Cardiomyocytes proliferate rapidly during fetal life but lose their ability of proliferation soon after birth. However, before terminal withdrawal from the cell cycle, cardiomyocytes undergo another round of cell cycle during early postnatal life in mice. While a transient wave of increased DNA synthesis in cardiomyocyte has been observed in postnatal mouse hearts, the molecular mechanisms describing cardiomyocyte cell cycle re-entry remain poorly understood. Atrial and B-type natriuretic peptides (ANP and BNP) are abundantly expressed in embryonic heart ventricles. After birth, the expression of both genes is strongly reduced in the ventricular myocardium. Forkhead O (FOXO) transcription factors are expressed in both embryonic and postnatal heart ventricles. Their transcriptional activity negatively affects cardiomyocyte proliferation. Upon phosphorylation, FOXO is translocated to the cytoplasm and is transcriptionally inactive. Despite these important findings, it remains largely unknown whether natriuretic peptides and FOXO cooperatively play a role in regulating cardiomyocyte cell cycle activity during early postnatal life.We observed that the expression of ANP and BNP and the level of phosphorylated FOXO were transiently increased in the postnatal mouse heart ventricles, which coincided with the burst of cardiomyocyte cell cycle re-entry during early postnatal life in mice. Cell culture studies showed that ANP/BNP signaling and FOXO cooperatively promoted cell cycle activity in neonatal mouse cardiomyocytes. The enhanced cell cycle activity observed in combined treatment of ANP/BNP and dominant-negative FOXO (DN-FOXO), which can bind FOXO recognition sites on DNA but cannot activate transcription, was primarily mediated through natriuretic peptide receptor 3 (Npr3). In mice, simultaneous application of ANP and DN-FOXO in postnatal hearts reactivated cell cycle in cardiomyocytes, resulting in reduced scar formation after experimental myocardial infarction.Our data demonstrate the cooperative effects of natriuretic peptide and DN-FOXO on promoting cardiomyocyte cell cycle activity and mouse cardiac repair and regeneration after injury.The online version contains supplementary material available at 10.1186/s12861-020-00236-y. Cardiomyocytes proliferate rapidly during fetal life but lose their ability of proliferation soon after birth. However, before terminal withdrawal from the cell cycle, cardiomyocytes undergo another round of cell cycle activity during early postnatal life in mice . While aCIP1) and p27 (p27KIP1) [Forkhead O (FOXO) transcription factors are known as important targets of insulin and growth factor action. They are under negative control by the PI3K-Akt/PKB pathway, which phosphorylates FOXO proteins and renders them transcriptionally inactive by promoting their nuclear exclusion –16. FOXOp27KIP1) . In contp27KIP1) . Thus, cIn this study, we report the expression of ANP and BNP and the level of phosphorylated FOXO in neonatal mouse heart ventricles during early postnatal life. We assess the effects of ANP/BNP signaling and FOXO transcription factors on cell cycle activity in primary neonatal mouse cardiomyocytes in culture. Treatment of infarcted mouse hearts with the natriuretic peptide and dominant-negative FOXO during early postnatal life provides in vivo evidence that natriuretic peptides and FOXO transcription factors work in an integrated manner to control cardiomyocyte cell cycle activity in early postnatal life.All litter sizes of wild-type mice  were adjusted to 8–10 pups per litter. The sex of newborn mice used in these studies was not determined. Generally, the sample size was chosen to use the least number of animals to achieve statistical significance, and no statistical methods were used to predetermine sample size. Animals were divided into experimental groups based on the type of treatment. We did not use exclusion, randomization, or blinding approaches. All the animal experiments were carried out by the NIH guidelines . All experimental procedures involving animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee of Temple University Medical Center.4 cells per well on a 96-well plate coated with laminin (10 μg/cm2). Twenty-four hours later, the culture medium was replaced with fresh medium  either with or without ANP , BNP , or adenovirus expressing dominant-negative FOXO (Ad-DN-FOXO) (1500 vp/cell), or Ad-Cre (1500 vp/cell). For lentiviral transduction, the culture medium was replaced with fresh medium containing Npr1 shRNA lentivirus (pLKO.1-Npr1 shRNA), Npr2 shRNA lentivirus (pLKO.1-Npr2 shRNA), Npr3 shRNA lentivirus (pLKO.1-Npr1 shRNA), or scramble shRNA lentivirus using 5 μg/ml polybrene . After 48 h, cardiomyocytes were fixed and processed for immunostaining with the indicated antibodies.The newborn mice (≤ 5 days of age) were euthanized by decapitation. Cardiomyocytes were collected using the method previously described . In shorMurine Npr1 shRNA, Npr2 shRNA, and Npr3 shRNA were synthesized and subcloned into pLKO.1 lentiviral vector . pLKO.1-scramble shRNA plasmid was used as previously described . LentiviThe dominant-negative FOXO (Δ256) protein contains the entire forkhead DNA binding domain but lacks the transactivation domain, thus inhibiting the transcriptional activity of FOXO1 and FOXO3 . InfectiHeart tissues were fixed in 4% formaldehyde and processed for paraffin histology and sectioned using routine procedures. Immunohistochemical staining was performed using the previously described protocol , 19. Pri9 vp). The muscle layer and skin were closed and allowed to recover. For EdU labeling, mice were injected with one dose of EdU 50 mg/kg via i.p. injection and sacrificed after 18 h. Mice were euthanized by inhalation of 100% CO2 followed by cervical dislocation.The myocardial infarction model was used as previously described . BrieflyIn situ hybridization was performed using Digoxigenin (DIG) Labeled In Situ Probe on 8-μm paraffin-embedded tissue sections. To synthesize antisense probes for ANP and BNP, part of ANP and BNP mRNA sequences were cloned into the pCR4-TOPO plasmid using the following primers: ANP forward primer TCAAGAACCTGCTAGACC, ANP reverse Primer CCTAGTCCACTCTGGGCT; BNP forward primer ATGCAGAAGCTGCTGGAGCTG, BNP reverse primer GTTACAGCCCAAACGACTGAC. After deparaffinization and rehydration, tissue slides from 2 (P2), 5(P5), and 7(P7) days old mice were treated with 10 μg/ml of proteinase K and fixed in 4% PFA. Pre-hybridization was performed incubating tissue samples in a solution containing formamide 50%, SSC 5x, 0.1% Tween-20, citric acid 10 mM, Yeast tRNA 50 mg/ml, and Heparin 50 μg/ml in a humidified chamber for 2 h. Hybridization was performed incubating tissue section in Hybridization solution containing the denatured digoxigenin-probe and incubating at 70 °C overnight in a humidified chamber. The day after, samples were incubated in blocking buffer  for 1 h and then in anti-DIG antibody conjugated to alkaline phosphatase (1:2000 in blocking buffer) overnight at 4 °C. Detection was performed using the substrate 5-Bromo-4-chloro-3-indolylphosphate/Nitro-blue tetrazolium (BCIP/NBT) in Alkaline Phosphatase Buffer . Tissue section images were taken on a Nikon Eclipse Ni using a Nikon digital sight ds-U3 camera.To produce a Lentiviral Npr1 shRNA expression construct, a sense (CCGGCCGTGCTTCTTCATA) and anti-sense (TATGAAGAAGCACGGCCGG) sequence were used to generate a short hairpin RNA in PLK1.0 lentiviral construct. To produce a Lentiviral Npr2 shRNA expression construct, a sense (TGCGGTTTGTCAGCTCCGA) and anti-sense (TCGGAGCTGACAAACCGCA) sequence were used to generate a short hairpin RNA in PLK1.0 lentiviral construct. To produce a Lentiviral Npr3 shRNA expression construct, a sense (TCAGTCTTGTGGACCGCGT) and anti-sense (ACGCGGTCCACAAGACTGA) sequence were used to generate a short hairpin RNA in PLK1.0 lentiviral construct.Total RNA was isolated using Trizol and was used to generate cDNA using random hexamer primers and SuperScript III RT (Invitrogen). Real-time thermal cycling was performed using the StepOne Plus cycler (Applied Biosystems) with SYBR green master mix. Transcript expression values were generated with the comparative threshold cycle (Delta CT) method by normalizing to the expression of the 18S gene. Sequences of the oligonucleotide primers used for qRT-PCR analysis are available in Table Mouse heart ventricular tissue lysis was obtained from heart ventricles homogenized using a tissue grinder on ice. Mouse plasma was obtained from blood samples by cardiac puncture. ANP and BNP levels of mouse hear ventricular tissue lysis and plasma were measured using a commercially available enzyme-linked immunosorbent assay kit  according to the manufacturer’s directions.Mouse heart ventricles were homogenized in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the BCA (Bicinchoninic Acid) Protein Assay Reagent Kit (Bio-Rad). Protein samples were separated on polyacrylamide gels (10% NuPAGE Bis-Tris Gel) and transferred to nitrocellulose membrane (Bio-Rad). The membranes were blocked with 5% BSA (Bovine Serum Albumin) and incubated with primary antibody diluted in 5% BSA at 4 °C overnight, followed by washing with TBST (TBS containing 0.1% Tween 20) solution. Membranes were incubated with near-infrared fluorophore-conjugated secondary antibodies diluted in TBST for 1 h at room temperature and followed by TBST washing three times. Target protein bands on the membranes were visualized using the Odyssey imaging system (LI-COR Biosciences). Primary antibodies include phospho-FOXO1 (Ser256)(E1F7T) Rabbit mAb , phospho-FOXO3 (Ser318/321) , FOXO1 (C29H4) Rabbit mAb , and FOXO3 (75D8) Rabbit mAb .P values are depicted as follows: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Results with P > 0.05 were considered not significant (n.s.). All analyses were performed with GraphPad Prism 7.Data are presented as mean ± standard error of the mean (s.e.m.). Multiple groups were compared by one-way ANOVA followed Tukey or Dunnett’s post hoc test. Student’s t test was used when comparing two experimental groups. Normality test was performed to determine whether parametric versus nonparametric testing is used. To determine DNA synthesis in cardiomyocytes, neonatal mice received a single intraperitoneal injection of 5-ethyl-2′-deoxyuridine (EdU) and were sacrificed after a 3-h labeling period. The frequency of EdU incorporation was determined on sectioned hearts by co-labeling with the antibody against cardiac troponin T (cTnT) Fig. a. ConsisNppa) and BNP  are important modulators during cardiomyocyte hypertrophy in adulthood [Anp mRNA increased by 3 and 4.5-fold on P3 and P5, respectively, compared to P2 , which inhibits transcriptional activity of FOXOs for 48 h. Cardiomyocyte cell cycle activity was evaluated by quantifying cell cycle marker (Ki67) expression and the frequency of EdU incorporation, the indicator of cells in the DNA synthetic phase. These studies showed that administration of either ANP, BNP, or DN-FOXO or Cre alone, had no significant impact on cardiomyocyte cell cycle activity compared to the control cultures Fig. a-d. NotaNatriuretic peptides bind to three natriuretic peptide receptors  . We inveMammalian mouse heart at P7 loses regenerative capacity and develops fibrotic scarring after myocardial infarction (MI) . To deteTo determine whether natriuretic peptides and Ad-DN-FOXO treatment affected cardiac fibrosis formation over the long term, we examined mouse hearts 14 days after MI Fig. a. AnalysIn this report, we establish a novel role for the cardiac natriuretic peptides and FOXO transcription factors in regulating cardiomyocytes cell cycle activity during early postnatal life in mice. Our data demonstrate that natriuretic peptides and FOXO transcription factors modulate cardiomyocyte cell cycle activity in a coordinated manner both in culture and in vivo. Specifically, combined treatment with ANP/BNP and DN-FOXO increased cardiomyocyte cell cycle activity, whereas single treatment of ANP/BNP or DN-FOXO did not significantly affect cell cycle activity compared with control cultures Fig. . The enhNatriuretic peptides are dynamically expressed during heart development , 3, 12. Npr1, Npr2, and Npr3 are expressed in cardiomyocytes during mammalian heart development . Both NpOur study indicates a new approach that delivers a proliferative stimulus to the injured heart using the simultaneous treatment of ANP and DN-FOXO. Although we did not observe any effects in non-cardiomyocytes, such an adenovirus approach of a proliferative stimulus could affect cellular homeostasis over the long term. The target application of ANP and DN-FOXO using bioengineered delivery approach may reduce the potential for off-target effects in the future.In summary, the current findings indicate that ANP/BNP signaling and FOXO nuclear activity cooperatively regulated cardiomyocyte cell cycle activity during early postnatal life in mice. Activation of ANP/BNP signaling and inhibition of FOXO nuclear activity promoted cardiomyocyte cell cycle activity in culture, which was primarily mediated through Npr3. Simultaneous application of ANP and DN-FOXO in postnatal hearts reactivated the cell cycle in cardiomyocytes, resulting in reduced scar formation after experimental myocardial infarction. Our data demonstrate the cooperative effects of natriuretic peptide signaling and FOXO nuclear activity on promoting cardiomyocyte cell cycle activity and mouse cardiac repair and regeneration after injury.Additional file 1: Supplementary Figure 1. Unprocessed versions of western blot images used in Fig.


H. pylori and antiproliferative activity against human gastric adenocarcinoma (AGS) cells of a large series of carvacrol-based compounds. Structural analyses consisted of elemental analysis, 1H/13C/19F NMR spectra and crystallographic studies. The structure-activity relationships evidenced that among ether derivatives the substitution with specific electron-withdrawing groups (CF3 and NO2) especially in the para position of the benzyl ring led to an improvement of the antimicrobial activity, whereas electron-donating groups on the benzyl ring and ethereal alkyl chains were not tolerated with respect to the parent compound (MIC/MBC = 64/64 µg/mL). Ester derivatives (coumarin-carvacrol hybrids) displayed a slight enhancement of the inhibitory activity up to MIC values of 8–16 µg/mL. The most interesting compounds exhibiting the lowest MIC/MBC activity against H. pylori  were also assayed for their ability to reduce AGS cell growth with respect to 5-Fluorouracil. Some derivatives can be regarded as new lead compounds able to reduce H. pylori growth and to counteract the proliferation of AGS cells, both contributing to the occurrence of gastric cancer.This study reports on the synthesis, structural assessment, microbiological screening against several strains of  Carvacrol is a naturally occurring monoterpene phenol abundant in several medicinal plants  which, besides its odoriferous and flavoring function, exhibits antimicrobial, food preserving, antioxidant and anticancer activities ,2. More Satureja spp., Thymus spp., and Origanum spp.) were also studied for their ability to inhibit Helicobacter pylori (H. pylori) growth, evidencing the possibility to introduce chemical modifications of this lead compound to improve this biological activity and/or to enhance its poor pharmacokinetic profile [Carvacrol and carvacrol-producing plants  cells in vitro and in Wistar rats in vivo [c cancer ; for thec cancer ,8 and eplignancy . The trihromycin  and levohromycin . Recentlhromycin ,13 as thsistance . The incsistance  induced caffolds . Moreovecaffolds ,18, whic in vivo .Starting from these premises and keeping in mind that the development of drugs from plant secondary metabolites is a topic of the most recent ongoing research and engages large-scale pharmacological screenings of extracts and active compounds, we aimed at designing a large library of carvacrol-based derivatives possessing multiple tuneable functional groups for their chemical modulation to desired properties and assuring the broadest chemical diversity. Indeed, natural product derivatives could shed light on new therapeutic agents against human diseases due to the modulation of the physical-chemical, toxicological and drug-like characteristics of their natural parent compound . This is1–46 we followed the synthetic approach reported in the 1–12, 14–34, 36, 37 and 40–42 , in the presence of potassium carbonate (K2CO3) and under nitrogen (N2) atmosphere. Some of the products obtained in this step were further employed for additional structural modifications obtained through ester hydrolysis, nitro reduction and sulfur oxidation.For the synthesis of the compounds nd 40–42 A have be12 was involved in a multistep synthesis. Firstly, it was hydrolyzed in mild conditions using lithium hydroxide (LiOH), in a mixture of water and methanol  at room temperature (RT), to provide the carboxylic acid derivative 13. Secondly, it was reacted with 3-nitrophenylhydrazine in ethanol to achieve the corresponding acetohydrazide 46 , leading to the p-NH2 derivative 35 phenyl)(methyl)sulfane, was treated with m-chloroperbenzoic acid (mCPBA) in dichloromethane (DCM). This reaction led to the two oxidized forms of sulfane  in the same step, by modulating the amount of the oxidant agent (mCPBA) added [43–45, we synthesized the coumarin-3-carboxylic acids at first, which were then used in a condensation reaction with carvacrol carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) as condensing agents in 4-methylmorpholine (NMM), gave the title compounds 43–45. The selection of this nucleus and its substituents was suggested by the good results obtained in the evaluation of H. pylori strains previously published by some of us [The NOative 35 C. DerivaA) added . For thearvacrol . Throughrol , whereas the purity of these compounds was confirmed by combustion analysis, X-ray diffraction studies (for compound 34), TLC parameters and melting point evaluation.The compounds were stable in their solid state at room temperature. The structures were confirmed by spectral studies  characterized by a different antibiotic susceptibility pattern was evaluated and the data reported in H. pylori strains.Disposing of all envisioned products, the in vitro inhibitory activity against nine strains of (1)1–13: In general, these derivatives were characterized by an increasing alkyl chain, linear or branched, saturated or unsaturated, functionalized with additional moieties . None of these modifications led to an improvement of the inhibitory activity with respect to the parent compound. Only derivatives 2 (OPr) and 3 (OBu) slightly presented MIC values comparable to carvacrol against two strains (F34/497 and F40/499), whereas compounds 6 (O-crotyl) and 9 (O-geranyl) were endowed with inferior MIC and MBC values up to 16 and 8 µg/mL, respectively, toward all the strains;Alkyloxy derivatives (2)14–40 and 46: The simplest representative of this class  had an anti-H. pylori activity comparable to carvacrol, whereas other substitutions on the aryl ring such as 3,4-diCl, 2,6-diF, 3-F, 3-OCH3, 2-Cl-4-OCH3, 2-Br, and 4-Br were detrimental or didn’t produce a strong increment of the antimicrobial activity. Conversely, some substituents, especially in the para position of the aryl ring, such as CF3, Ph, CN, NO2 and NH2 were promising to show improvements. Indeed, CF3 could act as a bioisostere of the NO2 group and in both series we can highlight the following activity order: p > m > o.Benzyloxy derivatives 3 in compound 22 didn’t synergistically contribute to an improved inhibitory action. As regards sulfur-based compounds (37–39) we highlighted a better activity with a higher sulfur oxidation state (ArSO2CH3 > ArSOCH3 > ArSCH3). Unfortunately, the introduction of bromine atoms in compounds 30 and 31 reduced the inhibitory effect likely due to their low ability to act as H-bond acceptors and their higher atomic radius, both determining a negative steric constrain. Moreover, electron-donating groups were not tolerated.The presence of a fluorine atom or a trifluoromethyl group into an organic scaffold can lead to changes in the physical, chemical and biological properties, often associated with an increase lipophilicity and electronegativity but a relatively small size, which can favour entry into the cell membranes. The presence of two CF(3)41 and 42: The change of the benzyl group into a naphthalene led to a total loss of inhibitory activity, whereas phthalimide can be tolerated;Bicyclic and heteroaryl derivatives (4)43–45: These compounds imparted a slight improvement of the anti-Helicobacter activity against all the strains with respect to carvacrol. These results were in accordance with those obtained with the same substitution pattern previously published by us [Coumarin ester derivatives ed by us ,27.The parent compound, carvacrol, displayed MIC and MBC values in the range 16–64 and 32–64 µg/mL, respectively. All the chemical modifications can be grouped into four classes to define robust structure-activity relationships (SARs):Regarding the mechanism of action, it is reasonable to consider these compounds as good bactericidal inhibitors, being the value of MBC/MIC ratio between 1 and 2.Helicobacter pylori activity (50 = 300 ± 6.5 μM) in a dose-dependent manner after treatment. Out of 17 carvacrol derivatives, only five  demonstrated a dose-dependent inhibitory effect on cell viability inferior to carvacrol. All of them possessed an IC50 value higher than the reference drug   .6 and 9) or coumarin rings (compounds 43–45) directly connected to the carvacrol oxygen led to a loss of inhibitory activity. The presence of a benzyl moiety, especially meta or para substituted, exerted some improvements. In particular, 4-CF3, 3-CH3, 4-SOCH3 and 4-SO2CH3 brought to compounds endowed with a stronger effect with respect to carvacrol. Other clear SAR trends are not observable. These small and easily accessible molecules are promising motifs in the development of dual agents able not only to reduce H. pylori growth, but also to counteract the proliferation of AGS cells at higher concentration.More in detail, carvacrol was a medium potency anti-proliferative agent against AGS cells. From the results shown in ®, Staffordshire, UK) and are uncorrected (temperatures are reported in °C). Structural analysis consisted of elemental analysis, 1H-/13C-/19F NMR spectra and crystallographic studies. 1H and 13CNMR spectra were mainly recorded at 300 MHz and 75 MHz , while some compounds were analysed at 400 MHz and 101 MHz on a Bruker spectrometer , using CDCl3 and DMSO-d6, as the solvents at room temperature. Conversely, 19F spectra were recorded on a Bruker AVANCE 600 spectrometer at 564.7 MHz, using CDCl3 as the solvent. All the compounds were studied at the final concentration of ~25 mg/mL. 1H and 13C chemical shifts are expressed as δ units (parts per millions) relative to the solvent signal, whereas 19F chemical shifts are expressed as δ units relative to an external standard . 1H spectra are described as follows: δH : chemical shift/ppm  in Hertz (Hz), number of protons, assignment). 13C spectra are described as follows: δC : chemical shift/ppm (assignment) and are fully proton decoupled. 19F spectra are described as follows: δF : chemical shift/ppm  in Hertz, number of fluorine, assignment). Multiplets are abbreviated as follows: br—broad; s—singlet; d—doublet; t—triplet; q—quartet; td—triplet of doublets; m—multiplet. The exchangeable protons  were assessed by the addition of deuterium oxide. The processing and analyses of the NMR data were carried out with MestreNova. Preparative chromatography was carried out employing silica gel . All the purifications and reactions were carried out by thin layer chromatography (TLC) performed on 0.2 mm thick silica gel-aluminium backed plates (60 F254). Spot visualization was performed under short- and long-wavelength  ultra-violet irradiation. Where given, systematic compound names were generated by ChemBioDraw Ultra 14.0 following IUPAC conventions. Microanalyses were performed with a Perkin-Elmer 260 elemental analyzer  for C, H and N and the results were within ±0.4% of the theoretical values. NMR spectra of all new compounds have been reported in the Unless otherwise indicated, all reactions were carried out under a positive pressure of nitrogen in washed and oven-dried glassware. All the solvents and reagents were directly used as supplied by Sigma-Aldrich  without further purification. Where mixtures of solvents are specified, the stated ratios are volume:volume. All melting points were measured on a SMP1 melting point apparatus . The suspension was stirred for 30 min at room temperature; then, the proper (substituted)benzyl, diarylmethyl, heteroarylmethyl or alkyl bromide (1.0 equiv.) was added and the reaction stirred until disappearance of the starting reagents, as detected by TLC. Once the reaction was completed, the mixture was poured into ice-cold water (100 mL) and extracted with dichloromethane . The organics were reunited and added with anhydrous sodium sulphate (Na2SO4) to remove water. The salt was filtered and washed three times with small amounts (5 mL) of dry DCM. The organic phase was evaporated in vacuo to afford the crude extract containing the target molecule that was recovered through column chromatography, employing silica gel (SiO2) and proper mixtures of n-hexane/ethyl acetate.To a stirring solution of carvacrol (1 equiv.) in dry DMF (10 mL) was added freshly ground and anhydrous potassium carbonate  in 10 mL of methanol was added dropwise lithium hydroxide (1.2 equiv.) dissolved in 10 mL of water. The reaction was stirred at room temperature for 24 h; then, the mixture was concentrated in vacuo to remove methanol and quenched with 3N HCl (15 mL). The precipitate was collected by filtration and washed with n-hexane to give the title compound 13, without further purification requirements.To a stirring solution of ethyl 2-(5-isopropyl-2-methylphenoxy) acetate  in 10 mL of THF was added triethyl amine (3.0 equiv.) and ethyl chloroformate (1.2 equiv.). After 30 min, 3-nitrophenylhydrazine (1.3 equiv.) was added and the reaction stirred at room temperature for 3 h. Once the reaction was completed, the mixture was poured on ice-cold water (100 mL) and the precipitate collected by filtration. Purification through column chromatography  afforded the title compound 46.To a stirring solution of ethyl 2-(5-isopropyl-2-methylphenoxy)acetic acid  in tetrahydrofuran  was added dropwise a freshly prepared solution of sodium dithionite (5.5 equiv.) dissolved in a basic solution made of water (15 mL) and sodium bicarbonate (5.5 equiv.). The reaction was stirred at room temperature until completion (assessed by TLC); then THF was evaporated in vacuo and the aqueous phase extracted with DCM (3 × 20 mL). The organics were reunited, dried over sodium sulphate and filtered to remove the salt. DCM was evaporated in vacuo to give the crude extract, that was purified by column chromatography  to afford the amino derivative 35 as an orange viscous oil.To a stirring solution of 4-isopropyl-1-methyl-2-((4-nitrobenzyl)oxy)benzene  in DCM (10 mL) placed on ice/water bath (0–5 °C), was added dropwise a freshly prepared solution of 3-chloroperbenzoic acid (1 equiv.) dissolved in 5 mL of DCM in an ice-bath. The reaction was followed by TLC and after 8 h another aliquot of 3-chloroperbenzoic acid (1 equiv. in 5 mL of DCM) was added and the reaction stirred at room temperature for further 24 h. Once the reaction completion was reached (appearance on TLC of the two spots relative to sulfoxide and sulfone derivatives), the mixture was concentrated in vacuo and the two compounds separated by column chromatography on silica gel  to give the title compounds 38 and 39.To a stirring solution of (4-((5-isopropyl-2-methylphenoxy)methyl)phenyl)(methyl) sulfane  was then dissolved in ethanol (10 mL) and hydrolyzed by using 20% NaOH solution (25 mL). After reaction completion assessed by means of TLC, the ethanol was evaporated in vacuo. The solution was quenched with 3N HCl (20 mL) leading to precipitation of the coumarin-3-carboxylic acid, that was collected by filtration and washed with n-hexane, affording the title compounds A1–C1 without further purification requirements.The corresponding ester  in 4-methylmorpholine  under nitrogen atmosphere, were added portionwise 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide  and 1-hydroxybenzotriazole . After 1 h, carvacrol (1.0 equiv.) was added and the reaction stirred for further 24 h. At the reaction completion (by TLC), the mixture was poured on ice-cold water. The precipitate was collected by filtration and washed with petroleum ether and n-hexane to afford title compounds 43–45 in good yield and purity.To a stirring solution of the proper coumarin-3-carboxylic acid (1). Colourless oil, 66% yield. 1H NMR : δ 1.29 , 1.46 , 2.23 , 2.87–2.94 , 4.06–4.11 , 6.74 , 6.76–6.78 , 7.10 . 13C NMR : δ 15.1 (CH3), 15.8 (CH3), 24.2 (2 × CH3), 34.2 (CH), 63.5 (OCH2), 109.6 (Ar), 117.9 (Ar), 124.2 (Ar), 130.4, 147.8 (Ar), 157.1 (Ar). Anal. Calcd for C12H18O: C, 80.85; H, 10.18. Found: C, 81.12; H, 10.14.2-ethoxy-4-isopropyl-1-methylbenzene (2). Colourless oil, 56% yield, mp 117–121 °C. 1H NMR : δ 1.12 , 1.31 , 1.84–1.93 , 2.26 , 2.89–2.96 , 4.00 , 6.75–6.79 , 7.11 . 13C NMR : δ 10.7 (CH3), 15.8 (CH3), 22.9 (CH2), 24.2 (2 × CH3), 34.2 (CH), 69.4 (OCH2), 109.5 (Ar), 117.8 (Ar), 124.2 (Ar), 130.4 (Ar), 147.9 (Ar), 157.2 (Ar). Anal. Calcd for C13H20O: C, 81.20; H, 10.48. Found: C, 81.33; H, 10.51.4-isopropyl-1-methyl-2-propoxybenzene (3). Yellow oil, 70% yield. 1H NMR : δ 1.37 , 1.41 , 2.36 , 2.94–3.01 , 4.66-4.69 , 5.37–5.60 , 6.14–6.27 , 6.83 , 6.86-6.89 , 7.19 . 13C NMR : δ 16.0 (CH3), 24.3 (2 × CH3), 34.3 (CH3), 68.8 (OCH2), 110.0 (Ar), 116.9 (Ar), 118.3 (=CH2), 124.3 (Ar), 130.6 (Ar), 133.9 (=CH), 147.9 (Ar), 156.8 (Ar). Anal. Calcd for C13H18O: C, 82.06; H, 9.54. Found: C, 81.87; H, 9.58.2--4-isopropyl-1-methylbenzene (4). Yellow oil, 74% yield. 1H NMR : δ 1.35 , 2.32 , 2.58 , 2.9–3.01 , 4.80 , 6.89 , 6.93 , 7.17 . 13C NMR : δ 15.9 (CH3), 24.2 (2 × CH3), 34.2 (CH), 56.01 (OCH2), 75.26 (Csp), 79.2 (CspH), 110.4 (Ar), 119.2 (Ar), 124.6 (Ar), 130.8 (Ar), 147.9 (Ar), 155.8 (Ar). Anal. Calcd for C13H16O: C, 82.94; H, 8.57. Found: C, 83.17; H, 8.54.4-isopropyl-1-methyl-2-(prop-2-yn-1-yloxy)benzene (5). Colourless oil, 78% yield. 1H NMR : δ 1.07 , 1.32 , 1.56–1.65 , 1.83–1.90 , 2.27 , 2.90–2.97 , 4.05 , 6.77–6.80 , 7.12 . 13C NMR : δ 14.0 (CH3), 15.9 (CH3), 19.5 (CH2), 24.2 (2 × CH3), 31.6 (CH2), 34.2 (CH), 67.6 (OCH2), 109.5 (Ar), 117.8 (Ar), 124.2 (Ar), 130.4 (Ar) 147.8 (Ar), 157.2 (Ar). Anal. Calcd for C14H22O: C, 81.50; H, 10.75. Found: C, 81.63; H, 10.77.2-butoxy-4-isopropyl-1-methylbenzene (6). Pale yellow oil, 69% yield. 1H NMR : δ 1.55–1.58 , 2.05 , 2.54 , 3.12–3.19 , 4.76 , 6.01–6.18 , 7.01 , 7.03 , 7.35 . 13C NMR : δ 16.2 (CH3), 18.1 (CH3), 24.4 (2 × CH3), 34.5 (CH), 68.8 (OCH2), 110.0 (Ar), 118.3 (Ar), 124.4 (Ar), 127.1 (=CH), 129.4 (=CH), 130.7 Ar), 147.9 (Ar), 157.2 (Ar). Mixture of E/Z isomers with 5.2:1 ratio. For sake of clarity, we reported only the signals related to the major isomer. Anal. Calcd for C14H20O: C, 82.30; H, 9.87. Found: C, 82.33; H, 9.88.2-(but-2-en-1-yloxy)-4-isopropyl-1-methylbenzene (7). Yellow oil, 71% yield. 1H NMR : δ 1.29–1.32 , 1.81–1.85 , 2.26 , 2.87–2.96 , 4.95 , 5.54–5.58 , 6.77 , 6.79–6.80 , 7.11 . 13C NMR : δ 16.0 (CH3), 18.3 (CH3), 24.2 (2 × CH3), 25.9 (CH3), 34.2 (CH), 65.0 (OCH2), 110.0 (Ar), 118.0 (-CH=), 120.5 (Ar), 124.4 (Ar), 130.4 (Ar), 137.0 (=C), 147.8 (Ar), 157.0 (Ar). Anal. Calcd for C15H22O: C, 82.52; H, 10.16. Found: C, 82.61; H, 10.13.4-isopropyl-1-methyl-2-((3-methylbut-2-en-1-yl)oxy)benzene (8). Colourless oil, 78% yield. 1H NMR : δ 1.01–1.06 , 1.35 , 1.45–1.61 , 1.86–1.97 , 2.29 , 2.92–2.99 , 4.06 , 6.78–6.82 , 7.14 . 13C NMR : δ 14.1 (CH3), 15.9 (CH3), 22.6 (CH2), 24.2 (2 × CH3), 28.5 (CH2), 29.2 (CH2), 34.2 (CH), 67.9(OCH2), 109.5 (Ar), 117.8 (Ar), 124.2 (Ar), 130.4 (Ar) 147.9 (Ar), 157.3 (Ar). Anal. Calcd for C15H24O: C, 81.76; H, 10.98. Found: C, 81.88; H, 11.01.4-isopropyl-1-methyl-2-(pentyloxy)benzene (9). Yellow oil, 74% yield. 1H NMR : δ 1.29–1.31 , 1.67 , 1.74 , 1.80 , 2.10–2.20 , 2.26 , 2.89–2.94 , 4.62 , 5.16–5.17 , 5.55–5.56 , 6.77 , 6.77–6.79 , 7.10 . 13C NMR : δ 16.0 (CH3), 16.7 (CH3), 17.7 (CH3), 24.2 (2 × CH3), 25.7 (CH3), 26.4 (CH2), 34.2 (CH), 39.6 (CH2), 65.0 (OCH2), 109.9 (Ar), 118.0 (Ar), 120.4 (=CH), 123.9 (Ar), 124.3 (=CH), 130.4 (Ar), 131.7 (=C), 140.1 (=C), 147.7 (Ar), 157.0 (Ar). Mixture of E/Z isomers with 2:1 ratio. For sake of clarity, we reported only the signals related to the major isomer. Anal. Calcd for C20H30O: C, 83.86; H, 10.56. Found: C, 84.01; H, 10.51.2-oxy)-4-isopropyl-1-methylbenzene (10). Colourless oil, 79% yield. 1H NMR : δ 1.30 , 2.33 , 2.97–3.06 , 4.79 , 6.89 , 6.97–7.00 , 7.21 . 13C NMR : δ 15.7 (CH3), 24.1 (2 × CH3), 34.1 (CH), 53.9 (OCH2), 110.5 (Ar), 115.8 (CN), 120.8 (Ar), 124.9 (Ar), 131.3 (Ar), 148.4 (Ar), 154.9 (Ar). Anal. Calcd for C12H15NO: C, 76.16; H, 7.99; N, 7.40. Found: C, 76.27; H, 8.00; N, 7.38.2-(5-isopropyl-2-methylphenoxy)acetonitrile (11). Yellow oil, 80% yield. 1H NMR : δ 1.27 , 2.31 , 2.35 , 2.84–2.94 , 4.55 , 6.60 , 6.82–6.84 , 7.13 . 13C NMR : δ 15.9 (CH3), 24.1 (2 × CH3), 26.7 (CH3), 34.1 (CH), 73.2 (OCH2), 109.3 (Ar), 119.1 (Ar), 124.1 (Ar), 130.9 (Ar), 148.1 (Ar), 155.9 (Ar), 206.5 (C=O). Anal. Calcd for C13H18O2: C, 75.69; H, 8.80. Found: C, 75.84; H, 8.77.1-(5-isopropyl-2-methylphenoxy)propan-2-one (12). Yellow oil, 77% yield. 1H NMR : δ 1.28–1.33 , 1.34 , 1.36 , 2.34 , 2.86–2.93 , 4.26–4.33 , 4.68 , 6.67 , 6.83 , 7.10 . 13C NMR : δ 14.2 (CH3), 15.8 (CH3), 24.1 (2 × CH3), 34.1 (CH3), 61.0 (CH2CH3), 65.7 (OCH2), 109.7 (Ar), 119.2 (Ar), 124.5 (Ar), 130.8 (Ar), 147.7 (Ar), 156.2 (Ar), 169.1 (Ar). Anal. Calcd for C14H20O3: C, 71.16; H, 8.53. Found: C, 71.22; H, 8.51.Ethyl 2-(5-isopropyl-2-methylphenoxy)acetate (13). Viscous white oil, 91% yield. 1H NMR : δ 1.23 , 2.26 , 2.85–2.95 , 4.70 , 6.61 , 6.81–6.83 , 7.08–7.11 . 13C NMR : δ 15.7 (CH3), 24.0 (2 × CH3), 34.0 (CH3), 65.3 (OCH2), 110.0 (Ar), 119.8 (Ar), 124.5 (Ar), 131.0 (Ar), 148.1 (Ar), 155.5 (Ar). Anal. Calcd for C12H16O3: C, 69.21; H, 7.74. Found: C, 69.11; H, 7.71.2-(5-isopropyl-2-methylphenoxy)acetic acid (14). Yellow oil, 88% yield. 1H NMR : δ 1.54 , 2.57 , 3.11–3.20 , 5.34 , 7.04–7.07 , 7.37 , 7.54–7.67 , 7.73 . 13C NMR : δ 16.3 (CH3), 24.4 (2 × CH3), 34.4 (CH), 70.1 (OCH2), 110.2 (Ar), 118.6 (Ar), 124.6 (Ar), 127.5 (2 × Ar), 128.0 (Ar), 128.7 (2 × Ar), 130.8 (Ar), 137.9 (Ar), 148.1 (Ar), 157.1 (Ar). Anal. Calcd for C17H20O: C, 84.96; H, 8.39. Found: C, 85.01; H, 8.42.2-(benzyloxy)-4-isopropyl-1-methylbenzene (15). Yellow oil, 86% yield. 1H NMR : δ 1.40 , 2.37 , 2.52 , 2.96–3.10 , 5.16 , 6.90–6.95 , 7.22 , 7.35–7.37 , 7.50–7.52 . 13C NMR : δ 16.1 (CH3), 19.1 (CH3), 24.3 (2 × CH3), 34.3 (CH), 68.5 (OCH2), 109.9 (Ar), 118.3 (Ar), 124.5 (Ar), 126.1 (Ar), 128.1 (Ar), 128.5 (Ar), 130.4 (Ar), 130.7 (Ar), 135.5 (Ar), 136.7 (Ar), 148.0 (Ar). Anal. Calcd for C18H22O: C, 84.99; H, 8.72. Found: C, 85.17; H, 8.70.4-isopropyl-1-methyl-2-((2-methylbenzyl)oxy)benzene (16). Pale yellow oil, 85% yield. 1H NMR : δ 1.58  2.60 , 2.67 , 3.16–3.21 , 5.33 , 7.07–7.10 , 7.38–7.43 , 7.56–7.58 . 13C NMR : δ 16.4 (CH3), 21.7 (CH3), 24.5 (2 × CH3), 34.5 (CH), 70.2 (OCH2), 110.2 (Ar), 118.6 (Ar), 124.7 (Ar), 128.3 (Ar), 128.7 (Ar), 128.8 (Ar), 130.8 (Ar), 137.9 (Ar), 138.3 (Ar), 148.1 (Ar), 157.2 (Ar). Anal. Calcd for C18H22O: C, 84.99; H, 8.72. Found: C, 85.11; H, 8.73.4-isopropyl-1-methyl-2-((3-methylbenzyl)oxy)benzene (17). Yellow oil, 73% yield. 1H NMR : δ 1.61 , 2.61 , 2.67 , 3.18–3.21 , 5.35 , 7.09–7.13 , 7.42 , 7.50 , 7.68 . 13C NMR : δ 16.4 (CH3), 21.5 (CH3), 24.6 (2 × CH3), 34.5 (CH), 70.0 (OCH2), 110.1 (Ar), 118.5 (Ar), 124.6 (Ar), 127.7 (2 × Ar), 129.5 (2 × Ar), 130.9 (Ar), 134.9 (Ar), 137.6 (Ar), 148.1 (Ar), 157.3 (Ar). Anal. Calcd for C18H22O: C, 84.99; H, 8.72. Found: C, 84.88; H, 8.69.4-isopropyl-1-methyl-2-((4-methylbenzyl)oxy)benzene (18). Yellow oil, 63% yield. 1H NMR : δ 1.27–1.30 , 2.29–2.30 , 2.38 , 2.89–2.93 , 5.05 , 6.79–6.83 , 7.00 , 7.13 . 13C NMR : δ 16.0 (CH3), 21.4 (2 × CH3), 24.2 (2 × CH3), 34.1 (CH), 70.1 (OCH2), 110.2 (Ar), 118.3 (Ar), 124.5 (Ar), 125.2 (2 × Ar), 129.4 (2 × Ar), 130.5 (Ar), 137.5 (Ar), 138.0 (Ar), 147.9 (Ar), 157.0 (Ar). Anal. Calcd for C19H24O: C, 85.03; H, 9.01. Found: C, 85.23; H, 9.05.2-oxy)-4-isopropyl-1-methylbenzene (19). Colourless oil, 84% yield. 1H NMR : δ 1.60 , 2.70 , 3.17–3.26 , 5.67 , 7.13–7.17 , 7.46 , 7.62–7.67 , 7.81–7.86 , 8.01 , 8.17 . 13C NMR : δ 16.3 (CH3), 24.3 (2 × CH3), 34.4 (CH), 66.1 (OCH2), 110.2 (Ar), 119.0 (Ar), 123.3 (CF3), 124.5 (Ar), 126.0 (Ar), 126.1 (Ar), 127.7 (Ar), 128.6 (Ar), 131.0 (Ar), 132.4 (Ar), 136.7 (Ar), 148.3 (Ar), 156.7 (Ar). 19F NMR : δ −58.63 . Anal. Calcd for C18H19F3O: C, 70.12; H, 6.21. Found: C, 69.98; H, 6.20.4-isopropyl-1-methyl-2-((2-(trifluoromethyl)benzyl)oxy)benzene (20). Colourless oil, 78% yield. 1H NMR : δ 1.58 , 2.59 , 3.14–3.23 , 5.36 , 7.09 , 7.11–7.12 , 7.40 , 7.69–7.74 , 7.84 , 7.90 , 8.06 . 13C NMR : δ 16.1 (CH3), 24.3 (2 × CH3), 34.4 (CH), 69.2 (OCH2), 110.0 (Ar), 119.0 (Ar), 124.0 (Ar), 124.0 (Ar), 124.1 (Ar), 124.6 (CF3), 124.7 (Ar), 129.2 (Ar), 130.6 (Ar), 131.0 (Ar), 139.0 (Ar), 148.3 (Ar), 156.8 (Ar). 19F NMR : δ –60.92 . Anal. Calcd for C18H19F3O: C, 70.12; H, 6.21. Found: C, 70.35; H, 6.22.4-isopropyl-1-methyl-2-((3-(trifluoromethyl)benzyl)oxy)benzene (21). Viscous colourless oil, 84% yield. 1H NMR : δ 1.46 , 2.49 , 3.00–3.12 , 5.30 , 6.96 , 6.98–7.01 , 7.30 , 7.74 , 7.82 . 13C NMR : δ 16.1 (CH3), 24.2 (2 × CH3), 34.3 (CH), 69.0 (OCH2), 110.0 (Ar), 118.9 (Ar), 124.5 (Ar), 125.5 (2 × Ar), 125.6 (Ar), 127.3 (Ar), 130.3 (CF3), 130.9 (Ar), 141.9 (Ar), 148.2 (Ar), 156.6 (Ar). 19F NMR : δ −60.77 . Anal. Calcd for C18H19F3O: C, 70.12; H, 6.21. Found: C, 70.17; H, 6.19.4-isopropyl-1-methyl-2-((4-(trifluoromethyl)benzyl)oxy)benzene (22). Yellow oil, 80% yield. 1H NMR : δ 1.26–1.28 , 2.30 , 2.87–2.94 , 5.21 , 6.78 , 6.83–6.85 , 7.14–7.16 , 7.88 , 7.97 . 13C NMR : δ 15.9 (CH3), 24.1 (2 × CH3), 34.1 (CH), 68.5 (OCH2), 109.9 (Ar), 119.3 (Ar), 121.7 (Ar), 121.9 (Ar), 124.4 (Ar), 124.6 (Ar), 127.1 (Ar), 130.9 (2 × Ar), 132.0 (2 × CF3), 140.3 (Ar), 148.1 (Ar), 156.1 (Ar). 19F : δ –66.85 . Anal. Calcd for C19H18F6O: C, 60.64; H, 4.82. Found: C, 60.80; H, 4.81.2-benzyl)oxy)-4-isopropyl-1-methylbenzene (23). Colourless oil, 82% yield. 1H NMR : δ 1.59–1.63 , 2.63 , 3.17–3.24 , 5.34 , 7.10–7.14 , 7.26–7.31 , 7.41–7.45 , 7.50–7.63 . 13C NMR : δ 16.3 (CH3), 24.4 (2 × CH3), 34.5 (CH), 69.2 (OCH2), 110.1 (Ar), 114.2 (Ar), 114.8 (Ar), 118.9 (Ar), 122.7 (Ar), 124.6 (Ar), 130.3 (Ar), 131.0 (Ar), 140.7 (Ar), 148.2 (Ar), 156.9 (Ar) 163.4 . 19F NMR : δ −111.26 . Anal. Calcd for C17H19FO: C, 79.04; H, 7.41. Found: C, 78.91; H, 7.39.2-((3-fluorobenzyl)oxy)-4-isopropyl-1-methylbenzene (24). Colourless oil, 80% yield. 1H NMR : δ 1.27–1.29 , 2.31 , 2.87–2.94 , 5.10 , 6.75 , 6.76–6.84 , 7.01–7.06 , 7.13–7.15 . 13C NMR : δ 16.0 (CH3), 24.1 (2 × CH3), 34.1 (CH), 68.6 (OCH2), 102.9 (Ar), 109.6 (Ar), 118.9 (Ar), 124.4 (Ar), 130.7 (Ar), 141.8 (Ar), 148.0 (Ar), 156.3 (Ar), 161.9 (Ar), 164.4 (Ar). 19F : δ –113.48 . Anal. Calcd for C17H18F2O: C, 73.89; H, 6.57. Found: C, 73.94; H, 6.58.2-oxy)-4-isopropyl-1-methylbenzene (25). Colourless oil, 89% yield. 1H NMR : δ 1.65 , 2.56 , 3.20–3.29 , 5.50 , 7.13–7.19 , 7.30 , 7.41 , 7.44–7.54 . 13C NMR : δ 16.0 (CH3), 24.4 (2 × CH3), 34.5 (CH), 58.4 (OCH2), 110.7 (Ar), 111.5 (Ar), 111.7 (Ar), 113.6 (Ar), 119.3 (Ar), 125.1 (Ar), 130.7 (Ar), 131.0 (Ar), 148.2 (Ar), 157.0 (Ar), 162.3 , 162.3 . 19F NMR : δ −112.80 . Anal. Calcd for C17H18F2O: C, 73.89; H, 6.57. Found: C, 74.00; H, 6.58.1,3-difluoro-2-((5-isopropyl-2-methylphenoxy)methyl)benzene (26). Colourless oil, 80% yield. 1H NMR : δ 1.26–1.29 , 2.30 , 2.87–2.92 , 3.86 , 5.10 , 6.81 , 6.89–6.91 , 7.07 , 7.12–7.14 , 7.32–7.36 . 13C NMR : δ 16.0 (CH3), 24.1 (2 × CH3), 34.1 (CH), 55.2 (OCH3), 69.8 (OCH2), 110.0 (Ar), 112.7 (Ar), 113.2 (Ar), 118.4 (Ar), 119.4 (Ar), 124.4 (Ar), 129.5 (Ar), 130.5 (Ar), 139.3 (Ar), 147.9 (Ar), 156.8 (Ar), 159.8 (Ar). Anal. Calcd for C18H22O2: C, 79.96; H, 8.20. Found: C, 80.11; H, 8.5.4-isopropyl-2-((3-methoxybenzyl)oxy)-1-methylbenzene (27). Pale yellow oil, 79% yield. 1H NMR : δ 1.52 , 2.52 , 3.11–3.20 , 5.24 , 7.01–7.04 , 7.34 , 7.54–7.61 . 13C NMR : δ 16.3 (CH3), 24.4 (2 × CH3), 34.4 (CH), 69.2 (OCH2), 110.2 (Ar), 118.8 (Ar), 124.5 (Ar), 128.7 (2 × Ar), 128.8 (2 × Ar), 130.9 (Ar), 133.7 (Ar), 136.4 (Ar), 148.1 (Ar), 156.9 (Ar). Anal. Calcd for C17H19ClO: C, 74.31; H, 6.97. Found: C, 74.43; H, 7.00.2-((4-chlorobenzyl)oxy)-4-isopropyl-1-methylbenzene (28). Colourless oil, 82% yield. 1H NMR : δ 1.26–1.28 , 2.28 , 2.87–2.92 , 3.84 , 5.13 , 6.79–6.81 , 6.86–6.89 , 6.99–7.00 , 7.10–7.12 , 7.50–7.52 . 13C NMR : δ 16.0 (CH3), 24.1 (2 × CH3), 34.1 (CH), 55.6 (OCH3), 66.9 (OCH2), 110.2 (Ar), 112.9 (Ar), 114.8 (Ar), 118.5 (Ar), 124.4 (Ar), 127.3 (Ar), 129.9 (Ar), 130.5 (Ar), 133.5 (Ar), 148.0 (Ar), 156.6 (Ar), 159.7 (Ar). Anal. Calcd for C18H21ClO2: C, 70.93; H, 6.94. Found: C, 71.10; H, 6.92.2-chloro-1-((5-isopropyl-2-methylphenoxy)methyl)-4-methoxybenzene (29). Colourless oil, 92% yield. 1H NMR : δ 1.59 , 2.58 , 3.16–3.21 , 5.22 , 7.05 , 7.10 , 7.39 , 7.48–7.52 , 7.64 , 7.79–7.80 . 13C NMR : δ 16.4 (CH3), 24.5 (2 × CH3), 34.5 (CH), 68.5 (OCH2), 110.0 (Ar), 119.1 (Ar), 124.5 (Ar), 126.6 (Ar), 129.2 (Ar), 130.7 (Ar), 131.1 (Ar), 131.8 (Ar), 132.8 (Ar), 138.3 (Ar), 148.2 (Ar), 156.7 (Ar). Anal. Calcd for C17H18Cl2O: C, 66.03; H, 5.87. Found: C, 65.94; H, 5.88.1,2-dichloro-4-((5-isopropyl-2-methylphenoxy)methyl)benzene (30). White powder, 61% yield, mp 51–52 °C. 1H NMR : δ 1.27 , 2.32 , 2.83–2.94 , 5.16 , 6.79–6.82 , 7.13 , 7.16–7.23 , 7.34–7.40 , 7.60–7.65 . 13C NMR : δ 16.1 (CH3), 24.2 (2 × CH3), 34.1 (CH), 69.2 (OCH2), 110.1 (Ar), 118.5 (Ar), 122.1 (Ar), 124.4 (Ar), 127.6 (Ar), 128.7 (Ar), 129.0 (Ar), 130.6 (Ar), 132.5 (Ar), 136.9 (Ar), 148.0 (Ar), 156.4 (Ar). Anal. Calcd for C17H19BrO: C, 63.96; H, 6.00. Found: C, 63.85; H, 5.99.2-((2-bromobenzyl)oxy)-4-isopropyl-1-methylbenzene (31). Colourless oil, 66% yield. 1H NMR : δ 1.55 , 2.56 , 3.12–3.18 , 5.25 , 7.04–7.09 , 7.38 , 7.55 , 7.71–7.75 . 13C NMR : δ 16.4 (CH3), 21.5 (CH3), 24.5 (2 × CH3), 34.5 (CH), 69.3 (OCH2), 110.0 (Ar), 118.8 (Ar), 121.9 (Ar), 124.5 (Ar), 129.1 (2 × Ar), 130.9 (Ar), 131.9 (2 × Ar), 136.9 (Ar), 148.1 (Ar), 156.9 (Ar). Anal. Calcd for C17H19BrO: C, 63.96; H, 6.00. Found: C, 63.99; H, 6.02.2-((4-bromobenzyl)oxy)-4-isopropyl-1-methylbenzene (32). White powder, 91% yield, mp 56–57 °C. 1H NMR : δ 1.25 , 2.32 , 2.86–2.91 , 5.50 , 6.78 , 6.80–6.83 , 7.13 , 7.50–7.53 , 7.69–7.75 , 7.98–8.01 , 8.17–8.20 . 13C NMR : δ 16.1 (CH3), 24.1 (2 × CH3), 34.1 (CH), 66.6 (OCH2), 110.0 (Ar), 118.8 (Ar), 124.2 (Ar), 124.9 (Ar), 128.2 (Ar), 128.5 (Ar), 130.7 (Ar), 134.0 (Ar), 134.4 (Ar), 146.9 (Ar), 148.2 (Ar), 156.1 (Ar). Anal. Calcd for C17H19NO3: C, 71.56; H, 6.71; N, 4.91. Found: C, 71.71; H, 6.70; N, 4.92.4-isopropyl-1-methyl-2-((2-nitrobenzyl)oxy)benzene (33). Yellow viscous oil, 72% yield. 1H NMR : δ 1.36 , 2.38 , 2.96–3.00 , 5.24 , 6.88–6.91 , 7.19 , 7.60–7.65 , 7.89 , 8.23 , 8.43 . 13C NMR : δ 16.1 (CH3), 24.2 (2 × CH3), 34.2 (CH), 68.5 (OCH2), 109.9 (Ar), 119.0 (Ar), 121.9 (Ar), 122.7 (Ar), 124.3 (Ar), 129.6 (Ar), 130.9, 133.1 (Ar), 139.9 (Ar), 148.1 (Ar), 148.4 (Ar), 156.3 (Ar). Anal. Calcd for C17H19NO3: C, 71.56; H, 6.71; N, 4.91. Found: C, 71.43; H, 6.73; N, 4.92.4-isopropyl-1-methyl-2-((3-nitrobenzyl)oxy)benzene (34). Yellow amber powder, 95% yield, mp 88–95 °C. 1H NMR : δ 1.23 , 2.28 , 2.84–2.88 , 5.19 , 6.71 , 6.80 , 7.11 , 7.64 , 8.26 . 13C NMR : δ 15.9 (CH3), 24.1 (2 × CH3), 34.1 (CH3), 68.5 (OCH2), 109.7 (Ar), 110.0 (Ar), 119.0 (2 × Ar), 123.8 (2 × Ar), 124.3 (Ar), 127.4 (Ar), 130.8 (Ar), 145.1 (Ar), 148.1 (C=O), 156.2 (C=O). Anal. Calcd for C17H19NO3: C, 71.56; H, 6.71; N, 4.91. Found: C, 71.63; H, 6.69; N, 4.90.4-isopropyl-1-methyl-2-((4-nitrobenzyl)oxy)benzene (35). Orange viscous oil, 70% yield. 1H NMR : δ 1.24–1.27 , 2.23 , 2.86–2.91 , 3.86 , 4.97 , 6.75–6.80 , 7.09 , 7.28 . 13C NMR : δ 16.1 (CH3), 24.2 (2 × CH3), 34.2 (CH), 70.1 (OCH2), 110.1 (Ar), 115.1 (2 × Ar), 118.2 (Ar), 124.5 (Ar), 124.6 (Ar), 128.9 (2 × Ar), 130.4 (2 × Ar), 130.8 (Ar), 146.0 (Ar), 147.8 (Ar), 157.1 (Ar). Anal. Calcd for C17H21NO: C, 79.96; H, 8.29; N, 5.49. Found: C, 80.14; H, 8.27; N, 5.51.4-((5-isopropyl-2-methylphenoxy)methyl)aniline (36). White powder, 79% yield, mp 74–76 °C. 1H NMR : δ 1.21–124 , 2.27 , 2.81–2.88 , 5.14 , 6.71 , 6.79 , 7.11 , 7.58 , 7.70 . 13C NMR : δ 16.0 (CH3), 24.1 (2 × CH3), 34.1 (CH), 68.9 (OCH2), 109.7 (Ar), 111.4 (CN), 118.8 (Ar), 124.3 (Ar), 127.3 (2 × Ar), 130.7 (Ar), 132.4 (2 × Ar), 143.1 (Ar), 148.1 (Ar), 156.2 (Ar). Anal. Calcd for C18H19NO: C, 81.47; H, 7.22; N, 5.28. Found: C, 71.56; H, 6.71; N, 4.91.4-((5-isopropyl-2-methylphenoxy)methyl)benzonitrile (37). White powder, 81% yield, mp 61–62 °C. 1H NMR : δ 1.35 , 2.35 , 2.56 , 2.92–2.99 , 5.19 , 6.86–6.88 , 7.18 , 7.35–7.38 , 7.65 . 13C NMR : δ 15.9 (SCH3), 16.2 (CH3), 24.4 (2 × CH3), 34.3 (CH), 69.6 (OCH2), 110.0 (Ar), 118.5 (Ar), 124.4 (Ar), 126.8 (2 × Ar), 128.0 (2 × Ar), 130.6 (Ar), 134.5 (Ar), 138.0 (Ar), 148.0 (Ar), 156.9 (Ar). Anal. Calcd for C18H22OS: C, 75.48; H, 7.74. Found: C, 75.37; H, 7.72.(4-((5-isopropyl-2-methylphenoxy)methyl)phenyl)(methyl)sulfane (38). Yellow viscous oil, 51% yield. 1H NMR : δ 1.24 , 2.27 , 2.77 , 2.85–2.87 , 5.15 , 6.75–6.80 , 7.09–7.12 , 7.65 . 13C NMR : δ 16.0 (CH3), 24.1 (2 × CH3), 34.1 (CH), 44.7 (SCH3), 69.1 (OCH2), 109.9 (Ar), 118.7 (Ar), 124.1 (Ar), 124.3 (2 × Ar), 128.0 (2 × Ar), 130.7 (Ar), 141.2 (Ar), 145.0 (Ar), 148.0 (Ar), 156.4 (Ar). Anal. Calcd for C18H22O2S: C, 71.49; H, 7.33. Found: C, 71.35; H, 7.30.4-isopropyl-1-methyl-2-((4-(methylsulfinyl)benzyl)oxy)benzene (39). Yellow viscous oil, 25% yield. 1H NMR  δ 1.21–1.22 , 2.27 ; 2.84–2.86 , 3.07 ; 5.17 , 6.72 , 6.79 , 7.09–7.12 ; 7.67 , 7.97 . Anal. Calcd for C18H22O3S: C, 67.89; H, 6.96. Found: C, 68.01; H, 6.99.4-isopropyl-1-methyl-2-((4-(methylsulfonyl)benzyl)oxy)benzene (40). White solid, 90% yield, mp = 99–100 °C. 1H NMR : δ 1.29–1.31 , 2.33 , 2.89–2.96 , 5.18 , 6.81–6.85 , 7.14–7.16 , 7.38–7.42 , 7.48–7.52 , 7.57–7.60 , 7.65–7.68 . 13C NMR : δ 16.1 (CH3), 24.2 (2 × CH3), 34.2 (CH), 69.7 (OCH2), 110.1 (2 × Ar), 118.4 (2 × Ar), 124.5 (Ar), 127.2 (2 × Ar), 127.3 (2 × Ar), 127.4 (2 × Ar), 127.7 (2 × Ar), 128.8 (Ar), 130.6 (Ar), 126.7 (Ar), 148.0 (Ar), 156.9 (Ar). Anal. Calcd for C23H24O: C, 87.30; H, 7.64. Found: C, 87.47; H, 7.65.4-((5-isopropyl-2-methylphenoxy)methyl)-1,1′-biphenyl (41). Yellow-brown sticky solid, 98% yield. 1H NMR : δ 1.29 , 2.21 , 2.88–2.97 , 5.52 , 6.80–6.83 , 6.95–6.96 , 7.48 , 7.50 , 7.51–7.59 , 7.66  7.86–7.94 , 8.10–8.14 . 13C NMR : δ 16.0 (CH3), 24.2 (2 × CH3), 34.2 (CH), 68.6 (OCH2), 110.0 (Ar), 118.4 (Ar), 123.9 (Ar), 124.6 (Ar), 125.3 (Ar), 125.8 (Ar), 126.2 (Ar), 126.3 (Ar), 128.6 (Ar), 128.8 (Ar), 130.6 (Ar), 131.6 (Ar), 132.9 (Ar), 133.7 (Ar), 148.0 (Ar), 156.9 (Ar). Anal. Calcd for C21H22O: C, 86.85; H, 7.64. Found: C, 86.97; H, 7.66.1-((5-isopropyl-2-methylphenoxy)methyl)naphthalene (42). Colourless viscous oil, 88% yield. 1H NMR : δ 1.23–1.26 , 2.18 , 2.80–2.93 , 5.67 , 6.79–6.82 , 7.06–7.04 , 7.74–7.78 , 7.88–7.92 . 13C NMR : δ 15.8 (CH3), 24.1 (2 × CH3), 34.0 (CH), 65.5 (OCH2), 112.6 (Ar), 120.3 (Ar), 123.8 (2 × Ar), 125.5 (Ar), 130.8 (Ar), 131.8 (Ar), 134.5 (2 × Ar), 148.0 (Ar), 154.3 (Ar), 167.2 (2 × C=O). Anal. Calcd for C19H19NO3: C, 73.77; H, 6.19; N, 4.53. Found: C, 73.69; H, 6.18; N, 4.55.2-((5-isopropyl-2-methylphenoxy)methyl)isoindoline-1,3-dione (H-chromene-3-carboxylate (43). White powder, 76% yield, mp 122–124 °C. 1H NMR : δ 1.24 , 2.22 , 2.44 , 2.89–2.95 , 7.01–7.07 , 7.17–7.19 , 7.28–7.31 , 7.44–7.51 , 8.69 . 13C NMR : δ 16.0 (CH3), 20.8 (CH3 coum.), 23.9 (2 × CH3), 33.6 (CH3), 116.7 (Ar), 117.4 (Ar), 117.6 (Ar), 119.8 (Ar), 124.5 (Ar), 127.2 (Ar), 129.4 (Ar), 131.0 (Ar), 134.8 (Ar), 136.0 (Ar), 148.2 (Ar), 149.0 (Ar), 149.9 (Ar), 153.6 (Ar), 156.8 (C=O), 161.6 (C=O). Anal. Calcd for C21H20O4: C, 74.98; H, 5.99. Found: C, 75.07; H, 6.00.5-isopropyl-2-methylphenyl 6-methyl-2-oxo-2H-chromene-3-carboxylate (44). White powder, 70% yield, mp 114–115 °C. 1H NMR : δ 1.24 , 2.21 , 2.87–2.92 , 7.00 , 7.04–7.08 , 7.19 , 7.34–7.37 , 7.60–7.65 , 8.64 . 13C NMR : δ 15.9 (CH3), 23.9 (2 × CH3) 33.6 (CH), 118.4 (Ar), 118.7 (Ar), 118.8 (Ar), 119.6 (Ar), 124.7 (Ar), 127.1 (Ar), 128.7 (Ar), 130.3 (Ar), 131.0 (Ar), 134.6 (Ar), 148.2 (Ar), 148.3 (Ar), 148.9 (Ar), 153.7 (Ar), 155.8 (C=O), 161.1 (C=O). Anal. Calcd for C20H17ClO4: C, 67.33; H, 4.80. Found: C, 67.40; H, 4.79.5-isopropyl-2-methylphenyl 6-chloro-2-oxo-2H-chromene-3-carboxylate (45). White powder, 73% yield, mp 134–138 °C. 1H NMR : δ 1.24 , 2.21 , 2.84–2.96 , 7.00 , 7.05–7.08 , 7.19 , 7.30 , 7.74–7.80 , 8.64 . 13C NMR : δ 15.9 (CH3), 23.9 (2 × CH3), 33.6 (CH), 117.5 (Ar), 118.6 (Ar), 118.8 (Ar), 119.3 (Ar), 119.6 (Ar), 124.7 (Ar), 127.1 (Ar), 131.1 (Ar), 131.7 (Ar), 137.4 (Ar), 148.2 (Ar), 148.2 (Ar), 148.9 (Ar), 154.2 (Ar), 155.8 (C=O), 161.0 (C=O). Anal. Calcd for C20H17BrO4: C, 59.87; H, 4.27. Found: C, 59.92; H, 4.28.5-Isopropyl-2-methylphenyl 6-bromo-2-oxo-2N′-(3-nitrophenyl)acetohydrazide (46). White solid; 62% yield, mp = 127–128 °C. 1H NMR : δ 1.26–1.28 , 2.34 , 2.88–2.95 , 4.76 , 6.41–6.42 , 6.73 , 6.89–6.91 , 7.15–7.18 , 7.37–7.39 , 7.69–7.71 , 7.77–7.79 , 8.33–8.34 . 13C NMR : δ 16.1 (CH3), 24.1 (2 × CH3), 34.1 (CH), 67.4 (OCH2), 108.0 (Ar), 109.9 (Ar), 116.0 (Ar), 119.1 (Ar), 120.2 (Ar), 123.8 (Ar), 130.0 (Ar), 131.2 (Ar), 148.7 (Ar), 148.9 (Ar), 149.2 (Ar), 155.1 (Ar), 169.0 (C=O). Anal. Calcd for C18H21N3O4: C, 62.96; H, 6.16; N, 12.24. Found: C, 63.05; H, 6.19; N, 12.29.2-(5-isopropyl-2-methylphenoxy)-−1, F(000) = 608. 9670 reflections were collected with a 4.356 < θ < 29.273 range with a completeness to theta 99.1%; 3395 were unique, the parameters were 190 and the final R index was 0.0619 for reflections having I > 2σI.C17H19NO3, M = 285.33, Monoclinic, space group P 21/c, a = 8.380(1), b = 15.416(1), c = 11.375(1)Å, β = 95.856(7), V = 1462.6(2)Å3, Z = 4 Dc = 1.296, µ = 0.089 mmA light yellow prismatic shaped crystal (0.07 × 0.06 × 0.03) was used for data collection. Hydrogen atoms were all assigned in calculated positions and refined as isotropic. No relevant hydrogen bonds were detected. CCDC 1980137 contains the supplementary crystallographic data for this molecule. Data can be obtained free of charge from the Cambridge Crystallographic Data Centre . CollectThe MIC determination was performed by modified broth microdilution assay as previously described . For MBCThe human adenocarcinoma gastric cell line (AGS), was derived from an untreated human adenocarcinoma of the stomach and retained the same cytological characteristics of the malignant cells obtained from Caucasian patients . The AGS6, 9, 16, 17, 20, 21, 29, 32–35, 38, 39, 42–45) (600 mM) were freshly prepared in dimethyl sulfoxide (DMSO) and in DMEM according to the experimental design by serial dilutions in complete culture medium. The final concentration of DMSO in experiments was 0.14%. No toxicity on AGS cells was observed (data not shown). 5-Fluorouracil was used as positive control.Working solutions of carvacrol and its derivatives (H-tetrazolium)) assay . The concentration of carvacrol and its derivatives for treatments was selected based on concentration—response curves constructed in preliminary experiments (data not shown). Briefly, AGS cells were seeded in 96-well plates (6 × 103 cells/well) and treated for 24 h with different concentrations (50–800 μM) of specific molecules (5 replica wells for each treatment condition).Cell viability was tested by MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-250 value was calculated from the concentration-response curves by nonlinear regression analysis [Briefly, cells were incubated with the MTS solution for at least 1 h and cell viability was determined colorimetrically by measuring the absorbance at 490 nm using GloMax-Multi Detection System . Cell viability was expressed as the percentage compared with the untreated cells designated as 100%. The ICanalysis .p value of 0.05 was considered statistically significant. IC50 values was calculated using the GraphPad Prism 7 software.A H. pylori and AGS cells) along with the assessment of robust structure-activity relationships. Several hits with required balance of activities were extrapolated as a result of this study. Moreover, the most active compounds displayed antimicrobial activity with similar MIC and MBC values toward H. pylori strains with a different antibiotic susceptibility pattern, thus suggesting a mechanism of action alternative to metronidazole, amoxicillin and clarithromycin. The most important result is that the anti-Helicobacter pylori activity was not only strictly related to the presence of an OH moiety, as reported for the general antibacterial activity of the parent compound. Among the compounds evaluated, some analogues exhibited MIC/MBC values in the low µg/mL range. In this regard, the most noteworthy compounds displaying the lowest MIC/MBC activity against H. pylori, such as compounds 16 and 39, also showed good activity against AGS cells . Further studies might explain the potential synergistic effects of the combination of these derivatives with the currently used therapeutics. In addition, the possibility to treat drug resistant H. pylori strains would be beneficial in the clinical setting due to the high development of resistance attributed to H. pylori. Finally, whereas the development of biofilms by H. pylori as well as the ability of the microorganism to enter the Viable But Non-Culturable (VBNC) state represent two different survival strategies that induce resistance/tolerance of the microorganism or the microbial community to antimicrobial drugs [H. pylori biofilm and VBNC state.Based on the shortlisted hits, a large series of carvacrol-based molecules were designed, synthesized and evaluated for their ability to act as dual agents (inhibitory action against the growth of al drugs , future


Support for rehabilitation research at the National Institutes of Health (NIH) is robust and evolving. Since the time of its Blue Ribbon Panel on Rehabilitation Research, NIH has participated in several initiatives to coordinate the science and advance the field.Collaborative teams must continue to address key limitations in the field, including the desire for broad application of rehabilitation interventions, the need for basic science and translational research, the support of clinical trials and standard approaches, and the integration of technology.Rehabilitation medicine is poised for critical advancements if interdisciplinary teams continue to work collaboratively to understand and address the needs of people with temporary or permanent functional limitations. Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), in 2015. It was an honor and a privilege to lead efforts at NIH, and none of the work performed over the ensuing years would be possible without the staff of the Center, our NIH colleagues, our federal partners, and an involved and engaged community of researchers, stakeholders, and advocates. This was truly an interdisciplinary team that evolved the guidance and direction of rehabilitation research funded by NIH; we achieved many goals. In this commentary, I will provide a review of the past four and a half years of change at the NIH with respect to rehabilitation research, the programs initiated by the NIH rehabilitation research community, what is needed to evolve the science of rehabilitation, and on the challenges we continue to face.In 2012, the National Institutes of Health (NIH) convened a Blue Ribbon Panel on Rehabilitation Research to advise the NIH Director on the changes needed in the coordination and funding of rehabilitation science, the necessary training and research infrastructure for the field, and the methods by which training of the next generation of scientists in this area could be encouraged [The Blue Ribbon Panel served as a catalyst for the community, and NIH recognized the need for a robust and coordinated response to the recommendations. First and foremost, a research agenda with clear priorities was required by legislation. But more than simply meeting a requirement, the Research Plan on Rehabilitation  allowed The scientific conference that supported the development of the research plan highlighted the key tenet that rehabilitation is applicable across diseases and conditions . It alsoAfter unifying the rehabilitation community at NIH with a set of shared priorities, disseminating the willingness for the NIH to support rehabilitation research across the Institutes and Centers through the conference and outreach at professional meetings, and regular communication with the field regarding research opportunities through communication efforts, there was a sense that the rehabilitation community could accomplish a great deal.The Medical Rehabilitation Coordinating Committee, which is a coordinating committee of 20 Institutes and Centers within NIH, began to pursue multiple lines of effort to further the work stemming from the Research Plan and the conference that are yielding results now . This enWithin NIH, the NCMRR, the National Institute of Neurological Disorders and Stroke (NINDS), and the Office of Disease Prevention will host a conference on the benefits of physical activity for individuals who use wheelchairs, with an accompanying systematic review completed by the Agency on Healthcare Research and Quality to support the inclusion of evidence-based recommendations in federal guidelines. NCMRR recently launched a Common Data Elements effort for rehabilitation in collaboration with the NINDS. The NIH community continues to sponsor funding announcements, most notably in community-based cardiac rehabilitation, rehabilitation research infrastructure, and training and career development.SM Initiative, or NIH HEAL InitiativeSM, and were successful in gaining support for multiple applications, including those focused on lower back pain, cancer rehabilitation, primary care provision of physical therapy, and new devices to treat or remediate pain [The NIH Medical Rehabilitation Coordinating Committee was integrally involved in large NIH efforts, such as the All of Us program, where we advocated for the inclusion of individuals with disability, successfully including a community partner for the program and establishing the evidence for the participant-provided information survey for disability . We engaate pain .NIH is consistently working with federal partners to advance the coordination of rehabilitation research efforts. The NCMRR funded an effort to advance the field of limb amputation and limb salvage by supporting the development of a registry in collaboration with the Department of Defense, that is moving toward an initial pilot in spring of 2020. NCMRR and colleagues from the National Institute of Diabetes and Digestive and Kidney Diseases are working with our federal colleagues from the Departments of Defense, Veterans Affairs, and throughout the Department of Health and Human Services to standardize common data elements in the limb loss field. This work to arrive at common definitions and standards across agencies, we hope, will serve as an exemplar to ease regulatory and reimbursement considerations for new approaches and devices for this population. NCMRR collaborated with the Centers for Disease Control and Prevention to fund the integration of disability questions into a national pregnancy surveillance effort to support evidence development for obstetric care for individuals with disability. NCMRR, the National Institute on Deafness and Other Communication Disorders and the Fogarty International Center worked collaboratively to support efforts on global implementation of rehabilitation with the US Agency for International Development and the World Health Organization and helped to establish global priorities for rehabilitation research, data, and infrastructure.Recent NIH-sponsored workshops and conferences have focused on innovative technologies for pediatric rehabilitation, clinical trials in rehabilitation, strategic directions for cerebral palsy and spinal cord injury research, integrative treatments, and medical device development and commercialization. The NIH Medical Rehabilitation Research Coordinating Committee is an engaged team, ready to pursue any avenue to promote rehabilitation as a critical area of science.As the field continues to evolve, I encounter several recurring themes when I attend conferences, workshops, meetings, and gatherings that occur in scientific, legislative, advocacy, and stakeholder domains. From my perspective, these themes can hamper progress, discourage young investigators from adopting new approaches, and limit innovation. I will highlight and rebut a few of these in the hope that the field can change the dogma.“Rehabilitation is intended only for people with chronic disability.” While it is true that individuals with chronic disability require evidence to support their need for continued access to rehabilitation over the lifespan, this should not be the only goal in our field. Rehabilitation and the science underlying rehabilitation approaches are critical to remediating temporary functional limitations from injury or surgery, to enable improved function after treatment for disease , and to engage older individuals who are on the cusp of aging into functional limitations. Imposing limits or curtailing a broader agenda limits the field and does not allow for broader applications.“Basic science is not a priority for rehabilitation research.” The basic science portfolio for rehabilitation, unlike many portfolios at NIH, is relatively small. However, concentrating only on interventions and outcomes misses a fundamental underpinning of science that contributes to advances that are well-known in other fields. The discovery of biomarkers, quantifiable variables that can be monitored, is critical to prognosis, diagnosis, and monitoring of recovery across conditions. Moreover, these biologic, imaging, or sensor-based measures allow for the development of interventions that are more precisely targeted to symptoms or individuals. The field is critically lacking endeavors in genomics, epigenomics, proteomics, transcriptomics, the microbiome, serum-based biomarkers, and imaging indicators that could contribute to more evidence-based care. This involves a continuum of research that is underemphasized in rehabilitation medicine and requires new partnerships with scientists in these fields to advance more quickly.“We cannot conduct large, adequately powered trials of rehabilitation interventions.” After working as a mental health clinician and researcher in the Department of Veterans Affairs, where psychotherapeutic interventions were randomized, tested, implemented, revised, and evaluated [“Each patient is different, you can’t have a standard approach,” and “There is no way to blind or control for exposure to the treatment.” Work currently ongoing in the field rebuts these contentions [valuated , there itentions . There i“It is hard to quantify rehabilitation, identify treatment targets, and measure progress”. Technology holds enormous promise for quantitative measurement of movement, discovery of biomarkers through high-throughput analyses, screening for potential treatment targets, outcome measurement, and monitoring social participation and community engagement. While technology is not the only solution for these challenges in rehabilitation research, the field needs to consider how to move to more rigorous and quantifiable metrics. Integration of sensors, videocapture, app-based tools, gaze technologies, voice analysis, new imaging approaches, innovative modeling, and machine learning approaches are critical to supporting the science and to engaging young investigators. Commercial off-the-shelf tools and research kits are available and the costs are lowering quickly for tools that were once prohibitively expensive. Collaborations are a key part of this work; technology companies, medical equipment providers, computer scientists, and engineers are increasingly interested in how their skills and abilities can assist individuals with temporary or chronic functional limitations. This is not to say that technology is the only solution to this set of problems, and there is a clear need to ensure that the solutions are cost-effective and accessible. However, the field of rehabilitation will miss opportunities and advancements if we do not continue to progress in this area.Leading the National Center for Medical Rehabilitation Research was an honor and a privilege. In this position, I had the opportunity to oversee new and emerging science, promote and support the field, and meet people all over the world who see rehabilitation as a global need, critical to enabling health. At every meeting or conference, there is a young investigator with passion, drive, and a sense of novelty that inspires others to keep advocating for rehabilitation.Rehabilitation is my professional mission. Throughout my career, starting with my undergraduate training, I found many ways to engage in the work. I came into this leadership role after serving in roles with individuals with chronic or temporary disability, from driving the van to the grocery and assisting with shopping, to managing a polytrauma care clinic, to conducting research on the effect of exercise on cognitive recovery. The one tenet that followed me throughout is “keep the person in the center.” My hope is that the next generation of basic scientists understands this tenet as well as the clinician scientist. Scientific work divorced from an end goal of improving health and optimizing function for a person can be an empty endeavor.There is an African proverb that reads “If you want to go fast, go alone; if you want to go far, go together.” In rehabilitation, different than some other disciplines, the work is fundamentally team-based. Throughout my career, I was honored to work with people contending with functional limitations, rehabilitation teams, interdisciplinary research collaborators, and my federal colleagues to advance a rehabilitation treatment plan, research project, or large policy or research agenda. The focus was to improve function, promote participation, and ensure that individuals with limitations are included in scientific research. Finding methods to recognize the efforts of these teams, especially in light of the challenges team science poses to investigators seeking career advancement, will be critical in the future. This will require effort to incorporate collaboration metrics rather than individual effort in evaluation and to de-emphasize traditional measures of success in favor of those that value the efforts that underly collaboration . I hope that the community continues to see the value in this team-based approach to overcome the challenges facing our field and to advance the science. Rehabilitation is critical to enabling healthy and optimal lives, and together, we will continue to support the evidence needed to achieve that goal.


OPA1‐Dominant Optic Atrophy (OPA1‐DOA) led to some degrees of visual improvement in seven patients. We here present the results of a cohort study, which investigated the effect of off‐label idebenone administration in a larger OPA1‐DOA group compared with untreated patients. Inclusion criteria were: OPA1‐DOA clinical and molecular diagnosis, baseline visual acuity (VA) greater than/equal to counting fingers and treatment duration greater than 7 months. We found a significant difference between the last visit and baseline VA in favor of stabilization/recovery in idebenone‐treated as compared to untreated patients. This effect was retained after controlling for confounders.We previously documented that idebenone treatment in  OPA1 haploinsufficiency heterozygous mutations, who were treated for at least 1 year with idebenone.OPA1‐linked DOA is characterized by defective mitochondrial fusion and cristae derangement, which in turn lead to defective oxidative phosphorylation with reduced ATP synthesis driven by complex I substrates.OPA1‐mutant DOA (OPA1‐DOA) patients pointed to a possible beneficial effect of idebenone therapy documenting the improvement of VA in these patients.The results of a double‐blind, placebo‐controlled, randomized clinical trial,OPA1 mutations leading to haploinsufficiency or missense point mutations have been treated with idebenone. Thus, through this observational cohort study, we aimed at investigating the effect of off‐label idebenone administration on visual outcome in OPA1‐DOA patients compared to untreated OPA1‐DOA individuals, considered as controls.After this pilot report, a larger group of DOA patients carrying either This study has a historical cohort design and follows the STROBE guidelines.OPA1‐DOA diagnosis were enrolled and divided in those who were treated with off‐label idebenone for at least 7 months between April 2007 and April 2017, compared with those untreated (controls). Similarly to our previous retrospective analysis of idebenone use in LHONhttps://www.ema.europa.eu/en/medicines/human/EPAR/raxone#authorisation-details-section). The dosage in individual cases was modulated to avoid the occurrence of specific clinical side effects or blood exam abnormalities , thus adjusting to maximal dosage without side effects, as required for off‐label drug administration. Patients with evidence or previous history of glaucoma or with any optic neuropathy other than DOA, and with a baseline best‐corrected‐visual acuity (VA) less than counting fingers (CF) were excluded.Patients satisfying inclusion criteria for a molecular and clinical defined OPA1‐DOA patients and stratified them into 37 untreated subjects and 50 idebenone‐treated.We identified 87 OPA1‐DOA is characterized by a relentless visual loss progression without spontaneous recovery.‐stabilization/recovery;‐worsening.The current knowledge of the natural history of For treated patients, baseline was considered as the last VA before the start of idebenone therapy, whereas in untreated baseline was the first available VA.logMAR VA: the subject’s eye with lower logMAR VA was the better‐seeing eye. If both eyes had an equal logMAR acuity, the following criterion was used;Mean Deviation (MD) for VF ;if the eyes were equal based on criteria 1 and 2, the best‐seeing eye was randomly selected.For statistical analysis, eyes were classified into the best‐ and worst‐seeing based on baseline visual examination. The pre‐defined algorithm for determining the best‐seeing eyes was the following:We followed a “one‐eye” approach by evaluating the best‐seeing eye as the one assumed to have more benefit from the idebenone therapy. We also assessed the worst‐seeing eye, to check for the degree of concordance of the two eyes as non‐independent variables.OPA1 mutation type, baseline VA, baseline age, observation time). Two‐sided P‐values and 95% CIs are presented. For statistical analyses, SPSS  and Stata SE  softwares were used.Chi‐square, Wilcoxon, Mann‐Whitney U‐ tests were used to compare variables among groups. We performed univariate and multivariate logistic regression analyses to study the association between idebenone administration (exposure), the outcome of interest (VA stabilization/recovery) and confounding factors . Most patients  took 540 mg/day of idebenone, ten 270 mg/day, ten 405 mg/day, five 675 mg/day, and only one 10‐year‐old patient received 135 mg/day. According to the established criteria we classified 74 eyes as stable/recovery and 13 as worsening. Considering the 74 stable/recovery best‐seeing eyes, 94.5% of the corresponding worst‐seeing eyes were stable/recovery as well (data not shown).In this study we included 87 patients from 69 unrelated pedigrees Table . Demograuntreated: Mdn, Q1–Q3 = 0.52, 0.2–0.9; VAidebenone: Mdn, Q1–Q3 = 0.52, 0.3–1.0) and last visit  time‐points was significantly different only in the treated group, which showed stability of visual acuity in most cases  which further confirms the tendency towards VA‐stabilization in idebenone‐treated group   Fig..OPA1‐DOA (Table We also used a univariate logistic regression model Table  to evaluOA Table .OPA1 pathogenic mutations was significantly associated with stabilization/recovery of visual acuity. In fact, DOA patients taking idebenone benefit four times more than untreated ones in terms of visual stabilization/recovery, even after controlling for confounders. Despite the major limitations due to the study’s retrospective nature, such as possible sample bias and not‐homogeneous between‐groups observational time and not‐homogeneous idebenone dosage, these findings point to a probable benefit of idebenone therapy for DOA patients.The key finding of our study is that off‐label idebenone administration in DOA patients carrying This study deserves a few comments. Data were collected from both eyes, but we did not combine them using a binary correlation, due to small sample size.OPA1‐DOA may be ameliorated by idebenone, as for LHON.OPA1‐related DOA would represent a very relevant option to fight blindness in the largest categories of inherited optic neuropathies. This is an important opportunity for this rare and currently untreatable disease while waiting for other therapeutic options such as gene therapyIn conclusion, this study strengthens the initial positive trend we have reported in 2013The authors declare that they have no conflict of interest related to the content of this article.MR, VC, wrote the first draft of the manuscript. MR, CLM, MC, PB, VC, contributed to the conception and design of the study. MR, CLM, MC, LDV, GA, MLC, PB, VC, contributed to acquisition of the data. MR, CZ, contributed to the statistical analysis of data. MR, CLM, MC, LDV, GA, MLC, PB, VC, contributed to the interpretation of the data. MR, CLM, MC, CZ, PB, VC, contributed to drafting a significant portion of the manuscript or figures or tables. MR, CLM, MC, LDV, GA, CZ, MLC, PB, VC, reviewed the manuscript and provided revisions for intellectual content.Figure S1. Visual acuity outcome for the worst‐seeing eyes in untreated and idebenone‐treated OPA1‐mutant DOA patients.Click here for additional data file.Table S1. Wilcoxon signed‐rank test to compare paired samples, before and after idebenone off‐label administration, in OPA1‐mutant DOA patients.Click here for additional data file.


Operational and functional reorganization from a cerebral point of view and motor recovery seem to require therapies that require an important use of the limb associated with an innovative type of learning and/or ability with regard to new motor skills.Based on the previous consideration, it is evident that simple movements do not lead to maximum recovery of the rehabilitated limb.Based on the first consideration, it is also clear that even the use of passive exercises does not lead to optimal recovery of the affected limb.The following problems have always existed in rehabilitation :OperatioMotivating and engaging rehabilitation exercises.Training that both optimizes and maximizes the functionality of the limb.An environment full of motivating stimuli.Hence the reasoning that led to the genesis and first use of robotics as practical and effective rehabilitation tools ,3 becausRehabilitation supported by the use of robotic systems can have numerous advantages . In partThere are two different types of robotic technological tool (RTT) in rehabilitation for both the lower and upper limbs. The first is based on exoskeletal instruments. The second is of the end-effector type.The exoskeletal robot, whether it is for the lower  or upperIn a robotic end-effector device, the input for carrying out the rehabilitation exercise comes directly from the distal part of the limb, allowing the natural kinematic activation of the movement without unnatural constraints. These systems are used for both lower  and uppeBoth the two RTTs produce patient benefits ,6,7,8,9.Improved trunk control.Improvement of the sleep-wake rhythm and reduction of perceived fatigue in carrying out daily life activities.Pain relief.Improvement in the state of mental health.Improvement of general anthropometric characteristics .Improvement of intestinal and bladder function.It is now well established that for the lower limbs the RTT produces various benefits, including:Some of these benefits are also obtained thanks to combination with specific software also based on virtual reality (VR) and/or augmented reality (AR), and also in defined protected immersive virtual environments where the rehabilitation scenarios called Cave Automatic Virtual Environment (better known with the acronym CAVE) take place.Neuromotor improvement of limb function.Pain relief.Improvement in the state of mental healthImprovement of general anthropometric characteristics Improvement of cognitive functions.It is now well established that for the upper limbs the use of an RTT shows several benefits, including:Some of these benefits are also obtained thanks to the combination with specific software that generally offer motivating GAME and recently, in some cases, also based on virtual reality (VR) and/or augmented reality (AR).Currently, robotics for rehabilitation are pushing a lot of research and development and numerous new interesting directions are opening both directly connected to the robotic tools mentioned above and in support of an even wider rehabilitation process.To assess the effects of using robots at different phases of recovery.To develop wearable robots easy and practical to wear and remove.To decrease the costs also by means of new models of care.To optimize and rethink the models of care based on robotics.To empower the synergy and collaboration between professionals of the rehabilitation team and designers through shared and properly designed projects.To make virtual reality, augmented reality, at home technologies, exoskeleton and artificial intelligence available for the treatment of cognitive and/or degenerative conditions.Some of these directions that more directly relate with motion rehabilitation ,10,11 arTo invest in social robots specifically designed to support during the rehabilitation phases (as for example in the care of the elderly).To invest in social robots specifically designed as cultural mediators to support during communication/therapy activity (as in the care of the autism).To face the problem of the empathy in robotics especially in relation to interaction with the social robots.Other directions, more focused to psychological support, in a wider approach to rehabilitation process are the following :To invesIn light of the above, the editorial aims to stimulate scholars to report their experiences relating to various aspects of innovation on the development and use of robotics in rehabilitation both from a technological and clinical point of view. For all the above listed issues, and in particular for the 6, 7 and 8, perspective articles are welcome.Despite the great development of robotics in the rehabilitation field, we are assisting to several different approaches in the use and in the relevant models of care. For example, both the rehabilitation therapies and the outcomes in the international panorama are often assessed in a different way. As in other sectors, such as telemedicine, robotics is often used very limited to pilot and/or research projects. Just like in telemedicine, all aspects that can strengthen the use of robotics in routine clinical activities must be addressed in the international panorama with strong dedicated initiatives. Through this approach, rehabilitation robotics will be able to be part of the portfolio of proposed healthcare offers in every state with a clear reimbursement of the indicated services. In light of the above, the editorial aims also to stimulate scholars to report their experiences related to these various aspects of the use of robotic technologies used in the rehabilitation centers and laboratories. From this collection obtained with heterogeneous methods, that presumably will range from the review to the mass survey, we expect to have important responses and stimuli for the international scientific community.


Adventitious root (AR) formation is a complex developmental process controlled by a plethora of endogenous and environmental factors. Based on fossil evidence and genomic phylogeny, AR formation might be considered the default state of plant roots, which likely evolved independently several times. The application of next-generation sequencing techniques and bioinformatics analyses to non-model plants provide novel approaches to identify genes putatively involved in AR formation in multiple species. Recent results uncovered that the regulation of shoot-borne AR formation in monocots is an adaptive response to nutrient and water deficiency that enhances topsoil foraging and improves plant performance. A hierarchy of transcription factors required for AR initiation has been identified from genetic studies, and recent results highlighted the key involvement of additional regulation through microRNAs. Here, we discuss our current understanding of AR formation in response to specific environmental stresses, such as nutrient deficiency, drought or waterlogging, aimed at providing evidence for the integration of the hormone crosstalk required for the activation of root competent cells within adult tissues from which the ARs develop. Ficus spp. and mangroves, crown and brace roots in maize), enhancing water capture , or allowing vegetative propagation of buried stems . In many dicots, however, the postembryonic root system is mainly composed of lateral roots (LRs), which originate from the pericycle cell layer of already formed roots  originate post-embryonically from tissues other than roots in response to different environmental signals, in a process that is highly regulated by hormonal crosstalk . During ed roots . In thesed roots .Selaginella moellendorffii) and Euphyllophytes (ferns and seed plants) have been proposed  genes are associated with stem cell regulation (WOX genes from the intermediate clade (IC-WOX genes) displayed evolutionary conserved functions in root organogenesis , while the other was recruited for PR specification during embryogenesis (WOX8/9)  gene, CrWUL, similar to the direct activation of WOX5 expression by WOX11 required for the root founder cell division that forms the root primordium during AR initiation in Arabidopsis thaliana (hereafter Arabidopsis) leaf explants (CrWOXA in Arabidopsis leaf explants enhanced adventitious rooting (WOX genes in the regulation of AR initiation in Euphyllophytes in two steps: (i) establishing the root founder cell downstream of the auxin signal and (ii) positioning the stem cell niche within the newly formed root primordia.In vascular plants, gulation . WOX genogenesis . In the (WOX8/9) . Additioichardii . In turnexplants . On the  rooting . These rde novo from vascular cells in adult leaves or stems in dicots after wounding (S. moellendorfii) and ferns (C. richardii), while AR formation in many angiosperms might be repressed through unknown mechanism(s) in the absence of the specific stress signal.We built an evolutionary consensus tree including 34 representative plant species in which the AR system has been studied in some detail . In mostwounding . In any Eucalyptus spp., and olive trees  pathways in response to a wide range of biotic and abiotic stimuli , brace roots are also produced from higher internodes at the stem ; in some species such as maize , which encodes an auxin-inducible LBD transcription factor  population from a cross of S. viridis, and S. italica was performed. As a result, two major QTLs for CR variation in response to water availability were identified; of the 380 genes in the confidence interval of one of these QTLs, 28 were differentially expressed in the CRs under water deficit conditions, thus identifying potential candidates for further study (S. viridis was confirmed in other Poaceae species: sorghum (Sorghum bicolor), switch grass (Panicum virgatum), and Brachypodium distachyon , which is closely related to waterlogging-intolerant tomato and eggplant crops, has been proposed as a model to study waterlogging-induced AR emergence  in deep-water rice when plants become submerged . In non-NSITIVE5  and BRANRANCHED1  might alTriticum aestivum), waterlogging induced the formation of nodal roots with aerenchyma tissues, a response that was associated with enhanced expression of some ethylene biosynthetic genes and RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) genes in the submerged stem , the waterlogging tolerant landrace, “Zaoer-N,” adapted to waterlogging stress by developing a large number of ARs in the hypocotyl, while almost no ARs were generated in the waterlogging-sensitive cultivar “Pepino”  and the study of haplotype diversity in a wide collection of cucumber inbred lines identified a causal polymorphism in the coding region of CsARN6.1 responsible for the dominant waterlogging tolerance of “Zaoer-N” (Gly) was not functional, and transgenic cucumber lines expressing the CsARN6.1Asp allele from “Zaoer-N” exhibited a significant increase in AR numbers under waterlogging conditions compared with that of the waterlogging-sensitive lines , GH3.5, and GH3.6 expression, which encode the acyl-acid-amido synthetases involved in the negative regulation of active JA levels that inhibit AR formation in the hypocotyl  seedlings, ARs usually emerge on the hypocotyls or at the internodes of the rhizome underground. Expression profile of miRNAs during different stages of AR formation in this species identified 240 upregulated and 70 downregulated miRNAs at the induction stage. Several of these miRNAs were related to plant hormone metabolism, particularly auxin and brassinosteroids  and ARFs (miR160a and miR9748), as well as auxin target genes, such as GH3 (miR171d-5p) and SAUR (miR162-3p)  .OsARF8 that, in turn, lowers OsGH3.2 function and increases the active auxin levels in the root, which might indirectly enhance CR formation  conditions in rice root and shoot tissues . Based oormation . On the ormation .35S::MIM156, which blocks the activity of miR156 and causes an increase in SPL expression, providing a plausible explanation for the observation that rooting capacity declines along with plant age  genes . A role lant age . Recent lant age . Here, the wound .crown root defect1 (crd1) mutant in rice , known to be important in miRNA biogenesis, function and transport  transcription factor family  and miRNA regulation. Despite all the recent advances in this field, little is known about the molecular mechanisms controlling the early specification of resident stem cells within adult tissues and the signals that maintain them in a quiescent state until their reprogramming is required for AR initiation.JMP-P was responsible for conceptualization and supervision, was involved in the review and editing of the manuscript, and provided the funding acquisition. MM and JMP-P performed the formal analysis and wrote the original draft. Both authors contributed to the article and approved the submitted version.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.


It is important to understand how to design AR content for surgical contexts to mitigate the risk of distracting the surgeons. In this work, we test information overlays for AR guidance during keyhole surgery. We performed a preliminary evaluation of a prototype, focusing on the effects of colour, opacity, and information representation. Our work contributes insights into the design of AR guidance in surgery settings and a foundation for future research on visualisation design for surgical AR. For abdominal surgery a rigid endoscope (known as a laparoscope) is used to visualise the patient’s internal anatomy and surgical instruments. The smaller incisions used allow a faster recovery time in comparison with open surgery. The adoption of keyhole surgery, however, reduces the amount of information available to the surgeon during surgery, due to the reduced field of view of the cameras used and the inability to directly touch the anatomy. The use of image guidance for keyhole surgery aims to overcome these limitations, making keyhole surgery more effective. This paper is principally concerned with enhancing keyhole surgery using augmented reality derived from pre-operative scans (CT and MRI).AR enables a composite image to be displayed on one screen through the combination of a computer-generated image and a live real world image , and hasThe majority of augmented reality systems for keyhole surgery propose projecting surface renderings of the internal anatomy onto the live video feed . A key tBecause the focus is usually on the technical difficulties of registration the majority of proposed surgical AR systems use fairly simplistic rendering, i.e. solid models and wireframes with various levels of opacity and various colours  10]. Th. Th10]. 2The majority of research on AR for keyhole surgery focuses on the engineering challenge of achieving an accurate and reliable overlay of the AR content onto the operation scene. Whilst some authors ,20,29 haThere is a significant body of work on the use of AR in both open and keyhole surgery. There are also several well designed studies looking at the impact of AR on certain clinical outcomes. These studies tell us little, however, about the best way to design AR visualisation to maximise the effectiveness of a given AR system. There are problems with generalising some studies plus the more specific problem of recruiting sufficient volunteers to study multiple design options when the tasks used require a high baseline skill level. Our aim is to create a more general methodology together with the tools  that can be applied to multiple studies in AR guidance for surgery.The original focus of this study was the occurrence of inattentional blindness (IAB) during surgery. IAB describes the situation in which important details are missed due to the user’s attention being held elsewhere, , 25. IABOne difficulty with the studies described by Dixon et al. and Pandit et al. , 21 is t2.1In this research, we explore how to utilise AR information overlay for guiding users through laparoscopic surgery tasks. We developed and evaluated an AR prototype which overlays points of interest over a phantom liver within a phantom patient. We performed a user study of the prototype where we were gaining participant feedback about the experience, particularly around the colour, opacity, and representation of the AR overlay. We also recorded task completion time to see if this was effected by overlay design, along with the occurrence of IAB. This work presents the first step towards better understanding how virtual information should be represented in AR systems for keyhole surgery. Our key contribution is design considerations of AR overlays for laparoscopic surgery, which can help optimise the design of future AR systems for this purpose.31 to mimic the setting of keyhole surgery , colour, and opacity. In order to reduce the effects of learnability, we randomised the colour  and opacity (low or high) of the overlay for each condition. There was no time limit and participants could stop at any time and move on to the next condition.The order was varied across subjects to give balanced order of use, to account for potential order effects. For each condition, participants were asked to grasp and remove a simulated tumour from the cut out area, under AR guidance. While participants were using each condition, we recorded the total time spent in each condition type. We collected qualitative feedback through interviews, at the very end of all the conditions, to seek participants’ perception of the AR tumour overlay for keyhole surgery.To test for the occurrence of IAB we also placed a black plastic spider withing the surgical scene, see We recruited 12 participants who were given a task designed to simulate laparoscopic surgery with AR guidance. The study was completed under ethical approval (Z6364106/Computer Science/2019/04/19) and all participants gave informed consent. Participants were asked about any visual impairments, but not specifically about colour blindness, which may be relevant given the contrast between the foreground and background images. Most of the volunteers were students studying Biomedical Engineering, so had some understanding of anatomy and interventional healthcare, but no surgical training. We created a simulated task that would represent a surgical task but at a level of abstraction relevant to the volunteers used.55.1Each participant performed the task 3 times, giving a total of 36 data points. Mean task completion time was 42 seconds with a standard deviation of 41 seconds, maximum of 188s, minimum of 9s. We observed a statistically significant (p = 0.011) increase in task completion time for first task completed by each participant (mean = 66 seconds) indicating a strong learning effect. Future studies should include a pre-trial training session. Our results indicate that it would be sufficient to include a single pre-trial training run into future studies.Our results do not demonstrate any statistically significant correlations between task completion time visualisation method or visualisation colour. Including outliers the red display gave a faster completion time on average which supports the qualitative results on the selection of overlay colour, however significantly more data would be required to demonstrate this.5.2After completing 3 tasks each participant was asked if they noticed anything out of place in the experiment. Only one participant noticed the spider. This surprised us, and demonstrates that IAB is a real problem that can be replicated in these studies. Given the small sample size it is not possible to extrapolate further at this point. Ideally we could design future studies with a finer balance between object prominence and task load to get a more useful result in terms of understanding IAB.5.3It was difficult to understand the depth of the tool”, “It was quite awkward looking at the screen and having to move my arm across here” and “It was tricky to know where things were in 3D and it took a few goes to know which direction you were moving in” were made. After the second task was completed, the majority of the participants also commented on their increased ability in determining the depth of the tool on screen. This performance increase was also seen in the time decrease taken to complete the second task when compared to the first. One participant stated “Finding the depth before we could actually extract the tumour was quite challenging. In a real-life scenario you won’t want to just move the tool around without knowing how deep it is going in the human body, until you find the organ itself”. This highlights the importance of simulated tools like our prototype for training student surgeons, allowing them to become accustomed to the controls in relation to the level of abstraction - in this case, users needed to focus on the external screen. Future research should investigate the use of head mounted displays such as the Microsoft HoloLens for providing a direct augmented view of the operating area.Our study included a qualities assessment using a questionnaire. The majority of participants commented that estimating depth perception was difficult. Several comments such as “the second time was easier compared to the first one, the lines on the screen helped me find the tumour, I found them very useful”. Similarly comments were made about the specific colours of the overlays like “It was easier to see the tumour this time due to the contrast between the background and the lines” and “the red colour was better as the tumour was marked clearly and hence it was easier to find”. While the colour of the overlay seems like an obvious aspect to consider for these systems, there may also be a need for the system to be adaptive based on the type of surgery in order for the content to stand out.Participants who chose not to comment on depth perception interpreted the question in another way and instead commented on other parts of the experiment like the bright green colour of the tumour and the lines of the wire frame visualisation technique. Comments were made about the general perceived assistance felt by having coloured lines from the wireframe overlay condition including, “6In this paper we presented an evaluation of an AR laparoscopy prototype to assist surgeons through keyhole surgery tasks. We ran a within-subject study of the prototype, exploring the effects of colour, opacity and information representation of the AR overlay. The results point to the potential of these systems to guide new surgeons and students through tasks while increasing their depth perception when using a laparoscopic camera and tools. At present we have insufficient data to draw any firm conclusions, however, we have demonstrated the potential of our approach and provide a firm basis for future work. Future work should aim to evaluate keyhole surgery using other AR hardware form factors, such as head mounted displays, and test AR’s performance with actual surgeons.