 392.1532, Found 392.1531 HPLC analysis: Daicel Chiralpak OD, hexane/iso‐propanol=95.5:4.5, flow rate=1.0 mL/min, λ=210 nm. Amended conditions of hexane/iso‐propanol 95:5 were used for the racemic sample.Yield: 0.055 g, 45% . Purified using column conditions: Pentane/Et1H NMR  δ 6.42–6.32 , 4.14–3.96 , 3.84 , 2.78 , 3.63–3.57 , 3.50–3.35 , 2.21 , 2.05–1.96 , 1.93–1.81 , 1.72 , 1.64–1.56 . 13C NMR  δ 170.35 (C=O), 158.69 (Ar), 157.26 (Ar), 140.19 (Ar), 103.02 (Ar), 95.52 (Ar), 61.20 (CH2OH), 55.98 (OMe), 55.61 (OMe), 41.85 (Cy), 36.21 (CH2CH2OH), 29.11 (Cy), 26.39 (Cy), 23.54 (C(O)CH3). [α]D20 −105.0 . IR (Diamond) υ 3408, 2937, 2878, 2839, 1634, 1607, 1048, 827 cm−1 HRMS (EI) Exact mass calculated for C15H21NO4 [M+H]+ 280.1549, Found 280.1546 HPLC analysis: Daicel Chiralpak OD, hexane/iso‐propanol=92.5:7.5, flow rate=1.0 mL/min, λ=210 nmYield: 0.14 g, 54% (yellow oil). Purified using column conditions: Hex/EtOAc, gradient, 75:25 to 9:1. 1H NMR  δ 6.34 , 4.23–4.15 , 3.85 , 3.79 , 3.61–3.56 , 3.50–3.43 , 3.42–3.30 , 3.17 2), 2.24 , 1.99–1.85 , 1.74–1.58 , 1.25 , 1.01  ppm; 13C NMR  δ 177.72 (C=O), 158.69 (Ar), 157.48 (Ar), 140.41 (Ar), 117.36 (Ar), 102.52 (Ar), 95.61 (Ar), 61.21 (CH2OH), 56.00 (OMe), 55.58 (OMe), 41.90 (Cy), 36.43 (CH2CH2OH), 31.48 (CH(CH3)2), 29.43 (Cy), 26.55 (Cy), 20.52 (CH3), 19.92 (CH3). [α]D20 −124.0 , m.p: 60.2‐62.3 °C. HRMS (Es‐Tof) IR (Diamond) υ 3405, 2956, 2937, 2872, 1653, 1634, 1494, 1046, 821 cm−1 Exact mass calculated for C17H25NO4 [M+H]+ 308.1862, Found 308.1864 HPLC analysis: Daicel Chiralpak AD‐H, hexane/iso‐propanol=92.5:7.5, flow rate=1.0 mL/min, λ=210 nmYield: 0.18 g, 64% (Yellow solid). Purified using column conditions: Hex/EtOAc, 2:3. 1H NMR  δ 7.30–7.18 , 6.12 , 5.67 , 4.08–3.94 , 3.76–3.67 , 3.58–3.52 , 3.42–3.36 , 3.20 , 2.32 , 2.07–2.01 , 1.92–1.74 . 13C NMR  δ 170.44 (C=O), 158.00 (Ar), 157.06 (Ar), 139.92 (Ar), 136.69 (Ar), 130. 19 (Ar), 128.49 (Ar), 128.30 (Ar), 115.76 (Ar), 103.58 (Ar), 95.61 (Ar), 61.39 (CH2OH), 55.94 (OMe), 55.23 (OMe), 42.51 (Cy), 35.48 (CH2CH2OH), 31.04 (Cy), 26.69 (Cy). [α]D20 −99.5  m.p: 103–104.5 °C IR (Diamond) υ 3429, 2939, 2843, 1637, 1611, 1586, 1250, 1043, 825 cm−1 HRMS (Es‐Tof) Exact mass calculated for C20H23NO4 [M+H]+ 342.1705, HPLC analysis: Daicel Chiralpak AD‐H, hexane/iso‐propanol=9:1, flow rate=1.0 mL/min, λ=210 nmYield: 0.13 g, 65% (Yellow solid). Purified using column conditions: Hex/EtOAc, 65:35. 2O/CH2Cl2, 3.5:4:2.5. 1H NMR  δ 6.64 , 6.37 , 4.15–4.12 , 3.85–3.78 , 2.95 , 2.12–2.01 , 1.83–1.74 , 1.62 ; 13C NMR  δ 148.7 (Ar), 148.6 (Ar), 143.2 (Ar), 116.5 (Ar), 112.2 (Ar), 100.95 (Ar), 63.5 (CH2OAr), 60.6 (CH2OH), 56.7 (OMe), 56.0 (OMe), 39.5 (CH2CH2OH), 29.9 (CHAr), 27.4 (CH2CH2OAr) ppm; [α] D20 +36.00 , IR (diamond) υ 3443, 2989, 2956, 2862, 2833, 1619, 1054, 1449, 1371, 1262, 1218, 1054, 1034, 855 cm−1 HRMS (EI) Exact mass calculated for C13H18O4 [M+H]+ 239.1283, Found 239.1293Yield: 0.005 g, 2% (brown oil). Purified using column conditions: Hex/EtCCDC‐1881707 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/structures/As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


The target fluorinated compound 12b, as the most active one, showed better anticancer activity compared to the reference drug sorafenib, with IC50 values of 11.5, 11.6, and 13 µM against the HepG-2, A2780CP, and MDA-MB-231 cell lines, respectively. Furthermore, compound 12b (IC50 = 0.092 µM) had VEGFR-2-inhibitory activity comparable to that of the standard inhibitor sorafenib (IC50 = 0.049 µM). Furthermore, the ability of compound 12b in modulating MAPK signaling pathways was investigated. It was found to decrease the level of total ERK and its phosphorylated form, as well as leading to the down-regulation of metalloproteinase MMP-9 and the over-expression of p21 and p27, thus leading to subG1 cell-cycle arrest and, thus, the induction of apoptosis. Additionally, compound 12b decreased the rate of wound healing in the absence of serum, in comparison to DMSO-treated cells, providing a significant impact on metastasis inhibition. The quantitative RT-PCR results for E-cadherin and N-cadherin showed lower expression of the neuronal N-cadherin and increased expression of epithelial E-cadherin, indicating the ability of 12b to suppress metastasis. Furthermore, 12b-treated HepG2 cells expressed a low level of anti-apoptotic BCL-2 and over-expressed proapoptotic Bax genes, respectively. Using the DAF-FM DA fluorescence probe, compound 12b produced NO intracellularly as efficiently as the reference drug JS-K. In silico molecular docking studies showed a structural similarity through an overlay of 12b with sorafenib. Interestingly, the drug-likeness properties of compound 12b met the expectations of Pfizer’s rule for the design of new drug candidates. Therefore, this study presents a novel anticancer lead compound that is worthy of further investigation and activity improvement.Over the past few decades, the development of broad-spectrum anticancer agents with anti-angiogenic activity has witnessed considerable progress. In this study, a new series of pyrazolopyrimidines based on a phenylfuroxan scaffold were designed, synthesized, and evaluated, in terms of their anticancer activities. NCI-60 cell one-dose screening revealed that compounds  Cancer is a large group of diseases, and is the result of the uncontrollable growth of abnormal cells . In 2018Vascular endothelial growth factor (VEGF) expression, through the expression of the cell-surface kinase VEGFR-2 (KDR/Flk-1), is strongly associated with both angiogenesis and tumor aggressiveness ,8. KDR iSunitinib, sorafenib, and cabozantinib were recently shown to be among the anti-angiogenic drugs that can inhibit VEGFR-2, and which are active against a vast array of cancer types . Their aN-oxide may serve as promising scaffolds for the design of a new class of anti-angiogenic agents through improved bioavailability and enhanced biological activity pyrimidine and 1,2,5-oxadiazole-activity ,21,22. Tvity pyrimidine derivatives exhibit potent anti-tumor activities ,30,31,32On the other hand, the nitric oxide (NO)-releasing furoxan moiety was shown to have potent anti-tumor activity in earlier reports ,22. It iA convenient way to achieve high levels of intracellular NO is through the use of NO donors . Among tEnlightened by these findings, we were inspired to design and synthesize a new series of pyrazolopyrimidines tethered to a NO-releasing furoxan moiety through a central aryl bridge. In this context, as shown in 3a–d. As depicted in 2a–h were prepared from alkylidyne malononitrile, through a multi-step reaction, with reasonable yields pyrimidine derivatives were synthesized as depicted in ves 8a–c . The 4-cchloride . The NO-o of 1:6 . Finallyves 9a–c .14a–c were obtained through the series of chemical reactions depicted in 13a–c were synthesized through intermolecular cyclization of the 5-amino-4cyano intermediates 7a–c, using chloroacetic acid and a catalytic amount of phosphorus oxychloride pyrimidine hybrids reported herein. A summary, based on the above discussion, of the preliminary structure–activity relationships (SARs) observed in this study is provided in The methyl substitution (EDG) at C3 and C6 , breast (MDA-MB-231 and MCF-7), Colon (HT-29), and ovarian  cancer cell lines. In order to determine the concentration that killed 50% of cells, serial dilutions were used in the treatment, and the determination of percentage of viability identified that 12b was the most active compound. The IC50 values are shown in 12b showed better anticancer activity, with IC50 values of 11.5, 11.6, and 13 µM against HepG-2, A2780CP, and MDA-MB-231 cell lines, respectively, while the IC50 for SKOV-3 was comparable to that of the reference drug sorafenib. In addition, the morphological changes in 12b-treated cancer cell lines at concentrations of 6.25 and 12.50 µM are shown in 15a and b were tested, but did not inhibit the proliferation of cancer cells at concentrations as high as 50 µM, indicating the significance of the phenylfuroxan moiety in the anticancer activity of 12b.The cell growth-inhibitory activities of the most active compound r HepG-2 ,53,54, or HepG-2 ,56, brea12b towards the various cancer cell lines was evaluated by detecting the required concentration to kill 50% of normal human skin fibroblast cells (HSF). The results revealed that compound 12b did not cause any morphological changes in HSF cells at a concentration as high as 50 µM, indicating its safety and selectivity towards cancer cells, rather than normal cells.Furthermore, the selectivity index (SI) of compound 12b) was evaluated for its VEGFRx  kinase inhibitory activities, using a VEGFR inhibitory assay with sorafenib as a reference drug. As shown in 12b possesses potency that is comparable to that of sorafenib, displaying noticeable selectivity against VEGFR-2 with an IC50 of 0.09 µM.The fluorinated pyrazolopyrimidine derivative based on the 1,2,5-oxadiazole-2-oxide scaffold , leading to the induction of apoptotic pathways , with a detection limit of ~3 nM. DAF-FM diacetate penetrates the cells and is deacetylated by esterase enzyme to yield a non-fluorescent dye, DAF-FM, which, upon reaction with NO, produces a fluorescent benzotriazole product , close to that of sorafenib (1.41 − log10[(mg/L)/(1000 × MW)]). Furthermore, the environmental toxicity rule for Tetrahymena pyriformis 50% growth inhibition concentration (IGC50) showed a higher value of 5.086 − log10[(mg/L)/(1000 × MW)], compared to that of sorafenib (4.478 − log10[(mg/L)/(1000 × MW)]). Overall, these findings shed light on the ADMET features of 12b, an intriguing target molecule.Prediction of the physicochemical, pharmacokinetic, and drug-likeness properties of compound Tlab 2.0  which ofTlab 2.0 . As showorafenib . In addi1H and 13C NMR spectra were obtained using a Bruker AVANCE and Varian Unity INOVA-400 MHz NMR spectrometer in DMSO-6d using solvent residual peaks as an internal standard; CDCl3 (δ = 7.27 ppm) and DMSO-6d (δ = 2.50 ppm) for 1H NMR and CDCl3 (δ = 77.23 ppm) and DMSO-6d (δ = 39.51 ppm) for 13C NMR. The signal multiplicities were reported as s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), or br (broad) and the J-coupling constants were in Hz. High-resolution mass spectra (HRESI-MS) were acquired on a Thermo Instruments MS system (LTQ XL/LTQ Orbitrap Discovery) linked to a Thermo Instruments HPLC system . Thin-layer chromatography (TLC) was performed for reaction monitoring on pre-coated silica gel 60 F254 (Merck) sheets and visualized by ultraviolet light (254 nm). Purification of synthesized compounds was achieved via either column chromatography using 150–250 μm silica gel or recrystallization from an appropriate solvent. Solvents and reagents were acquired from commercially available suppliers.2a–h 2a–h +: C20H17N5O: 344.1506; found: 344.1497.White powder; 65% yield; 1H NMR  δ = 9.96 , 8.45 , 8.20 , 7.71 , 7.55 , 7.34 , 7.01 , 6.11–6.02 , 5.42 , 5.27 , 4.58 , 2.53 . HRESI-MS m/z calcd for [M+H]+: C21H19N5O: 358.1662; found: 368.1655.White powder; 70% yield; 1H NMR  δ = 8.73 , 8.36 , 8.18 , 7.55–7.50 , 7.32 , 6.99 , 6.11–6.01 , 5.41 , 5.27 , 4.58 , 2.75 . 13C NMR  δ 14.81 (CH3), 68.38 (OCH2), 101.13 (C3a), 114.48 (C-Ar), 117.40 (OCH2CH=CH2), 120.51 (C-Ar), 125.57 (C-Ar), 125.84 (C-Ar), 129.10 (C-Ar), 131.37 (C-Ar), 133.81 (OCH2CH=CH2), 138.72 (C-Ar), 142.41 (C-Ar), 154.09 (C3), 155.27 (C7a), 155.85 (C4), 156.02 (C6).White powder; 78% yield; 1H NMR  δ = 8.54 , 8.19 , 7.59 , 7.51 , 7.29 , 6.97 , 6.11–6.01 , 5.41 , 5.27 , 4.57 , 2.70 , 2.46 . 13C NMR  δ 14.77 (3-CH3), 26.21 (6-CH3), 68.34 (OCH2), 99.25 (C3a), 114.39 (C-Ar), 117.34 (OCH2CH=CH2), 120.39 (C-Ar), 124.85 (C-Ar), 125.53 (C-Ar), 128.98 (C-Ar), 131.74 (C-Ar), 133.83 (OCH2CH=CH2), 138.93 (C-Ar), 142.09 (C-Ar), 154.92 (C3), 155.21 (C7a), 155.29 (C4), 165.22 (C6).White powder; 75% yield; 3a–d (0.015 mol) in glacial acetic acid (3 mL), saturated aqueous NaNO2 (0.045 mol) solution was added dropwise so that the temperature did not exceed 70 °C +: C20H16N6O3: 389.1357; found: 389.1347.Orange crystals; 55% yield; 1H NMR  δ = 10.48 , 8.32 , 8.18 , 7.58–7.53 , 7.40–7.32 , 7.34 , 7.01 , 6.11–6.03 , 5.45 , 5.32 , 4.71 , 2.39 . 13C NMR  δ 25.79 (6-CH3), 69.07 (OCH2), 100.31 (C3a), 110.30 (C-Ar), 118.06 (OCH2CH=CH2), 120.63 (C-Ar), 120.84 (C-Ar), 126.26 (C-Ar), 128.40 (C-Ar), 129.15 (C-Ar), 133.04 (C-Ar), 133.45 (OCH2CH=CH2), 138.81 (C-Ar), 144.88 (C-Ar), 153.98 (C3), 154.26 (C7a), 155.53 (C4), 165.08 (C6); HRESI-MS m/z calcd for [M+H]+: C21H18N6O3: 403.1513; found: 403.1503.Orange crystals; 60% yield; 1H NMR  δ = 9.59 , 8.40 , 8.20–8.15 , 7.67–7.32 , 6.07–6.04 , 5.44 , 5.31 , 4.71 , 2.79 . HRESI-MS m/z calcd for [M+H]+: C21H18N6O3: 403.1513; found: 403.1502.Orange crystals; 58% yield; 1H NMR  δ = 9.59 , 8.28–8.14 , 7.62–7.28 , 6.07–6.02 , 5.43 , 5.30 , 4.67 , 2.72 , 2.44 . HRESI-MS m/z calcd for [M+H]+: C22H20N6O3: 417.1670; found: 417.1659.Orange crystals; 53% yield; 2a–h (0.002 mol) in THF (15 mL), furoxan mesylate 10  and NaOH  were added and the mixture was heated at reflux for 2 h (TLC monitoring) pyrimidines based on a phenylfuroxan scaffold were detailed. One-dose screening of the NCI-60 cell revealed that compounds


Coronavirus disease 2019 (COVID-19) as a fatal epidemic has swept across the world, especially in India where the epidemic situation is the most serious. For COVID-19 patients, pulmonary rehabilitation training plays a significant role. However, it is still a controversial issue regarding the efficacy of WeChat APP-based pulmonary rehabilitation training in improving lung function, quality of life and bad mood of COVID-19 patients. To clarify this issue, a meta-analysis was conducted in this present study, so as to provide a basis for rehabilitation guidance of COVID-19 patients.We systematically searched PubMed, medRxiv, Web of Science, Scopus, Chinese Science Citation Database, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, Chinese Scientific Journal Database, and Wan-fang databases in May 2021 to identify randomized controlled trials and evaluate the effects of WeChat APP-based pulmonary rehabilitation training for COVID-19. Two researchers independently carried out data extraction. On the other hand, literature quality evaluation on the quality and meta-analysis of the included literature was performed with Revman5.3 software.The results of this meta-analysis will be submitted to a peer-reviewed journal for publication.This study will provide reliable evidence-based evidence on the effects of WeChat APP-based pulmonary rehabilitation training on lung function, bad mood, and quality of life in patients with COVID-19.Ethical approval was not required for this study. The systematic review will be published in a peer-reviewed journal, presented at conferences, and shared on social media platforms.DOI 10.17605/OSF.IO/MKXCH. Generally, the home lung rehabilitation training lasts for 8 to 12 weeks. However, to ensure the effectiveness of pulmonary rehabilitation, patients need long-term maintenance training. Pulmonary rehabilitation training has many limitations, including insufficient resources for pulmonary rehabilitation, low proportion of medical insurance allocation, and insufficient professional health care providers. Furthermore, other factors such as transportation, population movement, distance, and training location also prevent patients from requesting, participating in, and adhering to pulmonary rehabilitation training.Pulmonary rehabilitation training is a comprehensive rehabilitation with exercise as the core, which is mainly aimed at patients with lung diseases.–10 In recent years, WeChat has been applied in the continuous care of patients after discharge, thus strengthening doctor-patient communication and improving the compliance of treatment after discharge.–14Given that patients with COVID-19 need to be treated in isolation or isolated at home, it is particularly important to take appropriate guidance methods to train patients in lung rehabilitation. With the popularity of electronic products, mobile health management has been gradually applied. WeChat is a mobile Internet technology communication application launched by Tencent, and it can send text, voice, video and pictures over the network.There is not enough evidence to support that WeChat APP-based pulmonary rehabilitation training can improve lung function, adverse mood and quality of life in COVID-19 patients. Therefore, this study aimed to evaluate the effects of WeChat APP-based lung rehabilitation training on lung function, adverse mood and quality of life in COVID-19 patients, and to provide reference for non-drug intervention in COVID-19 patients.22.1The systematic review protocol has been registered in open science framework, and the registration number is DOI 10.17605/OSF.IO/MKXCH. The consent of this protocol report is based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMAP) Statement Guidelines.2.2Articles related to the pulmonary rehabilitation training based on WeChat App on COVID-19 patients will be included. All articles included are randomized controlled trial (RCT).2.2.1Patients with confirmed diagnosis of COVID-19.2.2.2Patients in the control group were given routine treatment, while patients in the experimental group accepted the pulmonary rehabilitation training based on WeChat App on the basis of routine treatment.2.2.31.Anxiety, Self-Rating Anxiety Scale or Hamilton Anxiety Scale;2.Depression, Self-rating depression scale or Hamilton Depression Scale;3.Life quality, related quality of life scales;4.Lung function, forced vital capacity (FVC), and forced expiratory volume in one second (FEV1) and FEV1/FVC.2.3We conducted a systematic search of databases  up to May 2021, using key terms. Besides, reference lists of relevant studies were identified. These search terms are summarized in Table 2.42.4.1The database search results will be imported into Endnote to delete duplicates, and then titles and abstracts will be screened to exclude references that are irrelevant to this study. Fully-text articles requiring further study will be evaluated using inclusion criteria. Two reviewers will screen the references independently and a third independent reviewer will be involved to resolve the differences in the screening process. The process of the selection is displayed in Figure 2.4.2Data extraction was conducted by 2 reviewers using a standardized data extraction form. The following data were extracted: first author, year of publication, number of patients, country, age, type, content and duration of life intervention, outcomes, result measurement data, intervention time and details, etc.2.4.3 Each item will be evaluated as “high,” “low,” or “unclear”.The quality of randomized controlled trials included was evaluated via Cochrane Handbook which is composed of 6 items: sequence generation, allocation concealment, blinding of participants, personnel and outcome assessors, incomplete outcome data, no selective outcome reporting and other sources of bias.2.4.4Standardized mean difference (SMD) is applied to measure the efficacy of 95% CI.2.4.5If there are insufficient or missing data in the literature, the authors will be contacted via email. If the data are still not available, only the current available data will be analyzed and the potential impacts will be discussed.2.4.62 test (the test level was α=0.1) and I2 to quantitatively determine the size of the heterogeneity. When P < 0.1 and/or I2 > 50%, the random effect model will be adopted for the combined analysis. Otherwise, the fixed effect model will be applied for the combined analysis.The heterogeneity included in the results of the study was analyzed by carrying out the χ2.4.7Publication bias was detected by funnel plots if enough articles (>10) were adopted for an outcome.2.4.8We will apply RevMan 5.3  for data analysis and quantitative data synthesis. If there are no findings of statistical heterogeneity, the fixed-effect model will be adopted for data synthesis. If there is significant statistical heterogeneity, the random effect model will be used.2.4.9A subgroup analysis will be conducted on the basis of intervention time (<8 weeks or ≥8 weeks).2.4.10We performed sensitivity analyses by re-estimating the pooled effects with fixed-effect or random-effect model.2.4.11The content of this article does not involve moral approval or ethical review and will be presented in print or at relevant conferences.3,18,19 However, to our knowledge, no meta-analysis has been conducted to evaluate the impacts of WeChat APP-based pulmonary rehabilitation training on patients with COVID19. Therefore, in order to provide new evidence-based medical evidence for rehabilitation guidance in COVID19 patients, we conducted a systematic review and meta-analysis to evaluate the impacts of WeChat APP-based pulmonary rehabilitation training on lung function, adverse mood, and quality of life in COVID19 patients. The results of this meta-analysis will provide evidence-based guidance for clinical pulmonary rehabilitation.Studies have revealed that WeChat APP-based pulmonary rehabilitation training can effectively promote lung rehabilitation, relieve adverse emotions and improve the quality of life.Conceptualization: Aihong Xu, Lili Ding.Data curation: Lili Ding, Zhiyu Xu.Formal analysis: Zhiyu Xu.Funding acquisition: Aihong Xu.Investigation: Zhiyu Xu.Methodology: Haiping Li.Project administration: Aihong Xu.Resources: Zhongyan Zhao.Software: Zhongyan Zhao.Supervision: Aihong Xu.Validation: Zhongyan Zhao, Haiping Li.Visualization: Haiping Li.Writing – original draft: Aihong Xu, Lili Ding.Writing – review & editing: Aihong Xu, Lili Ding.


The species included are from two Rhopalonematidae clades; they are Colobonema sericeum, Pantachogon haeckeli, Crossota millsae and two species of Benthocodon. Comparison is made with Aglantha digitale, a species from a third Rhopalonematidae clade brought to the surface by natural water movement. We find that although all Rhopalonematidae appear to have two swimming modes, there are marked differences in their neural anatomy, kinematics and physiology. Giant motor axons, known to conduct impulses during fast swimming in A. digitale, are absent from C. sericeum and P. haeckeli. Slow swimming is also different; in C. sericeum and its relatives it is driven by contractions restricted to the base of the bell, whereas in A. digitale it is driven by contractions in the mid-bell region. These behavioural differences are related to the position of the different clades on a ribosomal DNA-based phylogenetic tree. This finding allows us to pinpoint the phylogenetic branch point leading to the appearance of giant motor axons and escape swimming. They place the remarkable dual swimming behaviour of members of the Rhopalonematidae family into an evolutionary context.Although members of the Rhopalonematidae family  are known to exhibit unusually powerful jet swimming in addition to their more normal slow swimming behaviour, for the most part, reports are rare and anecdotal. Many species are found globally at depths of 600–2000 m, and so observation and collection depend on using remotely operated submersible vehicles. With a combination of  Summary: 18S ribosomal DNA data support anatomical, kinematic and electrophysiological evidence that identifies the phylogenetic branch point giving rise to giant-axon-based fast and slow swimming in the Rhopalonematidae. The phylum Cnidaria contains a diverse collection of life forms exhibiting a wide variety of behaviours. Some adult forms, such as the hydroids, sea anemones and corals, are sessile or remain so for long periods. By contrast, the Medusozoa are either fully motile or have a motile stage in their life cycle. Four classes of jellyfish make up the Medusozoa: the Scyphozoa, the Cubozoa, the Staurozoa and the Hydrozoa. Included in the Hydrozoa are several orders of jellyfish such as the Trachymedusae. Here, we examine swimming in a specific trachymedusan family, the Rhopalonematidae. This family is unique among jellyfish in having a dual system of swimming.Aglantha digitale as being of particular note because it gives “a single powerful contraction in response to a probe, which is considerably more effective than the normal type”. He suggested that this powerful contraction may be a means by which Aglantha can escape from a predator. In Aglantha, this substitutes for the slow ‘crumpling’ response of most hydromedusae to any form of threat (Aglantha by five body lengths with a maximum velocity of ∼40 cm s−1 (−1 (Gonionemus vertens) to 2.5 cm s−1 (Bougainvillia multitentaculata). The exceptional maximum velocity of A. digitale is associated with the presence of two sets of giant axons, which are responsible for conducting excitation around and up the animal so that the entire musculature contracts within 100 ms. This violent contraction means that the base of the bell-shaped body forms a nozzle through which the bell's water contents are expelled at high velocity . During a single slow swim, the animal moves forward ∼15 mm, about one body length  the Aglantha, Aglaura and Amphogona subclade ; and (3) the Colobonema, Pantachogon, Rhopalonema subclade .The Trachymedusae, together with three other hydrozoan orders – the Narcomedusae, the Limnomedusae and the Actinulida, are sometimes classed together as the Trachylina. This broad classification, based largely on anatomical and developmental considerations, has required fine-tuning as a consequence of recent DNA analysis . Such a Aglaura hemistoma and Amphogona apicata, have been observed to perform two distinct modes of swimming like A. digitale ”. In a single contraction, the initially motionless medusa (∼1 cm high) shoots ∼10 cm across the container. Although it is tempting to assume that all of these fast swims represent escape behaviour, this is by no means certain because it is not known whether they are elicited by specific external stimuli. Here, we find that freshly captured specimens of Pantachogon haeckeli and Colobonema sericeum are highly vibration sensitive and that, unlike the Aglaurinae, their fast swims do not depend on giant axons. We find that members of the Rhopaloneminae and Crossota clades are also capable of slow swimming, but that, unlike Aglantha, these contractions are confined to the bell margin. Thus, although all three Rhopalonematidae clades exhibit dual swimming, the myoneural basis is different. These findings allow us to pinpoint the phylogenetic branch point leading to these differences in organization.Members of the other Rhopalonematidae clades are less well studied because they occur at depths of 200–3000 m and are rarely brought to the surface. Nevertheless, it has been known anecdotally for some years that they exhibit unusually powerful swims. In b bench” . AccordiPrevious examinations of independently evolved behaviours within a clade provide important insights into their neural basis. Swimming has evolved multiple times in the nudipleura molluscs, for example. In some species, the swimming pattern is generated by entirely different sets of neurons; in others, homologous neurons play similar roles . Here, wAglantha digitale (O. F. Müller 1776) specimens were collected from surface water at the dock of Friday Harbor Laboratory, Friday Harbor, WA, USA. They were transferred to a narrow transparent chamber filled with natural seawater at 10°C. Swimming, which was confined to a narrow focal plane, was video recorded against a black background at 300 frames s−1. Escape swims were elicited with a glass probe.Colobonema sericeum E. Vanhöffen 1902 and a single Pantachogon haeckeli O. Maas 1893. In the laboratory, the animals were bathed with natural seawater collected at the appropriate depth and were kept in the dark at 6°C.Specimens of other Rhopalonematidae clades were collected from sites within Monterey Bay, CA, USA, at depths and temperatures that ranged from 200 to 600 m and from 6 to 7°C. The remotely operated submersible vehicle (ROV) Ventana lowered from the Monterey Bay Aquarium Research Institute (MBARI) vessel R/V Rachel Carson and equipped with a multiple-bin suction sampler  was usedColobonema and Crossota millsae E. V. Thuesen 2003 in their natural environment were filmed in situ with on-board cameras. For more details, see Benthocodon hyalinus (Benthocodon pedunculatus (H. B. Bigelow 1913) were measured from the supplementary material provided in Swimming behaviour of specimens of hyalinus  were obtColobonema or Pantachogon can swim normally in the laboratory, despite experiencing the large pressure change upon being brought to the ocean surface (up to 6.2 MPa), specimens were filmed on a cooled microscope stage (11–16°C). In the case of Pantachogon, the whole animal was small enough to be tethered and stimulated with a bipolar electrode. Colobonema specimens were too large to be treated in this way. However, the bell is made up of octants of muscle epithelium (myoepithelium) outlined by the eight radial canals that run from the margin to the bell apex. Sections of body wall consisting of an octant and its associated radial canals were pinned out in a Sylgard-coated dish, subumbrella side upward, using either steel pins or spines from the cactus Opuntia. In each case, a bipolar stimulating electrode was placed so as to lightly touch the myoepithelium without damaging it, and a 2 ms electrical stimulus was adjusted to be just supra-threshold.To discover whether Video recordings were converted to a series of sequential frames. Bell dimensions were measured using the image-analysis program ImageJ see  to proviSequences from 18S ribosomal DNA were obtained in the laboratory or via GenBank . They weC. sericeum (Movie 1), which suggests that it is normally a relatively rare occurrence. Although we have not found the released tentacles to be luminescent, they may nonetheless function to distract and discourage potential pursuers . It consists of six non-consecutive frames 60–120 ms apart. The time to maximum contraction varied from 120 to 180 ms, with the first of the series of swims being stronger and taking longer to reach its peak. Subsequent contractions often occurred before the bell had fully refilled and the time to peak was shorter. The contraction during a single strong swim, estimated from the change in bell diameter, is plotted in Aglantha digitale escape swim for comparison (filled blue circles). The immediate forward movement was about two body lengths, which is much the same as in A. digitale  and there is little or no contraction in the mid-bell region . In frame 1, the animal is seen resting with its tentacles spread ready to trap prey. Tentacle retraction starts as soon the reduction in bell diameter becomes measurable so that by frame 2 the average tentacle length is reduced by 25% (N=5). Tentacle withdrawal continues throughout the bell contraction and into the refilling phase. Note, however, that bell contraction brings relatively little forward movement, as is revealed by comparing the silhouettes of the bell margin taken from frames 1 and 5 . In the specimen shown in In The bell diameter was measured at a position mid-way up the bell, normalized to its resting value and plotted in Crossota millsae, like other Trachymedusae, is able to perform both fast and slow swims. It resembles Colobonema  in Monterey Bay. Towards the end of the sequence, one specimen initiates a slow swim, and this is immediately followed by a fast swim in a close neighbour. The animal travels about seven body lengths with just a single fast swim . Relaxation of the margin is faster than the mid-bell and the animal becomes distinctly cone shaped. B. hyalinus undergoing a slow swim. As with Colobonema, the bell contraction is only half that seen during an escape swim and it is confined to the basal region  confirms that R. krampi is shaped like A. digitale (15 mm high by 10 mm wide) and has a powerful swim. However Kramp's comment regarding the missing “characteristic bands along the radial canals” in R. krampi can only be referring to the giant axons that are such an obvious feature of the subumbrella in Aglantha. Thus, Ransonia has affinities with both Colobonema and Aglantha in that it is Aglantha shaped but may be Colobonema-like in neural circuitry. Unfortunately, there are no data for R. krampi in GenBank.Another species worthy of comment is A. digitale is not directly excitable and does not generate propagating action potentials. In this respect, it is unlike the myoid conduction seen in other hydromedusae, such as Polyorchis . This would allow pacemaker-driven slow swims to operate under the threshold for more general excitation. On the other hand, the different strengths of swim may reflect two different nerve inputs, one of them being limited to the marginal area. In this case, the subumbrella nerve plexus concerned may have evolved into the more organized system of giant axons and lateral nerves present in A. digitale . This would explain why the non-contracting half of the bell can start to contract so long after the start of the swim, once the inhibition has declined. In some other hydromedusae, such as Polyorchis, turning stems largely from an asymmetrical contraction of the velum. According to Colobonema, asymmetrical contractions of the velum also contribute to turning. Gonionemus, ‘are considerably more effective than the method relying strictly on velar action, which occurs in Polyorchis, for the turning radius …. is much smaller’.Another characteristic of slow swimming in A. digitale, the contraction phase of a slow swim lasts for ∼150 ms, each swim arising from a single pacemaker synaptic potential lasting no longer than 60 ms (Colobonema, the contraction builds over a course of ∼250 ms and may be the result of a series of summated events as it is in Aequoria victoria (Colobonema and Aglantha are similar in that only a limited region of the bell contracts; but, in Aglantha, it is a region mid-way up the bell (Colobonema, the contraction is restricted to the margin. The myoneural basis of slow swimming in Aglantha involves a low-threshold propagating Ca2+ spike, but the spike changes in shape as it travels towards the bell apex and provokes stronger contractions in the myoepithelium as it does so (In an 60 ms . In Colovictoria ; 2008. Sthe bell , whereas does so . The det


We found that ubiquitous foxo overexpression (OE) accelerated aging, induced the early onset of age-related phenotypes, increased sensitivity to thermal stress, and deregulated metabolic and proteostatic pathways; these phenotypes were more intense in transgenic flies expressing high levels of foxo. Interestingly, there is a defined dosage of foxo OE in muscles and cardiomyocytes that shifts energy resources into longevity pathways and thus ameliorates not only tissue but also organismal age-related defects. Further, we found that foxo OE stimulates in an Nrf2/cncC dependent-manner, counteracting proteostatic pathways, e.g., the ubiquitin-proteasome pathway, which is central in ameliorating the aberrant foxo OE-mediated toxicity. These findings highlight the differential dose- and tissue-dependent effects of foxo on aging, metabolic and proteostatic pathways, along with the foxo-Nrf2/cncC functional crosstalk.Aging is the gradual deterioration of physiological functions that culminates in death. Several studies across a wide range of model organisms have revealed the involvement of FOXO  transcription factors in orchestrating metabolic homeostasis, as well as in regulating longevity. To study possible dose- or tissue-dependent effects of sustained  Aging is a complex phenomenon caused by the time-dependent loss of cellular homeodynamics and, consequently, physiological organismal functions. The development of age-related phenotypes is a complex stochastic process characterized by the numerous evolutionary conserved hallmarks of aging, which (among others) include loss of energy balance and disruption of proteome homeostasis (or proteostasis) ,2. One oCaenorhabditis elegans and Drosophila melanogaster possess one FOXO gene , whereas, in mammals, there are four members  that regulate gene expression in a tissue-specific pattern  and foxoL [UAS foxo (BDSC 42221)] lines, along with flies expressing the muscle-specific (high transgene expression) Mef2Gal4 (BDSC 27390) driver were obtained from the Bloomington Drosophila Stock Center . The muscle-specific (weak expression) driver MhcGal4 was kindly provided by Dr. Orso . The cncC knockdown (UAS cncCRNAi), the cncC overexpressing (UAS cncC) line and the TubGal4 GeneSwitch  driver were a gift from Prof. Bohmann ; the conditional driver TubGal4 is ubiquitously activated upon dietary administration of 320 μΜ RU486 . Cardiomyocyte-specific tinC.Δ4Gal4 driver was donated by Prof. Frasch .d medium . Wild-ty2 anesthesia 24 h before the experiment, and an equal number of female and male flies were used per assay, unless otherwise stated. As described before, gonads display a distinct vs. somatic tissues aging rate and regulation of major proteostatic components , Akt1 , psggser21/9  and GABARAP (#13733S) were from Cell Signaling Technology, Inc. . Anti-ATP5a antibody  was purchased from Abcam ; anti-sgg antibody  was from Millipore , the anti-InR antibody was kindly provided by Prof. Ernst Hafen  and the antibody against Gapdh (#G9545) was from Sigma-Aldrich . Secondary antibodies, i.e., Alexa Fluor® 647 AffiniPure Donkey Anti-Rabbit IgG (#711-605-152) and Alexa Fluor® 647 AffiniPure Donkey Anti-Mouse IgG (#715-605-151) were from Jackson ImmunoResearch Europe Ltd. . Fluorescent dyes, Bodipy (#D3922), Phalloidin (#R415), and DAPI (#D1306) were purchased by Molecular Probes™ .The anti-dFOXO (#CAC-THU-A-DFOXO) antibody was purchased from Cosmo Bio Ltd. . The antibody against the Prosβ5 proteasome subunit was kindly provided by Maria Figueiredo-Pereira , and the antibody against PSMD11  was from Novus Biologicals . Primary antibodies against 20S-(α) , Rpn10  proteasome subunits and ubiquitin  were purchased by Santa Cruz Biotechnology, Inc. . Antibodies against t-test analysis or a two-way ANOVA test followed by Tukey’s multiple comparisons test. For graphical representation of data and statistical analyses, MS Excel , the Statistical Package for Social Sciences , and GraphPad Prism 8.00  were used. Significance was accepted at * p < 0.05 (shown in graphs by one asterisk); two asterisks denote ** p < 0.01.The presented experiments were analyzed at least in triplicates unless otherwise indicated. Data points correspond to the means of the independent experiments; error bars denote standard deviation (SD), and differences between compared groups were evaluated using the independent (unpaired) foxo in longevity and proteostasis, we sought to investigate the effects of inducible ubiquitous  foxo OE in Drosophila flies. We used two Drosophila lines, i.e., stock No 9575 (hereafter referred to as foxoH) and stock No 44,214 (hereafter referred to as foxoL) . Specifically, foxoH transgenic flies express significantly higher foxo mRNA and foxo protein levels as compared to foxoL flies  , which eoL flies –d, and tfoxoH and foxoL transgenic lines after inducible ubiquitous  foxo OE. Longevity assays revealed that foxo OE accelerated aging dose-dependently since foxoH flies had a ~80% reduction (vs. control) of their median lifespan, whereas foxoL showed a ~40% reduction in their median lifespan  in foxoH flies  susceptibility  of flies to both cold and heat  stress (foxoH line was more sensitive to either cold or heat stress.To further investigate the effects of ) stress ; again, Taken together, ubiquitous foxo overactivation induces (dose-dependently) the early onset of aging phenotypes and saturates the buffering capacity of stress response pathways.foxo OE on mitostatic and bioenergetic pathways. foxo expression levels did not significantly affect mitochondria structure in the flight muscle of young transgenic flies , dynamics , and energetics (blw) genes , Ilp2 inhibitory ecdysone (ImpL2), insulin receptor (InR), Akt1, and the gluconeogenesis related  genes; notably, the glycogen phosphorylase (GlyP) and glycogen synthase (GlyS) genes are only upregulated in the foxoL transgenic line (Ser505) and total Akt1; the inhibitory form of sgg (phosphorylated sggSer21/9) and total sgg, revealed the activation of the IIS pathway in foxoH flies and a trend towards activation in foxoL flies (foxo OE triggers (among others) a counteractive response which via activation of the IIS pathway aims to suppress its transcriptional activity [Since foxo has also been functionally implicated in energy regulation and metabolism ,13, we tic flies a; while w) genes b. Interenic line c. ConsisoL flies . Thus, factivity .foxoL flies (foxo OE suppressed (in both lines) GLU levels in somatic tissues and hemolymph, while glycogen (GLY) levels were found to decrease (likely due to increased breakdown) in foxoH and increase in foxoL transgenic flies  , a principal circulating sugar in hemolymph that serves as an efficient flight fuel due to its higher vs. glucose (GLU)  energy yoL flies e. Also, ic flies e. Moreovic flies f, showedenotypes g,h. Consas ATGL) i, which foxo overexpressing flies’ tissues and the fact that foxo is an important regulator of IIS in the peripheral tissues and in regulating food intake [foxo overexpressing flies. We found that foxo OE reduces feeding rates (foxo overexpressing flies to metabolic stress by firstly exposing them to starvation. We found that foxo OE increased (dose-dependently) flies’ sensitivity to nutrients deprivation  did not affect significantly their median lifespan (foxo overexpressing flies had reduced somatic and circulating sugar levels (see above), we then supplemented flies’ culture medium with additional sugar. Interestingly, sugar over-supplementation (SS) significantly increased (vs. control) by ~20% the median lifespan of foxoL, but not of foxoH, line , as well as in cardiomyocytes by employing the tinC.Δ4Gal4 driver (induces strong transgene expression).To study whether tissue-specific foxoH/MhcGal4 revealed both nuclear and cytoplasmic foxo localization in muscle fibers  longevity  longevity , while foxoL/MhcGal4 larvae completed development , where foxo is thought to play a crucial role in maintaining proteostasis [foxoH/tinC.Δ4Gal4 flies P, Atg8a) genes   genes d; also, , Rpn10) e and of , Rpn10) , as well in foxoH overexpressing flies  and foxoH in flies (foxoH/cncC OE resulted in more intense (vs. sole Nrf2/cncC or foxoH OE) 26S proteasome activities induction , also further support the notion of the detrimental effects of stress-responsive elements’ prolonged activation on organisms’ physiology and longevity.Biological aging results from the progressive accumulation of cellular damage and the diminishing buffering capacity of stress response mechanisms . Geneticfoxo induced longevity strongly relies on its expression dynamics. We found that ubiquitous foxo OE accelerated aging, induced the early onset of age-related phenotypes, and increased sensitivity to thermal stress in a dose-dependent manner. Similarly, ubiquitous high expression levels of other transcription factors functioning as stress sensors, e.g., Nrf2/cncC, have detrimental effects on flies’ longevity [Herein we report, that the ongevity . Severalongevity ,20,25.foxo OE in larvae alters animals’ feeding behavior and mimics the phenotypic effects of starvation [Sustained foxo activation deregulated nutrient and energy-sensing mechanisms in a dose-dependent manner, increased sensitivity to nutrient deprivation, and phenocopied acute starvation since it exhausted flies’ energy expenditure by disrupting mitochondrial homeostasis, increasing lipolysis and by inducing hypoglycemia. Supportively, FoxO1 has been shown to play a pivotal role in regulating cellular responses to fasting, as it regulates genes involved in both glucose and lipid metabolism , suggestarvation , causingfoxo OE mediated longevity is tightly affected by tissue-specificity and efficient inter-tissue communication [foxo OE mediated effects. Muscle-tissues  are central in the regulation of metabolic flexibility and plasticity in the fly, while studies show that effective interorgan signaling instigated by muscle-tissue responses plays a major role in promoting healthy aging [foxo OE is noxious for Drosophila’s physiology, probably due to intense muscle wasting, while moderate foxo OE delayed the onset of age-related phenotypes and increased healthspan. Accordingly, strong muscle-specific foxo OE in healthy mice reduces skeletal muscle mass and disturbs glycemic control [foxo OE in Drosophila flies was found to preserve muscle integrity and function during aging by modulating the accumulation of protein aggregates and nutrient-sensing mechanisms [Since nication , we alsohy aging ,49,50. O control , thereby control . On the chanisms , thus suDrosophila suggest that moderate heart-specific foxo OE is involved in the amelioration of cardiac age-associated function decline [C. elegans and D. melanogaster [foxo OE induces the upregulation of proteostatic modules in a dose-dependent manner. More specifically, although UPP upregulation was not always associated with foxo OE, increased UPP activity was a key feature of long-lived tissue-specific foxo overexpressing flies, further supporting the notion that the ability of the organism to maintain proteostasis correlates strongly with a delay in tissue aging. In support, it was shown that FOXO directly regulates the activity of 19S proteasome by upregulating the Rpn6 proteasome subunit, promoting resistance to various types of exogenous stressors [β5 proteasome gene’s promoter [foxo overexpressing Drosophila lines is Nrf2/cncC-mediated. It was shown previously that the functional crosstalk between stress-responsive elements, upon, e.g., starvation-like conditions, precisely orchestrates genome-wide changes to induce specific gene expression programs and balance aberrant cellular damage, thereby impacting lifespan [In addition, FOXO isoforms have been shown to have divergent functions in cardiac response to stress signals, and targeting FOXO proteins could inhibit or even reverse cardiac hypertrophy. More specifically, and in line with our findings, studies in mice show that FOXO1/FOXO3 activation decreases cardiomyocytes, while sustained FOXO3 activity leads to reversible heart atrophy ,54,55. M decline , and our decline . FOXO-innogaster . On the nogaster . Our dattressors ,57; alsopromoter . Furtherlifespan . Furtherlifespan .foxo upregulation exhausted energy expenditure, triggered proteostasis, in a coordinated action with Nrf2/cncC, and affected lifespan in a dose-dependent manner, supporting the notion that prolonged intense activation of stress sensors is not a favorable condition. On the other hand, depending on the metabolic profile of the targeted tissue, a specific dose of foxo OE improved flies’ healthspan by delaying the onset of age-related functional decline. Thus, defining that context-depended activity  may be required to unleash FOXO’s beneficial potential as a strategy to prevent age-related diseases and improve life quality.Taken together, our findings suggest the differential dose- and tissue-dependent effects of foxo on longevity-associated signaling cascades, such as bioenergetic and proteostatic pathways. Sustained ubiquitous


During the COVID-19 pandemic, stroke remains a leading cause of death and disability. Since the approach towards patients, treatments, and follow-up regimens has changed, tele-rehabilitation became a pillar for patients with ongoing recovery processes and rehabilitation strategies, especially for post-stroke patients. We describe the case of a 50-year-old male, suffering from right spastic hemiplegia and patellar clonus, for whom rehabilitation services were delivered both in‐person  as well as with the aid of a tele-rehabilitation program. Assessments were conducted remotely via a self-adapted treadmill and stabilometric equipment, both connected to the Internet. At discharge and at 20 weeks follow-up, the patient showed decreased spasticity grade, improvement in sensorimotor function, balance, functional mobility, clonus score, ambulation distance, and decreased pain intensity. The case highlights the utility of tele-rehabilitation strategies in complementing and enhancing the effects of conventional physical therapy and radial extracorporeal shock wave therapy (rESWT) in post-stroke spasticity. Stroke is one of the main causes of death and disability in adults worldwide -2. AccorWe present the case of a 50-year-old post-stroke male for whom the integration of a tele-rehabilitation program proved its effectiveness for adapting and optimizing the neurorehabilitation strategies and therapies used during hospitalization, namely conventional physical therapy and radial extracorporeal shock wave therapy (rESWT).The patient consented to participate in this case report, which was conducted in accordance with the Declaration of Helsinki.A 50-year-old male with post-stroke spasticity and right hemiparesis due to an ischemic stroke was transferred from the Neurology Unit to our Physical and Rehabilitation Medicine Department for the initiation of a neurorehabilitation program. At admission, according to the ratings criteria of the Modified Ashworth Scales (MAS), the patient suffered from grade three spasticity in the right upper limb and grade two spasticity in the right lower limb. Clinical examination also showed a right-sided hemiparesis, hemihypesthesia and patellar clonus. Biologic investigations depicted a mild inflammatory syndrome and moderate anaemia. Based on the clinical and paraclinical grounds, we initiated a conventional physical therapy program combined with rESWT. We define conventional physical therapy as any rehabilitation therapy and technique performed according to each rehabilitation centre or department. Therefore, therapies involving interventions for post-stroke patients  are the components of the conventional physical therapy program. The rESWT  was applied as follows: 2000 shots with a frequency of 10 Hz, and energy density of 60 mJ (1 bar). The intervention site was the myotendinous junction of the gastrocnemius and the soleus muscles. The patient underwent two rESWT sessions once a week for two weeks.Clinical and stabilometric assessments were performed at baseline (T0), at the end of the rehabilitation program and after the second rESWT session (T1), and 20 weeks after patient discharge (T2). Overall, the clinical outcome measures showed significant improvement at different assessment times, proving long-term effectiveness of rESWT as a non-invasive treatment for post-stroke patients with spasticity, clonus, pain, mobility, balance, functionality, and gait deficits. Due to the COVID-19 pandemic, the patient could not benefit from a new adapted neurorehabilitation program, including rESWT sessions, botulinum toxin type A injections (BoNT-A), or assessments implying hospital stay but, to ensure the continuity of the rehabilitation plan, tele-rehabilitation sessions with a physical therapist were initiated after discharge. Prior to discharge, the patient was provided with a custom-tailored rehabilitative exercise program and properly instructed about both the clinical and virtual assessments that were to be conducted.Clinical assessmentsThe spasticity grade decreased by one point for the MAS, and the effect was maintained at 20 weeks follow-up. Also, for knee and ankle passive range of motion (PROM), the gain in terms of degrees was maintained at 20 weeks after discharge (T2) in comparison to T0, even though it showed a small decrease at T2 compared to the assessment at T1. For the pain intensity assessment by Visual Analogue Scale (VAS), the score decreased by two points at T1 and T2. Concerning the clonus score, it decreased by two points and maintained the same level at T1 and T2. The patient also showed significantly improved mobility, functionality, balance, gait, ambulation speed and safety even in the long term. The Tinetti Assessment Tool, Functional and Ambulation Categories (FAC), Fugl-Meyer Assessment for Lower Extremity (FMA-LE), and Timed Up and Go Test (TUG) were significantly improved. Additionally, TUG scored better results at long-term follow-up, and functional mobility improved compared to the initial and the second assessment. A complete review is provided in Table Stabilometric assessmentsThe stabilometric assessment and analysis were carried out by using ProKin 252  and Walker View system . The ProKin system incorporates a force-sensitive stabilometric platform that provides the evaluation of trunk, stance, static and dynamic balance, and the Walker View system assists with a full postural and walking analysis. The two systems provide real-time visual feedback, which facilitates the improvement of stance during static, dynamic balance, and walking. As for the stabilometric outcomes assessed through the ProKin system, the results were consistent with the clinical parameters, presenting satisfying short-term and long-term effects of rESWT sessions during conventional physiotherapy programs and complemented through tele-rehabilitation strategies. Dynamic balance, trunk analysis, and limits of stability were much improved at T1 compared to the initial assessment and continued to show amelioration at 20 weeks, correlating with clinical parameters. Regarding static balance with eyes-open (EO) and eyes-closed (EC), all parameters scored improved results at T2 compared to T1 and T0, except perimeter with EC in the static balance test. However, the improvement was significant compared to T0 and scored only a mild difference compared to T2. A complete review is provided in Table After the stabilometric assessment was performed by a skilled physical therapist, the results were transmitted automatically via the internet to the assessor’s tablet, which integrated the stabilometric outcome measures with the clinical evaluation, as presented in Figure Tele-rehabilitation helped maintain and further improve the results obtained during the hospital stay. At discharge, the patient demonstrated improved physical functional independence with satisfying functional mobility, decreased spasticity grade, reduced clonus and pain symptoms. Ambulation speed, precision, and safety during gait were all improved. At 20 weeks follow-up, after the weekly tele-rehabilitation program, these parameters persisted within the same limits, and the patient also scored better results for TUG, ambulating with more reassurance, mobility, and precision. No adverse events were reported during or after the rehabilitation program. No falls were registered either during the assessments, tests, or hospital stays.The aim of this case report was to highlight the utility of an adapted tele-assessment and tele-rehabilitation strategy for a recovering patient who also underwent conventional physical therapy and rESWT during the hospital stay. During the COVID-19 pandemic, tele-assessment and tele-rehabilitation techniques were delivered for rehabilitation plan progression and real-time results, providing an objective evaluation in correlation with clinical parameters. Telemedicine for chronic neurological conditions could become part of everyday practice both for practitioners and patients, not only during the COVID-19 pandemic but also in less challenging times . For manThe patient presented significant improvement of all outcome measures on both short and long-term, with the aid of effective tele-assessment and tele-rehabilitation strategies. The static and dynamic balance were improved, as well as the Tinetti Assessment Tool score. In the eyes-open (EO) setting, area and perimeter decreased significantly, and the results were maintained at 20 weeks follow-up. The only stabilometric parameter that showed few changes in score was the static assessment in the eyes-closed (EC) setting. However, it showed improvement at T2 compared to T0. The clinical improvement of MAS grade showed the effectiveness of the rESWT in the short and long term, and the results are consistent with those from various studies -8. Knee During the COVID-19 pandemic, the follow-up process of post-stroke patients was highly affected. To promote the rehabilitation continuity and reassessment of all outcome measures, we proposed a tele-assessment and tele-rehabilitation approach. Through this approach, we were able to further reassess spasticity grade, clonus, pain intensity, balance, mobility, functional capacity, and gait and to correlate the new findings with the already collected data during the hospital stay. Integrating clinical examination and virtual assessment provided both the patient and the rehabilitation team with objective data by tracking progression landmarks, further leading to a more adapted, individualized therapeutic approach.Another extremely important aspect is that classic rehabilitation programs for stroke survivors imply high costs, thus representing a huge burden on healthcare systems worldwide, especially in low-and-middle income countries. In the management of post-stroke patients, tele-rehabilitation was shown to be cost-effective when compared to in-clinic intervention . It alsoThe case highlights the usefulness of tele-rehabilitation and tele-assessment in enhancing the effects of conventional physical therapy and rESWT in post-stroke spasticity. This strategy was essential for our patient during the pandemic when access of post-stroke patients to their usual in-clinic rehabilitation program and follow-up regimen is limited. The patient presented improved lower limb spasticity grade and trunk control, decreased pain intensity and clonus score, improved static and dynamic balance, enhanced sensorimotor performance, functionality, and gait. Through tele-rehabilitation sessions, treatment effectiveness lasted up to 20 weeks, showing that tele-rehabilitation programs are key factors for both practitioners and patients, especially in these unprecedented COVID-19 times. In addition, rESWT showed a good safety profile and long-term efficacy for all the outcome measures. Future research is needed concerning adapted approaches of tele-rehabilitation techniques, therapies, and assessment tools.


While the Player Load is a widely-used parameter for physical demand quantification using wearable accelerometers, its calculation is subjected to potential errors related to rotational changes of the reference frame. The aims of this study were (i) to assess the concurrent validity of accelerometry-based Player Load against force plates; (ii) to validate a novel metric, the Accel’Rate overcoming this theoretical issue. Twenty-one recreational athlete males instrumented with two triaxial accelerometers positioned at the upper and lower back performed running-based locomotor movements at low and high intensity over six in-series force plates. We examined the validity of the Player Load and the Accel’Rate by using force plates. Standard error of the estimate was small to moderate for all tested conditions . Accel’Rate displayed trivial to small mean biases (−1.0 to 6.1 a.u.) while the Player Load displayed systematic very large to extremely large mean biases (17.1 to 226.0 a.u.). These findings demonstrate a better concurrent validity of the Accel’Rate compared to the Player Load. This metric could be used to improve the estimation of whole-body mechanical load, easily accessible in sport training and competition settings. During the last few decades, the physical demand of team sports has greatly increased, as reflected by the increasing game frequency over a season, competition level and rules modifications . Becausex, y and z axes , on both tested metrics validity.Twenty-one recreational team-sport players  volunteered to take part in this study. Participants were informed regarding the nature, aims and risks associated with the experimental procedure. This study was conformed to the standards of the Declaration of Helsinki.The experimental procedure is depicted in Locomotor tasks were carried out over six individual force plates connected in series . The second force plate was turned by 90 degrees such that the total length of the force plates area was 6.6 m  FigureB. This sDuring the experimental protocol, participants were instrumented with two triaxial accelerometers  sampled at 100 Hz . The first device was placed in the center of the upper back and worn in a standard manufacturer harness (Catapult) designed to minimize artifact movements between the device and the body. The sensor was located ~5 cm lower than the base of the neck . The second device was securely attached directly on the skin by a bi-adhesive support on the middle of the lower back over the horizontal axis determined by the posterior superior iliac crests. The sensors have been calibrated by the manufacturer and not specifically re-calibrated/validated for this study. A study using similar accelerometers (MinimaxX 2.0) previously reported acceptable within- and between-device reliability in the lab and on the field in both static and dynamic conditions using a hydraulic testing machine . In thea is the center-of-mass acceleration, g is the vertical acceleration due to gravity (9.81 m·s2), f is the force applied, obtained from force plates, and m is the mass of the participant. The x, y, and z axes are oriented in the medial-lateral, anteroposterior and vertical directions in the terrestrial reference frame of force plates, respectively  while GRF signals were exported using Bioware (Kistler). All analyses were then performed using custom written scripts . To synchronize force plate signals with accelerometer signals, we asked the participants to make three successive jumps at the beginning of each trial. Then, after GRF data were down sampled at 100 Hz, force plate and accelerometer signals were synchronized by cross-correlation analyses . The thrils, see ). We theTriaxial accelerometers were used to provide the direct measurement of the acceleration of the body part to which it was attached, also named segmental acceleration . Medial-As previously mentioned, we aimed to evaluate the concurrent validity of the Player Load and to assess the concurrent validity of a new computation, the Accel’Rate. First, we calculated Player Load using its classical equation (Equation (1)) based on the instantaneous acceleration variation of each component. The computation of Player Load provided by the manufacturer software comprises additional data filtering techniques . In a piFor Player Load and Accel’Rate metrics, the agreement between data obtained using accelerometers  and force plates (criterion measure) was examined . The levConcurrent validity of both Player Load and Accel’Rate are presented in For the Player Load metric, Pearson’s correlation coefficient showed a moderate to very large relationship (0.53 to 0.90) between the accelerometer  and the force plates (criterion measure) depending on the type of locomotor task . SpecifiThe Accel’Rate was calculated from the accelerometer data  and compared against the Accel’Rate calculated from the force plates (criterion measure). Pearson’s correlation coefficients ranged from 0.71 to 0.97 . We founWe observed an influence of the locomotor task intensity on the concurrent validity of the metrics, especially for Player Load. Mean biases  and SEEs (0.45 to 0.97 vs. 0.51 to 0.97) were systematically larger, and correlation coefficients were lower at high intensity compared to low intensity for Player Load . Regarding Accel’Rate, mean biases were similar between low and high intensities . CorrelaAt both upper and lower back locations, we observed large to very large correlations between Player Load and the criterion measure for GLM, RS and 1 vs. 1 (0.68 to 0.90). However, the upper back position showed better correlations for RCV at low and high intensity compared to the lower back position . Similar SEEs data were also found for the two sensor locations using Player Load . Each teWe observed less influence of sensor positions on the Accel’Rate concurrent validity. Specifically, we found a higher bias for the lower back in comparison with the upper back position . These mean biases were considered as trivial to small for the upper back position and as moderate to large for the lower back position except for 1 vs. 1 at high intensity . Moreover, the SEEs were larger in the lower back position compared to the upper back position except for RS at high intensity .In this investigation, we sought to assess the concurrent validity of the Player Load metric, and to overcome theoretical issues related to its calculation. We therefore developed and quantified a novel metric, the Accel’Rate, with the ultimate goal of improving the estimation of the whole-body mechanical load using triaxial accelerometers. The present study has four novel findings: first, the Player Load inferred from the common accelerometry-based computation method did not match the whole-body mechanical load applied to the player’s center-of-mass in ecological conditions using in-series force plates. Second, the Accel’Rate measured with wearable accelerometers presented good to very good validity with the criterion measure  for various team-sport locomotor tasks executed at different intensities. Third, the locomotor task intensity substantially altered the accuracy and agreement of Player Load evaluation compared to Accel’Rate, which remained robust regardless of the task intensity. We posit that the novel Accel’Rate metric, based on instantaneous variations of the modulus of the 3-axes acceleration vector, is more suitable to reflect the whole-body mechanical load elicited by various locomotor tasks and intensity. Fourth, the use of classical accelerometer body-worn position between scapulae was shown to be a better alternative than trunk position (lumbar) during running-based locomotor tasks and may therefore be recommended during training and competitions.To our knowledge, while previous studies aimed to quantify the validity of the Player Load  versus the same device fixated onto a belt worn. We noticed a major issue with using a belt because of pelvis motion moved up and down the belt, thereby eliciting artificial accelerations. Furthermore, even firmly attached, the belt moved from its original location throughout the protocol. Using the bi-adhesive support, the accelerometer undoubtedly held better. To prevent for large skin and soft tissues motions during high intensity actions, we covered the device with the elastic band of the participant’ shorts. Sweating effect on fixation was controlled during the protocol by checking the location and firmness of the adhesive double-tape support between trials. In exceptional cases where sweating affected the fixation, we removed the device, dried the lower back area and put the device back at the exact same location with new double-side adhesive tape. Previously, Barrett et al. (2014) found a 15.7 ± 9.7% lower Player Load at the upper back location compared to the lower back location (pouch fitted in a garment) when running on a treadmill. In our study, we observed similar results with higher Player Load at the lower back but with larger differences compared with Barrett et al. (2014)  are co-inventors of the European Patent no. (FR18/4435592) which is owned by the French Institute of Sport (INSEP) and contains scientific content related to that presented in the manuscript. Priority Date: 2017/08/28; International Filing Date: 2018/08/28; International Publication Date: 2019/03/07.


Adv. Sci. 2021, 8, 210140210.1002/advs.202101402DOI: In the originally published version of article, the affiliations of Haidong Zhou are incorrect. Please find the correct affiliations here:L. L. Kish, A. Thaler, A. V. Zakrzewski, D. Reig‐i‐Plessis, B. A. Wolin,X. Wang, R. Budakian, G. J. MacDougallDepartment of Physics and Materials Research LaboratoryUniversity of Illinois at Urbana‐ChampaignUrbana, IL 61801, USAlazark2@illinois.edu; gmacdoug@illinois.eduE‐mail: M. Lee, V. S. ZapfNational High Magnetic Field LaboratoryLos Alamos National LaboratoryLos Alamos, NM 87544, USAD. Reig‐i‐PlessisDepartment of Physics and Astronomy and Quantum Matter InstituteUniversity of British ColumbiaVancouver, British Columbia V6T 1Z1, CanadaH. ZhouDepartment of Physics and Astronomy University of TennesseeKnoxville, Tennessee 37996, USAR. BudakianDepartment of Physics and AstronomyUniversity of WaterlooWaterloo, Ontario N2L 3G1, CanadaZ. GaiCenter for Nanophase Materials SciencesOak Ridge National LaboratoryOak Ridge, TN 37831, USAA. Thaler, K. C. Littrell, M. D. Frontzek, A. A. Aczel, L. DeBeer‐SchmittNeutron Scattering DivisionOak Ridge National LaboratoryOak Ridge, TN 37831, USAdebeerschmlm@ornl.govE‐mail:


In these studies, we observed that heteromerization between two receptor partners may depend on the presence and the expression levels of other partnering receptors. To test this hypothesis and to gain initial insight into the formation of these receptor heteromers in native cells, we utilized proximity ligation assays in hVSMCs to visualize receptor-receptor proximity and systematically studied how manipulation of the expression levels of individual protomers affect heteromerization patterns among other interacting receptor partners. We confirmed subtype-specific heteromerization between endogenously expressed α1A/B/D-ARs and detected that AVPR1A also heteromerizes with α1A/B/D-ARs. siRNA knockdown of CXCR4 and of ACKR3 resulted in a significant re-arrangement of the heteromerization patterns among α1-AR subtypes. Similarly, siRNA knockdown of AVPR1A significantly increased heteromerization signals for seven of the ten receptor pairs between CXCR4, ACKR3, and α1A/B/D-ARs. Our findings suggest plasticity of seven transmembrane helix (7TM) receptor heteromerization in native cells and could be explained by a supramolecular organization of these receptors within dynamic clusters in the plasma membrane. Because we previously observed that recombinant CXCR4, ACKR3, α1a-AR and AVPR1A form hetero-oligomeric complexes composed of 2–4 different protomers, which show signaling properties distinct from individual protomers, re-arrangements of receptor heteromerization patterns in native cells may contribute to the phenomenon of context-dependent GPCR signaling. Furthermore, these findings advise caution in the interpretation of functional consequences after 7TM receptor knockdown in experimental models. Alterations of the heteromerization patterns among other receptor partners may alter physiological and pathological responses, in particular in more complex systems, such as studies on the function of isolated organs or in in vivo experiments.Recently, we reported that the chemokine (C-X-C motif) receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) heteromerize with α Seven-transmembrane-helix (7TM) receptors, of which the majority are G protein-coupled receptors (GPCRs), play essential roles in many aspects of human physiology and in numerous disease processes. GPCRs are important drug targets, and a large proportion of Federal Drug Administration approved drugs are agonists or antagonists of various GPCRs , 2.While GPCRs were originally thought to function as individual protomers or homodimers, accumulating evidence suggests that many GPCRs can also form heterodimers or hetero-oligomers with other receptor partners, which exhibit pharmacological properties distinct from the individual protomers –9.2-adrenergic receptor (AR), δ-opioid receptor, cannabinoid receptor 2, protease-activated receptor 1, or the virally-encoded GPCR of Herpesvirus saimiri, which is thought to alter the pharmacological properties of the receptor partners [1A/B/D-ARs and arginine vasopressin receptor 1A (AVPR1A) in recombinant systems and in rodent and human vascular smooth muscle cells (hVSMCs), through which the receptors cross-talk [1A/B/D-ARs, and AVPR1A are interdependent and demonstrate plasticity of their heteromerization patterns.Chemokine (C-X-C motif) receptor 4 (CXCR4), which is a prototypical GPCR, has been reported to exist as a monomer, dimer, and within nanoclusters comprised of more than three protomers in cells , 11. Morpartners , 12–21. oss-talk , 22–27. oss-talk , which coss-talk  and systhVSMCs  were purchased from American Type Culture Collection. As described previously , 25, hVS1A-AR (Abcam Ab87990) or rabbit anti-α1A-AR (Abcam Ab137123), rabbit anti-α1B-AR (Abcam Ab169523) or goat anti-α1B-AR (Santa Cruz SC27136), and goat anti-α1D-AR (Santa Cruz SC27099) at 37°C for 105 min in a humidifying chamber. To visualize receptor–receptor interactions, slides were incubated with a combination of two antibodies raised in differing species as appropriate at 37°C for 105 min in a humidifying chamber. All antibodies were used in dilutions of 1: 500. Slides were then washed with PBS and incubated for 60 min at 37°C in a humidifying chamber with secondary species-specific antibodies conjugated with plus and minus Duolink II PLA probes (1:5), as appropriate. Negative control slides were incubated with omission of one primary antibody and two differing species-specific secondary antibodies. Slides were washed again with PLA wash buffer A (Duolink II) and then incubated with ligation-ligase solution for 30 min at 37°C in a humidifying chamber and also washed with PLA wash buffer A. Finally, slides were incubated with amplification polymerase solution for 100 min at 37°C in a humidifying chamber. Slides were then washed twice with PLA wash buffer B (Duolink II), once with 0.01× PLA wash buffer B and allowed to dry. Slides were then mounted with a minimal volume of Duolink II mounting medium with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) overnight, and PLA signals (Duolink In Situ Detection Reagent Red (λexcitation/emission 598/634 nm) were identified as fluorescent spots under a fluorescence microscope (Carl Zeiss Axiovert 200M with EC Plan-Neofluor objective lenses (40 × /1.30 oil) equipped with Axio CamMRc5 (Carl Zeiss) and AxioVision Rel. 4.9.1 (Carl Zeiss) acquisition software) at room temperature. For each vision field 10 z-stack images in 1 μm sections were acquired and compressed. PLA signals were quantified using the Duolink Image Tool software (Sigma-Aldrich). Images were imported in merged.tiff formats containing both signal and nuclei channels. Merged images were visually verified for analytical quality. Comparisons and statistical analyses were performed only when PLA assays were performed on the same day in parallel experiments, and fluorescence microscopy was performed with the identical settings. For each experiment and condition, 10 randomly selected non-overlapping vision fields were analyzed.PLAs were performed as described in detail previously –25, 29. AVPR1A, CXCR4, and ACKR3 siRNA gene silencing was performed as described previously , 25, 30.Data are expressed as mean ± standard error. Student’s t-test or One-way analyses of variance (ANOVA) with Dunnett’s multiple comparison post hoc test for multiple comparisons were used to assess statistical significance, as appropriate. A two-tailed p<0.05 was considered significant. All analyses were calculated with the GraphPad Prism 8, Version 8.4.0 software.1A/B/D-ARs and AVPR1A in recombinant systems and in hVSMCs [1-AR subtypes, as assessed by intermolecular bioluminescence energy transfer assays in HEK293T cells [1A/B/D-ARs has not been reported, we first tested whether AVPR1A heteromerizes with any of the α1-AR subtypes in hVSMCs. Consistent with our previous findings [1-AR subtypes could be visualized in hVSMCs when PLA was performed to detect each protomer individually . PLA signals for proximity between α1A-AR and α1D-AR were not significantly different from negative control PLA signals (p = 0.17), which is in agreement with previous findings in a recombinant system [Homodimerization of αeceptors . To conft system .1A/B/D-ARs in hVSMCs, we tested whether depletion of CXCR4 or ACKR3 from the cell surface by gene silencing via RNA interference influences the heteromerization patterns between α1A/B/D-ARs. We showed previously that siRNA knockdown of CXCR4 or ACKR3 does not influence cell surface expression levels of α1A/B/D-AR in hVSMCs [Because CXCR4 and ACKR3 heteromerize with αn hVSMCs , 24. As 1A-AR:α1B-AR heteromers, but did not affect PLA signals for α1B-AR:α1D-AR or α1A-AR:α1D-AR heteromers (Figs 1A-AR:α1B-AR heteromers, but significantly increased PLA signals for α1B-AR:α1D-AR and α1A-AR:α1D-AR heteromers (Figs 1D-AR compete for heteromerization interfaces with α1A-AR and α1B-AR, which are occupied by α1D-AR upon depletion of ACKR3. The finding that CXCR4 knockdown reduces α1A-AR:α1B-AR heteromers, however, could be explained by a higher-order receptor complex, in which CXCR4 facilitates proximity between α1A-AR and α1B-AR.We observed that depletion of CXCR4 from the cell surface significantly reduced PLA signals corresponding to αers Figs  and 6. Iers Figs  and 6. T1A/B/D-AR protomers in hVSMCs, we tested whether depletion of AVPR1A from the cell surface would also affect heteromerization patterns between all other receptor pairs. As compared with hVSMCs incubated with NT siRNA, PLA signals for AVPR1A in hVSMCs after incubation with AVPR1A siRNA were reduced by 66±4% (1A/B/D-ARs were indistinguishable in hVSMCs incubated with AVPR1A-siRNA and NT-siRNA (Because our previous studies, in combination with the findings of the present study, suggest that AVPR1A heteromerizes with each of the CXCR4, ACKR3, and αby 66±4% . PLA sigNT-siRNA .1A/B/D-ARs showed significant increases after incubation with AVPR1A siRNA for the following receptor pairs, as compared with hVSMCs incubated with NT-siRNA (Figs 1A-AR– 91±38%; CXCR4:α1B-AR– 72±30%; ACKR3:α1A-AR– 91±25%; α1A-AR:α1B-AR– 101±17%; α1A-AR:α1D-AR– 238±53%; α1B-AR:α1D-AR– 141±29%; CXCR4:ACKR3–59±21%. PLA signals suggestive of heteromerization between CXCR4 and α1D-AR and between ACKR3 and α1B/D-ARs were not significantly affected by AVPR1A knockdown.Quantification of the PLA signals for all combinations of heteromeric complexes between ACKR3, CXCR4, and αRNA Figs  and 9: C2-AR and CXCR4, which were found to exist in a dynamic equilibrium of monomers, dimers, and higher-order homomers in the plasma membrane [1A-AR, CXCR4:α1A-AR, and CXCR4:α1B-AR heteromers [Our observations further support the concept that heteromeric receptor complexes exist in a dynamic equilibrium on the cell surface, in which depletion of one receptor heteromerization partner leads to the re-arrangement of multiple heteromeric receptor complexes among other partnering receptors , 25. Sucmembrane , 32 and teromers . The fin1-AR subtypes and on heteromerization between AVPR1A and α1-AR subtypes in hVSMCs in the present study confirm previous observations in recombinant expression systems and validate that findings on these recombinant receptors are applicable to their endogenously expressed counterparts. Furthermore, we demonstrate interdependency of the heteromerization patterns among six distinct endogenously expressed 7TM receptor protomers. Irrespective of the underlying molecular mechanisms, these findings suggest plasticity of 7TM receptor heteromerization in native cells and could be explained by a supramolecular organization of these receptors within dynamic clusters in the plasma membrane. The latter is consistent with our recent findings in recombinant systems and with the idea that such receptor hetero-oligomers form larger and possibly metastable signaling complexes [Taken together, our findings on heteromerization between αomplexes , 9.1-ARs and AVPR1A is well documented [1-ARs and AVPR1A on the molecular, cellular, and organ levels previously [The present study is limited in scope because it was not designed to explore functional consequences associated with the observed re-arrangements of the receptor heteromers. Nevertheless, functional cross-talk between αcumented –35 and weviously , 22–26. eviously , 37.in vivo experiments.In addition, our observations advise caution in the interpretation of functional consequences after GPCR knockdown in experimental models, because alterations of the heteromerization patterns among other receptor partners may alter physiological and pathological responses, in particular in more complex systems, such as studies on the function of isolated organs or in


Haliaeetus albicilla) is strictly protected in Poland due to its threat of extinction. This study’s main goal was to assess their exposure to indirect poisoning by anticoagulant rodenticides (AR). This study presents the investigation results of 40 white-tailed eagles’ suspected poisoning cases in the years 2018–2020 in Poland. In all tested liver samples, using a liquid chromatography–mass spectrometry method, at least one of the AR  was detected and confirmed. The other tested AR compounds  were not detected. The mean concentration of the sum of rodenticides was 174.4 µg/kg (from 2.5 to 1225.0 µg/kg). In 20 cases, the sum concentration was above 100 µg/kg and in 10 cases it was above 200 µg/kg. Interpretation of cases of AR poisonings should take into account their concentration in the liver, anatomopathological lesions, circumstances of death/finding of the animal, and elimination of other possible causes of poisoning. Based on this study, AR was the direct cause of death in 10% of incidents. Extensive use of rodenticides generates a high risk of poisonings of white-tailed eagles in Poland.The white-tailed eagle ( The discovery of anticoagulant rodenticides and their high efficacy significantly changed the game in the field of rodent control and led to their widespread and extensive use worldwide . Their mHaliaeetus albicilla) in Poland based on analyses of test results of 40 liver samples. For toxicological investigations, we used a previously published method that allows the analysis of compounds from the groups belonging to rodenticides, carbamate, and organophosphate pesticides, coccidiostats and mycotoxins [This research allows us to estimate the prevalence of, and assess exposure to, AR poisonings of white-tailed eagles  is the largest predatory and scavenging free-living species of bird in Poland, also generally widely distributed across Eurasia [Vulpes vulpes), and occasionally carrion [Haliaeetus albicilla) exposure to AR in Poland.The white-tailed eagle . After that, 0.7 mL of each supernatant were added to a new centrifuge tube with 150 mg of MgSO4, 50 mg of C18, and 50 mg of Primary Secondary Amine (PSA), mixed with a vortex mixer for 1 min, and centrifuged for 10 min at 2930× g rcf. The supernatant solutions were transferred to centrifuge filters (0.2 µm), centrifuged at 9447× g rcf for 10 min, and transferred to autosampler vials.Liver samples were defrosted and homogenized. First, 2 g of sample was mixed with 5 mL of acetonitrile using a vortex mixer for 30 s. Then, 0.5 g of sodium acetate was added and further mixed for 1 min. The resulting suspension was sonicated for 15 min in an ultrasonic bath, mixed with a vortex mixer for 1 min, and centrifuged at 2930× Extracts from liver samples were analyzed on a Shimadzu liquid chromatography system connected to an ABSciex API 5500 Qtrap mass spectrometer. The chromatography was performed on a C8 column (75 mm × 2.1 mm × 3 μm) with a mobile phase consisting of two solutions: 5% isopropanol in ethanol and 0.5% isopropanol in 0.1% acetic acid in water. For the AR analysis, the mass spectrometer was operating in negative ionization mode and the transition reactions monitored are shown in 2) were between 0.98–1.00. The average apparent recoveries for AR were in the range of 90–110%, with average repeatability below 12% and average within-laboratory reproducibility below 15%. The limit of quantification (LOQ) was established at 1.00 µg/kg of wet liver sample for bromadiolone, brodifacoum, coumachlor, coumatetralyl, difenacoum, flocoumafen, and warfarin and 5.00 µg/kg for chlorophacinone, diphacinone, and difethialone. Detailed data for calibration ranges, recovery, and limit of quantification of the other analyzed compounds are available in Sell et al. [The whole procedure for the determination of AR was validated according to the requirements of SANTE/11945/2015. The calibration curves were prepared for each batch by fortifying fresh blank turkey liver samples from 1.00 to 250 µg/kg. The correlation coefficients , anatomopathological lesions, observed in varying degrees of intensity, were symptomatic for blood clotting disorders and included hemorrhages, petechiae, absence of blood clots in the heart and major blood vessels, or bleeding into body cavities . At leasHaliaeetus albicilla) liver samples.In contrast, the levels of rodenticides are presented in Based on toxicological and anatomopathological investigations, AR were identified as the direct cause of death of white-tailed eagles in four cases . The mean concentration of the sum of AR in these birds’ livers was 695.6 µg/kg (from 219.7 to 1225.0 µg/kg). From carbamate pesticides, in twenty-seven cases carbofuran (from 19 to 5428 µg/kg) and in five bendiocarb (from 2722 to 19487 µg/kg) were detected.Haliaeetus albicilla) exposure to AR in Poland. If we take into consideration different species of birds of prey, the situation is similar to other countries, concluding that the main hazards from AR are SGARs, including bromadiolone and brodifacoum [Vulpes vulpes), especially during the winter period when the availability of fish is limited. The fox population in Poland is estimated at 200,000–250,000 individuals [The obtained results represent the first report about white-tailed eagles’  and those introduced in their place (bendiocarb), are still the main cause of poisoning in Poland. There are many documented indirect poisonings by acetylcholinesterase inhibitors ,28,37,38In situations when we detect AR concentrations above 100 or 200 µg/kg combined with symptoms of blood-clotting disorders, another compound can be determined as the direct cause of death. For example, bird #20 was found dead near numerous feathers of a domestic pigeon. There was a copious mucous discharge from the beak and single drops of noncoagulated blood. The anatomopathological lesions also included noncoagulated blood flowing from the severed blood vessels and heart, numerous hemorrhages, and a small amount of bloody fluid in the abdominal cavity. In the liver sample of this white-tailed eagle, the sum of AR was 172.0 µg/kg. At first sight, it appears that the cause of death may have been AR poisoning, yet the use of the multicomponent method allowed us to detect bendiocarb at a concentration of 19,487 µg/kg. In this case, bendiocarb was found to be the direct cause of death. Another example was white-tailed eagle #2, where the use of a multicomponent method allowed identifying carbofuran as the direct cause of death . The detWhite-tailed eagles had a prevalence of 100% for AR, with 50% of the eagles exceeding concentrations of 100 µg/kg in their livers. This demonstrates an extreme exposure in a top predator caused by an unregulated and intensive use of rodenticides in Poland. A likely source of exposure for white-tailed eagles may be the red fox, which is exposed to both indirect and intentional poisoning. AR products are very widely available and freely used without an appropriate level of monitoring. Specific regulations are needed in this field to limit the influence on wildlife health. Interpretation of toxicological test results in case of suspected AR poisoning should take into account not only the AR concentration and anatomopathological lesions, but also the circumstances of the death/finding of the animal followed by exclusion of other possible causes of poisoning, e.g., carbofuran, which in Poland is the leading cause of death of white-tailed eagles.