The geranyl acetophenone 2,4,6-trihydroxy-3-geranyl acetophenone (tHGA), an in-house synthetic non-steroidal compound, attenuates airway hyperresponsiveness and remodeling in murine models of asthma. The effect of tHGA upon human ASM proliferation, migration and survival in response to growth factors was assessed and its molecular target was determined. Following serum starvation and induction with growth factors, proliferation and migration of human bronchial smooth muscle cells (hBSMCs) treated with tHGA were significantly inhibited without any significant effects upon cell survival. tHGA caused arrest of hBSMC proliferation at the G1 phase of the cell cycle with downregulation of cell cycle proteins, cyclin D1 and diminished degradation of cyclin-dependent kinase inhibitor (CKI), p27Kip1. The inhibitory effect of tHGA was demonstrated to be related to its direct inhibition of AKT phosphorylation, as well as inhibition of JNK and STAT3 signal transduction. Our findings highlight the anti-remodeling potential of this drug lead in chronic airway disease.Increased airway smooth muscle (ASM) mass is a prominent hallmark of airway remodeling in asthma. Inhaled corticosteroids and long-acting beta The asthmatic airway undergoes remodeling as a healing process which involves increased airway smooth muscle (ASM) mass, sup-epithelial fibrosis, epithelium mesenchymal transition (EMT), goblet cell and myofibroblast hyperplasia4. As a consequence of these structural changes, thickening of the airway wall causes lumen narrowing that ultimately leads to airway obstruction4.Airway remodeling, a collective term describing the structural changes in the asthmatic airway, occurs in conjunction with, or as a result of, chronic airway inflammation2-agonists that provide minimal beneficial effects upon airway remodeling6. It has been suggested that airway remodeling may not be reversed by steroid treatment but rather prevented7. Hence there seems to be alternative molecular targets that may be directly responsible for airway remodeling which are independent of proinflammatory processes. Furthermore, repeated allergen challenge in murine models have been shown to result in persistent airway remodeling following resolution of airway inflammation and hyperresponsiveness (AHR)9. Hence, treatments that target single or multiple components of pathways that induce airway remodeling may be useful in the management of asthma.Current asthma treatment regimens employ a combination of inhaled corticosteroids (ICS) and beta11. Furthermore, tHGA-treated mice were found to have reduced expression of α-SMA and thinner layers of smooth muscle surrounding the airways in comparison to untreated mice11. Thickening of the airway wall, primarily due to increased ASM mass, reduces the diameter of the airway as it contracts and causes significant airflow limitation and AHR12. Another study of ours recently demonstrated that tHGA attenuated eosinophil-induced epithelial-mesenchymal transition (EMT) of bronchial epithelial cells in a concentration-dependent fashion through its suppression of transforming-growth factor-β (TGF-β) synthesis via both PI3K and JNK pathways13. Hence, we are interested to explore further the pharmacological effects of tHGA in modulating various elements of tissue remodeling.Our previous studies demonstrated that 2,4,6-trihydroxy-3-geranyl acetophenone (tHGA) is effective in attenuating AHR in response to methacholine challenge as well as reducing inflammatory cell infiltration in both acute and chronic murine models of asthma12. ASM hyperplasia can be defined as an increased number of ASM cells in the asthmatic airway. This increase in cell number is either due to increased cellular proliferation, reduced apoptosis or/and increased cellular migration towards the airway lumen in response to proinflammatory mediators release15. Proinflammatory mediators such as growth factors and cytokines activate several signal transduction pathways through binding to tyrosine kinase receptor (RTK) and G protein-coupled receptors (GPCRs) that culminate in proliferation and migration of ASM19. In this communication, we describe the inhibitory effect of tHGA upon growth factor-induced ASM cell proliferation and migration in an established cellular model. This effect was found to be related to the inhibition of AKT phosphorylation, a downstream signaling molecule of the PI3K pathway that plays a regulatory role in smooth muscle cell proliferation, migration and apoptosis21.ASM mass is increased through hyperplasia and hypertrophyTo determine the maximum non-cytotoxic concentration of tHGA for further experiments, lactate dehydrogenase (LDH) release from growth factor-induced hBSMCs following tHGA treatment was measured. tHGA concentrations of 20 µM and below were not cytotoxic Fig. , and theP value < 0.001) in the percentage of bromodeoxyuridine (BrdU)-positive hBSMCs after 48 hours compared to non-induced hBSMCs. Co-treatment with tHGA showed a significant decrease in BrdU-positive cells at 20 µM (47 ± 5.2%) and 10 µM (80 ± 3.4%) compared to growth factor-induced hBSMC (100%)  Fig. . tHGA wa0%) Fig. . ForskolMigration of hBSMCs in both scratch and transwell migration assays are shown in Fig. vs induced: 41 ± 1.0%)  was significantly higher than that observed in untreated induced hBSMCs. The assay control forskolin also caused a significant increase in the percentage of cells in the G1 phase and reduced the percentage of cells in the S phase in comparison to induced cells.To further understand the mechanism of tHGA in inhibiting the proliferation of hBSMCs, its effect upon cell cycle regulation was assessed. Cell cycle analysis demonstrated that growth factors induced a significant increase in the percentage of hBSMCs entering the S phase as compared to non-induced hBSMCs (non-induced: 7 ± 1.0% 0%) Fig. . A signiSince ASM hyperplasia could be due to increased cell proliferation or reduced apoptosis, the effect of tHGA upon hBSMC apoptosis following growth factor induction was also examined. Non-induced hBSMCs demonstrated a higher percentage (11.7 ± 0.9%) of apoptotic cells as compared to induced hBSMCs (5.3 ± 0.6%). Conversely, 20 µM tHGA caused a slight, yet insignificant increase (8.5 ± 1.6%) in the percentage of apoptotic cells Fig. . SimilarKip1 are key regulators of G1/S progression in hBSMCs22. As cells progress through the G1 phase, cyclin D1 is rapidly expressed in response to growth factors. Conversely, p27Kip1, a cyclin kinase inhibitor, will be degraded during the G1 phase23. Therefore, expression of cyclin D1 and p27Kip1 were examined to determine whether tHGA had any effect upon these key regulatory molecules. tHGA significantly inhibited growth factor-induced cyclin D1 expression in hBSMCs at 20 and 10 µM . However, tHGA failed to exert any significant effect , which served as the substrate for PDK1 and mTORC2. mTORC2 was immunoprecipitated from the protein lysate by using an anti-Rictor antibody; Rictor is an essential component of mTORC228. The integrity of the mTORC2 complex was preserved following tHGA treatment as shown by the presence of mTOR, which was detected with an anti-mTOR antibody  phosphorylation . LY294002, which is PI3K inhibitor, significantly reduced kinase activity of mTORC2.Since tHGA affected the phosphorylation of AKT without affecting the phosphorylation of upstream molecules, it can be postulated that AKT could be the potential molecular target of tHGA. In order to evaluate AKT as the potential molecular target of tHGA, key regulatory components that are involved in the phosphorylation of AKT were studied. In response to growth factor, mTORC2 will phosphorylate AKT at the Ser473 phosphorylation site, while PDK1 will phosphorylate AKT at the Thr308 phosphorylation sitein vitro AKT(Thr308) phosphorylation .Growth factors significantly increased the kinase activity of PDK1 as demonstrated by an increase of Since tHGA demonstrated an inhibitory effect upon AKT phosphorylation without any effect upon upstream molecules, it is likely that AKT may act as a potential target of tHGA. Therefore, we attempted to validate the role of AKT as a target for tHGA by using a constitutively active AKT construct (myr-AKT). Transfection conditions were optimized with a positive control pmaxGFP. hBSMCs transfected with program FF-130 demonstrated a 72% transfection efficiency and 86% cell viability, hence program FF-130 was used for subsequent transfections Fig. . HA-tagg30. Increased ASM mass, a hallmark of airway remodeling, causes airway narrowing and airflow obstruction31. The effect of the most widely used asthma therapies, corticosteroids and β2-agonists, in reducing the increased ASM mass remains controversial32. Therefore, treatment that can prevent or reverse airway remodeling is much sought after in the management of asthma. Our previous study revealed that tHGA exhibited significant inhibitory effects upon AHR as well as airway remodeling in a chronic murine model of asthma. In particular, treatment of tHGA was shown to inhibit accumulation of the ASM layer, which is demonstrated by reduced expression of α-SMA in chronic ovalbumin (OVA)-induced asthmatic mice11. In this study, an attempt was made to determine whether tHGA had any effect upon the proliferation/migration/apoptosis of bronchial smooth muscle cells and to dissect the mechanism of action.Airway remodeling has been recognized as a major factor contributing to AHR and reduced lung functiontHGA was shown to attenuate ASM proliferation induced by growth factors as indicated in the BrdU proliferation assay. This was further supported by the significant decrease in the expression of proliferation marker, Ki-67 following tHGA treatment, providing a strong indication of the anti-proliferative role of tHGA in growth factor-induced ASM proliferation. To further understand the underlying mechanism of tHGA action, three major pathways relevant to proliferation, namely MAPKs, JAK2/STAT3 and PI3K were studied. It was found that tHGA inhibited the phosphorylation of several key pathway regulators, namely JNK, STAT3 and AKT.33. However, tHGA only inhibited the phosphorylation of STAT3 while not affecting JAK2 phosphorylation. STAT3 inhibition was reported to attenuate house dust mite (HDM)-induced airway inflammation and remodeling in a murine model of asthma34. The inhibition of STAT3 phosphorylation by tHGA highlights its anti-proliferative potential since knockdown of STAT3 expression in ASM has been reported to significantly reduce ASM proliferation35. Several reports have demonstrated that activation of the MAPKs and PI3K pathways will induce the activation of transcription factors, including STAT3, that are required for cyclin D1 expression38. This study demonstrated reduced cyclin D1 expression following tHGA treatment. This study demonstrates that tHGA inhibits STAT3 phosphorylation, but did not inhibit the phosphorylation of JAK2, a STAT3 upstream activator. Therefore, further investigations are required to determine whether tHGA’s inhibitory effect on ASM proliferation may also involve its inhibitory effect on other upstream activators of STAT3 including PI3K or MAPKs39.The JAK2/STAT3 pathway has been described recently as another important pathway involved in the development of allergic asthma40. In the case of tHGA, it was demonstrated that despite no effect whatsoever upon ERK expression, the treatment was still able to induce an anti-proliferative effect. A recent study demonstrated the role of JNK in ASM proliferation, revealing that this pathway regulates human ASM proliferation partly through its regulation of cyclin D1 and cell cycle progression25. This suggests that JNK inhibition by tHGA could be one of the factors for the reduced level of cyclin D1 in our current findings. Together with MAPKs, PI3K is another main signaling pathway activated by mitogenic stimuli in ASM. tHGA showed significant inhibition upon AKT phosphorylation, a downstream signaling molecule of the PI3K pathway that plays a critical role as a regulator in cell proliferation, migration and apoptosis41. Upon mitogenic stimulation, PI3K phosphorylates phosphatidylinositol membrane lipids, thus producing the second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3)42. AKT will be recruited to the plasma membrane by PIP3 where its conformation is altered to allow subsequent phosphorylation by PDK1 and cell cycle progression through regulation of cyclin D1 and p27Kip1 expression44.Amongst the MAPKs, the ERK pathway is suggested to dominate the regulation of ASM proliferation45. A model employing dominant-negative AKT transfected human bronchial epithelial cells demonstrated AKT acts as an upstream regulator of JNK in arsenite-mediated proliferation via PI3K/AKT/JNK/c-Jun/cyclin D1 signaling46. Another study using PTEN null cells demonstrated JNK and AKT to be independently activated by PI3K, indicating that PI3K instead of AKT acted as the upstream effector of JNK47. These findings point to a possible inhibitory effect of tHGA upon PI3K or AKT activation leading to the inhibition of JNK phosphorylation, thus making JNK more of a downstream effector instead of the molecular target of tHGA.Based on the findings obtained, the identification of the molecular targets of tHGA was narrowed down to either AKT or JNK. This is not surprising since there is crosstalk between both PI3K and MAPK pathways48. Since tHGA was shown to inhibit AKT phosphorylation at Ser473 and Thr308, this finding points to the possibility that tHGA is causing inhibition of AKT activation.Since the major inhibitory effect of tHGA was upon AKT phosphorylation, effects upon upstream molecules of AKT, including PI3K, PDK1 and mTORC2, were investigated. The specificity of AKT as a target is suggested since no effects upon phosphorylation of PI3K and PDK1 as well as the kinase activities of PDK1 and mTORC2 was shown. Inhibition of AKT is attained through various ways, namely direct inhibition of AKT kinase activity, inhibition of AKT phosphorylation, activation of AKT dephosphorylation or prevention of AKT translocation to the plasma membrane49. tHGA treatment caused a significant reduction in phosphorylation of AKT at both Ser473 and Thr308 in myr-AKT-expressing cells. This indicates that tHGA could possibly block the phosphorylation site of AKT that leads to AKT inactivation.There are basically two prominent cell regulatory events involved in AKT activation, which include phosphorylation of the AKT kinase active site and translocation of AKT kinase from the cytoplasm to the cell membrane via the PH domain of AKT. By using myr-AKT construct, the mechanism of tHGA action upon AKT was further explored. Myr-AKT is a constitutively active mutant of AKT. The myristoylation signal constructed in this AKT mutant allowed the recruitment of AKT to the plasma membrane without its PH domain, thus causing an increase of its basal level of phosphorylation even in the absence of any growth factorKip1 expression can be explained by the fact that AKT mediates cell cycle progression from the G1 to S phase through inactivation of GSK-beta, which leads to an increase in cyclin D1. Furthermore, inhibition of forkhead family transcripition factors and the tumor suppressor tuberin (TSC2) by active AKT results in the reduction of p27Kip1 50. The protein stability of p27Kip1 is also reported to be mediated by AKT-dependent phosphorylation of Skp251. Blocking AKT activity allows p27Kip1 to continuously inhibit the activity of CDKs, thus promoting cell cycle exit.The effect of tHGA upon both cyclin D1 and p27Kip1 is also involved in regulating cell migration via the Rho kinase pathway. Binding of p27Kip1 to RhoA interferes with the interaction between RhoA and its activator, thus inhibiting its activation52. It is possible that through inhibition of p27Kip1 reduction, tHGA could suppress ASM migration in response to growth factors through indirect effects upon the RhoA pathway. The inhibition of AKT by tHGA could also impact cellular migration through disruption of the PI3K pathway. PTEN, a tumour suppressor, was reported to inhibit human airway smooth muscle cell migration through AKT signaling pathway53. A study employing AKT null cells showed lack of polarization and reduced migration in response to cAMP54. Furthermore, AKT mediates the regulation of PAKa which is required for myosin assembly, thus in part, controlling cell polarity and chemotaxis55. A study demonstrated that tetrandrine (Tet), a calcium blocker isolated from Stephania tetranda, inhibited cell migration through its inhibition on AKT and JNK activation. In addition, their findings also revealed that AKT inhibitor (triciribine) and JNK inhibitor (SP600125) significantly attenuated cell migration56. These findings further imply that tHGA inhibition on AKT and JNK phosphorylation could be the reason that lead to the anti-migratory effect in hBSMCs.Apart from its role in cell cycle regulation, p2757. Hence one would suspect inhibition of AKT would lead to an increased rate of apoptosis. However, tHGA failed to cause any effect upon apoptosis in growth factor-induced hBSMCs. This could be due to the fact that Bcl-2 can be regulated through several other upstream activators, such as ERK and p38 of which were not targeted by tHGA, hence enabling normal regulation of Bcl-2 leading to prevention of apoptosis58. In addition, a study also suggested that ERK cooperate with other survival signaling pathways, such as PKC activation, to ensure that Bcl-2 function properly59. Furthermore, another study had demonstrated that AKT inhibition alone was not sufficient to induce apoptosis. Instead, inhibition of PI3K, which is the upstream activator of AKT, led to the decrease of the phosphorylation of AKT and ERK and subsequently causes apoptosis60. This further explains the possible reason that AKT inhibition by tHGA in this study did not lead to apoptosis induction.In describing the mechanism involved in tHGA-induced cell cycle arrest, possible effects upon ASM apoptosis were evaluated in which tHGA was demonstrated to have no effect upon apoptosis. It has been reported that AKT upregulates expression of Bcl-2, an anti-apoptotic protein, through cAMP-response element-binding protein in the apoptotic pathway63. Proliferation of both asthmatic and non-asthmatic ASM were reported to be regulated via identical pathways, which includes the PI3K/AKT signalling pathway that was reported to be dominant in asthmatic ASM cells in response to strong mitogenic stimulation62. Furthermore, several studies had also demonstrated that the PI3K/AKT signalling pathway is integral in regulating proliferation of asthmatic ASM, in which proliferation is inhibited through inhibition of signalling molecules of the PI3K/AKT pathway65. It is highly likely that the inhibition demonstrated by tHGA in non-asthmatic ASM in this communication would occur through similar pathways in asthmatic ASM. Further work employing asthmatic ASM is envisaged in which the effect of tHGA upon established pathological changes can be assessed.Previous studies have reported that asthmatic ASM cells proliferate at a higher rate as compared to non-asthmatic ASM cellsAlthough the current study has determined that inhibition of AKT phosphorylation to be the major effector mechanism of tHGA upon bronchial smooth muscle proliferation and migration, the possibility of multiple targets still exists and would require further experimentation. Further investigations should examine the role of JNK and STAT3 inhibition and an understanding of alterations to cross-talk between these molecules and AKT following tHGA treatment. The role of tHGA on other upstream activators of JNK, including MEK4 and MEK7, as well as the activity of PI3K can be further examined. Nevertheless, in this communication, our findings showed that tHGA inhibits hBSMCs proliferation and migration without inducing apoptosis through inhibition of AKT, JNK and STAT3 signaling molecules. AKT was suggested to be the major molecular target of tHGA in hBSMCs upon growth factor stimulation  was prepared by dissolving the compound in 100% DMSO. The final concentrations of DMSO in all assays was 0.1%.tHGA was synthesized as reported previously2. Cells between passages 4 and 8 were used for all experiments. Prior to tHGA treatment on hBSMCs, hBSMCs (1.56 × 104 cells/cm2) were seeded in complete media for 24 hours, followed by serum starvation for 24 hours with 0.1% FBS SmBM. Serum starved cells were then induced with growth factor-enriched media containing 5% (v/v) fetal bovine serum (FBS), 5 ng/ml insulin, 2 ng/ml human bFGF and 50 ng/ml human EGF and co-treated with/without tHGA at indicated time points. For assay controls, hBSMCs were treated with 10 µM forskolin, 5% DMSO or respective inhibitors.hBSMCs were obtained from Lonza  and were maintained in smooth muscle basal medium (SmBM) supplemented with 5% (v/v) fetal bovine serum (FBS), 5 ng/ml insulin, 2 ng/ml human bFGF, 50 ng/ml human EGF, 50 µg/ml gentamicin and 50 ng/ml amphotericin at 37 °C with 5% COhBSMCs were serum starved for 24 hours prior to induction with growth factor-enriched media and co-treatment with/without tHGA for 48 hours in 96-well culture plates with a seeding density of 5000 cells per well. The LDH cytotoxicity assay was performed according to the manufacturer’s protocol . Non-toxic concentrations of tHGA were determined and used for all subsequent assays.The BrdU cell proliferation assay was carried out according to the manufacturer’s instructions . Briefly, hBSMCs were seeded at 5000 cells per well in 96-well culture plates and were serum starved for 24 hours prior to induction with growth factor-enriched media and co-treatment with/without tHGA for 48 hours. Cell proliferation was determined through the measurement of the BrdU incorporation during DNA synthesis, thus BrdU reagent was added 24 hours prior to the end of treatment. Anti-BrdU monoclonal antibody was then added to detect the BrdU-incorporated cells, followed by addition of IgG-peroxidase conjugated secondary antibody, substrate and stop solution. Lastly, the BrdU-incorporated cells were measure through colorimetric detection at dual wavelength of 450/550 nm.Serum starved hBSMCs were induced with growth factor and co-treated with/without tHGA for 48 hours in T25 flasks. RNA was extracted with the RNeasy Mini kit  and RT-PCR was carried out using Qiagen OneStep RT-PCR kit . Primers used for the Ki-67 gene were: forward, 5′-CTA CTC CAA AGA AGC CTG TG-3′; reverse, 5′-AAT GAA GTT GTT GAG CAC TCT G-3′ while for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene primers used were: forward, 5′-GCC GCA TCT TCT TTT GCG TC-3′; reverse, 5′-TCG CCC CAC TTG ATT TTG GA-3′. The thermal cycler  was programmed for 30 minutes reverse transcription at 50 °C followed by 30 minutes PCR activation step at 95 °C and 32 cycles with 30 seconds denaturation at 94 °C, 30 seconds annealing at 58 °C and 1 minute elongation at 72 °C. The elongation step for the final cycle was extended for another 10 minutes at 72 °C. The PCR products were analyzed on a 2% agarose gel with ethidium bromide staining. Densitometry of the PCR bands was quantitated by using Image J Image Processing Software  and normalized to GAPDH.Serum starved hBSMCs were induced with growth factor-enriched medium and co-treated with/without tHGA for 48 hours in T25 flasks. Cells were trypsinized and washed twice with phosphate-buffered saline (PBS), followed by fixing and permeabilization with 70% ethanol for 30 minutes at 4 °C. Cells were then spun down and incubated with RNase A (100 µg/ml) for 5 minutes. Next, propidium iodide (50 µg/ml) was added and cells incubated for 15 minutes at room temperature in the dark. Stained cells  were analyzed with a BD LSR Fortessa flow cytometer .Serum starved hBSMCs were induced with growth factor-enriched medium and co-treated with/without tHGA for 48 hours in T25 flasks. Cells were trypsinized and washed twice with PBS prior to annexin V/propidium iodide staining. The annexin V/propidium iodide assay was performed using a FITC annexin V apoptosis detection kit according to the manufacturer’s instructions . Stained cells  were analyzed with a BD LSR Fortessa flow cytometer .66.hBSMCs were seeded in 12-well culture plates, cultured until confluent and serum-starved for 24 hours. Linear scratches were made on cell monolayers with sterile pipette tips and cell debris was removed by washing with PBS. Serum starved hBSMCs were induced with growth factor-enriched medium and co-treated with/without tHGA. Scratched areas were marked with a permanent marker and images were captured with Dino-Eye eyepiece camera  attached to an inverted light microscope  at 0, 24 and 48 hours post-treatment. The total number of migrated cells per area was ccounted based on 3 representative images captured for each treatment condition67. Media was removed from the lower chamber followed by addition of 500 µL of fresh media and 100 µl of MTS solution and incubated for 4 hours followed by measurement of absorbance at 490 nm through microplate reader 68.hBSMCs were serum-starved for 24 hours prior to seeding into 8 µM pore membrane inserts (transparent PET membrane)  (5000 cells per well), pre-coated with 1.5 mg/mL type 1 rat-tail collagen . Cells were maintained in serum-free media with or without tHGA/forskolin. Growth factor-enriched media was added into transwell lower chambers (24 well-plate) with or without tHGA/forskolin. Lower chambers containing media with 0.1% FBS were included to serve as negative controls. Following a 6 hour incubation period, non-migrated cells (upper chamber) were removed with a sterile cotton swabSerum starved hBSMCs were induced with growth factors and co-treated with/without tHGA in T75 flasks at indicated time. Following treatment, cells were lysed with RIPA lysis buffer containing 150 mM NaCl, 50 mM Tris HCL, 0.1% SDS, 1% Triton X, 0.5% sodium deoxycholate, protease inhibitor (1:100) and phosphatase inhibitor (1:100). Then, lysates were shaken on ice for 15 minutes prior to centrifugation at 14,000 × g for 10 minutes. Protein concentration was estimated using a BCA protein assay kit . Equal amount of whole cell lysates (10 µg/lane) were separated by 8–12% SDS-PAGE electrophoresis for 1–2 hours at 100 V and transferred onto 0.2 µm polyvinylidene difluoride (PVDF) membranes using a semi-dry transfer system  at 0.25 mA for 8–10 minutes. Membranes were blocked with 5% bovine serum albumin (BSA) for 1 hour followed by an overnight incubation with primary antibodies. Membranes were then washed thrice with tris-buffered saline/Tween 20 (TBST) followed by incubation with secondary antibodies for 1 hour. Antibodies were prepared in 5% BSA according to the recommended dilution  and were used before in other studies. Bands were visualized by enhanced chemiluminescence (ECL) and imaged on a Vilber Fusion gel documentation system . Membrane were stripped and reprobed as required. Densitometry of bands was conducted with Image J Image Processing Software  and normalized to β-actin.Pierce crosslink magnetic IP/Co-IP kit  was used for immunoprecipitation (IP) according to the manufacturer’s instructions. An anti-PDK1 antibody  was used to extract cellular PDK1 from the total protein lysates. Immunoprecipitates were then incubated with PDK1 kinase buffer containing 1× kinase buffer, 200 µM adenosine triphosphate (ATP)  and 500 ng of AKT substrate  at 30 °C for 30 minutes. Kinase activity was stopped with 1× Laemmli loading buffer. Proteins were separated on 8% SDS-PAGE for 1–2 hour at 100 V and transferred onto PVDF membranes using a semi-dry transfer system  at 0.25 mA for 10 minutes. Membranes were then blocked with 5% bovine serum albumin (BSA) for 1 hour followed by an overnight incubation with primary antibodies against phospho-AKT and AKT . Membranes were then washed thrice with tris-buffered saline/Tween 20 (TBST) followed by incubation with secondary antibodies for 1 hour. Antibodies were prepared in 5% BSA according to the recommended dilution . Bands were visualized by enhanced chemiluminescence (ECL) and imaged on a Vilber Fusion gel documentation system . Densitometry of bands was conducted with Image J Image Processing Software .Pierce crosslink magnetic IP/Co-IP kit  was used for IP according to the manufacturer’s instructions. However, the IP lysis buffer was replaced with CHAPS lysis buffer (pH 7.4) containing 40 mM HEPES, 120 mM NaCl, 2 mM EDTA, 0.3% CHAPS, 10 mM pyrophosphate, 10 mM glycerophosphate, protein inhibitor cocktail (1:100) and phosphatase inhibitor cocktail (1:100). An anti-rictor antibody  was used to co-immunoprecipitate the mTORC2 complex from total cellular protein. An anti-mTOR antibody  was used to detect the mTORC2 complex via western blot analysis. Protein lysate was separated on 8% SDS-PAGE and immunoblotted with antibodies against Rictor and mTOR  and transferred onto PVDF membrane through wet transfer system  . Antibodies were prepared in 5% BSA according to the recommended dilution . Membranes were then blocked with 5% bovine serum albumin (BSA) for 1 hour followed by an overnight incubation with primary antibodies against mTOR and Rictor . Bands were visualized by enhanced chemiluminescence (ECL) and imaged on a Vilber Fusion gel documentation system . Membrane were stripped and reprobed as required. Densitometry of bands was conducted with Image J Image Processing Software .Immunoprecipitates were incubated in mTORC2 kinase buffer containing 25 mM HEPES, 100 mM potassium acetate and 1 mM MgCl at 30 °C for 30 minutes. Kinase activity was stopped with 1× Laemmli loading buffer and proteins were separated on 10% SDS-PAGE and immunoblotted with antibodies against phospho-AKT (Ser473) and AKT . Antibodies were prepared in 5% BSA according to the recommended dilution . Bands were visualized by enhanced chemiluminescence (ECL) and imaged on a Vilber Fusion gel documentation system . Membrane were stripped and reprobed as required. Densitometry of bands was conducted with Image J Image Processing Software .49 (Addgene plasmid # 10841) while the empty vector (pECE) was a gift from William Rutter69 (Addgene plasmid # 26453). Plasmids were received as bacteria in agar stabs. Bacterial colony isolation was carried out by streak plating on Luria Bertani (LB) agar for 16 hours followed by inoculation into LB broth and 16 hour incubation at 37 °C on an orbital shaker (200 rpm) . Plasmids were extracted with EndoFree® Plasmid Maxi Kit  according to the manufacturer’s instructions. Transient transfection of hBSMCs with myr-AKT or pECE (1.5 µg) was performed via NucleofectionTM, an electroporative technique developed by Lonza Group Ltd. . Prior to this, transfection condition was optimized using the pmaxGFPTM vector, provided with the nucleofector kit, according to the manufacturer’s instruction to determine the transfection efficiency and cell viability post transfection. Optimization of transfection condition was performed with 6 different NucleofectorTM programs plus 1 negative control (no program). Briefly, hBSMCs (1 × 106 cells) were resuspended in 100 µL of 4D-NucleofectorTM mastermix  and run in a 4D-NucleofectorTM System  for each transfection condition. Cells were then seeded in T25 flask (cell viability) and Falcon CultureSlides (8-well) (transfection efficiency)  with complete media for 48 hours. hBSMCs in Falcon CultureSlides was fixed with 4% paraformaldehyde followed by Hoechst 33342 DNA staining. GFP-expressing hBSMCs were visualized under fluorescence microscope at excitation 488 nm and emission 509 nm while Hoechst stained cells were detected at excitation 352 nm and emission 455 nm. Transfection efficiency was determined by counting GFP-expressing cells over total cells. hBSMCs viability post transfection was determined through trypan blue exclusion method. The optimum NucleofectorTM program was determined and was used for pECE or Myr-AKT transfection in hBSMCs. Myr-AKT expressing hBSMCs were treated with/without 20 µM tHGA for 1 hour. Following treatment, cells were lysed with RIPA lysis buffer and protein lysate was then separated with 10% SDS-PAGE  and immunoblotted with antibodies against HA-tag, phospho-AKT (Ser473), phospho-AKT (Thr308) and β-actin. Antibodies were prepared in 5% BSA according to the recommended dilution . Bands were visualized by enhanced chemiluminescence (ECL) and imaged on a Vilber Fusion gel documentation system . Membrane were stripped and reprobed as required. Densitometry of bands was conducted with Image J Image Processing Software .The plasmid myrAkt delta4–129 (myr-AKT) was a gift from Richard RothP-value < 0.05 was considered to be statistically significant.Data are expressed as means ± SEM and analyzed by one way ANOVA followed by Dunnett’s post hoc test  by comparison to growth-factor-induced control. A Supplementary Information


Using the Iowa Gambling Test (IGT), we demonstrated previously impaired decision- making process in young tattooed women. The purpose of the present study was to explore the associations among the three facets of impaired inhibition  and decision-making processes in this population.To this end, the participants of the previous study (60 tattooed women and 60 non-tattooed women) were assessed in the Go/NoGo task, a measure of response inhibition, the Matched Familiar Figure Test (MFFT), a measure of reflection inhibition and the Stroop task a measure of interference inhibition.Tattooed women were significantly slower than non-tattooed women in the Go/NoGo performance; however, no differences were detected in the MFFT and the Stroop task. A hierarchical regression analysis did not reveal any significant main effects of these inhibition measures on the IGT performance.These findings do not support the hypothesis that risky decision in young tattooed women is due to impaired inhibitory control. Further studies are needed to identify the cognitive mechanisms involved in the tendency to risky decisions in young tattooed women. Young people are likely to engage in risky behaviors, such as drinking alcohol, taking illegal drugs, having unprotected sex, engaging in delinquent activity, and driving recklessly . Empiricresponse inhibition and cognitive inhibition or resistance to distractor interference. In addition, Kagan [reflection inhibition as a capacity to choose the correct course of action from a number of possibilities prolonging the time before action is taken. We assessed these three inhibition capacities in an attempt to identify which inhibition facet is associated with getting tattoo.Are tattooed women exhibit risky behavior as a result of impaired inhibition capacities? It should be noted that inhibition is a complex concept. Bari and Robbins  suggesten, Kagan  introducIs there an effect of inhibition capacities on decision making process in young women with tattoo? It is expected that in tattooed population there is an association between risky decision and limited inhibition resources. The current study attempted to analyze influence of different inhibition mechanisms on the IGT performance in a sample of tattooed young women assumed to exhibit high level of impulsivity trait compared to a non-tattooed population , 37, 38.Thus, it is unclear whether tattooing behavior can be independently predicted by a weak inhibitory control or risky decisions or both. The current study attempts to answer two main issues:To our knowledge, there are no studies on the evaluation of inhibition capacities among tattooed population and the relation between decision-making process and inhibition capacities in women with tattoos.As described in previous publications , 39 the The link between risky decision and inhibition capacities was analyzed using three laboratory measures.A semi-structured interview  with a 2The 60 non-tattooed women (control group) did not include anyone with current or past DSM-IV-TR axis I psychiatric disorder. Participants were of similar ages, 18–35 . The education level for this group was as follows: high school diploma or lower – 25%, first university degree – 28.3%, second university degree – 41.7% and philosophy degree – 5%. The percentage of smokers in this group was only 10%.We used one decision-making measure (IGT) and three inhibition measures  for our analysis. The IGT was used in our previous study .As a simulation of real-life decision-making, the IGT involves weighing expected, but uncertain, rewards and penalties (for review ). DiminiThe IGT requires choosing between several different alternatives. Each trial has four decks of cards to choose from. The four decks of cards A, B, C, D were placed one beside the other simultaneously on a computer screen. Participants knew that teach deck gives them virtual money, however, they were unaware that A and B were termed risky, and their long-term results are negative, while C and D were safe decks with a more positive overall outcome. They gains were also varied. Participants were instructed that they had 100 options to choose from and their aim was to earn as much virtual money as possible. The choices (selection of cards from different decks) are either advantageous or disadvantageous, but each choice is associated with ambiguity regarding the outcome, since it is difficult for the subject to keep track and remember the gains and losses from previous trials . In the Response inhibition can be defined as a cognitive action that enables a person to repress certain behaviors or reactions. The purpose of the Go/NoGo task is to estimate whether a behavior that is not appropriate can be controlled . In thisThe Matching Familiar Figures Task (MFFT)  was condWe use a manual key-press variant of the Stroop task  with unlt-test) and non-parametric (χ2) tests were performed to compare group differences in demographic and behavioral parameters. For evaluation of differences between groups in the inhibition performance, the multivariate analysis of covariance (MANCOVA) was conducted with performance variables  as dependent measures and group (tattooed women and controls) as between-subject measures, with education and smoking as covariates. A hierarchical regression was conducted to examinee if variables of our interest: (i) Stroop interference reaction time (response time in the incongruent condition minus response time in the congruent condition), (ii) the Go/NoGo mean of response time (as sum of mean responses time in the blocks of the Go/NoGo task), and (iii) the total number of errors in the MFFT explain a statistically significant amount of variance in the IGT performance . Since participants with high impulsivity perform poorly in the IGT due to a robust learning component [Data were analyzed using SPSS (v. 19) software for Windows. All analyses used two-tailed levels of significance. The parametric  and with a higher of rate of smoking status  than non-tattooed women. No differences in age were found between the two groups . Thus education and smoking habit were considered as covariates.Women with tattoos were significantly less educated  was considered as the dependent variable. Three inhibition measures: (i) response inhibition (as sum of response time in the two blocks of the Go/NoGo task), (ii) the total number of errors in the MFFT and (iii) Interference Index (Response Time of Incongruence Condition – Response time of Congruent Condition) in the Stroop task were considered as independent measures. The hierarchical regression analysis did not reveal significant main effects of any inhibition measures on the risky decision process . Analysis of the different inhibition processes may lead to a better understanding of risky behavior. Tattooed women did not display deficits in tasks measured reflexive inhibition and interference control, but impaired response inhibition as measured by the slow response time in the Go/NoGo task. In the Go/NoGo task, response slowness was a sensitive measure of inhibition impairment . ImpairmThe second hypothesis of the current study was that tattooed women would exhibit risky decisions as a result of impaired inhibition capacity. Impulsive responses (faster with more errors) reflect reduced behavior monitoring efficiency and inhibition control that are expected when greater reward sensitivity is required . In our Tattooed young women shows a preserved reflexive inhibition and interference control while express the impaired response inhibition. In current study, we aimed to assess the role of impaired inhibition control in risky decisions among tattooed persons. Although, it is likely that several of inhibition facets may contribute differentially to risky behavior all three investigated inhibition pathways not accounted for the variance in the relation between risky decision and tattooing behavior.This study defined the behavioral and impulse-dimension attributes of young women with tattoos. The strengths of the present study include the evaluation of a population of young women with limited number of tattoos, absence of: criminal or antisocial behavior, unemployment and substance use disorders, as well as using standard cognitive performance tasks that compared risk-taking decisions with different inhibition-related tasking challenges. The major limitation of the present study is the inclusion of healthy population of women only, which confers the advantage of relative homogeneity, but limits the generalizability to the general tattooed population. Unfortunately, only three inhibition paradigms were used in this study. More measures/domains of inhibition  should be assessed in an attempt to clarify the association between inhibition control and risky decision in tattooed women..This study was designed to clarify the associations between inhibition ability and decision making process among young tattooed women. Inhibition capacities of tattooed women are preserved except slowness in the Go/NoGo task. Thus, it appears that risky decision in tattooed women is not a direct consequence of impaired inhibition capacitiesIt may be concluded that risk-taking decisions as assessed in the IGT (hot EF) and inhibition abilities (cool EF) are different and independent aspects of self-control, as was suggested in previous studies in non-tattooed population –31, 41. The limited research in tattooing persons, justifies further studies that will explore the multiple aspects of possible association between inhibition capacities and risky decisions, including variables such as the number of tattoos, their size and body localization, as well as their psychological meaning.


Single-crystal X-ray analysis revealed that the three compounds are isotypic and belong to space-group type P63/m. Ca2+ and Y3+ cations are distributed over two crystallographic sites  are condensed into an isolated [Si719] unit, in which an [Si]4 tetrahedron is located at the center of a 12-membered oxynitride ring with composition [Si6O15N3]. The present compounds are the first to have such an [Si719] unit in their structures.Single crystals of the solid solution series Ca Figs. 11–3. There are five sites in the structure associated with oxygen and/or nitrogen positions. Two sites at Wyckoff position 12i, O1 and O2, and one 6h site, O3, are solely occupied by oxygen, and one 6h site, N5, is solely occupied by nitrogen, irrespective of the value for x. Oxygen and nitrogen atoms are disordered for 4 situated on a 4f site. For 2 and 3, the O:N ratio at this site amounts to 0.25:0.75 for 2 and 0.5:0.5 for 3, respectively, whereas for 1 this site is exclusively occupied by nitrogen atoms.Compounds 2+ and Y3+ occupy two sites, viz. M1 with site symmetry b and M2 with site symmetry 1 at Wyckoff position 12i. M1 is coordinated by six oxygen atoms in the structures of 1–3 whereas the coordination environment of M2 depends on the value of x. In the structure of 1, six oxygen and two nitrogen atoms define the respective coordination sphere, but there are two possible coordination environments for 2 and 3 because of the disorder of the anionic 4f site, i.e. two nitrogen and six oxygen atoms, and one nitrogen and seven oxygen atoms, respectively. Site occupancies of Ca2+ were refined as 0.1379 (14) (1), 0.1338 (14) (2) and 0.2572 (14) (3) for M1, and 0.6437 (2) (1), 0.7277 (2) (2) and 0.7905 (2) (3) for M2. Thus, Ca2+ prefers to occupy the larger M2 site, in agreement with the larger ionic radius of Ca2+ compared to Y3+ for six- (1.00 versus 0.90 Å) and eight-coordination 8] dodecahedra are linked through their edges, resulting in the formation of a layer structure extending parallel to (001). Adjacent layers are connected along [001] through corner-sharing and by silicon atoms in the interstices. There are three Si sites in the crystal structure: Si1, Si2 and Si3 at Wyckhoff positions 6h (site symmetry m..), 6h, and 4f (3..), respectively. The latter site is disordered and shows half-occupancy. At both 6h sites, [SiO3N] tetrahedra are present that are condensed into a 12-membered ring, [Si6O15N3], by corner-sharing. As a result of the disorder and associated splitting of the 4f site, tetrahedra above and below the ring are present that share three corners with the ring, resulting in the formation of isolated [Si719] units, as shown in Fig. 4z = ±0.25 and are situated between the layers formed by layers of [M1O6] octahedra and [M28] dodecahedra.Ca4] tetrahedra are collated in Table 1Bond lengths of the [Si1–3 were obtained from powders synthesized by a solid-state reaction method. CaCO3 , Y2O3 , SiO2 , Si3N4 , and CeO2  in the molar ratio of CaCO3:Y2O3:SiO2:Si3N4 = 5.76:0.62:2.8:1.4 for 2 and of CaCO3:Y2O3:CeO2:SiO2:Si3N4 = 5.76:0.61:0.01:2.8:1.4 (2 mol% Ce to Y) for 1 and 3 were ground in the presence of 20 wt% CaF2  as a flux. The mixtures were pelletized at 20 MPa, put on an alumina boat with a carbon sheet dish  and calcined at 1733 K for 4 h under 100 ml min−1 of flowing nitrogen. The reaction mixtures were slowly cooled under different conditions: to 1373 K at a rate of 30 K h−1, to 1173 K at a rate of 100 K h−1 and to RT by turning off the power for 1, and to 1373 K at a rate of 60 K h−1, to 1173 K at a rate of 100 K h−1 and to RT by turning off the power for 2 and 3. After roughly grinding the recrystallized fused pellets, the powders obtained were washed with 5 M HCl(aq.) and distilled water, followed by drying at 353 K. Colourless platelet-like single crystals were selected from the reaction products. Each crystal was cut into two portions. One was affixed to a Mitegen(R) micro-mount device with a drop of Paratone N oil for single-crystal X-ray analysis. The other part was used for elemental analysis by energy dispersive X-ray (EDX) spectrometry using a scanning electron microscope  equipped with an EDX detector . EDX analysis indicated a Ca:Y:Si ratio of 0.266 (9):0.237 (4):0.497 (9) for 1, of 0.325 (9):0.183 (5):0.492 (5) for 2, and of 0.386 (19):0.146 (10):0.468 (10) for 3. The ideal ratios according to the formulae of the three title compounds are 0.286:0.214:0.500, 0.321:0.179:0.500, and 0.357:0.143:0.500, respectively.Single crystals of d site, corresponding to a triangular environment of three N5 atoms. As a result of the strong anisotropy of the displacement ellipsoids along the c axis, the Si3 sites were subsequently refined as being split with half occupancy and a mirror symmetry element at z = ±0.25. When the occupancies of the disordered Ca2+ and Y3+ sites were refined freely, the ratios of Ca:Y were 7:86:6.14 for 1, 10.07:3.93 for 2, and 9:88:4.12 for 3. Reliability factors for these refinements are summarized in Table 31 and 3 was confirmed by their photoluminescence (the data are not shown). However, the contamination was ignored because of the small amount . Actually, consideration of the presence of Ce had only a marginal effect on refinement parameters and refined structures.Crystal data, data collection and structure refinement details are summarized in Table 2et al., 2010et al., 2017h is coordinated by three Si atoms and thus should be occupied by nitrogen (N5) alone. The relatively long bond length of Si3—4, 1.804 (6), 1.769 (8), and 1.765 (7) Å for 1, 2, and 3, respectively, indicate that the 4 site at the 4f position also might be occupied by nitrogen. Under consideration of charge neutrality for the different compositions in 1–3, this site was refined as being occupationally disordered by oxygen and nitrogen for 2 and 3.Five sites around the silicon atoms were detected. Although it is difficult to distinguish between oxygen and nitrogen atoms by XRD analysis alone, site occupancies of oxygen and nitrogen sites were determined from coordination environments, bond lengths, and bond-valence sums  global, 1, 2, 3, Publ_Block. DOI: 10.1107/S2056989019001257/wm54831sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989019001257/wm54832sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989019001257/wm54833sup4.hklStructure factors: contains datablock(s) 3. DOI: 1892876, 1892875, 1892874CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The underlying mechanisms by which cystatin C affects cardiovascular disease (CVD) are not very clear. Metabolic syndrome (MetS) is a cluster of risk factors that increase the risk of CVD. Here, we aimed to investigate the association of cystatin C with metabolic syndrome and cardiovascular outcomes in non-ST-segment elevation acute coronary syndrome (NSTE-ACS) with preserved renal function. In total, 422 NSTE-ACS patients with preserved renal function were enrolled to examine the association of cystatin C with MetS. MetS was defined based on the NCEP-ATP-III guidelines. Major adverse cardiovascular events (MACEs) were also evaluated, which included cardiac death, nonfatal myocardial infarction (MI), target vessel revascularization (TVR), heart failure, and nonfatal stroke. All patients underwent a 12-month follow-up for MACEs after admission. Cystatin C was significantly correlated with metabolic risk factors and inflammation markers. The prevalence of MetS and MACEs correlated with cystatin C levels. Cystatin C showed a strong diagnostic performance for cardiovascular risk factors and outcomes in ROC analysis. After adjustment for multiple risk factors, cystatin C level was independently associated with MetS . During a 12-month follow-up, the patients with high cystatin C level and MetS had higher incidence of MACEs  and cardiac death  compared to the others. Multivariate Cox analysis indicated that cystatin C level was an independent predictor of MACEs . Cystatin C may be an independent predictor of metabolic syndrome and therefore valuable for management of NSTE-ACS patients. Further multicenter, large-scale studies are required to assess the implication of these results. Cystatin C is an endogenous inhibitor of cathepsin cysteine proteases and is generally considered to be constantly secreted and be freely filtered by the glomerulus but be neither secreted by the renal tubule nor reabsorbed into circulation , 2. TherRecent studies have described cystatin C as a prominent predictor of cardiovascular diseases (CVD) that is significantly associated with high risk of cardiovascular outcomes in acute coronary syndrome (ACS) , 6. UntiOf note, several studies have reported that hypertension, dyslipidemia, and diabetes were associated with cystatin C level, which were the components of metabolic syndrome (MetS) and cardiometabolic risk factors –17. InteThe study protocol complied with the Declaration of Helsinki and was approved by Xinqiao Hospital Ethics Committee, Army Military Medical University . All patients provided informed consent.∗ 1.73 m2 at admission. The exclusion criteria were nonobstructive coronary disease, primary cardiomyopathy and valvular heart disease, primary kidney disease, severe hepatic dysfunction, significant infection, thyroid and adrenal cortex dysfunction, autoimmune diseases, use of steroids and immune inhibitors, hematologic disorders, surgery or trauma 3 month prior to participation, and malignant diseases. Finally, a cohort of 422 patients was enrolled in this study.This was a prospective observational study consisting of 797 consecutive patients with NSTE-ACS patients with preserved renal function who were admitted between January 2017 and September 2017. The inclusion criteria were as follows: (1) with complete clinical information; (2) all patients underwent coronary angiography; and (3) preserved renal function defined as estimated glomerular filtration rate (eGFR) ≥ 60 mL/min Clinical data were collected from medical records by trained physicians. These included demographic data, medical history, laboratory parameters, and basic medication information. The venous blood samples were collected after overnight fasting before coronary angiography, and routine biochemical indicators were measured by automatic biochemical analyzer . Concentration of serum cystatin C was determined by particle-enhanced turbidimetric immunoassay (PETIA) method. The angiographic data were obtained from the cardiac catheterization laboratory records. The synergy between percutaneous coronary intervention with TAXUS and cardiac surgery (SYNTAX) scores for quantifying coronary lesions was assessed by experienced interventional cardiologists using the score calculator (version 2.28) in SYNTAX score website. Primary outcomes were major adverse cardiovascular events (MACEs) defined as the combination of cardiac death, nonfatal myocardial infarction, target vessel revascularization (TVR), heart failure, and nonfatal stroke . All pat∗ 1.73 m2) = 186 × Scr−1.154× age−0.203× 1.233 × 0.742  [μg/mL as the cutoff value of elevated CRP level and 100 pg/mL as the cutoff value of elevated BNP level [The definition of NSTE-ACS complied with the current guidelines of the European Society of Cardiology (ESC) . Global  female) . C-reactNP level , 32. KilNP level , 28. t test was used if continuous variables were normally distributed, while the Mann–Whitney U test was applied if continuous variables were not normally distributed. Differences in categorical variables were evaluated by the Chi-squared test or Fisher exact test. Correlation was assessed using the Spearman rank correlation test. Diagnostic performances of cystatin C on cardiovascular risk factors and outcomes were assessed by the receiver operating characteristic (ROC) curve analysis. Logistic regression was applied to explore the association between cystatin C and MetS, and the variables with unadjusted P value of < 0.1 were selected as potential risk factors and included in the multivariate model. Event-free survival time was defined as from the date of admission to the date of cardiovascular events as verified during the follow-up. Survival curves or cumulative risk curves for cardiovascular outcomes were constructed using the Kaplan-Meier method, with differences assessed using the log-rank test. Cox regression analyses were performed to evaluate the association of cystatin C with MACEs. Multivariate Cox models were adjusted for established cardiovascular risk factors , clinical variables affecting cardiovascular outcomes , medication , and variables that might affect cystatin C levels . Adjusted model 1 included age, gender, BMI, smoking, hypertension, diabetes, LDL-C, and HDL-C; and adjusted model 2 same as model 1 plus eGFR, CRP, Killip class > 1, GRACE scores, SYNTAX scores, ACEI/ARB, β-blocker, and PCI/CABG. P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS software version 22.0 .Continuous variables were expressed as mean ± SD or median (IQR) according to the presence or absence of normal distribution, and categorical data were expressed as frequencies and percentages. To compare the baseline characteristics, the study population was divided into two groups by the median cystatin C level (0.90 mg/L) of the cohort: low cystatin C group (≤ 0.90 mg/L) and high cystatin C group (> 0.90 mg/L). TheThe characteristics of the study population are described in Spearman rank correlation analysis was used to examine the correlation of cystatin C, creatinine and eGFR-MDRD with other clinical variables. Compared to creatinine and eGFR-MDRD, cystatin C showed greater correlation with metabolic parameters, inflammation markers, and other cardiovascular risk factors .Furthermore, we stratified the subjects with or without MetS into the following four groups based on cystatin C levels: low cystatin C/MetS (-) (n = 160), low cystatin C/MetS (+) (n = 51), high cystatin C/MetS (-) (n = 128), and high cystatin C/MetS (+) (n = 83). Elevated CRP level, elevated BNP level, elevated Killip class, high GRACE score, multivessel disease, high SYNTAX score, and MACEs were significantly different among the four groups (P < 0.001), and the high cystatin C/MetS (+) group had more cardiovascular risk factors and events than the other three groups . In addiIn ROC curve analysis, the predictive cutoff values of cystatin C were constructed according to the ROC curves for identifying the patients with more cardiovascular risk factors, and for predicting the occurrence of MACEs. According to the area under the curve (AUC), cystatin C showed a powerful diagnostic performance for cardiovascular risk factors and outcomes. Using the cutoff points, the predictive values of cystatin C for MetS and MACEs were 1.01 mg/L and 0.87 mg/L, respectively .Potential risk factors for metabolic syndrome were chosen in univariate logistic regression analysis (P < 0.1) and were used as the variables of multivariate model. The results indicated that cystatin C level was independently associated with metabolic syndrome  .Cox proportional hazard model was used to examine the association between cystatin C and cardiovascular outcomes during the 12-month follow-up. Univariate analysis showed that cystatin C was significantly associated with MACEs and its components. Confounders were included in the multivariate model for adjustment, and cystatin C remained to be an independent predictor of MACEs in adjusted models 1  and 2  . In the Our study focused on the link between non-GFR determinants of cystatin C and cardiometabolic risk factors and highlighted the predictive role of non-GFR determinants in NSTE-ACS. To the best of our knowledge, we for the first time examined the relationship between cystatin C and MetS in NSTE-ACS with preserved renal function. In our study, the main findings were as follows: (1) cystatin C was a powerful diagnostic indicator for cardiovascular risk factors; (2) cystatin C level was independently associated with MetS; and (3) cystatin C level was an independent predictor of MACEs during 12-month follow-up.Renal impairment reflected by decreased GFR is known as an important cardiovascular risk factor . ConsidePrevious evidence revealed the significant association between cystatin C and metabolic risk factors, such as dyslipidemia, diabetes, and hypertension , 20, 37.In previous studies, Magnusson et al.  reportedIn the current study, we found that cystatin C was a strong independent predictor of MACEs in NSTE-ACS, even after adjustment for CRP, MetS, eGFR, and SYNTAX scores. The results indicated that non-GFR determinants of cystatin C were associated with cardiovascular outcomes, which was supported by previous studies that directly measured GFR thus avoiding errors of eGFR , 48. SimOur study had several limitations. Firstly, this was a single center and observational study with potential selection bias. Second, our sample size was relatively small, and follow-up duration was short. Third, laboratory parameters were only measured once at admission with potential bias due to measurement error. Fourth, our study failed to rely on direct GFR measurement to assess the association of GFR with cardiovascular risk factors and outcomes that could not completely exclude the compounded effect of GFR on the predictive role of cystatin C . Fifth, Our study indicated that non-GFR determinants of cystatin C might be an independent predictor of MetS and cardiovascular outcomes in NSTE-ACS. Cystatin C links renal impairment, insulin resistance, vascular remodeling, and cardiovascular risk as a cardiorenal metabolic marker. The detection of cystatin C may be helpful for management of NSTE-ACS to decrease cardiovascular events. Given the limitations of current study, further multicenter, large-scale, long follow-up period studies are required to assess the implication of these results.


The seabird community of Prince Leopold Island had a broader isotopic niche during lower sea‐ice conditions, thus having a more divergent diet, while the opposite was observed during years with more extensive sea‐ice conditions. Species' trophic position was influenced by sea ice; in years of lower sea‐ice concentration, gulls and kittiwakes foraged at higher trophic levels while the opposite was observed for murres and fulmars. For murres and fulmars over a longer time series, there was no evidence of the effect of sea‐ice concentration on species' isotopic niche. Results suggest a high degree of adaptation in populations of high Arctic species that cope with harsh and unpredictable conditions. Such different responses of the community isotopic niche also show that the effect of variable sea‐ice conditions, despite being subtle at the species level, might have larger implications when considering the trophic ecology of the larger seabird community. Species‐specific responses in foraging patterns, in particular trophic position in relation to sea ice, are critical to understanding effects of ecosystem change predicted for a changing climate.Sea‐ice coverage is a key abiotic driver of annual environmental conditions in Arctic marine ecosystems and could be a major factor affecting seabird trophic dynamics. Using stable isotope ratios of carbon (δ Sea ice is fundamental to the cycle of biomass abundance in Arctic marine ecosystems, and its annual phenology of development and breakup plays a dominant role in ecosystem structure and function , and fish , sea ice is an essential factor contributing to the highly productive food web on which they rely. The reductions in or absence of sea ice induced by earlier breakup of the pack could decouple trophic linkages between primary and secondary producers in the food web , thick‐billed murre (Uria lomvia), black‐legged kittiwake (Rissa tridactyla), and glaucous gull (Larus hyperboreus) at Prince Leopold Island (PLI), Nunavut, a colony of major importance for both diversity and abundance of seabirds in the Canadian high Arctic. The prelaying period at the breeding site is a critical time for Arctic‐breeding migratory seabirds, as the females are principally income breeders, acquiring energy and nutrients required for egg production from local prey resources  and invertebrates such as euphausiids  and carbon (δ13C) in those eggs were measured since the late 1990s. These metrics are suitable proxies of trophic position and some aspects of foraging location  for birds during egg formation  in this study because eggs of that species were not collected during the same 3 years as for black‐legged kittiwake and glaucous gull. Fifteen eggs were collected each year with the following exceptions: 2003—12 black‐legged kittiwake eggs; 2008 and 2013—nine and 12 glaucous gull eggs, respectively; 2015—six northern fulmar eggs.We collected eggs during the last week of June or first week of July at Prince Leopold Island , Nunavut. Eggs were collected from active nests in 1998, 2003, and 2005–2015 for northern fulmars, and 2003 and 2005–2014 for thick‐billed murres. Black‐legged kittiwake and glaucous gull eggs were also collected in 2003, 2008, and 2013. We did not include black guillemot , Ottawa, Ontario, for processing and chemical analyses  were consistent within measurement error; that is, mean values for δ15N between the two laboratories differed by <0.2‰ and a t test comparison of results for the duplicate samples indicated no significant difference (p > 0.05).The 1998–2011 samples were analyzed on a Europa 20:20 continuous‐flow isotope ratio mass spectrometer (CFIRMS) interfaced with a Robo‐Prep elemental analyzer. Within each analytical run, five unknowns were separated by two albumen laboratory standards. The 2012–2015 samples were analyzed using an Isotope Cube Elemental Analyser (Elementar) interfaced with a Delta Advantage continuous‐flow isotope ratio mass spectrometer (Thermo) using a ConFlo III (Thermo). A glutamic acid laboratory standard was included for every 10 unknown samples. Quality control was maintained by running sample duplicates. All measurements are reported in standard δ‐notation in parts per thousand (‰) relative to the AIR international standard. Replicate measurements of internal laboratory standards  indicated measurement errors of ±0.3‰ and ±0.2‰, respectively. Interlaboratory comparisons of duplicate samples  versus C:N to normalize the measured δ13C values:Strong linear negative relationships between δ15N and C:N for any of the species, so the δ15N values did not require correction.As lipids contain negligible nitrogen, as expected, there was no relationship between egg δ2.3https://nsidc.org/data/NSIDC-0051/versions/1. The data were imported in ArcGIS , and δ15Nbase is that of the primary consumer or primary herbivore (TP = 2) at the base of the food chain. Here, we used a mean δ15N of 7.8‰ for Calanus hyperboreus sampled in Lancaster Sound . We theb) for each species each year, based on a posteriori distribution of standard ellipses obtained following 10,000 iterations. In addition to the northern fulmar and thick‐billed murre, we estimated using the same probabilistic method the isotopic niche of the black‐legged kittiwake and glaucous gull in 3 years during which eggs of the four species were collected: 2003, 2008, and 2013. To better infer the effect of various sea‐ice conditions on isotopic niche at the community level, we also used a Bayesian probabilistic method  SEAb that are smaller (or greater) than the number of iterations for the compared group SEAb:We determined the isotopic niche of northern fulmar and thick‐billed murre using Stable Isotope Bayesian Ellipses in R—the “SIBER” package  to look at the significance of the fixed terms.We calculated the average distance to the floe edge, which we defined as the 70% sea‐ice concentration in June and used that parameter along with the overall sea‐ice concentration in the Barrow Strait—Lancaster Sound area to determine the ice condition in 2003, 2008, and 2013. Based on those parameters, we considered 2003 as a “Low” ice year, 2008 as an “Extensive” ice year, and 2013 as a “Moderate” ice year  and on the annual mean trophic position of both species using again GLM followed with likelihood ratio test (F‐statistic) to look at the significance of the fixed terms. We chose to add the interaction between average sea‐ice concentration and species in our models since the period of the prelaying season differs between the species leading to a biological interaction a priori between species and sea‐ice concentration since sea ice is more extensive in May (northern fulmar prelaying period) than in June  in Lancaster Sound in interaction with species and species alone on the annual mode of isotopic niche . In contrast, thick‐billed murre isotopic niche was broader during the year of extensive sea ice than in the two other years . The difference between low and extensive ice years was not as marked for glaucous gulls for which the isotopic niche was significantly smaller in the year of moderate ice conditions compared with the two other years  of the four species (Table bp < 0.03), while 2008 and 2013 had similar community isotopic niche areas ; black‐legged kittiwakes and glaucous gulls occupied a higher trophic position in 2003, the year of low ice, compared with the other 2 years  or June . The average trophic position of both thick‐billed murres and northern fulmars was positively influenced by average sea‐ice concentration , as suggested for the thick‐billed murre in the four species model  nor fulmar  trophic position exhibited a significant trend through time. We did not find any significant temporal trend in thick‐billed murre isotopic niche , although fulmar isotopic niche slightly narrowed over time   or primary producer  collected over multiple years during the ice melting and postmelting season in Lancaster Sound preclude us ignoring the hypothesis of possible changes in basal food web δ15N. For example, Søreide, Hop, Carroll, Falk‐Peterson, and Nøst Hegseth (15N in particulate organic matter (POM) from ice algae compared to pelagic water phytoplankton. Such differences suggest that depending on the phenology of ice breakup, and its associated ice algal bloom followed by a delayed pelagic primary productivity in the spring, secondary producers such as C. glacialis and C. hyperboreus might have access to different food sources of different quality (sympagic vs. pelagic) influencing their biomass development and growth  . Stable isotopes are accessible tools to investigate trophic aspects within and sources of primary productivity to a community, and combined with other techniques, would enhance our overall comprehension of community dynamic under variable and changing environmental factors. Nonetheless, the isotopic space approach we have adopted provides a first look at potential variability in seabird foraging ecology related to sea ice in this system.Collectively, we found that sea‐ice conditions affected the stable isotope values of prebreeding high Arctic seabirds and that this trophic response varied across species. The increased distance between the four species' isotopic niches suggests that reduction in sea‐ice cover might increase pressure on this Arctic seabird community, although this response could be due to effect size (only 3 years were used to assess the influence of sea‐ice conditions on the community isotopic niche). In that regard, the absence of a distinct relationship between isotopic niche and sea‐ice concentration when considering multiple years (murre and fulmar) stresses the relevance of investigating the community‐wide response to changes in the ocean and in sea‐ice conditions over time. Despite being subtle at the species level, we suggest that the effect of variable sea‐ice conditions might have larger implications when considering the trophic ecology of the community. Consequently, our study emphasizes the importance of considering a variety of organisms employing various foraging tactics  to better use seabirds and generally marine top predators Gulka, , as indiNone declared.IP and MLM conceptualized the study, interpreted the results, and drafted the manuscript. IP analyzed the data. BB and KAH contributed with data and to laboratory analyses, revised, and improved the manuscript. All authors approved the final version of this manuscript.


The addition of BPAH-AGEs or BPCH-AGEs to quinine led to significant (p < 0.05) reductions (up to 38%) in bitterness intensity of quinine. The use of 3% hydrolysate to react with glucose yielded glycated peptides with a stronger ability to reduce quinine bitterness than when 1% was used. Calcium release from HEK293T cells stably expressing the T2R4 human bitter taste receptor was significantly (p < 0.05) attenuated by BPAH-AGEs (up to 96%) and BPCH-AGEs (up to 92%) when compared to the BPAH (62%) and BPCH (3%) or quinine (0%). We concluded that BPAH-AGEs and BPCH-AGEs may be used as bitter taste blockers to formulate better tasting foods.Being averse to bitter taste is a common phenomenon for humans and other animals, which requires the pharmaceutical and food industries to source compounds that can block bitterness intensity and increase consumer acceptability. In this work, beef protein alcalase hydrolysates (BPAH) and chymotrypsin hydrolysates (BPCH) were reacted with glucose to initiate Maillard reactions that led to the formation of glycated or advanced glycation end products (AGEs), BPAH-AGEs and BPCH-AGEs, respectively. The degree of glycation was higher for the BPAH-AGEs (47–55%) than the BPCH-AGEs (30–38%). Analysis by an electronic tongue instrument showed that BPAH-AGEs and BPCH-AGEs had bitterness scores that were significantly ( So far, there are a few bitter taste inhibitors -L-lysine, homoeriodictyol and 4-butanoic acid, probenecid, 6-Methoxylflavanones) that have been reported to act through interactions with bitter taste receptors [AGEs are a heterogeneous group of compounds generated from non-enzymatic reactions between the carbonyl group of a reducing sugar and an amino group of a protein, which is termed the Maillard reaction. The reaction process includes three stages, which are initiation, propagation and an advanced stage. The early glycation and oxidation processes form Schiff bases and Amadori products while further glycation of amino groups of peptides or proteins results in molecular rearrangements that lead to the generation of AGEs. The types and yields of final products are attributed to the selected reducing sugar types, pH, temperature, and heating time . The proIn addition to the aforementioned effects, AGEs are also undoubtedly important substances for generating a unique aroma and taste for thermally processed foods. For example, 4-hydroxy-2 (or5)-ethyl-5(or2)-methyl-3(2H)-furanone, 2-hy-droxy-3-methyl-2-cyclopenten-1-one, and 3-hydroxyl-4,5-dimethyl-2(5H)-furanone were reported to enhance the sweetness perception of sugar ,14. MoreMeat has multiple proteins  and enzy®  and Alcalase®  were purchased from Sigma-Aldrich . BPAH and BPCH were produced as previously described [Ground beef was bought from the local market . D-glucose, chymotrypsing at 4 °C) for 30 min and the resulting supernatant collected while the residue was used to repeat the extraction procedure. Finally, the supernatants were pooled and evaporated using the vacuum rotary evaporator maintained at a temperature range between 35 and 45 °C. The concentrated supernatant was filtered through a 0.2 μm disc to remove insoluble materials and then freeze dried.The Maillard reaction was carried out based on the protocol reported by Dong et al. . PreviouBased on previous reports ,23, the Diagnostic solutions including 0.1 M HCl, 0.1 M NaCl and 0.1 M monosodium glutamate (MSG) as well as the calibration solution (1 M HCl) were purchased from Alpha M.O.S . Known bitter score substances such as acetaminophen, caffeine monohydrate, quinine hydrochloride, leporamide hydrochloride and femotidine were purchased from MP Biomedicals . Each freeze-dried AGE sample was dissolved in distilled water to give 0.5, 1.0, 2.0, 5.0 and 10.0 mg/mL concentrations followed by filtration first through a 0.45 μm polytetrafluorethylene (PTFE) filter disc and then a 0.2 μm PTFE filter. Bitter scores of AGEs were evaluated using the Astree II e-Tongue system . This system is a completely automated taste analyzer equipped with seven sensors, BD, EB, JA, JG, KA, OA, and JE, based on the ChemFET technology  to analyze liquid samples . Firstly2+ mobilization using a Fluo-4 NW calcium assay kit [5 viable cells/well were plated in the 96-well clear bottom black-walled BD falcon biolux plates, which were then incubated at 37 °C in a CO2 incubator for 16 h. Following this, the culture medium was substituted with Fluo-4 NW dye (lyophilized dye in 10 mL of assay buffer and 100 µL 2.5 mM probenecid added to prevent dye leakage from the cytosol) for 40 min at 37 °C in the CO2 incubator and followed by 30 min incubation at room temperature. Calcium mobilization was measured after the addition of AGEs to the cells in the presence or absence of agonist quinine and relative fluorescence units (RFUs) using a Flexstation-3 microplate reader  at 525 nm were recorded, following 494 nm excitation. Data were collected from two independent experiments each done in triplicate and PRISM software version 6.0  was used to generate the graphs.Determination of the ability of AGEs to activate or block T2R4 was carried out by measuring intracellular Cassay kit . Stable p < 0.05.Data analyses were performed using one-way analysis of variance (ANOVA) with an IBM SPSS Statistical package, version 20 . Mean values were compared using the Duncan Multiple Range Test, and significant differences were accepted at p < 0.05) less values than the BPAH-AGEs. The DG improved with the increase of D-glucose concentration; thus, the AGEs produced with 0.5 M D-glucose had significantly (p < 0.05) higher values than the other AGEs from lower glucose concentrations. The relationship between the hydrolysate concentration and the DG of the AGEs was not linear. For example, the 1% (w/v) hydrolysate AGEs did not always have a lower DG than those produced from 3%. Therefore, degree of glycation was more dependent on glucose concentration rather than protein hydrolysate concentration.The degree of glycation (DG) of AGEs is summarized in p < 0.05) higher bitterness scores than that of BCML but significantly lower than that of quinine. Based on the acceptability of humans for bitter taste as shown in p < 0.05) reduced. For a combination of BPAH-AGEs and quinine, AGEs from 0.3 M, 0.4 M, 0.5 M D-glucose concentrations combined with 1% BPAH had significantly (p < 0.05) lower bitter scores than other AGEs. In contrast, the effect of glucose concentrations on the reduction of quinine bitterness intensity by BPCH-AGEs was not significant. However, BPCH-AGEs prepared with a 3% hydrolysate concentration had significantly (p < 0.05) stronger suppression of quinine bitterness intensity than AGEs from 1%. Predicted bitter score of BPAH-AGEs and BPCH-AGEs are shown in The unglycosylated AH was more effective in reducing quinine bitterness when compared to some of the AGEs produced with 0.1 and 0.2 M glucose. However, the addition of 0.3 or 0.4 M glucose produced AGEs with a significantly higher suppression of quinine bitterness than the unglycosylated AH. In contrast, with the exception of the CH-AGEs from 0.1 M glucose, there were no significant differences in the quinine bitter taste suppressing ability of the AH-AGEs when compared to the unglycosylated CH.p < 0.05) lower calcium release when compared to their respective hydrolysates, suggesting that the Maillard reaction reduced the bitter taste of beef protein enzymatic hydrolysates. Consistent with the electronic tongue data, calcium mobilization by BPAH-AGEs prepared with 3% hydrolysates was significantly lower than those of the 1% concentration. The BPAH-AGEs from 1% hydrolysate + 0.4 or 0.5 M glucose were especially found to be the least effective suppressors of quinine bitterness based on their significantly (p < 0.05) higher calcium releases when compared to other AGEs. The BPCH-AGEs also significantly (p < 0.05) decreased quinine-dependent calcium mobilization although this occurred with greater efficiency when prepared with 0.1–0.3 M glucose than 0.4 and 0.5 M glucose. In this study, HEK293T cells expressing T2R4-Ga16/44 were used as the experiment group, while HEK293 T cells expressing only Ga16/44 were used as the control group. Results of the calcium assay of AGEs are shown in p < 0.05) lower DG than that of BPAH, suggesting that in addition to peptide size, the type and arrangement of amino acids on the peptides may be important in determining the degree of glycation. Li et al. [Glycation is a common method used to modify proteins, and the properties and characteristics of the resulting AGEs are closely related to the degree of glycation ,26, whici et al.  reportedi et al. . Howeveri et al. . Glycatii et al. ,31. Thus2 value of 0.94 when the electronic tongue data were plotted against the human sensory panel [Bitter scores of BPAH-AGEs and BPCH-AGEs were estimated by the electronic tongue. The ability of the AGEs to suppress quinine bitterness was stronger when 3% BPCH hydrolysate concentration was used but not BPAH. The greater bitter taste-suppressing activity of BPAH-AGEs from 0.3 M or 0.4 M glucose suggested the presence of complex glycosylated peptides with structures that promote better interactions with the T2R4 receptor when compared to the other AGEs. However, the BPCH-AGEs did not show a similar effect on glucose concentration. Therefore, it is possible that the peptide composition differed between BPAH and BPCH, which would explain the variation in the bitter taste-suppressing ability of the AGEs even when the same concentration of peptides was reacted with glucose. However, most importantly, the results suggest that these AGEs could serve as useful agents in blocking bitter sensation in the human mouth. The results are reliable indications of the potential use of the AGEs as bitter taste blockers. This is because the use of this instrumental approach was based on a previous report that showed the prediction of bitter taste of dairy protein hydrolysates had a Rry panel . Althougry panel ,34. Howery panel . This syry panel ,37. Thisry panel . In ordeBased on the mechanism of the bitter taste transduction pathway, the activation of T2Rs will stimulate calcium release from intracellular stores, hence the calcium assay is used to identify bitter taste agonists and antagonists of T2Rs . FurtherThis study showed that the Maillard reaction efficiently reduced the bitterness of beef protein hydrolysates, which may be attributed to increased electrophilic properties of the glycated peptides. The increased electrophilic properties may be responsible for the decreased intrinsic bitterness of the glycated peptides and their enhanced ability to reduce quinine bitterness intensity as measured by the electronic tongue. The higher charged state of the glycated peptides could have reduced hydrophobic interactions with the T2R4 protein, hence the weak activation that was measured as a poor receptor response to quinine-induced calcium release (a measure of reduced bitterness intensity). The stronger ability of BPAH-AGEs to block quinine-dependent bitterness intensity may be due to the high state of glycation and poorer hydrophobic interactions with T2R4 (reduced receptor activation) when compared to the less glycated BPCH-AGEs. Future studies that are focused on structural elucidation of the AGEs will be required to determine the structural basis for the bitter taste-blocking ability. The use of human taste evaluation panels will be necessary to confirm the potential role of peptide AGEs as bitter taste-blocking ingredients in food and nutraceutical products development.


Hypsignathus monstrosus) in the Congo basin was first described in the 1970s. More recently molecular testing implicated this species and other African bats as potential reservoir hosts for Ebola virus and it was one of only two fruit bat species epidemiologically linked to the 2008 Luebo, Democratic Republic of Congo, Ebola outbreak. Here we share findings from the first pilot study of hammer-headed bat movement using GPS tracking and accelerometry units and a small preceding radio-tracking trial at an apparent lekking site. The radio-tracking revealed adult males had high rates of nightly visitation to the site compared to females (only one visit) and that two of six females day-roosted ~100 m west of Libonga, the nearest village that is ~1.6 km southwest. Four months later, in mid-April 2018, five individual bats, comprised of four males and one female, were tracked from two to 306 days, collecting from 67 to 1022 GPS locations. As measured by mean distance to the site and proportion of nightly GPS locations within 1 km of the site (percent visitation), the males were much more closely associated with the site , than the female . Despite the small sample size, our tracking evidence supports our original characterization of the site as a lek, and the lek itself is much more central to male than female movement. Moreover, our pilot demonstrates the technical feasibility of executing future studies on hammer-headed bats that will help fill problematic knowledge gaps about zoonotic spillover risks and the conservation needs of fruit bats across the continent.The biology and ecology of Africa’s largest fruit bat remains largely understudied and enigmatic despite at least two highly unusual attributes. The acoustic lek mating behavior of the hammer-headed bat ( Hypsignathus monstrosus, or the hammer-headed bat, and he conducted the first detailed observational, acoustic, growth-rate, and radio-tracking movement study of H. monstrosus at a research station near Makoko, Gabon [Lek mating behavior is a very rare mating strategy among the more than 1200 known species of bats [o, Gabon . CuriousH. monstrosus at cacophonous lekking sites is an opportunity to reliably sample large numbers of individuals. Importantly, this species was epidemiologically linked to a human outbreak of Ebola virus  [Zaire ebolavirus antibodies [In Central Africa, the visitation of demographically mixed f Congo) , is one f Congo) , and hastibodies –8, thougtibodies . An inhatibodies . Such agtibodies ,12.H. monstrosus from the Congo river upstream to the Lulua river in the Democratic Republic of Congo (DRC) [There is one study that reported visual observations of a month-long seasonal migratory movement of go (DRC) , but litgo (DRC) . Furthergo (DRC) –17.Hypsignathus monstrosus (ML170632). We began with an affordable VHF radio-tracking study that helped inform the selection of a base station methodology that we then used for the GPS-based second pilot. With data collections completed, here we describe our findings and revisit Dr. Bradbury’s definition of a lekking species to support or reject our initial hypothesis of the site as lek. As pilot efforts our sample sizes are low but our findings begin to reveal some insights on the unique lek-associated movement ecology of male and female hammer-headed bats and present future opportunities to characterize bat-human spillover interfaces. We also aim to help inform and improve future movement ecology studies focused on this species.Here we present an analysis of hammer-headed bat movement in northern Republic of Congo from two pilot studies involving a limited number bats. The purpose of the pilots was to begin elucidating the movement ecology of a population of hammer-headed bats already the focus of a longitudinal virological study that began in 2011. That study includes monthly tree phenology observations that began in 2016 and local daily rainfall and temperature data collections since 2015. The distinctive honking sounds made by the congregations of males and the amount of bat activity during the initial and subsequent missions led us to believe the site is a lek [All hammer-headed bats were mist netted at a site  near the village of Libonga in northern Republic of Congo in December 2017 and April 2018.We conducted a preliminary tracking effort in December 2017, using basic VHF trackers . Following capture and sample collection, 10 bats  were hand restrained and 100% ethyl isocyanoacrylate glue, which cured within minutes, was used to attach the trackers. Hair was layered over to improve the attachment. Nine of the bats were released and monitored for 10 days and one adult male was monitored for nine days.In April 2018, we tagged 11 bats with Bird Solar 15 g units  programmed to collect a burst of five GPS points every 30 minutes and accelerometry measurements every 10 min on three axes in the evening between approximately 18h00 West Africa Time  and 06h00 WAT. A subset of the heaviest male and female bats were tagged beginning on April 12 and ending April 21.™ VX21) to fit neck circumferences of 100–125 mm in ~5 mm increments (™) and cured 24 hrs in a clamp. Fabric areas that received Seam Grip were pre-treated with 99% alcohol and abrasion to remove the fabric’s durable water-repellent finish that could reduce bond strength. On site with hand restraint, capes were custom-fit to each individual, allowing enough room for a handler to insert a finger between the collar and neck but small enough to attempt to prevent the collar from slipping over the head and ears. Once fit, the cape was removed and the bat was placed in a cloth bag. The unit was glued to the cape with a mix of UV Aquaseal and Cotol-240™ (to accelerate curing to time to ~2 hours) along with clamping. After curing the unit was labeled with a recovery phone number and then reattached to the bt with the buckle. Total cape and unit mass was 18 g.The Bird Solar 15 g units were attached to a pre-made ‘capes’ cut from soft and pliable six ounce mouse grey fabric  for observation in an isolated and shaded mesh cage (5.0 m x 5.0 m x 1.8 m). The cage  permitted short distance flight and was enriched with large leaves for cover, roosting branches, as well as food and water ad libitum. Care was taken to avoid disturbing bats in the cage and behavioral observations were made from a distance. Bats were initially clearly stressed by the cage and flew repeatedly into the mesh walls of the cage, apparently unaware it was there, until they established a roost on the cage roof. Beyond the cage stress, we observed no adverse impacts of the collar on behaviors in the initial eight bats that were collared and three bats that were included as controls. As no adverse collar issues were noted and substantial cage stress was observed we opted to release the last three bats without any cage observation period.Data from the units were remotely downloaded from each individual bat when in proximity of a base station . We fixed a permanent base station with a large antenna (Omni-Antenna 868MHz 3dBd) in a small clearing used for netting the bats in the lek. Mid-mission we temporarily placed the fixed base station and large antenna on a vehicle that drove at 5 km/hr and surveyed Route N2 15 km northeast and southwest of the site. In addition, during the 10 day deployment mission we patrolled Route N2 ~3 km southwest or northeast of the lek. Then each day through May 28, 2018, we used a hand-held base station to survey along the road between the lek and the southern extent of Libonga village. Between 14–16 February, 2019, the team macheted their way through the forest to visit male and female frequently used day-roosts with a hand-held base station. A return visit to the female’s primary day roost was made on 7 June, 2019.For analysis, we selected one of the five GPS burst points so that each reported ‘GPS location’ was spaced approximately 30 min apart. Individuals with 24 hours or less of movement (i.e from the night of release) were excluded from further analyses. We classified flying and non-flying behavior from the accelerometry data according to Abedi-Lartey et al. . We tranwww.movebank.org) under study name ‘Hypsignathus monstrosus Olson Central Africa’.To visualize bat movement between 12 April and 31 May, we created a movie using the R package moveVis . The utiAll male detections occurred at our lek site . After rEleven bats, comprised of four females and seven males, were collared and released . We recoOur lek was a more central feature of the movements and utilization distributions of male bats compared to the female bat . The lekOn average these hammer-headed fruit bats showed fidelity to certain day-roost sites within a few hundred meters . They alFor the majority of the males, site visitation was daily. Three of the male bats visited the site every night, totaling 7, 8, and 18 consecutive nights for bats COG0246, COG0174, and COG0223 respectively. Although the GPS tracking data for the fourth male (COG0247) does not show any re-visitation to the site, the base station at the lek site logged communications and incomplete download attempts with COG0247 on April 20, 25, 26, and 28.The female (COG0287) appeared to not visit the site for at least a period of 35 days from 21 April through 26 May, when 30 minute nightly GPS logging capability was lost due to low battery power. With the decline of tag power, the tag’s base station communication remains functional, but the tag decreases the number of GPS fixes and eventually shuts off GPS. The lek base station downloads revealed she visited the site in 2018 on 12 June, 24 July, 30 July, and 24 August. Though her two initial visits were detected by GPS and not the base station, a rough estimate of mean site visitation frequency is 22 days with standard deviation 22 days. All data from her unit was last downloaded on 16 February 2019 when the team detected her at an intensely used day roosting site .Overall flight activity for both male and female bats, whether detected by displacement or accelerometry, appears greatest early in the evening and trends downwards until a spike in activity before dawn . ClassifOur small pilots over short time spans provide a glimpse into hammer-headed bat movement ecology and further support our original assessment of the site as a lek for this fascinating species. The VHF and GPS pilots both indicated males frequently visit and occupy the site and that the site is visited, albeit much less frequently, by female bats. We found 51% of fixes for all males combined and 2.2% of the fixes for the female were within 1 km of the site and the latter’s 80% KDE showed no overlap with the site. Hence, we infer the lek does not contain ‘significant resources required by females except the males themselves’ [In general, movement patterns matched the magnitude of those observed by Bradbury’s radio-tracking study . His popEven though our unit performance was similar to other studies, interpretation of our findings should carefully consider our study limitations . We collIn general we attributed the premature GPS unit failures, which resulted in large differences in the number of days we were able to track individuals, to a combination of factors. Our recovery of two intact GPS collars with no indication of a bat carcass suggested our pre-cautionary principle-based collar sizing was likely inappropriate for some bats. Future collar improvements, such as use of an elastic degradable material would be best evaluated in a confined population. With our system the unit needed to come within range of a download station for us to acquire data stored on the unit, so if bat movement patterns did not overlap with our download stations no data was collected. Scheduling time to visit frequently used day roosts sites may address this issue, but the bat needs to be picked up by at least one download station after several days of movement for us to identify a frequently used day roost site. Another explanation is the technical failure of the unit or the ability of the download station to acquire the data from the unit. As O’Mara et al. 2014 point out the impact of GPS units on bat behavior and pre-mature mortality is yet another explanation for the failures we observed and an aspect of bat tracking needing further research . The higICARUS Initiative or Motus, combined with the growing number of bat movement studies can help support their conservation and protect human health.Our findings will help future GPS-based movement ecology studies be better equipped and prepared to maximize their data collections for different research questions and tracking methodologies. This can help mitigate the already often high transaction costs of conducting research in remote corners of Africa. For example, we can now make more appropriate staffing and logistical plans for future deployments to visit the location of each bat’s regular day roost site. In addition, species-specific programming can improve and extend the performance of GPS units , and so S1 FigContours of 80 (darker with outline) and 95 (lighter) percent probability of utilization for each bat.(PNG)Click here for additional data file.S2 FigDay roost location is defined as the first GPS location of the evening typically around 18h00 (time 0 in the figure). Displacement is observed over 30 min intervals for all males combined and separately for the female.(PNG)Click here for additional data file.S3 FigThe female’s nightly displacement is negatively correlated with nightly lunar illumination . There is no correlation of displacement and daily lunar illumination among the males . Data excludes 13 bat nights when less than 10 GPS locations were collected.(PNG)Click here for additional data file.S1 File(c) Mapbox, (c) OpenStreetMap, and (c) DigitalGlobe; improve this map.The single female bat, COG0287, is tracked in pink. Each individual’s track fades after 24 hrs. Local time (WAT) is displayed. Route N2 is the faintly visible diagonal road along a southwest and northeast axis. Basemap data from Mapbox Satellite (MOV)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.