Mycobacterium tuberculosis (Mtb) infection, particularly drug-resistant strains, are urgently required to tackle the global burden of this disease. Herein, we reported the synthesis of a novel series of N-substituted amino acid hydrazides, utilising a scaffold hopping approach within a library of anti-tubercular agents. Efficacy and selectivity were evaluated against three strains of Mtb , and cytotoxicity against macrophages in vitro. The antibacterial activity and therapeutic index of these molecules were significantly affected by modifications with the N-substituents. Introduction of a 3,5-dinitroaryl moiety demonstrated enhanced antibacterial activity against all three strains of Mtb. In contrast, the inclusion of an imidazo pyridine-3-carboxy moiety resulted in enhanced activity towards isoniazid mono-resistant Mtb relative to wild-type Mtb. Consequently, this scaffold hopping approach showed significant promise for exemplification of novel molecules with specific activity profiles against drug-resistant tuberculosis.Discovery and development of new therapeutic options for the treatment of  N′s (WHO) global tuberculosis report in 2019 recognises tuberculosis (TB) as a communicable disease responsible for a major cause of long term illness and continues to be one of the top ten causes of death worldwide from a single infectious agent . Th. Th10]. 0-ATPase ,12. Wher0-ATPase . More ree clinic . Furthere clinic . Consequmycolata over Gram-positive and Gram-negative bacteria pyridine-3-carboxy moiety of Q203 5  to exhibit the highest activity when conjugated within these hydrazides -diazole moiety scaffold with those from other molecules with known anti-TB activity in order to appraise the therapeutic impact of substituents at this position and the potential for exploitation towards the development of molecules with demonstrated activity against drug-resistant N-Boc amino acids and the corresponding hydrazide  -diazoles, we observed a level of antibacterial activity in the wild-type strain similar to or better than our lead compounds  ,21. Consompounds . Notably6 or 4-trifluoromethylphenyl methionine (Met) hydrazide 7. However, encouragingly, the Boc-protected 3-chlorophenyl Ala and 3-chlorophenyl Met hydrazide 8 and 9 demonstrated an observable increase in activity relative to the 4-trifluorophenyl amino acid hydrazides 6–7  hydrazide ides 6–7 .10–17, in most cases, did not provide enhanced antibacterial activity  in this protein, which was generated and identified in this study, analogous to previously published laboratory-generated and clinical strains pyridine-3-carboxy moiety has been identified as the active component of several novel anti-tubercular drugs, including the pantothenate synthetase inhibitor GSK358607A and Q203 1, which targets the cytochrome bcc-aa3 supercomplex pyridine-3-carboxy-substituted hydrazides 36–37, the inclusion of Met over Ala resulted in a two-fold increase in antibacterial activity. In contrast to the 4-trifluoromethylphenyl-substituted hydrazides, the 3-chlorophenyl-substituted hydrazides showed exquisite selectivity toward INH-resistant Mtb with MIC values <10 µM for both the Ala 36 and Met 37 containing compounds. However, although showing potent antibacterial activity, all of the imidazopyridine-3-carboxy-substituted hydrazides exhibited significant toxicity against macrophages, implying their limited utility as viable drug molecules. Despite this, a greater understanding of the structure-activity relationship (SAR) and identification of the targets of these compounds is an important concept in improving our understanding of principles associated with their selectivity towards drug-resistant Mtb.The Q203 analogues rcomplex ,28,29. A34–37, it was conceivable that the target of these compounds could include either the cytochrome supercomplex of proteins, as is the case with Q203 1, or pantothenate synthetase, the reported target of GSK358607A. Nevertheless, our results implied that the target was unlikely to be pantothenate synthetase since the Mtb mc27000 strains used in this study possessed ΔpanCD gene deletions and relied on media supplementation with pantothenate for survival and growth, thereby abrogating dependency for growth upon this enzyme. Inclusion of the imidazopyridine-3-carboxy moiety resulted in equivalent or greater activity than the initial Boc-protected compounds 6–9. To further confirm this hypothesis, the activity of compounds 34–37 was evaluated against Mycobacterium bovis BCG as this constitutively expressed the pantothenate pathway. No difference in activity was detected with these compounds when in the presence or absence of pantothenate in the growth media with this mycobacterium, reinforcing pantothenate synthetase as unlikely to be the therapeutic target for these compounds. Furthermore, this target would also not explain the selectivity of these compounds for INH-resistant Mtb.With respect to the imidazopyridine-3-carboxy-derived hydrazides 36–37 towards INH-resistant Mtb is one analogous to that of Q203. This drug targets the cytochrome bcc-aa3 supercomplex and disrupts the ETC in mycobacteria pyridine-3-carboxy-substituted hydrazides bacteria . Recentlbacteria . In thisbacteria . Further254) and visualised by UV radiation at 254 nm, or by staining with phosphomolybdic acid in ethanol or potassium permanganate in water. All melting points were determined using a Gallenkamp melting point apparatus and are uncorrected. Infrared spectra were recorded using a Diamond ATR  accessory (Golden Gate) or as a solution in chloroform via transmission IR cells on a Bruker Tension 5 spectrometer. 1H NMR spectra were recorded in deutero solvent on Bruker Avance III 300, Bruker Avance II 400, Bruker Avance III HD 500 or Bruker Avance III HD 700 instruments and are reported as follows; chemical shift δ (ppm) , assignment). Residual protic solvent CHCl3 (δH = 7.26) or DMSO (δH = 2.50) were used as the internal reference. 13C NMR spectra were recorded using the central resonance of CDCl3 (δC = 77.0 ppm) or DMSO (δC = 39.5 ppm) as the internal reference. All chemical shifts are quoted in parts per million relative to tetramethylsilane (δH = 0.00 ppm) and coupling constants are given in Hertz to the nearest 1 Hz. Assignment of spectra were carried out using COSY, HSQC, HMBC and NOESY experiments and all 1H and 13C NMR for new compounds are provided for reference (S) were obtained on a Micromass LCT mass spectrometer. High resolution mass spectra (HRMS) were obtained using a Thermo LTQ mass spectrometer (ES).All reactions were carried out under a nitrogen atmosphere in glassware dried in the oven overnight (>75 °C) or under high vacuum by a heat-gun unless otherwise stated. Ether refers to diethyl ether. Solvents were obtained dry from Sigma Aldrich, Gillingham, UK or another appropriate chemical supplier and used as supplied. In cases where mixtures of solvents were utilised, the ratios refer to the volumes used. Reagents were used as supplied unless otherwise stated. Flash chromatography was carried out using silica gel 40–63 μ 60Å. Analytical thin layer chromatography (TLC) was performed using precoated aluminium or glass-backed plates  in dichloromethane (DCM) (25 mL) was treated with N,N-Diisopropylethylamine (DIPEA) (1.2 equiv.) and N-aryl hydrazine (1.2 equiv.), followed by 4--4-methyl-morpholinium chloride (DMTMM) (1.2 equiv.), and the solution was stirred at room temperature for 3 h. The reaction was mixed with DCM (20 mL), and the organic layer was washed with sat. aq. NH4Cl (20 mL), sat. aq. NaHCO3 (20 mL) and brine (20 mL). The organic layer was dried over MgSO4, filtered, evaporated and dried in vacuo to afford the desired N-Boc amino acid hydrazides.A solution of 6 synthesis is previously reported hydrazine carbonyl}propyl]carbamate, 7, following the standard procedure A, Boc-Met-OH  and 4-trifluoromethylphenylhydrazine were transformed following flash chromatography  to afford the title compound as a pale yellow solid ; Rf 0.32 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); m.p. 102–108 °C; νmax (ATR) 3321, 2984, 1680, 1662, 1515, 1327, 1306, 1156, 1102, 830, 589 cm−1; δH  8.82 , 7.41 , 6.81 , 6.53 , 5.43 , 4.44 , 2.56 ), 2.09–2.14 , NHCH(CH2CH2SCH3)), 1.49 3); δC  172.4 (CHCONH), 156.1 (NHCOOC(CH3)3), 150.5 (Ar–C), 126.3 (Ar–C), 124.2 , 122.7 , 112.7 (Ar–C), 80.9 (NHCOO-C), 51.9 (NHCHCO), 30.9 (NHCH(CH2CH2SCH3)), 30.24 (NHCH(CH2CH2SCH3)), 28.3 (NHCOOC(CH3)3), 15.4 (NHCH(CH2CH2SCH3)); m/z (ES+) 408 (MH+); HRMS (ES+) found MH+, 408.1573 (C17H25F3N3O3S requires 408.1569).tert-butyl (S)-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)carbamate, 8, following the standard procedure A, Boc-Ala-OH  and 3-chlorophenylhydrazine were transformed to afford the title compound as a deep-yellow solid ; Rf 0.46 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); m.p. 110–114 °C; νmax (ATR) 3428, 3278, 3078, 2979, 2933, 1678, 1504, 1367, 1158 cm−1; δH  8.44 , 7.03 , 6.76 , 6.71 , 6.60 , 5.07 , 4.22–4.19 , 1.39 3)), 1.31 ; δC  192.7 (OC=ONH), 173.0 (CHC=ONHNH), 155.9 ((CH3)3COC=O), 149.1 (Ar–C), 135.0 (Ar–C), 130.2 (Ar-CH), 121.0 (Ar–C), 113.4 (Ar-CH), 111.69 (Ar-CH), 48.7 (NHCHC=O), 28.3 (C(CH3)3), 17.5 (NHCH(CH3)CO); m/z (ES+) 314 ([35Cl]MH+), 316 ([37Cl]MH+); HRMS (ES+) found ([35Cl]MH+), 314.1271 (C14H21N3O335Cl requires 314.1278).tert-Butyl N-[(1S)-1-[N′-(3-chlorophenyl)hydrazinecarbonyl]-3-(methylsulfanyl)propyl] carbamate, 9, following the standard procedure A, Boc-Met-OH  and 3-chlorophenylhydrazine were transformed following flash chromatography (DCM/EtOH/ sat. aq. NH3 [400:8:1] and [200:8:1]) to afford the title compound as a pale yellow solid ; Rf 0.30 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); m.p. 94–97 °C; νmax (ATR) 3331, 3272, 2985, 1683, 1658, 1521, 1163, 768 cm−1; δH  8.73 , 7.10 , 6.85 , 6.80 , 6.67 , 6.34 , 5.42 , 4.43 , 2.57 , 2.09–2.13 , 1.47 3); δC  172.2 (CHCONH), 156.9 (NHCOO(CH3)3), 149.0 (Ar–C), 134.9 (Ar–C), 130.2 (Ar–C), 121.0 (Ar–C), 113.4 (Ar–C), 111.6 (Ar–C), 80.8 (NHCOOC(CH3)3), 51.9 (NHCHCO), 31.0 (NHCHCH2CH2SCH3), 30.2 (NHCHCH2CH2SCH3), 28.32 (NHCOOC(CH3)3), 15.5 (NHCHCH2CH2SCH3); m/z (ES+) 374 ([35Cl]MH+), 376 ([37Cl]MH+), 396 ([35Cl]MNa+), 398 ([37Cl]MNa+); HRMS (ES+) found ([35Cl]MNa+), 396.1141 (C16H24N3O3S35ClNa requires 396.1125).S)-N-(1-Oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)isonicotinamide, 10, following the standard procedure B, tert-butyl (S)-(1-oxo-1-(2-(4-(trifluoromethyl) phenyl)hydrazineyl)propan-2-yl)carbamate  and isonicotinic acid were transformed to afford the title compound as a pale-yellow solid ; Rf 0.15 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); νmax (ATR) 3261 (N–H), 3095, 2973 (C–H), 1638 (C=O), 1617, 1536, 1327 (C–F), 1158 (C–N), 1102, 1065 (C–F) cm−1; δH  10.05 , 8.98 , 8.73 , 8.39 , 7.82 , 7.44 , 6.83 , 4.50 CO), 1.44 CO); δC  172.6 (CONHNH), 165.4 (PyCONH), 152.9 (ipso-Ar–C), 150.6 (Py-C), 141.4 (ipso-Py-C), 126.6 (Ar–C), 124.4 , 122.0 (Py-C), 118.5 , 111.9 (Ar–C), 48.7 (NHCH(CH3)CO), 18.0 (NHCH(CH3)CO); δF  -59.28 ; m/z (ES+) 353 (MH+); HRMS (ES+) found MH+, 353.1237 (C16H16F3N4O2 requires 353.1225).(S)-N-(4-(methylthio)-1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)butan-2-yl)isonicotinamide, 11, following the standard procedure B, tert-Butyl N-[(1S)-3-(methylsulfanyl)-1-{N′-[4-(trifluoromethyl)phenyl]hydrazine carbonyl}propyl]carbamate  and isonicotinic acid were transformed to afford the title compound as a white solid ; m.p. 179–181 °C; νmax (ATR) 3264, 1665, 1639, 1615, 1531, 1480, 1410, 1323, 1236, 1158, 1105, 1065, 1010, 878, 836, 757 cm−1; δH  10.12 , 8.98 , 8.74 , 8.43 , 7.84 , 7.45 , 6.82 , 4.61 ), 2.67–2.53 ), 2.15–2.09 ; δC  171.7 (CONHNH), 165.3 (PyCONH), 150.6 (Py-C), 126.6 (Ar–C), 122.1 (Py-C), 111.9 (Ar–C), 52.3 (NHCH(CH2CH2SCH3)), 31.3 (NHCH(CH2CH2SCH3)), 30.4 (NHCH(CH2CH2SCH3)), 15.1 (NHCH(CH2CH2SCH3)); δF  -59.28 ; m/z (ES+) 413 (MH+); HRMS (ES+) found MH+, 413.1261 (C18H20N4O2S requires 413.1259).(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)isonicotinamide, 12, following the standard procedure B, tert-butyl (S)-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)carbamate  and isonicotinic acid were transformed to afford the title compound as a white solid ; Rf 0.13 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); νmax (ATR) 3302, 3254 (N–H), 3035 (Ar–CH), 2983, 2930 (C–H), 1668, 1638 (C=O) 1599, 1537, 1511 (C=C) 1106 cm−1; δH  9.97 , 8.99 , 8.75 , 8.08 , 7.84 , 7.14 , 6.75–6.66 , 4.53 CO), 1.44 CO); δC  172.6 (CONHNH), 165.4 (Py-CO), 151.3 (ipso-Ar–C), 150.6 (Py-C), 141.3 (ipso-Py-C), 133.9 (ipso-Ar–C), 130.5 (Ar–C), 122.0 (Py-C), 118.3 (Ar–C), 111.9 (Ar–C), 111.3 (Ar–C), 48.7 (NHCH(CH3)CO), 17.9 (NHCH(CH3)CO); m/z (ES+) 319 ([37Cl]MH+), 321 ([35Cl]MH+); HRMS (ES+) found [35Cl]MH+, 319.0962 (C15H16N4O235Cl requires 319.0962).(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-4-(methylthio)-1-oxobutan-2-yl)isonicotinamide, 13, following the standard procedure B, tert-Butyl N-[(1S)-1-[N′-(3-chlorophenyl)hydrazinecarbonyl]-3-(methylsulfanyl)propyl] carbamate  and isonicotinic acid were transformed to afford the title compound as a brown solid ; m.p. 78–81 °C; νmax (ATR) 3259, 2919, 1659, 1622, 1597, 0521, 1475, 1407, 1385, 1310, 1229, 1151, 1096, 1065, 992, 834 cm−1; δH  10.03 , 8.98 , 8.75 , 8.09 , 7.84 , 7.15 , 6.78–6.62 , 4.60 ), 2.67–2.53 ), 2.09–1.98 ; δC  171.6 (CONHNH), 165.8 (PyCONH), 151.3 (ipso-Ar–C), 150.6 (Py-C), 141.4 (ipso-Ar–C), 134.0 (ipso-Ar–C), 131.3 (ipso-Ar–C), 130.8 (Ar–C), 122.1 (Py-C), 118.3 (Ar–C), 111.9 (Ar–C), 52.3 (NHCH(CH2CH2SCH3)), 31.2 (NHCH(CH2CH2SCH3)), 30.4 (NHCH(CH2CH2SCH3)), 15.1 (NHCH(CH2CH2SCH3)); m/z (ES+) 379 ([35Cl]MH+), 381 ([37Cl]MH+); HRMS (ES+) found [35Cl]MH+, 379.0994 (C17H20N4O2S35Cl requires 379.0995).(S)-N-(1-Oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)pyrazine-2-carboxamide, 14, following the standard procedure B, tert-butyl (S)-(1-oxo-1-(2-(4-(trifluoromethyl) phenyl)hydrazineyl)propan-2-yl)carbamate  and pyrazinoic acid were transformed following flash chromatography ; Rf 0.19 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); νmax 3255 (N–H), 3098, 2981 (C–H), 1638 (C=O), 1617, 1534, 1326 (C–F), 1156 (C–N), 1101, 1065 (C–F) cm−1; δH  10.10 , 9.22 , 8.91 , 8.87 , 8.77 , 8.43 , 7.48 , 6.84 , 4.60 CO), 1.50 CO); δC  172.2 (CONHNH), 163.1 (PyzCO), 152.8 (ipso-Ar–C), 148.2 (Pyz-C), 144.9 (ipso-Pyz-C), 143.99 (Pyz-C), 143.85 (Pyz-C), 126.6 (Ar–C), 123.3 , 118.5 , 111.9 (Ar–C), 47.9 (NHCH(CH3)CO), 18.7 (NHCH(CH3)CO); δF  -59.31 ; m/z (ES+) 354 (MH+); HRMS (ES+) found MH+, 354.1188 (C15H15F3N5O2 requires 354.1178).(S)-N-(4-(methylthio)-1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)butan-2-yl)pyrazine-2-carboxamide, 15, following the standard procedure B, tert-Butyl N-[(1S)-3-(methylsulfanyl)-1-{N′-[4-(trifluoromethyl)phenyl]hydrazine carbonyl}propyl]carbamate  and pyrazinoic acid were transformed to afford the title compound as a yellow semisolid ; νmax (ATR) 3264, 1661, 1616, 1519, 1404, 1322, 1157, 1105, 1064, 1019, 838, 832, 771 cm−1; δH  9.36 , 9.06 , 8.78 , 8.53 , 8.43 , 7.40 , 6.82 , 4.99 ), 2.63 ), 2.35–2.10 ; δC  171.2 (CONHNH), 163.7 (Pyz-CONH), 150.4 (ipso-Ar–C), 147.9 (Pyz-C), 145.0 (ipso-Pyz-C), 144.3 (Pyz-C), 143.4 (ipso-Ar–C), 142.8 (Pyz-C), 126.6 (Ar–C), 122.6 , 112.5 (Ar–C), 50.7 (NHCH(CH2CH2SCH3)), 30.94 (NHCH(CH2CH2SCH3)), 30.15 (NHCH(CH2CH2SCH3)), 15.4 (NHCH(CH2CH2SCH3)); m/z (ES+) 414 (MH+); HRMS (ES+) found MH+, 414.1207 (C17H19F3N5O2S requires 414.1212).(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)pyrazine-2-carboxamide, 16, following the standard procedure B, tert-butyl (S)-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)carbamate  and pyrazinoic acid were transformed to afford the title compound as a white solid  as a mixture of rotamers [6:1]; Rf 0.21 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); νmax (ATR) 3383, 3293 (N–H), 3029 (Ar-CH), 2922, 2865 (C–H), 1681, 1659 (C=O) 1596, 1536, 1092, 1018, 886 cm−1; NMR data given for major rotamer δH  9.35 , 8.89 , 8.76 , 8.53 , 8.34 , 7.06 , 6.79 , 6.76 , 6.65 , 4.85 CO), 1.57 CO); δC  172.1 (CONHNH), 163.5 (Pyz-CONH), 149.0 (ipso-Ar–C), 147.8 (Pyz-C), 144.3 (Pyz-C), 143.6 (ipso-Pyz-C), 142.8 (Pyz-C), 135.0 (ipso-Ar–C), 130.2 (Ar–C), 121.1 (Ar–C), 113.4 (Ar–C), 111.7 (Ar–C), 47.58 (NHCH(CH3)CO), 17.7 (NHCH(CH3)CO); m/z (ES+) 320 ([35Cl]MH+), 322 ([37Cl]MH+); HRMS (ES+) found [35Cl]MH+, 320.0914 (C14H15N5O235Cl requires 320.0914).(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-4-(methylthio)-1-oxobutan-2-yl)pyrazine-2-carboxamide, 17, following the standard procedure B, tert-Butyl N-[(1S)-1-[N′-(3-chlorophenyl)hydrazinecarbonyl]-3-(methylsulfanyl)propyl] carbamate  and pyrazinoic acid were transformed to afford the title compound as a white solid ; m.p. 99–102 °C; νmax (ATR) 3333, 3290, 2921, 1708, 1678, 1652, 1596, 1585, 1523, 1482, 1438, 1425, 1404, 1375, 1318, 1299, 1272, 1234, 1221, 1188, 1163, 1114, 1091, 1071, 1052, 1023, 991, 958, 946, 914, 864, 809 cm−1; δH  10.03 , 9.22 , 9.00 , , 8.91 , 8.78 , 8.10 , 7.16 , 6.74–6.62 , 4.70 ), 2.59–2.50 ), 2.18–2.03 ; δC  171.2 (CONHNH), 163.7 (Pyz-CONH), 151.3 (ipso-Ar–C), 148.1 (Pyz-C), 145.0 (ipso-Pyz-C), 144.1 (Pyz-C), 143.8 (Pyz-C), 134.0 (ipso-Ar–C), 130.8 (Ar–C), 118.4 (Ar–C), 111.9 (Ar–C), 111.3 (Ar–C), 51.7 (NHCH(CH2CH2SCH3)), 31.6 (NHCH(CH2CH2SCH3)), 30.2 (NHCH(CH2CH2SCH3)), 15.1 (NHCH(CH2CH2SCH3)); m/z (ES+) 380 ([35Cl]MH+), 382 ([37Cl]MH+); HRMS (ES+) found [35Cl]MH+, 380.0948 (C16H1935ClN5O2S requires 380.0948).(S)-N-(1-Oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)-2-naphthamide, 18, following the standard procedure B, tert-butyl (S)-(1-oxo-1-(2-(4-(trifluoromethyl) phenyl)hydrazineyl)propan-2-yl)carbamate  and 2-napthoic acid were transformed to afford the title compound as a white solid ; Rf 0.24 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); νmax (ATR) 3329, 3264 (N–H), 3051, 2995, 2854, 1674, 1618 (C=O), 1525, 1325 (C–F), 1160 (C–N), 1095, 1065 (C–F), 834 (C–C) cm−1; δH  10.04 , 8.82 , 8.55 , 8.42 , 8.08–7.97 , 7.66–7.58 , 7.47 , 6.87 , 4.60 ), 1.49 ); δC  173.1 (CONHNH), 166.9 (NpCONH), 153.0 (ipso-Ar–C), 134.7 (ipso-Ar–C), 132.5 (ipso-Ar–C), 131.7 (ipso-Ar–C), 129.3 (Np–C), 128.4 (Np–C), 128.4 (Np–C), 128.2 (Ar–C), 128.1 (Np–C), 127.2 (Np–C), 126.6 (Np–C), 125.0 (Np–C), 123.7 , 118.6 , 112.0 (Ar–C), 48.7 (NHCH(CH3)), 18.2 (NHCH(CH3)); δF  -59.3 ; m/z (ES+) 402 (MH+); HRMS (ES+) found MH+, 402.1427 (C21H19F3N3O2 requires 402.1429).(S)-N-(4-(methylthio)-1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)butan-2-yl)-2-naphthamide, 19, following the standard procedure B, tert-Butyl N-[(1S)-3-(methylsulfanyl)-1-{N′-[4-(trifluoromethyl)phenyl]hydrazine carbonyl}propyl]carbamate  and 2-naphthoic acid were transformed to afford the title compound as a white solid ; m.p. 163–166°C; νmax (ATR) 3264, 1666, 1635, 1615, 1520, 1503, 1432, 1325, 1232, 1159, 1103, 1065, 1011, 955, 897, 865, 838 cm−1; δH  10.12 , 8.83 , 8.56 , 8.44 , 8.10–7.94 , 7.65–7.55 , 7.60 , 6.87 , 4.69 ), 2.74–2.55 ), 2.19–2.06 ; δC  172.1 (CONHNH), 167.4 (CONH), 152.9 (ipso-Ar–C), 134.7 (ipso-Ar–C), 134.0 (ipso-Ar–C), 132.5 (ipso-Ar–C), 131.8 (ipso-Ar–C), 129.3 (Np–C), 128.4 (Np–C), 128.2 (Np–C), 128.1 (Np–C), 127.2 (Np–C), 126.6 (Ar–C), 125.0 (Np–C), 118.8 , 111.9 (Ar–C), 52.3 (NHCH(CH2CH2SCH3)), 31.5 (NHCH(CH2CH2SCH3)), 30.5 (NHCH(CH2CH2SCH3)), 15.2 (NHCH(CH2CH2SCH3)); δF  59.26 ; m/z (ES+) 462 (MH+), 484 (MNa+); HRMS (ES+) found MH+, 462.1465 (C23H23F3N3O2S requires 462.1463).(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)-2-naphthamide, 20, following the standard procedure B, tert-butyl (S)-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)carbamate  and 2-napthoic acid were transformed to afford the title compound as a white solid ; Rf 0.32 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); νmax (ATR) 3331, 3236 (NH), 3062 (Ar-CH), 2983, 2926 (CH), 1634, 1626 (C=O),1599, 1536, 1536 (C=C), 1334 (C–N) cm−1; δH  9.96 , 8.82 , 8.56 , 8.07–7.95 , 7.67–7.57 , 7.14 , 6.77 , 6.73–6.69 , 4.58 CO), 1.47 CO); δC  173.0 (CHCONHNH), 166.9 (Np-CONH), 151.4 (Ar–C), 134.7 (Np–C), 134.0 (ipso-Ar–C), 132.5 (Np–C), 131.8 (Np–C), 130.8 (Ar–C), 129.3 (Np–C), 128.3 (Np–C), 128.2 (Np–C), 128.1 (Np–C), 127.2 (Np–C), 125.0 (Np–C), 118.2 (Ar–C), 111.9 (Ar–C), 111.3 (Ar–C), 48.7 (NHCH(CH3)CO), 18.1 (NHCH(CH3)CO); m/z (ES+) 368 ([35Cl]MH+), 370 ([37Cl]MH+); HRMS (ES+) found .(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-4-(methylthio)-1-oxobutan-2-yl)-2-naphthamide, 21, following the standard procedure B, tert-Butyl N-[(1S)-1-[N′-(3-chlorophenyl)hydrazinecarbonyl]-3-(methylsulfanyl)propyl] carbamate  and 2-naphthoic acid were transformed to afford the title compound as a white solid ; m.p. 205–208 °C; νmax (ATR) 3224, 1636, 1625, 1597, 1527, 1494, 1452, 1440, 1335, 1303, 1268, 1231, 1201, 1076, 989, 955, 892, 863, 839 cm−1; δH  10.04 , 8.85 , 8.56 , 8.11 , 8.10–7.97 , 7.68–7.54 , 7.15 , 6.77 , 6.73–6.65 , 4.67 ), 2.68–2.56 ), 2.12–2.06 ; δC  172.0 (CHCONHNH), 167.4 (Np-CONH), 151.4 (Ar–C), 134.7 (Np–C), 134.0 (ipso-Ar–C), 132.5 (Np–C), 131.8 (Np–C), 130.8 (Ar–C), 129.3 (Np–C), 128.3 (Np–C), 128.2 (Np–C), 128.1 (Np–C), 127.7 (Np–C), 125.0 (Np–C), 118.3 (Ar–C), 111.9 (Ar–C), 111.3 (Ar–C), 52.3 (NHCH(CH2CH2SCH3)), 31.4 (NHCH(CH2CH2SCH3)), 30.5 (NHCH(CH2CH2SCH3)), 15.1 (NHCH(CH2CH2SCH3)); m/z (ES+) 428 ([35Cl]MH+), 430 ([37Cl]MH+); HRMS (ES+) found [35Cl]MH+, 428.1201 (C22H2335ClN3O2S requires 428.1200).(S)-N-(1-Oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)-1-naphthamide, 22, following the standard procedure B, tert-butyl (S)-(1-oxo-1-(2-(4-(trifluoromethyl) phenyl)hydrazineyl)propan-2-yl)carbamate  and 1-napthoic acid were transformed to afford the title compound as a white solid ; Rf 0.24  3315, 3242 (N–H), 3074 (C–H), 2999, 2865 (C–H), 1667, 1633 (C=O), 1617, 1536, 1324 (C–F), 1159 (C–N), 1095, 1065 (C–F), 828, 787, 776 (C–C) cm−1; δH  10.10 , 8.83 , 8.47 , 8.28 , 8.03 , 7.98  7.68 , 7.59–7.50 , 7.47 , 6.91 , 4.60 CO), 1.44 CO); δC  173.1 (CONHNH), 167.3 (NpCONH), 153.0 (ipso-NHNHAr–C), 134.1 (Np–C), 134.0 (Np–C), 133.9 (Np–C), 130.2 (Np–C), 128.6 (Np–C), 126.9 (Np–C), 126.7 (Np–C), 126.6 (Ar–C), 126.1 (Np–C), 125.9 (Np–C), 125.3 (Np–C), 118.4 , 112.0 (Ar–C), 48.5 (NHCH(CH3)CO), 18.0 (NHCH(CH3)CO); δF  −59.25 ; m/z (ES+) 402 (MH+); HRMS (ES+) found MH+, 402.1433 (C21H19F3N3O2 requires 402.1429).(S)-N-(4-(methylthio)-1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)butan-2-yl)-1-naphthamide, 23, following the standard procedure B, tert-Butyl N-[(1S)-3-(methylsulfanyl)-1-{N′-[4-(trifluoromethyl)phenyl]hydrazine carbonyl}propyl]carbamate  and 1-naphthoic acid were transformed to afford the title compound as a white solid ; m.p. 189–192 °C; νmax (ATR) 3239, 1671, 1635, 1616, 1592, 1521, 1439, 1414, 1378, 1325, 1256, 1231, 1182, 1104, 1065, 1011, 966, 939, 886, 834 cm−1; δH  10.18 , 8.86 , 8.50 , 8.27 , 8.08–7.94 , 7.66–7.52 , 7.47 , 6.92 , 4.70 ), 2.74–2.55 ), 2.15–2.01 ; δC  172.1 (CONHNH), 167.3 (NpCONH), 153.0 (ipso-Ar–C), 134.7 (ipso-Ar–C), 133.6 (ipso-Ar–C), 130.4 (ipso-Ar–C), 130.3 (Np–C), 128.6 (Np–C), 127.0 (Np–C), 126.7 (Ar–C), 126.5 (ipso-Ar–C), 126.1 (Np–C), 126.0 (Np–C), 125.4 (Np–C), 125.4 (Np–C), 118.3 , 112.0 (Ar–C), 52.1 (NHCH(CH2CH2SCH3)), 31.4 (NHCH(CH2CH2SCH3)), 30.4 (NHCH(CH2CH2SCH3)), 15.1 (NHCH(CH2CH2SCH3)); δF  59.27 ; m/z (ES+) 462 (MH+), 484 (MNa+); HRMS (ES+) found MH+, 462.1468 (C23H23F3N3O2S requires 462.1463).(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)-1-naphthamide, 24, following the standard procedure B, tert-butyl (S)-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)carbamate  and 1-napthoic acid were transformed to afford the title compound as a white solid ; νmax (ATR) 3404, 3237 (NH), 3052 (Ar-CH), 2995, 2946 (CH), 1634 (C=O), 1595 (C=C), 1324 (C–N) cm−1; δH  10.00 , 8.83 , 8.32 , 8.15 , 8.03 , 7.98 , 7.68 , 7.60–7.50 , 7.16 , 6.83 , 6.76–6.71 , 4.59 CO), 1.42 CO); δC  173.1 (CONHNH), 169.2 (Np-CONH), 151.4 (Ar–C), 134.6 (Np–C), 134.0 (Ar–C), 133.5 (Np–C), 130.8 (Ar–C), 130.3 (Np–C), 128.5 (Np–C), 127.0 (Np–C), 126.6 (Np–C), 126.2 (Np–C), 126.0 (Np–C), 125.3 (Np–C), 118.3 (Ar–C), 111.9 (Ar–C), 111.3 (Ar–C), 48.4 (NHCH(CH3)CO), 18.0 (NHCH(CH3)CO); m/z (ES+) 368 ([35Cl]MH+), 370 ([37Cl]MH+); HRMS (ES+) found .(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-4-(methylthio)-1-oxobutan-2-yl)-1-naphthamide, 25, following the standard procedure B, tert-Butyl N-[(1S)-1-[N′-(3-chlorophenyl)hydrazinecarbonyl]-3-(methylsulfanyl)propyl] carbamate  and 1-naphthoic acid were transformed to afford the title compound as a pale yellow solid ; m.p. 183–186 °C; νmax (ATR) 3239, 1667, 1635, 1594, 1524, 1471, 1435, 1313, 1255, 1216, 1074, 992, 885, 851, 765 cm−1; δH  10.07 , 8.87 , 8.27 , 8.15 , 8.05–7.93 , 7.69 , 7.63–7.50 , 7.17 , 6.82 , 6.76–6.70 , 4.68 ), 2.70–2.58 ), 2.15–2.01 ; δC  172.1 (CONHNH), 169.5 (NpCONH), 151.4 (ipso-Ar–C), 134.6 (ipso-Ar–C), 134.0 (ipso-Ar–C), 130.4 (ipso-Ar–C), 130.3 (Np–C), 128.6 (Np–C), 127.1 (Np–C), 126.7 (Ar–C), 126.7 (ipso-Ar–C), 126.1 (Np–C), 125.4 (Np–C), 118.3 (ipso-Ar–C), 112.0 (Ar–C), 111.4 (Ar–C), 52.1 (NHCH(CH2CH2SCH3)), 31.4 (NHCH(CH2CH2SCH3)), 30.4 (NHCH(CH2CH2SCH3)), 15.1 (NHCH(CH2CH2SCH3)); m/z (ES+) 428 ([35Cl]MH+), 430 ([37Cl]MH+); HRMS (ES+) found [35Cl]MH+, 428.1192 (C22H23N3O2S35Cl requires 428.1200).(S)-N-(1-Oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)quinoline-3-carboxamide, 26, following the standard procedure B, tert-butyl (S)-(1-oxo-1-(2-(4-(trifluoromethyl) phenyl)hydrazineyl)propan-2-yl)carbamate  and 3-quinolinecarboxylic acid were transformed to afford the title compound as a white solid ; Rf 0.15 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); νmax (ATR) 3259 (N–H), 2998, 2935 (C–H), 1637, 1617 (C=O), 1522, 1450, 1325, 1158, 1103 1065 (C–F) cm−1; δH  10.09 , 9.34 , 9.06 , 8.93 , 8.43 , 8.11 , 7.89 , 7.71 , 7.47 , 6.87 , 4.63 CO), 1.50 CO); δC  172.8 (CONHNH), 165.6 (Quin-CO), 152.9 (ipso-Ar–C), 149.7 (Quin-C), 148.9 (ipso-Ar–C), 136.4 (Quin-C), 131.7 (Quin-C), 129.6 (ipso-Ar–C), 129.2 (Quin-C), 127.9 (Quin-C), 127.1 (ipso-Ar–C), 126.6 (ipso-Ar–C), 124.2 , 118.6 , 111.9 (Ar–C), 48.8 (NHCH(CH3)CO), 18.1 (NHCH(CH3)CO); δF  -59.28 ; m/z (ES+) 403 (MH+); HRMS (ES+) found MH+, 403.1367 (C20H18F3N4O2 requires 403.1382).(S)-N-(4-(methylthio)-1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)butan-2-yl)quinoline-3-carboxamide, 27, following the standard procedure B, tert-Butyl N-[(1S)-3-(methylsulfanyl)-1-{N′-[4-(trifluoromethyl)phenyl]hydrazine carbonyl}propyl]carbamate  and 3-quinolinecarboxylic acid were transformed to afford the title compound as a beige solid ; m.p. 138–141 °C; νmax (ATR) 3258, 3001, 1636, 1616, 1538, 1496, 1418, 1328, 1231, 1185, 1158, 1098, 1066, 1009, 959, 917, 902, 865, 832 cm−1; δH  10.15 , 9.34 , 9.06 , 8.93 , 8.45 , 8.12 , 7.88 , 7.71 , 7.47 , 6.85 , 4.70 ), 2.75–2.56 ), 2.20–2.05 ; δC  171.9 (CONHNH), 166.0 (Quin-CO), 152.9 (ipso-Ar–C), 149.7 (Quin-C), 148.9 (ipso-Ar–C), 136.4 (Quin-C), 131.7 (Quin-C), 129.6 (ipso-Ar–C), 129.3 (Quin-C), 127.9 (Quin-C), 127.1 (ipso-Ar–C), 126.9 (Ar–C), 123.7 , 118.6 , 112.0 (Ar–C), 52.4 (NHCH(CH2CH2SCH3)), 31.4 (NHCH(CH2CH2SCH3)), 30.5 (NHCH(CH2CH2SCH3)), 15.1 (NHCH(CH2CH2SCH3)); δF  -59.27 ; m/z (ES+) 463 (MH+); HRMS (ES+) found MH+, 463.1416 (C22H22F3N4O2S requires 463.1416).(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)quinoline-3-carboxamide, 28, following the standard procedure B, tert-butyl (S)-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)carbamate  and 3-quinolinecarboxylic acid were transformed to afford the title compound as a white solid ; Rf 0.29 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); νmax (ATR) 3324 (N–H), 2988, 2947 (C–H), 1646, 1613 (C=O), 1513 (C=N), 1151, 1103, 1065 (C–F) cm−1; δH  10.00 , 9.35 , 9.08 , 8.93 , 8.15–8.07 , 7.89 , 7.72 , 7.15 , 6.78 , 6.71 , 4.60 ), 1.48 ); δC  172.7 (CONHNH), 165.6 (Quin-CONH), 151.4 (ipso-Ar–C), 149.7 (Quin-C), 149.0 (ipso-Quin-C), 136.4 (Quin-C), 134.0 (ipso-Ar–C), 131.7 (Quin-C), 130.8 (Ar–C), 129.6 (Quin-C), 129.3 (Quin-C), 127.9 (Quin-C), 127.1 (ipso-Ar–C), 126.9 (ipso-Ar–C), 118.3 (Ar–C), 111.9 (Ar–C), 111.3 (Ar–C), 48.7 (NHCH(CH3)), 18.1 (NHCH(CH3)); m/z (ES+) 369 ([35Cl]MH+), 371 ([37Cl]MH+); HRMS (ES+) found [35Cl]MH+, 369.1120 (C19H18N4O235Cl requires 369.1118).(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-4-(methylthio)-1-oxobutan-2-yl)quinoline-3-carboxamide, 29, following the standard procedure B, tert-Butyl N-[(1S)-1-[N′-(3-chlorophenyl)hydrazinecarbonyl]-3-(methylsulfanyl)propyl] carbamate  and 3-quinolinecarboxylic acid were transformed to afford the title compound as a white solid ; m.p. 141–144 °C; νmax (ATR) 3211, 3029, 2913, 1664, 1638, 1595, 1570, 1534, 1492, 1424, 1374, 1307, 1272, 1229, 1203, 1155, 1136, 1112, 1080, 1019, 993, 981, 960, 948, 914, 900, 876, 855 cm−1; δH  10.08 , 9.35 , 9.07 , 8.93 , 8.15–8.07 , 7.89 , 7.72 , 7.15 , 6.77 , 6.70 , 4.67 ), 2.71–2.59 ), 2.16–2.06 ; δC  171.8 (CONHNH), 166.0 (Quin-CONH), 151.4 (ipso-Ar–C), 149.7 (Quin-C), 149.0 (ipso-Quin-C), 136.4 (Quin-C), 134.0 (ipso-Ar–C), 131.7 (Quin-C), 130.8 (Ar–C), 129.6 (Quin-C), 129.3 (Quin-C), 127.9 (Quin-C), 127.1 (ipso-Ar–C), 126.9 (ipso-Ar–C), 118.3 (Ar–C), 111.9 (Ar–C), 111.4 (Ar–C), 52.4 (NHCH(CH2CH2SCH3)), 31.4 (NHCH(CH2CH2SCH3)), 30.4 (NHCH(CH2CH2SCH3)), 15.1 (NHCH(CH2CH2SCH3)); m/z (ES+) 429 ([35Cl]MH+), 431 ([37Cl]MH+); HRMS (ES+) found [35Cl]MH+, 429.1148 (C21H2235ClN4O2S requires 429.1152).(S)-3,5-Dinitro-N-(1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)benzamide, 30, following the standard procedure B, tert-butyl (S)-(1-oxo-1-(2-(4-(trifluoromethyl) phenyl)hydrazineyl)propan-2-yl)carbamate  and 3,5-dinitrobenzoic acid were transformed to afford the title compound as a white solid ; νmax (ATR) 3250 (NH), 3101 (Ar-CH), 2896 (CH), 1641 (C=O), 1617 (Ar C–C), 1535, 1342, 1330, 1160, 1107, 1066 (C–F) cm−1; δH  10.13 , 9.47 , 9.14 , 8.98 , 8.43 , 7.47 , 6.84 , 4.61 CO), 1.49 CO); δC  172.4 (CONHNH), 163.0 (ArCONH), 152.9 (ipso-Ar–C), 148.6 (ipso-Ar–C), 137.1 (ipso-Ar–C), 128.4 (Ar–C), 126.7 (Ar–C), 121.5 (Ar–C), 111.9 (Ar–C), 49.2 (NHCH(CH3)CO), 18.0 (NHCH(CH3)CO); δF  -59.34 ; m/z (ES+) 442 (MH+); HRMS (ES+) found MH+, 442.0958 (C17H15F3N5O6 requires 442.0974).(S)-N-(4-(methylthio)-1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)butan-2-yl)-3,5-dinitrobenzamide, 31, following the standard procedure B, tert-Butyl N-[(1S)-3-(methylsulfanyl)-1-{N′-[4-(trifluoromethyl)phenyl]hydrazine carbonyl}propyl]carbamate  and 3,5-dinitrobenzoic acid were transformed to afford the title compound as a beige solid ; m.p. 97–100 °C; νmax (ATR) 3464, 3212, 3125, 3075, 1662, 1633, 1614, 1541, 1440, 1342, 1322, 1251, 1162, 1115, 1063, 1010, 933, 837 cm−1; δH  10.20 , 9.45 , 9.15 , 8.99 , 8.44 , 7.47 , 6.84 , 4.68 ), 2.74–2.53 ), 2.19–2.04 ; δC  171.4 (CONHNH), 163.4 (ArCONH), 152.8 (ipso-Ar–C), 148.6 (ipso-Ar–C), 137.0 (ipso-Ar–C), 128.5 (Ar–C), 127.3 (ipso-Ar–C), 126.6 (Ar–C), 121.5 (Ar–C), 118.7 , 111.9 (Ar–C), 52.7 (NHCH(CH2CH2SCH3)), 31.2 (NHCH(CH2CH2SCH3)), 30.4 (NHCH(CH2CH2SCH3)), 15.1 (NHCH(CH2CH2SCH3)); δF  -59.30 ; m/z (ES+) 502 (MH+); HRMS (ES+) found MH+, 502.1010 (C19H19F3N5O6S requires 502.1008).(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)-3,5-dinitrobenzamide, 32, following the standard procedure B, tert-butyl (S)-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)carbamate  and 3,5-dinitrobenzoic acid were transformed to afford the title compound as a light brown solid ; Rf 0.21 (DCM/EtOH/ sat. aq. NH3 [200:8:1]); νmax (ATR) 3264 (NH), 3094 (Ar-CH), 2983, 2926 (CH), 1651, 1597 (C=O), 1537, 1342 (N–O), 1334 (C=C) cm−1; δH  10.05 , 9.48 , 9.15 , 8.99 , 8.10 , 7.14 , 6.76 , 6.73–6.66 , 4.57 CO), 1.48 CO); δC  172.3 (CONHNH), 163.0 (ArCONH), 151.3 (ipso-Ar–C), 148.5 (ipso-Ar–C), 137.1 (ipso-Ar–C), 134.0 (ipso-Ar–C), 130.7 (Ar–C), 128.4 (Ar–C), 118.3 (Ar–C), 111.9 (Ar–C), 111.3 (Ar–C), 49.2 (NHCH(CH3)CO), 17.9 (NHCH(CH3)CO); m/z (ES+) 408 ([35Cl]MH+), 410 ([37Cl]MH+); HRMS (ES+) found [35Cl]MH+, 408.0701 (C16H15N5O635Cl requires 408.0711).(S)-N-(1-(2-(3-chlorophenyl)hydrazineyl)-4-(methylthio)-1-oxobutan-2-yl)-3,5-dinitrobenzamide, 33, following the standard procedure B, tert-Butyl N-[(1S)-1-[N′-(3-chlorophenyl)hydrazinecarbonyl]-3-(methylsulfanyl)propyl] carbamate  and 3,5-dinitrobenzoic acid were transformed to afford the title compound as a light brown gum ; m.p. 87–89 °C; νmax (ATR) 3259, 3071, 1649, 1640, 1596, 1536, 1476, 1341, 1340, 1320, 1076, 1041, 918, 847 cm−1; δH  10.12 , 9.44 , 9.15 , 8.99 , 8.10 , 7.15 , 6.78–6.62 , 4.68 ), 2.74–2.53 ), 2.19–2.04 ; δC  171.4 (CONHNH), 163.4 (ArCONH), 151.2 (ipso-Ar–C), 148.6 (ipso-Ar–C), 137.1 (ipso-Ar–C), 134.0 (ipso-Ar–C), 130.7 (Ar–C), 128.5 (Ar–C), 121.3 (Ar–C), 118.5 (Ar–C), 111.9 (Ar–C), 111.3 (Ar–C), 52.8 (NHCH(CH2CH2SCH3)), 31.2 (NHCH(CH2CH2SCH3)), 30.4 (NHCH(CH2CH2SCH3)), 15.1 (NHCH(CH2CH2SCH3)); m/z (ES+) 468 ([35Cl]MH+), 470 ([37Cl]MH+); HRMS (ES+) found [35Cl]MH+, 468.0743 (C18H1935ClN5O6S requires 468.0745).(S)-6-chloro-2-ethyl-N-(1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)imidazopyridine-3-carboxamide, 34, following the standard procedure B, tert-butyl (S)-(1-oxo-1-(2-(4-(trifluoromethyl) phenyl)hydrazineyl)propan-2-yl)carbamate  and 6-chloro-2-ethylimidazopyridine-3-carboxylic acid were transformed to afford the title compound as a pale yellow solid ; m.p. 221–224 °C; νmax. (ATR) 3252, 2967, 2932, 1667, 1609, 1541, 1522, 1493, 1453, 1442, 1391, 1329, 1281, 1251, 1220, 1160, 1097, 1066, 1009, 995, 952, 906 cm−1; δH  10.10 , 9.01 , 8.47 , 8.33 CO), 7.68 , 7.51–7.45 , 6.91 , 4.60 CO), 3.00 , 1.46 CO), 1.26 ; δC  172.8 (CONHNH), 161.1 (CONH), 153.0 (ipso-Ar–C), 151.8 (ipso-Ar–C), 143.7 (ipso-Ar–C), 127.5 (ipso-Ar–C), 126.6 (Ar–C), 125.2 (Ar–C), 120.1 (ipso-Ar–C), 117.7 (Ar–C), 112.0 (Ar–C), 48.3 (NHCH(CH3)CO), 22.2 (CH2CH3), 18.2 (NHCH(CH3)CO), 13.5 (CH2CH3); m/z (ES+) 454 ([35Cl]MH+), 456 ([37Cl]MH+); HRMS (ES+) found [35Cl]MH+, 454.1256 (C20H2035ClF3N5O2 requires 454.1258).(S)-6-chloro-2-ethyl-N-(4-(methylthio)-1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)butan-2-yl)imidazopyridine-3-carboxamide, 35, following the standard procedure B, tert-Butyl N-[(1S)-3-(methylsulfanyl)-1-{N′-[4-(trifluoromethyl)phenyl]hydrazine carbonyl}propyl]carbamate  and 6-chloro-2-ethylimidazopyridine-3-carboxylic acid were transformed to afford the title compound as a white solid ; m.p. 227–230 °C; νmax (ATR) 3248, 1664, 1612, 1526, 1487, 1417, 1394, 1374, 1328, 1291, 1189, 1158, 1108, 1066, 1013, 1000, 924, 897, 838 cm−1; δH  10.16 , 8.96 , 8.48 , 8.42 , 7.67 , 7.52–7.45 , 6.92 , 4.70 ), 3.00 , 2.75–2.56 ), 2.20–2.05 , 1.28 ; δC  171.9 (CONHNH), 161.6 (CONH), 152.9 (ipso-Ar–C), 151.8 (ipso-Ar–C), 143.7 (ipso-Ar–C), 127.5 (ipso-Ar–C), 126.5 (Ar–C), 125.1 (Ar–C), 120.1 (ipso-Ar–C), 117.7 (Ar–C), 116.3 (ipso-Ar–C), 112.0 (Ar–C), 52.4 (NHCH(CH2CH2SCH3)), 31.2 (NHCH(CH2CH2SCH3)), 30.4 (NHCH(CH2CH2SCH3)), 22.2 (CH2CH3), 15.1 (NHCH(CH2CH2SCH3)), 13.4 (CH2CH3); δF  -59.32 ; m/z (ES+) 514 ([35Cl]MH+), 516 ([37Cl]MH+); HRMS (ES+) found [35Cl]MH+, 514.1290 (C22H2435ClF3N5O2S requires 514.1291).(S)-6-chloro-N-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)-2-ethylimidazopyridine-3-carboxamide, 36, following the standard procedure B, tert-butyl (S)-(1-(2-(3-chlorophenyl)hydrazineyl)-1-oxopropan-2-yl)carbamate  and 6-chloro-2-ethylimidazopyridine-3-carboxylic acid were transformed to afford the title compound as a white solid ; m.p. 213–215 °C; νmax. (ATR) 3258, 2978, 1666, 1608, 1605, 1538, 1492, 1455, 1417, 1394, 1370, 1317, 1269, 1248, 1218, 1159, 1139, 1110, 1075, 1044, 993, 950, 906, 850, 802 cm−1; δH  10.00 , 9.07 , 8.31 CO), 8.13 , 7.68 , 7.48 , 7.15 , 6.85 , 6.74–6.70 , 4.54 CO), 3.00 , 1.46 CO), 1.28 ; δC  172.8 (CONHNH), 161.1 (CONH), 151.9 (ipso-Ar–C), 151.3 (ipso-Ar–C), 143.7 (ipso-Ar–C), 134.1 (ipso-Ar–C), 130.7 (Ar–C), 127.6 (Ar–C), 125.3 (Ar–C), 120.1 (ipso-Ar–C), 118.3 (ipso-Ar–C), 117.7 (Ar–C), 116.3 (ipso-Ar–C), 112.0 (Ar–C), 111.4 (Ar–C), 48.3 (NHCH(CH3)CO), 22.2 (CH2CH3), 18.1 (NHCH(CH3)CO), 13.4 (CH2CH3); m/z (ES+) 420 , 422 , 424 ; HRMS (ES+) found MH+, 420.0992 .(S)-6-chloro-N-(1-(2-(3-chlorophenyl)hydrazineyl)-4-(methylthio)-1-oxobutan-2-yl)-2-ethylimidazopyridine-3-carboxamide, 37, following the standard procedure B, tert-Butyl N-[(1S)-1-[N′-(3-chlorophenyl)hydrazinecarbonyl]-3-(methylsulfanyl)propyl] carbamate  and 6-chloro-2-ethylimidazopyridine-3-carboxylic acid were transformed to afford the title compound as a white solid ; m.p. 188–191 °C; νmax (ATR) 3334, 2916, 1666, 1603, 1519, 1489, 1417, 1396, 1341, 1305, 1288, 1212, 1151, 1120, 1048, 998, 963, 872, 847, 814 cm−1; δH  10.07 , 9.04 , 8.41 , 8.15 , 7.70 , 7.48 , 7.16 , 6.86 , 6.76–6.68 , 4.59 ), 3.00 , 2.68–2.56 ), 2.12–2.06 , 1.28 ; δC  172.0 (CONHNH), 161.6 (CONH), 152.0 (ipso-Ar–C), 151.3 (ipso-Ar–C), 143.8 (ipso-Ar–C), 134.1 (ipso-Ar–C), 130.7 (Ar–C), 127.6 (Ar–C), 125.3 (Ar–C), 120.1 (ipso-Ar–C), 118.3 (ipso-Ar–C), 117.7 (Ar–C), 116.4 (ipso-Ar–C), 112.0 (Ar–C), 111.4 (Ar–C), 52.0 (NHCH(CH2CH2SCH3)), 31.2 (NHCH(CH2CH2SCH3)), 31.1 (NHCH(CH2CH2SCH3)), 22.3 (CH2CH3), 15.1 (NHCH(CH2CH2SCH3)), 13.6 (CH2CH3); m/z (ES+) 480 , 482 , 484 ; HRMS (ES+) found MH+, 480.1026 ..Mycobacterial species were cultured in either Middlebrook 7H9 broth or Middlebrook 7H10 agar media supplemented with albumin–dextrose–catalase (ADC) or oleic acid–albumin–dextrose–catalase (OADC) enrichments and 0.2% glycerol, 0.2% casamino acids, 24 μg/mL pantothenate, 1 μg/mL penicillin G, 10 μg/mL cyclohexamide, purchased from BD Biosciences. All reagents were purchased from Sigma-Aldrich, Gillingham, UK unless stated otherwise.Mtb mc27000 mono-resistant mutants were generated by plating 1 × 108 mid-log cells on solid media  containing 0.1, 0.2, 0.4, 1.6 and 3.2 μg/mL of RIF or INH. Plates were incubated at 37 °C, 5% CO2 until the growth of colonies was visible. Spontaneous resistant mutants were confirmed by minimum inhibitory concentration (MIC) testing in liquid media as described below (REMA). The genomic DNA from validated mutants was extracted, and Next Generation Sequencing (NGS) analysis was performed to confirm mutation locations.Mycobacterium was cultured in Middlebrook 7H9 broth supplemented with 10% OADC and 0.2% glycerol, 0.2% casamino acids, 24 μg/mL pantothenate, 1 μg/mL penicillin G, 10 μg/mL cyclohexamide and 0.05% Tween 80 for one week at 37 °C in an atmosphere of 5% CO2. The concentration was adjusted at McFarland 0.5 and diluted to 1 × 106 CFU/mL and diluted in growth media 1:25. A 100 μL of the inoculum was added to each well of a 96-well microplate together with 100 μL of the compounds. The plate was incubated at 37 °C in an atmosphere of 5% CO2. After 5 days, 10 μL 0.2% (w/v) resazurin (solubilized in sterile water) was added. MIC99 was defined as the lowest concentration resulting in 99% inhibition of growth of Mycobacterium resulting in no colour change. Samples were set up in quadruplet and tested in two independent assays.Bacterial minimum inhibition concentrations (MIC) were determined using the resazurin microtiter assay (REMA) method, according to Palomino et al. . Stock s2. Cells were passaged after reaching 90% confluence.RAW 264.7 cell lines were purchased from American Type Culture Collection, Middlesex, UK (ATCC1 TIB-71™). Cells were cultured in DMEM high glucose medium, supplemented with 10% FBS, 2 mM L-Glutamine and 1% penicillin/streptomycin, at 37 °C in a humidified incubator containing 5% CO4 cells/well in 200 µL of media and incubated for 24 h. The media on the cells was removed and replaced with 180 µL of fresh media. To each respective well, 20 µL of test compound at a concentration of 1 mM was added, resulting in a final compound concentration of 100 µM. To control wells, DMSO drug solvent was added. In all wells, the final concentration of DMSO did not exceed 0.1%. Following incubation for a further 96 h at 37 °C in a humidified incubator containing 5% CO2, all media was removed and replaced with 100 µL of fresh media. To each well, 20 µL of 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay solution  was added, and the plate was incubated in the dark at 37 °C for 4 h. Finally, the absorbance of each well was recorded at 490 nm using a microplate reader. The percentage of cell viability was calculated relevant to the compound solvent (DMSO) control.Cells, in logarithmic growth, were plated in 96-well plates at a density of 1 × 10Mtb (With the end TB strategy faltering and the pipeline for new TB drugs being minimal, the requirement for novel architectures to combat the rise in drug-resistant TB infections is of paramount importance. The existing benzoxa--diazole amino acid hydrazide scaffolds were modified in order to demonstrate that the removal of the benzoxa--diazole moiety and replacement, utilising a scaffold hopping approach with relevant pharmacophores from existing anti-tubercular drugs, was a feasible tactic. The use of known chemical moieties present in molecules with anti-TB activity, such as quinoline or imidazo pyridine-3-carboxy substituents, have provided a variety of antibacterial activities similar or greater to that with the benzoxa--diazole moiety and molecules with defined selectivity against drug-resistant strains of N-substituted amino acid hydrazides have provided a versatile scaffold for elaboration with known pharmacophores from existing drugs to provide active and clinically translatable anti-tubercular drugs. Whilst the exact mechanism of action is not understood, phenotypic observations have shown that these compounds favour isoniazid-resistant Mtb strains and so may target a pathway upstream of the site of INH activity or one involving perturbation of the ETC within mycobacteria. This hypothesis is yet to be tested and is currently under investigation.Overall,


Body integrity identity disorder (BIID) is a rare condition characterised by a discrepancy between specific areas of an individual’s perceived body image and body schema which causes the individual to disassociate those physical areas of their body from their internal representation. There are currently no efficacious, ethically unambiguous means for achieving long-lasting symptom reductions. In the case we present, two patients with BIID underwent an augmented reality (AR)-based simulation that virtually amputated their alienated limbs, allowing them to experience their ideal selves. During the exposure, both patients reported reductions in BIID-related complaints. These preliminary results suggest the existence of a possible therapeutic and diagnostic potential that AR possesses, which warrants further consideration within clinical healthcare settings. Diagnostic and Statistical Manual of Mental Disorders. The inability to establish a clear consensus on the diagnostic criterion of this disorder ultimately stems from the dearth of studies and diagnostic tools currently available. As such, no standard psychopharmacological or psychotherapeutic treatments for this disorder have been devised. Though controversial, the only method that has been reported to result in definite symptom improvement is to surgically remove the alienated limbs4 16 17Body integrity identity disorder (BIID) is a rare and secretive condition characterised by the feeling of a lack of bodily ownership over a certain healthy limb or limbs, which subsequently causes patients to report a desire to obtain an amputation in order to appear as their ideal self. Due to the taboo nature surrounding this condition, most individuals do not electively present their symptoms to medical professionals and are instead found on internet forums which are mostly private. This current state of affairs makes it especially difficult to provide an accurate assessment of the prevalence of BIID. Individuals with this condition often speak of having an ideal self, which refers to the physical appearance that they feel best reflects their true body schema. It is common that patients are very specific regarding the position of the feeling of undesirability of the limb. The primary motivation for these individuals to seek an amputation is to feel complete or to feel satisfied inside, with sexual motivations often being secondary.Two adult male patients were recruited from a patient group located at the Amsterdam Universitair Medische Centra , Amsterdam, Netherlands). Both patients were diagnosed with BIID with a primary motivation to feel complete or satisfied inside by a certified psychiatrist working in the outpatient clinic who is specialised in body image-related disorders. Neither of the patients had any relevant comorbid diagnoses. One of the patients desires a single below-the-knee amputation on the right leg (patient 1), while the other desires a double above-the-knee amputation (patient 2).Patient 1 reported that the onset of his desires began in early childhood after seeing another child in a wheelchair with a stump amputation. His persistent desires to feel complete inside led him to feeling alone and depressed throughout his childhood and into adulthood. He currently simulates his ideal self by seating himself in a wheelchair and tying his leg down to the chair in a way where his limb folds, creating the appearance of a stump. He simulates his ideal self as often as possible during the day but is only able to perform a simulation for around 1 hour at a time due to his alienated limb becoming temporarily paraesthetic. He has, and still does, consider self-amputation; however, he has never made an attempt due to recognising the danger associated with doing so.Patient 2 similarly reported that the onset of his desires began in his early childhood after meeting a family friend with a one-sided lower leg amputation. As a child, he simulated his desired appearance through limb binding without having any understanding that others had similar desires to his own. Only after the advent of the internet did he discover the extent of his own desires, both socially and clinically. He has also always considered self-amputation, but has never taken any concrete actions to accomplish this.At the time of the current investigation, patient 1 had previously sought medical treatment elsewhere from a physiatrist, an orthopaedic surgeon, and a professor who is specialised in amputations and complex regional pain syndrome with regard to seeking an amputation for his alienated limb. He was never prescribed any medications to help treat his BIID-related complaints. He previously received hypnosis therapy; however, it proved unsuccessful.Patient 2 has never sought medical treatment for his BIID, even though his primary physician has been made aware of his condition. Therefore, he has never been prescribed any medication nor has he undergone any form of psychotherapy. Rather than seeking medical attention, the patient has sought to self-manage his BIID by trying to adapt it into his life, although he still struggles with being able to do so.Neither patient has any family history of BIID or body image-related disorders.In the present study, we investigated the effects of virtual amputation in non-amputated patients in order to determine whether or not this could influence the way they experienced their BIID. We accomplished this by administering an augmented reality telepresence to the ideal-self (ART-IS). Augmented reality (AR) is one particular medium within a larger spectrum of other emerging technologies that are collectively referred to as virtuality technologies. Currently, the two most prevalent virtuality technologies are AR and virtual reality (VR). AR infuses or omits features from an individual’s real environment with digital content in a way that gives the individual an impression that these modifications exist as natural features of their real environment. AR content can be presented on a hand-held device or by using a head-mounted display (HMD), which is a specialised pair of non-transparent glasses that display the virtual content to the user. VR, on the other hand, is an entirely digital experience whereby individuals are completely separated from their real environment and are immersed into a digitally created virtual environment. VR content is necessarily presented on an HMD. While no previous research has specifically investigated the use of virtuality technologies to alter body ownership in patients with BIID, VR has been previously used in different ways to alter the sense of body ownership in healthy populations.1 4We assessed the efficacy of ART-IS by performing and comparing symptom provocations in two different tasks, AR on and AR off , using aEach task consisted of three sequences. Each sequence contained four blocks, each of which represented a different anatomical region. The blocks were presented pseudorandomly, such that each block could not be repeated back-to-back across or within sequences. The anatomical regions were (A) above the left knee, (B) below the left knee, (C) above the right knee and (D) below the right knee. Within each block, we administered six stimuli, each of which had a duration of 0.2 ms. The interstimulus interval was set to 20 s, which allowed us to obtain a clear and analysable physiological signal. The intersequence interval was set to 2 min in order to allow us a sufficient amount of time to administer a verbal Likert scale (VLS), which assessed the patients’ subjective experience of the symptom provocation. Each VLS consisted of five questions with answers ranging from 1 to 5: 1 indicated not at all; 2 indicated slightly; 3 indicated moderately; 4 indicated substantially; and 5 indicated extremely. These questions asked (1) how tense they were, (2) how much their BIID symptoms were disturbed or effected in the moment, (3) how much physical discomfort they were in, (4) how fearful they were and (5) how angry they were. We further evaluated the patients’ experiences by measuring heart rate (HR), respiratory sinus arrhythmia (RSA) and pre-ejection period (PEP) to assess autonomic nervous system activity. HR is an indicator of combined parasympathetic and sympathetic control.26ART-IS was presented on an HMD (Oculus Rift DK2) from a fixed third person perspective, which was created by a video feed from a stereoscopic camera (Zed Mini) . In ordeDuring AR off and AR on, both patients indicated low VLS scores (1 and 2) for questions addressing whether they had any physical discomfort, how fearful and how angry they were.Patient 1 reported a score of 4 when asked how much his BIID was disturbed during the last AR off sequence and the last two AR on sequences, compared with reporting a score of 1 for the other sequences during both AR off and AR on. Due to the nature of his scoring, he was asked to further clarify his scoring rationale while responding. He provided further insight into his answer by saying that it was currently affecting his BIID in ‘a positive and welcoming way’ unlike AR off. Moreover, several days after testing, he reported a reduction in his symptoms via unprompted follow-up emails.Patient 2 reported a decrease of ‘how tense he was’ from the first sequence to the third sequence during AR off with a score of 3 and 2, respectively. During AR on, his response of a 5 during the first sequence reduced to a score of 3 during the third sequence. Similar to patient 1, he was asked to provide further clarification during his response. He described his high level of tension ‘positively’ by stating that it was ‘emotionally overwhelming to finally see himself like this’.The primary, within-subjects, physiological outcome measures are depicted in In this study, we investigated whether ART-IS influences the way BIID patients experience their alienated limbs. During testing, the self-report data from both patients indicated that ART-IS was well tolerated. This was confirmed directly after the exposure as both patients expressed the desire to experience ART-IS again in the future. When asked if the exposure increased their desire to obtain an amputation, both patients replied that it did not. Patient 1 added to this question by stating that this exposure validated his desires to obtain an amputation. Moreover, patient 1 also reported a reduction in his symptoms for several days after testing, indicating that there may have been a carry-over effect from the exposure. This could be interpreted as a treatment option rather than a mere carry-over effect. In both patients, we found increased activity of PEP and decreased activity of RSA during AR on compared with when it was off, indicating a physiological stress response. Our observed interactions between sympathetic and parasympathetic nervous system activity are in line with results from other studies that used the Trier Social Stress Task, which is a well-documented task that has been shown to reliably elicit psychological distress and a physiological stress response.28During data collection for patient 1, stimulation was applied incorrectly for the first two stimuli of the first B block in the AR on task by applying stimulation to D. This error was quickly identified and corrected during testing. Due to this, we decided that rather than excluding these data, we would instead reassign these data under the correct label.One of the limitations surrounding the current research is that the VLS we administered during our exposure has not been used or validated. Because we are unaware of a validated questionnaire that assesses the effects of virtual amputation in non-amputated individuals, we resorted to creating our own. In order to better interpret the physiological signals of interest, we had to better understand what factors could be contributing to a potential stress response while administering uncomfortable stimuli. Therefore, our questions also reflected other, non-BIID-related, factors that would allow us to better explain potentially confounding variables. Therefore, future investigations should seek to establish and validate a standardised questionnaire that is suitable for assessing the effects of virtual amputation in non-amputated patients. Another limitation of this study was that we were restricted to observing individuals with the desire for an amputation of their lower extremities . While lower extremity amputation wishes are common variants within this population,While more research is still needed, it is possible that this method may suffice as a stand-alone application that is able to gradually ameliorate BIID symptoms. Conversely, it could be used as an adjunct during neuromodulation therapyEmerging virtuality applications have the potential to act as a therapeutic adjunct to treatment for individuals with body integrity identity disorder (BIID).Emerging virtuality applications have the potential to enhance diagnostic insight into this medically and ethically complex disorder.Augmented reality telepresence to the ideal-self can be used for any variation of BIID in both clinical or at-home settings.Future iterations of this application should focus on the longitudinal effects of longer and more repetitive exposures to the ideal self in virtual environments.


Organic viticulture can generate a range of ecosystem services including supporting biodiversity, reducing the use of conventional pesticides and fertilizers, and mitigating greenhouse gas emissions through long-term carbon (C) storage. Here we focused on aboveground C storage rates and accumulation using a one-year increment analysis applied across different winegrape varietals and different-aged vineyard blocks. This produced a chronosequence of C storage rates over what is roughly the productive lifespan of most vines (aged 2–30 years). To our knowledge, this study provides the first estimate of C storage rates in the woody biomass of vines. Additionally, we assessed C storage in wildland buffers and adjacent oak-dominated habitats over a 9-year period.Carbon storage averaged 6.5 Mg/Ha in vines. We found the average annual increase in woody C storage was 43% by mass. Variation correlated most strongly with vine age, where the younger the vine, the greater the relative increase in annual C. Decreases in C increment rates with vine age were more than offset by the greater overall biomass of older vines, such that C on the landscape continued to increase over the life of the vines at 18.5% per year on average. Varietal did not significantly affect storage rates or total C stored. Carbon storage averaged 81.7 Mg/Ha in native perennial buffer vegetation; we found an 11% increase in mass over 9 years for oak woodlands and savannas.Despite a decrease in the annual rate of C accumulation as vines age, we found a net increase in aboveground C in the woody biomass of vines. The results indicate the positive role that older vines play in on-farm (vineyard) C and overall aboveground accumulation rates. Additionally, we found that the conservation of native perennial vegetation as vineyard buffers and edge habitats contributes substantially to overall C stores. We recommend that future research consider longer time horizons for increment analysis, as this should improve the precision of C accumulation rate estimates, including in belowground  reservoirs. Agriculture plays a major role in the storage, release and cycling of greenhouse gases –3. From In Mediterranean-type climates, vineyard cropping systems can make up a significant fraction of agricultural lands. In California, for example, table and wine grapes are the second leading agricultural commodity in sales after dairy, occupying 359,360 hectares  of cultivated land , 11. To Vineyards are proven long-term bellwethers of environmental conditions . With thWhile the woody biomass of vines represents a smaller carbon reservoir in the vineyard landscape compared to that of soil , it is mThis study makes progress in that direction by providing the first field-based estimates of how annual carbon increment varies in woody biomass for grape vines of different ages and varietals. We present results from a northern California organic vineyard with hopes that similar studies emerge in other vineyard and perennial woody crop landscapes across a range of management types  to give greater context to these findings. Together, such studies will improve our understanding of how geography, environment and management can be harnessed to advance the much-needed goal of expanding the carbon mitigation potential of agricultural landscapes , 21 and Vineyard lands sampled in this study are located in the Russian River watershed near the town of Hopland (38°58′23″N 123°06′59″W) in Mendocino County, California. The region is characterized by a Mediterranean-type climate with cool, wet winters and hot, dry summers where the average monthly temperature high/low for the coldest month (December) is 14°/8° C, and for the warmest month (July) is 35°/21° C. Annual rainfall is approximately 1010 mm, with most of that falling between the months of December and March. Vineyard elevations vary from a low of 150 m asl at the valley bottom to roughly 800 m asl at the highest site.www.ccof.org), while the ranches under biodynamic management  follow protocols set and certified by Demeter (www.demeter-usa.org). Wildland buffers were classified into five vegetation types ).To estimate woody C increment in wildlands and in order to compare 2009 C stocks with 2018 stocks as precisely as possible, we measured aboveground C in the same 2009-established plots (identified by geographic coordinates recorded on a handheld GPS) using the same methodology used in 2009 [All statistical analyses were conducted in R 3.4.4 .Total C stored in perennial vine woody biomass averaged 6.5 Mg/ha 2018 data) across all properties (vineyard blocks). Carbon stocks in adjacent uncultivated wildland woody biomass varied from 48.4 to 120.2 Mg/ha across the 5 vegetation types sampled (Table 8 data acp <  < 0.001) of annual C increment in woody biomass across the vineyard blocks and varietals we measured. There was considerable variation in the per-vine increment, as indicated by the high standard deviation (57%). Much of the variability in annual increment came from differences in vine age: on average, young vines, which tended to be both small and fast-growing, added a larger percentage of biomass each year, and had higher variability in growth rates than older vines. For example, vines aged 2–5 years had standard deviation values more than three times any other 5-year grouping.For the individual vines measured, the average estimated annual increase in woody C content was 38% over the course of the year considered. We found vine age to be a significant predictor (2 = 0.64). We found that on an annual basis different aged vines stored similar amounts of C and showed no clear trend with respect to age and the total amount of C added , further analysis with Tukey HSD multiple comparisons showed no significant differences between paired sites at the p = 0.05 level.An evaluation of the effect of site, specifically the ranch property that a given vineyard block belonged to, was inconclusive. Although two-way ANOVA indicated “site” had a significant effect on increment  carbon storage. Carbon storage in 5 native vegetation types averaged 81.7 Mg/Ha, more than an order of magnitude greater than the capacity of vines. Over a 9-year increment, C storage increased by 11.1% in oak dominated vegetation. Conservation of native perennial vegetation in buffers amidst vineyard blocks or adjacent to wetlands offers significant C accumulation benefits in addition to ecosystem services such as biodiversity, water quality, and erosion control.Essentially, we combined a one-year increment analysis applied across different-aged vineyard blocks to produce a chronosequence of carbon storage rates over what is roughly the productive lifespan of most winegrape vines aged 2–30 years), collectively represented as the curve in Fig. –30 yearsDespite the limitations of the chronosequence approach and the absence of multiyear rate data, we nevertheless found vine age to be an excellent predictor of annual C increment in woody biomass across the vineyard blocks and varietals measured, especially after the first six years when the annual variation in growth was highest. Our data show that despite the decrease in the annual rate of C accumulation, there is a net increase in aboveground woody biomass in vines over time that amounts to 18.5% per year on average.Our research shows clearly that carbon in the woody biomass of vineyard landscapes increases over time—a pattern obviously not observed in annual crops. Given that perennial crops such as grape vines, with their relative longevity on the landscape and historical importance, have provided and continue to provide a range of ecosystem services at multiple spatial scales , 22, 33,The flattening of the curve in the annual decrease in carbon accumulation in vines roughly between 15 and 30 years of age Fig. a has posFinally, while our analysis of soil organic C was inconclusive, this is not surprising given the short time period (one year) considered. Based on what other researchers have found (e.g., , 37, we In conclusion, the findings presented here offer the first published estimates of C accumulation rates in vineyard ecosystems. Other studies have quantified C in vineyard systems, surrounding wildlands, soils, and in the vines themselves , 40–42, Evidence from this study suggests that organic viticulture has the potential to generate a range of ecosystem services at multiple scales including mitigation of greenhouse gas emissions through long-term carbon storage. Advancing research in newly emerging areas, such as regenerative practices, would benefit from establishing baseline assessments of ecosystem services both within and adjacent to vineyards. This is especially true for quantifying C, but also for other ecosystem services such as nutrient cycling and biodiversity. As Biasi et al.  conclude


COVID-19 has rapidly become a pandemic emergency, distressing health systems in each affected country. COVID-19 determines the need for healthcare in a large number of people in an extremely short time and, like a tsunami wave, overruns emergency, infectious diseases, and pneumology departments as well as intensive care units, choking healthcare services. Rehabilitation services are also affected by this epidemic which forces radical changes both in the organization and in the operating methods. In the absence of reference literature on this issue, this report aims to provide a background documentation to support physicians and healthcare personnel involved in neurorehabilitation and rehabilitation care. The spread of coronavirus disease 2019 (COVID-19—COrona VIrus Disease 2019) has become unstoppable and in the last few weeks has reached the epidemiological criteria to be declared a pandemic by the World Health Organization, having widely exceeded 400,000  infected people in the world in over 100 countries  and non-European countries, shows that the challenge is similar for everyone. Nonetheless, the absence of a common pandemic plan in Italy and Europe, with Regions and individual countries that are still adopting different methods of managing the epidemic, runs the concrete risk of dissolving the effectiveness of the stringent measures implemented in different areas or countries. This can also be observed in China, where, in the face of an internal growth of new cases equal to zero over the past few days, new infections have been recorded due to the presence of the so-called “re-entry cases.” It must also be taken into account that the epidemic spread will occur with peaks at different times and areas, within and between the various countries, so the overall consequences will be related to the effectiveness of the different health systems.Numerous mathematical models for predicting the progress of the epidemic are being proposed in Italy and in the world in an attempt to provide useful tools to decision makers . Howeverwait-and-see attitude, but should rather induce us to learn from this “temporal advantage,” promptly implementing all the social distancing measures essential to reduce the circulation of the virus.The currently still limited number of cases, albeit growing, in the central-southern regions, should teach us not to repeat the error of a One of the most worrying aspects of the COVID-19 epidemic is linked to the involvement of frail and vulnerable people, in particular the elderly, subjects who suffer from multiple comorbidities or chronic diseases and people with disabilities. For this reason, it is essential to address and focus on prevention, health interventions and care in chronically ill patients staying in health care facilities, as well as patients suffering recent functional limitation requiring rehabilitation (or admitted to rehabilitation facilities).In fact, in an extremely short time compared to the spread of the epidemic, Rehabilitation Facilities need to enact prompt remodellling of the health organization both in the internal structure and in the strategy and approach of care delivery, which presents unique peculiarities within healthcare organizations , 10.Furthermore, in some areas due to the lack of beds, rehabilitation facilities are now being occupied with other patients, with consequent difficulty in hospitalizing patients, for example, discharged from stroke units.Therefore, in the need to reorganize hospital and outpatient rehabilitation activities , 12, thiThe following indications are suggested in order to make the reorganization of rehabilitation activities homogeneous, whether they are carried out in hospitalization or outpatient settings or at home, with the main aim of limiting patient flows within the facilities and maintaining staff safety. For some structures, these indications must take into account territorial and network needs, as well as the possible increase in hospital admission in order to favor discharge from acute care facilities.The definition of dedicated territorial and local pathways for patients from acute care facilities, separating noCOVID or negative COVID patients from suspected positive or positive Covid cases.Admissions to hospital wards must be limited to only those that are essential, and in any case supervised by personnel equipped with personal protective equipment. All other accesses must be closed.Posting of notices with behavioral rules at the entrances to and within all the departments [see ]Posting of hand hygiene recommendations near hand-sanitizing gel dispensers [see ]Staff of external companies are required to comply rigorously and systematically with standard precautions in addition to those provided by air, by droplets, and by contact, as indicated in the behavioral rules.Preparation of extraordinary plans for daily cleaning and sanitization of the rooms.Preparation of plans for extraordinary sanitization and cleaning in cases of access or identification of a “suspected case.”Reorganization of work shifts  with reduction of activities in order to reduce contact and movements. Where possible, encourage staff to work from home . Obligation for everyone to report any symptoms that have arisen recently; in this case, respect home isolation.Strengthening of patient and caregiver support networks, also through information technologies.Emphasis on the role of remote assistance and/or tele-rehabilitation in particular as remote home monitoring for patients who are unable to access rehabilitation hospitals or must be discharged in advance as well as for consulting activities in hospitals or in case of consultation for out-patientsFor all, monitoring of body temperature <37.5°C—Even in the absence of fever, it is necessary to subject people to careful triage by explicitly asking for their place of origin and detecting potential contacts.Suspension of caregiver visits to hospitalized patients . Exceptional cases must be authorized by clinicians according to the rules of the health management; access in derogation will be managed by the staff in order to avoid any contact and for a limited time.Rehabilitation units will have to keep the doors closed in order to control and direct the flow . for all health personnel and visitors with permission, monitoring of body temperature <37.5°C For discharges (see rules for access in derogation and compliance with point 2)Suspension of all meeting activities, replaced by the use of telephone or email contact.Clinical interviews with family members by phone or email only.Remodeling of rehabilitation programs, identifying the most relevant goals, among the short-term achievable onesSuspension of all rehabilitation activities that require internal flow (movement between floors or to reach gyms)Carrying out of rehabilitation activities in the patients' room where possible; in the case of gym activities, strictly keep the distance of at least 2 m between the patients.Reduction of the rehabilitation team's activities Education and empowerment of all healthcare professionals involved in rehabilitation team, by means of specific targeted training For all healthcare professionals, it is recommended to “enhance hand hygiene by following the WHO instructions, before and after each patient and whenever the hands move from the patient to another surface” Remember that “Masks with greater protection  are indicated only in suspicious or full-blown cases, therefore it is suggested not to abuse these devices, so as not to reduce their availability for cases of real need”In the management of suspicious cases (cases with not deferrable treatment) , remembepatient with respiratory symptoms (no COVID19): the patient is recommended to wear the surgical mask; keep a distance of at least 1 m or wear a surgical mask;patients suspected or affected by Covid-19: the use of FFP2 or FFP3 mask, protective gown, gloves, eye protection (goggles or face shield) is recommended;patients suspected or affected by Covid-19 during the execution of procedures capable of generating aerosols: the use of FFP3 mask, protective gown, gloves, eye protection (goggles or face shield) is recommendedEven in the absence of fever, people must be subjected to careful triage by explicitly asking where they came from and detecting potential contacts.NOTE: Remember that it is possible to carry out “…respiratory physiotherapy in hospitalization and re-education settings in recent outcomes of surgery, in trauma with fractures and the immediately post-acute phase of cardiac and neurological disabling pathologies  …,” for this specific area, please refer to the joint document of the Association of Rehabilitators of Respiratory Insufficiency and the Italian Association of Physiotherapists  (update of 16/03/2020) , if they are not affected or suspected by Covid-19.with appropriate personal protective equipment due to the impossibility of maintaining a distance of <1.5 m)Patients suspected or affected by Covid-19 with neurological impairments requiring rehabilitation and according to clinical conditions  should be treated in the room  without fever and cough suggestive of Covid-19 infection.Neurorehabilitation/rehabilitation Unit located outside general and multi-disciplinary Hospitals should only admit patients with sub-acute neurological disorders negative for Covid-19 infections in order to facilitate prompt availability of intensive care unit.Suspension of all outpatient and/or Day-hospital rehabilitation activities and/or with access from the outside Suspension of all activities including those of a freelance type (re-scheduling of visits and activities)Suspension of all home rehabilitation activities except those that cannot be postponed according to clinical judgment.The greatest difficulty in applying these indications in the field of neurorehabilitation/rehabilitation is related to the need to find the right balance between the provision of services useful to the patient (in case of not deferrable treatment) and the reduction of the risk of spreading the virus. In this phase of maximum spreading speed, priority must be given to reducing the risk of spreading the infection.The carrying out of rehabilitation activities in hospital stays, and in services in general, can only be continued in compliance with the needs of the patients and the protection of the health of all staff, as activities require close contact with the patient, or with the production of aerosols and secretions as for respiratory rehabilitation interventions  . FinallyIt must be reiterated that given the specificity of Rehabilitation/Rehabilitative interventions, which necessarily have to take into account the territorial contexts, the above indications must be adapted to the specific realities, while adhering as closely as possible to the recommendations.In fact, it seems useful to reiterate that the indications and measures to be taken are valid for all and should be applied in all countries and in the different areas as simultaneously as possible, because a non-uniform alignment would also lead to the risk of an inevitable misalignment in the desirable economic restart, with heavy long-term relapses.The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.MB conceived the study. MB, DI, CL, GS, SP, and MZ also on behalf of the Authors listed in the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Inorganic arsenic oxides have been identified as carcinogens in several human tissues, including epidermis. Due to the chemical similarity between trivalent inorganic arsenic (arsenite) and antimony (antimonite), we hypothesized that common intracellular targets lead to similarities in cellular responses. Indeed, transcriptional and proteomic profiling revealed remarkable similarities in differentially expressed genes and proteins resulting from exposure of cultured human epidermal keratinocytes to arsenite and antimonite in contrast to comparisons of arsenite with other metal compounds. These data were analyzed to predict upstream regulators and affected signaling pathways following arsenite and antimonite treatments. A majority of the top findings in each category were identical after treatment with either compound. Inspection of the predicted upstream regulators led to previously unsuspected roles for oncostatin M, corticosteroids and ephrins in mediating cellular response. The influence of these predicted mediators was then experimentally verified. Together with predictions of transcription factor effects more generally, the analysis has led to model signaling networks largely accounting for arsenite and antimonite action. The striking parallels between responses to arsenite and antimonite indicate the skin carcinogenic risk of exposure to antimonite merits close scrutiny. Many mechanisms have been proposed for these effects, indicating possible interference of arsenic with a variety of signaling pathways2 to which epigenetic changes may contribute3. Proteins with vicinal thiols, including zinc finger DNA repair proteins4, are particularly attractive as potential targets with deleterious downstream effects. Continuing exposure of large populations has evoked considerable effort to elucidate the broad consequences for health and the mechanisms that lead to their manifestation.Chronic exposure to inorganic arsenic, primarily in water supplies, has numerous deleterious effects on humans, including cancer at several anatomic sites5, considerable exposure occurs to inorganic antimony, a metalloid immediately below arsenic in the periodic table to which it exhibits chemical similarity6. The presence of antimony is increasing in the environment through use in small arms ammunition, as a catalyst in plastic, as a flame retardant, and through watershed pollution by mining waste or by recycling operations. This has raised concern for public health, sustainable agriculture and ecosystem effects8. Inasmuch as antimony trioxide is a rodent carcinogen9, finding commonalities in actions of antimonite (SbIII) and arsenite (AsIII) would assist in understanding their mechanisms of action in vivo.Also of concern, including human therapeutic treatment for leishmaniasis13. Recently, protein and transcriptional profiling experiments using SIK demonstrated that changes due to treatment with these two agents occur in parallel14. The initial targets and mechanisms of action, and how similar these are for arsenite and antimonite, remain to be determined. For this purpose, we have analyzed profiling data to predict cell signaling pathways and transcription factors that appear to mediate downstream effects. Global analyses of changes in proteins and mRNAs now permit a more general approach to the identification of signaling pathways and transcription factors that drive the responses. As a result, three previously unsuspected signaling pathways for these metalloids have been revealed and validated in keratinocytes, and model signaling networks largely accounting for arsenite and antimonite action are presented.Human epidermis is a known target for carcinogenic effects of arsenic. Cultured human epidermal keratinocytes, a model system for studies of effects of chemicals on epidermis, have been shown to respond similarly to treatment with arsenite and antimonite. As shown using the normal cells and a minimally deviated line of spontaneously immortalized human epidermal keratinocytes (SIK), both metalloids suppress many aspects of the cell differentiation program while preserving the proliferative potential of the cells14, can be visualized as a scatterplot of log fold change of genes differentially expressed after arsenite treatment vs. log fold change of those genes after antimonite treatment  and arsenite or antimonite treatments, respectively, while the number of differentially expressed mRNAs comparing samples from arsenite- and antimonite-treated cells was 49, an order of magnitude fewer. This relationship, consistent with our previous reportent Fig. . Not preent Fig. . A scattent Fig. .Figure 115, such studies showed poor concordance in response to arsenic treatment compared to cadmium, chromate or vanadate  software was used to explore signaling pathways and upstream regulators resulting from arsenite and antimonite exposure. Results indicate that arsenite and antimonite produce similar effects in keratinocytes because they target many of the same metabolic pathways and regulators, leading to significant overlap in differentially expressed genes and proteins.NFE2L2) activation. Proteomic analysis also predicted the involvement of the Nrf2-mediated oxidative stress response as a top affected pathway , a transcription factor induced by arsenite treatment, and both are predicted to be activated. The predicted activation of these two regulators after antimonite treatment also provides strong support for the hypothesis that arsenite and antimonite overlap in their mechanisms.The list of top five regulators, predicted by analysis of differentially expressed genes due to arsenite and antimonite treatments, overlap for four of the top five predicted molecules Table . Two of 17. Sorted by z score (activation score), 45 and 85 regulators are predicted to be activated by arsenite and antimonite, respectively (24 in common), and 33 and 30 predicted to be inhibited (11 in common)  and tumor necrosis factor alpha (TNFα) may help to explain keratinocyte-specific effects since these cytokines are known to target this cell typeThe predicted activities of upstream regulators show remarkable similarity for arsenite and antimonite treated samples, as displayed in a heat map of top regulators  , GLI2, retinoic acid receptors and ligands (including isotretinoin), corticosteroids (including dexamethasone) and OSM.Overall, these predicted upstream regulators from analysis of arsenite and antimonite treated samples display extensive overlap and suggest potential shared targets for exploration to explain downstream effects. The impact of arsenite on NFE2L2 and its consequences have been well documented in many cell typesSince OSM is the top predicted upstream regulator of transcriptional changes induced by arsenite and antimonite, experiments were designed to determine whether treatment of cells with OSM would elicit responses similar to those observed after treatment with arsenite and antimonite, especially effects on differentiation and proliferative potential. Because we have been investigating effects of chronic treatment (days rather than hours) with arsenite and antimonite, we used similar treatment times for our experiments with OSM, with treatments starting at confluence, just as the cells begin differentiating.11, chronic OSM treatment increased the proliferative potential of cultured keratinocytes after confluence compared to untreated cultures as measured by colony forming efficiency . Chronic exposure of keratinocytes to OSM increased phospho-Stat1, -Stat3, and -Erk, as expected  and Erk signaling pathways , the fold suppression by arsenite and antimonite was altered, but this was predominantly due to changes induced by hydrocortisone alone. The suppressive effects of corticosteroids on these genes have also been observed in vivo28. For all of these genes, hydrocortisone affected the responses to arsenite and antimonite similarly. Responses to dexamethasone were similar to those obtained with hydrocortisone.The prediction of dexamethasone as an upstream regulator suggests that responses to arsenite and antimonite may be different in the presence and absence of corticosteroids. Under standard culture conditions, arsenic oxides have been shown to suppress various markers of differentiationlar Fig. . Presentlar Fig. . Most dr29) and were identified by IPA as upstream regulators after arsenite and antimonite treatment, we examined effects of arsenite and antimonite on levels of ephrin and ephrin receptor mRNA expression. Seven ephrin mRNAs and seven ephrin receptor mRNAs were expressed at moderate levels (>1 RPKM). Three ephrin receptor mRNAs  and one ephrin mRNA (EFNB2) were decreased two fold or more by arsenite. These decreased receptor mRNAs led to decreases in the encoded proteins by both arsenite and antimonite as demonstrated by Western blotting  makes ascribing particular functions to individual receptors difficult. While responses to particular ephrins are largely overlapping, some differences have been demonstrated22, suggesting that receptors distinguish among the ligands and initiate different downstream signaling. Perhaps EphA2 is of particular importance for regulation of TGM1, which was suppressed by arsenite and antimonite, but increased by OSM, while regulation of KRT1, KRT10, DSG1 and DSC1, which were decreased by all three agents, may be under the control of EphA1 and/or EphA4. All of these markers have been shown to be in the set of ephrin up-regulated genes22.Since ephrins and their receptors have significant impacts on keratinocyte differentiation . To this end, we filtered the gene expression data for transcription factors that were differentially expressed at the statistical significance of p ≤ 0.05. Supplementary Table A network of this first group of transcription factors, demonstrating predicted regulation of several downstream differentiation markers, is shown in Fig. 15. Perhaps this sustained response helps rationalize their unique abilities to suppress keratinocyte differentiation while maintaining proliferative potential. However, hyperactivation of a single signaling pathway could produce a contrasting result. For example, unopposed activity of the Nrf2-mediated oxidative stress response by KEAP1 ablation leads to epidermal hyperkeratosis and increased expression of keratinocyte differentiation markers in mice52.Examination of affected canonical pathways revealed that many of the same differentially expressed genes led to the predicted perturbation of the top pathways, suggesting connections among these pathways. Among several compounds that induce Nrf2-mediated oxidative stress response in keratinocytes, judging by their abilities to induce HO1, only arsenite and antimonite induce a robust and sustained response30, while MYC has been shown to drive cells from the stem cell compartment and initiate the differentiation process53. FOS and JUN have been identified as transcription factors important for expression of several keratinocyte differentiation markers, reviewed in54. Finally ROCK inhibitors prolong the proliferative capacity of cultured keratinocytes25. The predicted inhibition of ROCK2 by arsenite and antimonite could rationalize their abilities to maintain proliferative potential.The lists of top upstream regulators predicted by transcriptional and proteomic analysis are intriguing and provide some validation of the predictive process. That arsenic trioxide is predicted to be activated by both arsenite and antimonite is especially supportive. Most of the other regulators have already been identified as important regulators of keratinocyte differentiation and proliferative potential. OSM, ephrins (e.g. EFNA3 and EFNA4) and dexamethasone all have been demonstrated to affect transcription of differentiation markers28) while antagonizing arsenite and antimonite suppression of several other differentiation markers. These mixed responses are consistent with a z-score that predicted neither activation nor inhibition. Finally, ephrins and their receptors have been shown to promote keratinocyte differentiation22 and were predicted upstream regulators of arsenite and antimonite downstream effects. We have shown here that amounts of several ephrin receptors were decreased by these metalloids.Three of the predicted upstream regulators have been experimentally validated. First, OSM, predicted to be activated by antimonite, did indeed demonstrate many similarities to arsenite and antimonite. All three agents effectively suppressed a subset of differentiation markers while maintaining proliferative potential. Second, corticosteroid agonists such as dexamethasone and the endogenous agonist hydrocortisone acted similarly to arsenite and antimonite by suppressing FLG, LOR and IVL expression  and potassium antimony tartrate (6 µM), as shown previously for transcriptional responses and confirmed in this work for survival , transcriptomic analysis using RNA-Sequencing technology and proteomic analyses were performed as described14; to permit accurate signaling pathways analysis, two additional experiments were processed to add to the two previously analyzed. Thus a total of 12 samples, four each from untreated control (n = 4), arsenite-treated (n = 4) and antimonite-treated (n = 4) human epidermal keratinocyte cultures, were analyzed by RNA-sequencing. Each of the four pairs of arsenite and antimonite-treated samples, along with the untreated control, was derived from a separate experiment performed at different times. An average of 35 million sequence reads was obtained for each individual sample. These were assembled and mapped to the annotated human genome assembly (release 80). In all samples, ~90% (88–91%) of the reads were mapped to the human reference sequence. Differential gene expression analysis between untreated (control) and treated (arsenite or antimonite) human epidermal keratinocytes was performed by t-testing with correction for false discovery rate.These experiments used a line of spontaneously immortalized epidermal keratinocytes (SIK)http://proteomecentral.proteomexchange.org (ID # PXD011259).For access to original transcriptional data, the sequencing results  have been deposited into the NCBI SRA repository (Temporary Submission ID: SUB4618061) with accession #PRJNA510150. For access to the original proteomic data, the raw data were deposited in the Massive Proteomics repository (massive.ucsd.edu ID #MSV000082992) and ProteomeExchange Transcriptomic datasets comparing arsenite or antimonite treated samples to untreated samples were uploaded into the Qiagen Ingenuity Pathway Analysis program. Core analysis was performed, filtering for fold change ≥2, expression intensity (RPKM) ≥0.2 and false discovery rate ≤ 0.2.15. Fragmented cRNA was hybridized to U-133A arrays , and the hybridization was detected with streptavidin-labeled phycoerythrin. After measurement of hybridization intensities, the.CEL files were imported into an R language environment using Bioconductor software and subjected to quantile normalization. An arithmetic average of 16 normalized intensity values from PM probes were transformed using the general logarithm transformation (glog)57. The processed data are available in the Dryad Digital Repository (10.5061/dryad.t4q2nc8). Prior to analysis, probes with present calls in fewer than 2 samples were filtered, leaving 11,952 probes. Differential expression analyses were conducted using the Bioconductor package limma, version 3.38.358, with expression modeled by group using a single factor linear model. The analysis incorporated variance weights for log expression calculated using the function vooma within limma59. Analyses were conducted using R, version 3.5.3 . Each treatment was analyzed in two independent cultures. The duplicates of arsenic consisted of one arsenite and one arsenate culture, previously shown to be equivalent in transcription15. A multi-dimensional scaling plot of the results showed that the arsenic samples were well separated from the cadmium, chromate and vanadate samples, but the last two were not clearly separated from each other . cDNA was synthesized using total RNA with oligo dT priming followed by second strand RNA synthesis using biotin-labeled nucleotides as previously described60 in the presence and absence of combinations of 10 µM hydrocortisone, 3 µM arsenite and 6 µM antimonite. RNA was prepared using Trizol reagent (Life Technologies), followed by cDNA preparation using an Applied Biosystems reverse transcription kit. cDNA was analyzed by qPCR with Taqman assays (Applied Biosystems), normalizing to MAPK1, a gene product demonstrated by transcriptomic analysis not to be changed by arsenite and antimonite treatments.SIK cultures were treated at confluence with 50 ng/ml Oncostatin M (OSM) (R&D Systems), 3 µM sodium arsenite, 6 µM potassium antimony tartrate (antimonite) or not treated. In some experiments cell cultures were pretreated for 30 min with 10 µM U1026 (an inhibitor of MEK1 and MEK2) (LC Laboratories) or 1 µM JAK Inhibitor I  before addition of OSM, arsenite or antimonite. To test the influence of hydrocortisone on arsenite or antimonite effects, SIK cultures were grown to confluence in normal culture medium, then switched to medium containing serum depleted of steroids by charcoal/dextran treatment2 with antibodies obtained from Cell Signaling Technologies: EphA1 , EphA2 , EphA3/A4/A5 , Erk1/2 , phospho-Erk1/2 , Stat1 , phospho-Stat1 , Stat3 , phospho-Stat3  and cleaved Notch1 . Antibodies were used at a 1:1000 dilution of the supplied stock following the protocols specified by the supplier. Blots were developed using Thermo Fisher ECL Plus and visualized with a My ECL imager. Image acquisition time was adjusted to visualize all bands without saturating the brightest bands (from 30 seconds to 30 minutes depending on the efficacy of the antibody). Data were analyzed using Thermo My Image Analysis software, version 2.0. Images were inverted to display dark bands on a light background and in some cases brightness or contrast were adjusted to show lighter bands more distinctly, again without saturating the darkest bands. Images were then cropped, arranged and converted to TIFs with Photoshop, version CS6.Immunoblotting was performed as described61, colonies of approximately 50 cells or more were counted. Results were normalized to the untreated sample. The experiment was performed in triplicate.Confluent SIK cultures were untreated or treated with 50 ng/ml OSM or 3 µM arsenite in the presence and absence of 10 µM U1026 or 1 µM JAK Inhibitor I. After 9 days, cells were trypsinized, counted and 3000 cells from each sample were inoculated on 6 cm dishes in the presence of irradiated 3T3 feeder cells. Cultures were grown for 2 weeks, when most colonies were 2–5 mm in diameter. After fixation and staining with Rhodanile blueSupplementary information.