The charge/discharge curves exhibited clear plateaus in the high voltage range of 3.3–3.7 V. The capacities of the plateau regions were comparable to the calculated capacities corresponding to the one-electron redox of the molecules. The amount of the active compound 3b could be increased up to 30 wt% in the electrode composite, and fast charge/discharge performance was also observed.Hydrazine-embedded heterocyclic compounds with dimeric dimethylacridine ( In recent years, we have reported novel hydrazine-embedded polyheterocyclic compounds including hydrazinohelicenes 1 and 2 ] than 1a and 2a9. Low solubility of the active materials in the electrolyte of a battery is generally desired for maintaining the capacity and recycle performance of the battery. Compounds 1b and 2b were prepared in 8 steps from N-phenylanthranilic acid and in 5 steps from carbazole according to our previous reports, respectively9. Phenothiazine dimer 3b6 was synthesized by bromination of phenothiazine, Ni-mediated coupling of bromophenothiazine, and aerobic oxidation under basic conditions in 3 steps  measurements ] of the monocation radical of 1b-3b and 4 were conducted to obtain information about the electronic states of the monocation radical species. The calculated spin density distributions and the electrostatic potential (ESP) maps are shown in Figs 4, t-butyl and ethyl groups were replaced with methyl groups. The calculated spin densities of all the monocation radicals of 1b-3b and 4 are delocalized over the entire skeleton, leading to the stability of the monocation radicals. The calculated ESP maps of 1b-3b show that the inside part of the molecules around the nitrogen atoms is positively charged in addition to the aromatic hydrogen atoms. In contrast, the calculated ESP map of 4 indicated that the alkyl groups on the nitrogen atoms directed to the outside of molecules are positively charged.The electrochemical oxidation potentials of N-methyl-2-pyrrolidone (Method A). A lithium organic battery was assembled with a Li metal foil anode and the prepared cathode with a porous polymer film separator in an electrolyte solution composed of 1 M LiPF6 in ethylene carbonate/diethylcarbonate (1:1) in a coin-shaped cell (2032-type). The charge/discharge performances of the batteries were measured at a constant current  for 20 cycles between 2.5–4.0 V (1b-3b) or 2.0–4.2 V (4) on a charge/discharge device (HOKUTO DENKO HJ1010mSM8A). The selected charge/discharge curves and the cycle performance of discharged capacities at 2.5 V (1b-3b) or 2.0 V (4) are shown in Figs 1b-3b exhibited clear plateaus in the range of high voltage = 3.3–3.7 V in both charge and discharge processes. Compared with the voltages of reported Li-ion organic batteries using representative active materials, the voltages of 1b-3b are higher than those of quinone molecules  and comparable to those of nitroxide molecules (around 3.6 V)15. The plateau regions correspond to the chemical one-electron redox processes in the CV, since the obtained capacities are consistent with the calculated ones from one-electron reduction and the one-electron redox voltage in CV (vs. Li+/Li) corresponds to the plateau voltage. The voltage of the plateau region of 1b is lower than that of 2b, which is consistent with their oxidation potentials  in CV. The plateau voltage of 3b in the discharge process gradually decreased in the range of 3.6–3.2 V, while the plateau for the thin film cathode by using method B was more flat (vide infra). The gradual decrease of plateau voltage of 3b in the pellet cathode is probably due to the higher resistance in the pellet than in the thin film. In contrast to 1b-3b, the curve of 4 did not show a plateau in either charge or discharge processes [Fig. 4 was not due to the decomposition of the compound. This suggests that its redox reaction gradually occurs when the voltage decreases, which is probably caused by the difference in molecular skeleton. The calculated capacities per weight of compounds 1b-3b were 65, 81, and 68 mAhg−1 for one-electron redox, respectively. For 3b, larger capacity was observed in the first discharge process, which is probably due to non-Faradic reaction and SEI in the first discharge process. The observed capacities of 1b and 3b were gradually decreased by the cycling charge/discharge process  were prepared by mixing the compound (30 wt%), polyvinylidene difluoride (PVDF) (10 wt%), and conductive carbon black (TOKA BLACK 5500 of TOKAI CARBON) (60 wt%) with N-methyl-2-pyrrolidone on an aluminum sheet. Compared with method A, the amount of 3b was increased up to 30 wt% and the thickness of the cathode electrode composite was decreased to 0.1 mm. Lithium batteries were assembled with the cathode electrode composite, and the performance was investigated. The charge/discharge performance of the batteries was measured at a constant current (200 mAg−1 or 500 mAg−1) for 20 cycles between 2.5–4.2 V. Selected charge/discharge curves and the cycle performance of discharged capacities at 2.5 V are shown in Figs −1 [Fig. −1 [Fig. 3b even if the active material concentration was high.We then applied these compounds as cathode-active materials for Li-ion batteries. Cathode electrode composites (0.5 mm thickness) were prepared by mixing the compound (10 wt%), polyvinylidene difluoride (PVDF) (20 wt%), and conductive carbon black (TOKA BLACK 5500 of TOKAI CARBON) 70 wt%) with 0 wt% witses Fig. . Since t −1 Fig.  to 500 m −1 Fig. . Since t1b-3b with dimethylacridine, carbazole, and phenothiazine skeletons to cathode active materials of rechargeable lithium organic batteries and investigated the performance of the assembled batteries. The charge/discharge curves exhibited clear plateaus in the high voltage range of 3.3–3.7 V. The capacities of the plateau regions were comparable to the calculated capacities corresponding to the one-electron redox of the molecules. The amount of the active compound 3b could be increased up to 30 wt% in the electrode composite, and fast charge/discharge performance was also observed. This study demonstrated the applicability of hydrazine-embedded heterocyclic compounds to the cathode active materials of a high voltage rechargeable lithium organic battery as a first example. We expect that hydrazine-embedded heterocyclic compounds with excellent reversible redox properties can be applied as a component of a variety of other redox systems including organic electronic devices.In summary, we have applied hydrazine-embedded heterocyclic compounds 17. A solution of Ni2 , 2,2′-bipyridyl , and 1,5-COD  in dry THF (20 mL) was heated at 70 °C under an Ar atmosphere for 10 min. To the solution, 1-bromophenothiazine  in dry THF (10 mL) was added dropwise over 1 min. The solution was stirred at 70 °C for 1 h. The solution was poured in CHCl3 and silica gel was added. The suspension was filtered through a silica gel pad, washed by CHCl3, and evaporated. The residue was dissolved in CH2Cl2 (5 mL) and precipitated by addition of hexane (20 mL). The precipitate was filtered to afford 1,1′-biphenothiazine  as a colorless solid.1-Bromophenothiazine was prepared according to the literature−1. 1H NMR (CDCl3): δ 7.08 , 6.99 , 6.95–6.91 , 6.87 , 6.80 , 6.27 , 5.78  ppm. 13C NMR (CDCl3): δ 141.0, 139.6, 129.1, 127.4, 127.2, 126.6, 123.0, 122.8, 121.8, 119.8, 118.2, 115.3 ppm. HRMS (EI) (m/z) for C24H16N2S2 (M+): calculated 396.0755, found 396.0745.Mp: 250–252 °C. IR (ATR): ν 3364, 2359, 1474, 1425, 1283, 1249, 777, 751, 719, 667 cmt-butoxide  in THF (10 mL) under air at room temperature was added 1,1’-biphenothiazine . The solution was stirred for 4 h. The reaction mixture was quenched by aq. NH4Cl at 0 °C and extracted by CH2Cl2 (20 mL × 3). The combined organic layers were dried over Na2SO4, filtered through Celite, and evaporated. The residue was dissolved in CH2Cl2: hexane = 1: 1 and filtered through a silica gel pad. After evaporation of solvent, the residue was suspended in hot MeOH (100 mL) and stored in a fridge overnight. The precipitate was filtered to afford 1,1′,10,10′-biphenothiazine  as a yellowish solid. Mp: 183 °C (decomp.). IR (ATR): ν 1468, 1442, 1414, 1220, 1029, 894, 772, 744, 644 cm−1. 1H NMR (CDCl3): δ 7.57 , 7.43 , 7.25 , 7.17 , 7.14 , 7.06 , 6.98  ppm. 13C NMR (CDCl3): δ 146.2, 140.3, 127.9, 127.5, 127.3, 125.3, 124.8, 123.9, 123.4, 121.8, 121.0, 113.7 ppm. HRMS (FAB) (m/z) for C24H14N2S2 (M+): calculated 394.0598, found 394.0601.To a solution of potassium N-methyl-2-pyrrolidone (Aldrich). The mixture was spread at a thickness of 0.5 mm onto a polypropylene sheet, cut into a disc with a diameter of 1.6 cm, and dried in vacuo after removal of the polypropylene sheet. Under an argon atmosphere, the cathode, a Li metal foil (0.2 mm thickness) , and a porous polymer film separator (polyolefin)  were placed in a coin-shaped cell (2032-type) with an electrolyte solution composed of 1 M LiPF6 in ethylene carbonate/diethylcarbonate (1:1) .Method A: The cathode-active compound (50 mg), polyvinylidene difluoride  (100 mg), and conductive carbon black (TOKA BLACK 5500 of TOKAI CARBON) (350 mg) were mixed using N-methyl-2-pyrrolidone. The mixture was spread at a thickness of 0.1 mm onto an aluminum sheet using a doctor blade, dried in vacuo, and cut into a disc with a diameter of 1.6 cm. Under an argon atmosphere, the cathode, a Li metal foil (0.2 mm thickness), and a porous polymer film separator (polyolefin) were placed in a coin-shaped cell (2032-type) with an electrolyte solution composed of 1 M LiPF6 in ethylene carbonate/diethylcarbonate (1:1).Method B: The cathode-active compound (90 mg), polyvinylidene difluoride (PVDF) (30 mg), and conductive carbon black (180 mg) were mixed using


It was recently shown that the conditioned medium (CM) of mesenchymal stem cells can enhance viability of neural and glial cell populations. In the present study, we have investigated a cell-free approach via CM from rat bone marrow stromal cells (MScCM) applied intrathecally (IT) for spinal cord injury (SCI) recovery in adult rats. Functional in vitro test on dorsal root ganglion (DRG) primary cultures confirmed biological properties of collected MScCM for production of neurosphere-like structures and axon outgrowth. Afterwards, rats underwent SCI and were treated with IT delivery of MScCM or vehicle at postsurgical Days 1, 5, 9, and 13, and left to survive 10 weeks. Rats that received MScCM showed significantly higher motor function recovery, increase in spared spinal cord tissue, enhanced GAP-43 expression and attenuated inflammation in comparison with vehicle-treated rats. Spared tissue around the lesion site was infiltrated with GAP-43-labeled axons at four weeks that gradually decreased at 10 weeks. Finally, a cytokine array performed on spinal cord extracts after MScCM treatment revealed decreased levels of IL-2, IL-6 and TNFα when compared to vehicle group. In conclusion, our results suggest that molecular cocktail found in MScCM is favorable for final neuroregeneration after SCI. Spinal cord injury (SCI) is one of the major traumas of the central nervous system (CNS) leading to devastating neurological outcomes ,2. UnforIndeed, in recent years, convincing immunomodulatory and neurotrophic properties have been ascribed to mesenchymal stem cells (MSc) derived from the bone marrow showing partial beneficial impact on locomotor function ,14,15,16Furthermore, we have confirmed therapeutic efficacy of intrathecal administration of PKH67-labelled MSc by improving motor function of the hind paws after SCI . HistoloAs opposed to cell therapies, the local delivery of secretomes gets to the forefront of interest for possible treatment of CNS injuries. These secretomes must supply the injury site with bioactive molecules for a sufficient time to achieve optimized clinical outcomes. Therefore, frequent delivery during longer periods from about two weeks should be considered.Supporting this concept, here we determine whether localized, intrathecal delivery of the MScCM affects tissue sparing, axonal regrowth, immune response and functional outcome after SCI. The designed CM treatment represents a promising alternative to traditional stem cell therapy for the treatment of acute spinal cord injury.2) continuously multiplied and formed neurosphere-like structures during one week. Immunohistochemistry using TUJ1 confirmed that these spheres were interconnected via extended neurites and formed almost confluent neuronal networks (Primary cultures of DRGs (PCDRGs) plated at a high density . Under this condition, the surface area outside the cell body that was covered with delicate neurites was determined , six weeks (* p < 0.05), 8–10 weeks (** p < 0.01) post-injury with the following BBB scores: 9.2 ± 1.3/two weeks, 11.8 ± 1.5/three weeks, 13.3 ± 1.4/four weeks, 13.9 ± 1.1/five weeks, 14.9 ± 1.4/six weeks, 15.7 ± 1.8/seven weeks, 17.2 ± 1.5/eight weeks, 17.8 ± 1.1/nine weeks, 18.1 ± 1.4/ten weeks (SCI + MScCM); and 4.9 ± 1.4/two weeks, 6.2 ± 1.9/three weeks, 8.8 ± 1.4/four weeks, 11.2 ± 1.2/five weeks, 10.7 ± 1.4/six weeks, 11.7 ± 1.9/seven weeks, 13.1 ± 1.3/eight weeks, 13.3 ± 1.6/nine weeks, 13.1 ± 1.2/ten weeks (SCI + V)  and treated group (SCI + CM) was detected at two weeks, three weeks, four weeks (** SCI + V) .p < 0.01) and −caudal sections 5,6,7,8,9 mm (* p < 0.05); CM treated group: +cranial 9,8,7,6,5 mm = 2.6, 2.5, 2.5, 2.3, 2.0/±0.1–3 mm; −caudal 5,6,7,8,9 mm = 1.9, 1.9, 2.1, 2.4, 2.4, 2.1/±0.2–5 mm. Compared to the vehicle treated group; +cranial 9,8,7,6,5 mm = 1.5, 1.3, 1.1, 0.8, 0.7/±0.2–0.5 mm; −caudal 5,6,7,8,9 mm = 0.8, 1.1, 1.3, 1.5, 1.5/±0.2–5 mm)   was done with luxol fast blue/cresyl violet staining on coronal spinal cord sections taken from rats processed to SCI + V and SCI + CM treatment B,C. Stai < 0.05) .p < 0.01. CM-mediated treatment promoted growth of GAP-43-labeled axon fibers that were infiltrating the lesion site and adjacent segments B,E,F. ThCytokine arrays confirmed the time-dependent synthesis of chemokines and cytokines after SCI. Compared with control, CINC-2a/b, IL2, IL6, MIP-1α, Rantes, CXCL3, TNFα, TIMP-1 and VEGF were significantly overexpressed 15 days after SCI A. At theWe have previously reported that IT delivered MSc migrated and incorporated into the central lesion leading to higher motor function recovery . Thus, iBased on these results, we developed a regimen for IT CM delivery. We had to consider the optimal dose and therapeutic window to ensure a continuous supply of trophic molecules to the site of injury. Since we were delivering stem cell products that have limited biological activity, we needed to continually supply the injury site with sufficient amount of trophic molecules. Therefore, we decided to administer CM at 4-day intervals from 1 day to 13 days after injury. Four-day interval was selected due to our previous in vitro data, which showed that the supplementation of neural progenitors with trophic factors  every 3–4 days is sufficient to maintain their biological activity and to stimulate their neurogenic behavior (growth and proliferation of neurospheres). Furthermore, the pro–regenerative and inflammatory processes are initiated within the first week after SCI, which outline this period as a potential therapeutic window for treatment intervention ,26. In context with this strategy, our behavioral tests confirmed a progressive improvement of the motor function in the SCI + CM treated group compared to the untreated one (SCI + V). We noticed the most significant changes during the first 2–3 weeks and then from 8–10 weeks, although in the vehicle group there was small number of animals that survived up to 10 weeks. Therefore, for future studies we need to work with larger groups in order to support behavioral data. This beneficial outcome correlates with the spared white and gray matter, which was approximately 30% higher in the SCI + CM group. Furthermore, conditioned medium-induced sparing of spinal cord tissue should logically correlate with enhanced regenerative processes. For this reason, we have studied the expression of GAP-43-positive axons, which correspond to newly outgrowing axons with the capacity of re-building the neural connections after CNS damage. The regeneration in adult tissue can be initiated only for a few days when GAP-43-positive fibers are able to grow over short distances ,28. HoweAnother important factor influencing regeneration that needs to be considered is the severe inflammatory response after SCI. In the CM treated group, we have experienced a significant decrease in IL6 and TNFα and an increase in the trophic factors of CNTF and VEGF, which were gradually reduced with time to the levels observed in the untreated group. This points to the fact that MScCM is capable of temporarily modulating limited immune response as we previously demonstrated on microglia cells . This isIndeed, these results positioned the MScCM treatment on the level of cell based therapy, where similar beneficial outcomes were observed ,20. The 2 in uncoated tissue culture flasks, as these cells readily adhere to the plastic. The cells were incubated in a humidified atmosphere with 5% CO2 at 37 °C. Non-adherent cells were removed after 3–4 days by medium change and the remaining cells were fed twice per week. When the cultures reached 80% of confluence, the MScs were passaged with 0.25% trypsin/0.53 mM Ethylene diamine tetraacetic acid , centrifuged, and re-plated at a density of 5000 cells/cm2. The MScs were propagated for three passages and then characterized as previously described [MScs were isolated from the bone marrow of four 10-week old male Wistar rats (300 g), collected from the long bones (femur and tibia) ,20. The escribed . 2 at 37 °C for 24 h and used for MScCM collection, using a similar protocol as in the previous study [Cells at passage 3 cultured in Dulbecco’s modification of Eagle’s medium  with low glucose and without fetal bovine serum were incubated in a humidified atmosphere with 5% COus study . n = 50) Wistar rats following decapitation. Under sterile conditions and using a stereomicroscope the DRGs were cut into smaller pieces and incubated at 37 °C in 3 mL collagenase dissolved in a dissociation solution consisting of HBSS supplemented with collagenase , hyaluronidase . The digestion process was stopped by adding an equal volume of HBSS, and the suspension was filtered through a stainless mesh sieve (pore size 100 μm), centrifuged for 5 min (1000× g) and the supernatant was discarded. Cells were divided into 3 groups: (1) NGF+ group, grown in DMEM F12 medium with 1% fetal bovine serum and nerve growth factor ; (2) NGF− group, grown in DMEM F12 medium with 1% fetal bovine serum; and (3) MScCM group, grown in MScCM in DMEM F12 medium and 1% fetal bovine serum (ratio 3:1). All three groups were supplemented with 1% penicillin and streptomycin and allowed to adhere overnight onto poly-dl-ornithine (500 μg/mL) and laminin (10 ng/mL) coated glass coverslips in 12-well tissue culture plates  at 37 °C for 7days in vitro (7DIV). For all PCDRGs, we have used standard IHC procedures to visualize neurite outgrowth. PCDRGs were incubated in mouse anti-Neuron-specific class III beta-tubulin (TUJ1)  for 24 h at 4 °C. Afterwards, they were washed in 0.1 M PBS and incubated for 2 h with goat anti-mouse IgG . For nuclear staining, we used 4-6-diaminidino-2-phenylindol  and finally the slides were coverslipped with Vectashield mounting medium . Digitized images of PCDRGs/per treatment (n = 6) were captured and saved with NIS-Elements Imaging Software . Neurite outgrowth was analyzed at identical sampling fields, for each CM experiment by ImageJ software according to the above-mentioned method applied for primary antibodies quantification, according to our previous study [Dorsal root ganglia (DRGs) located in cervical to sacral spinal levels were dissected from newborn , us study .Intrathecal (IT) catheter implantation was performed according to our previously published procedure . The ratn = 24) weighing between 300 and 320 g, according to our previous study [All experimental procedures were approved by the institutional ethical committee for animal research, and were in accordance with the Slovak Law for Animal protection . SCI was induced using the modified balloon compression technique in adult male Wistar rats  and at ten weeks after SCI .All SCI rats were randomly divided into the following groups: (1) MSc conditioned medium (CM) group receiving four daily injections of 30 µL MScCM (SCI + CM) at 1, 5, 9 and 13 days; or (2) a vehicle group (SCI + V) receiving 30 µL of DMEM per rat at the same intervals. A maximum dose of 30 µL CM was flushed with 5 µL of saline over 30 min through a calibrated PE10 catheter , which was connected to a 500-µL Hamilton syringe attached to a mechanical mini-pump. This procedure was performed in each rat of the SCI + CM and vehicle groups. Implantation of the IT catheter was performed on the same day as IT delivery, at 1 day after SCI. Animals were intracardially perfused at 15 days and four weeks  21-point open field locomotor scale . BBB scoCytokines profile of CM derived from control, SCI + V, SCI + CM after 15 days and 30 days for the segment R1 was performed by using a Rat Cytokine Array Panel A according to the manufacturer’s instructions . Conditioned media from rostral segments from spinal cord tissue  were obtained according to our previous study and afterwards processed for Cytokine profile detection . We have2. To analyze the amount of spared spinal cord tissue, we have performed modified luxol fast blue histological staining according to our procedure . A 1.8 cExperimental and control rats were deeply anesthetized with a ketamine-xylazine cocktail (15 mg/kg of xylazine and 150 mg/kg of ketamine), and transcardially perfused with 0.1 M PBS, followed by 2% paraformaldehyde in PBS. The spinal cord and externalized IT catheter was removed, post-fixed in the same fixative solution overnight at 4 °C, and embedded in the gelatin albumin substrate in 2% paraformaldehyde in PBS. Frozen spinal cord sections were cut from a 1.5-cm-long spinal cord segment positioned on the injury epicenter, embedded in embedding medium, dissected into three 0.5-cm-thick blocks , and stored at −20 °C. Sections 10-µm thick were then serially cut from the epicenter and mounted directly onto slides. From the rostral and caudal regions, 40-µm-thick cryostat (Leica) sections were cut and collected in 24-well plates with 0.1-M PBS containing 0.1% sodium azide. Sections were taken at 200 µm intervals and 20 sections per block were obtained. Ten sections per block were stained with hematoxylin and eosin (H&E) to assess tissue morphology and determine the injury epicenter. For immunohistochemistry, free-floating sections (40 μm) were immersed in PBS  containing 10% normal goat serum (NGS), 0.2% Triton X-100 for 2 h at room temperature to block non-specific protein activity. This was followed by overnight incubation at 4 °C with primary antibodies: mouse anti-GAP-43  for 24 h. Afterwards sections were washed in 0.1 M PBS and incubated with secondary fluorescent antibodies goat anti-mouse conjugated fluorescein isothiocyanate (FITC) (Alexa Flour 488) at room temperature for 2 h. For general nuclear staining, DAPI (1:200) was added to the final secondary antibody solutions. Finally, sections were mounted and coverslipped with Vectashield mounting medium. Immunochemically stained sections were analyzed using Olympus BX-50 fluorescent microscope at 10× and 20× magnifications, captured with digital camera HP Olympus and analyzed by Image J software according to the previous protocol . Quantifp < 0.05, ** for p < 0.01, and *** for p < 0.001.Obtained data from tissue analyses and behavioral testing were reported as mean ± SEM. Mean values among different experimental groups were statistically compared by one-way ANOVA and Tukey’s post hoc tests using GraphPad PRISM software. Significant values were denoted with * for Our results confirm the pro-regenerative effects of MScCM on tissue sparing, axonal growth, immunomodulation and final functional outcome. These results suggest that multiple IT delivery of stem cell products may improve behavioral function when the dose and timing are optimized. Taken together, the molecular cocktail found in MScCM is favorable for final neuroregeneration in vivo.


We sought to analyze the relationship of 31p recovery time constant (PCr) and exercise capacity in a population with PAD.Patients with PAD who have intermittent claudication have reduced exercise tolerance, however, the mechanisms are not well understood. Ankle-brachial index (ABI) correlates neither with walking distance nor the degree of claudication or functional limitation as it is only able to measure flow at the macrovascular level. 31p MR spectroscopy using a 3 T Siemens Trio MR scanner during recovery after peak exercise. A single-pulse, surface coil localized 512 msec free induction decay acquisition with 20 averages centered on the midcalf was used. A standard 31p surface coil in the patient table is employed. PCr was then calculated using a monoexponential fit of phosphocreatine concentration versus time, beginning at cessation of exercise. Patients later exercised on a treadmill (Gardner exercise protocol) and completed a 6 min-walk protocol. Peak VO2 (peak oxygen consumption) was measured. METS , total distance (feet), total exercise time and start of claudication time were also recorded.Twenty-three (23) patients with PAD (ABI < 0.9) were prospectively enrolled. All performed supine plantar flexion exercise at 50 rpm using a pedal ergometer until exhaustion or limiting symptoms. PCr was measured by 2 13.4 ± 3.7 (ml/kg/min), METS 3.8 ± 1.1, treadmill exercise time 5.8 ± 4.4 min. PCr correlated with 6-min walk total distance , total METS  and peak VO2 . No correlation was seen between PCr and claudication distance (p = 0.27) nor start of leg discomfort (p = 0.24). ABI did not correlate with any of these exercise parameters.The mean age was 67 ± 11 years, 63% were male, 63% were Caucasian, the mean ABI was 0.69 ± 0.09. The mean logPCr was 1.68 ± 0.25, 6-min walk distance 1024.7 ± 351.9 feet, 6-min walk start of claudication 330.0 ± 274.2 feet, peak VO2 in patients with PAD whereas ABI does not. PCr recovery kinetics could be used a therapeutic target in novel interventions in patients with PAD as it correlates better with exercise parameters than ABI.PCr recovery time constant correlates significantly with total distance, METS and peak VO


SugPOP-1–3) have been synthesized by Friedel–Crafts reaction using formaldehyde dimethyl acetal as an external cross-linker. Three perbenzylated monosugars with similar chemical structure were used as monomers in order to tune the porosity. These obtained polymers exhibit microporous and mesoporous features. The highest Brunauer–Emmett–Teller specific surface area for the resulting polymers was found to be 1220 m2 g−1, and the related carbon dioxide storage capacity was found to be 14.4 wt % at 1.0 bar and 273 K. As the prepared porous polymer SugPOP-1 is based on hemiacetal glucose, Ag nanoparticles (AgNPs) can be successfully incorporated into the polymer by an in situ chemical reduction of freshly prepared Tollens’ reagent. The obtained AgNPs/SugPOP-1 composite demonstrates good catalytic activity in the reduction of 4-nitrophenol (4-NP) with an activity factor ka = 51.4 s−1 g−1, which is higher than some reported AgNP-containing composite materials.Porous hypercross-linked polymers based on perbenzylated monosugars ( Hypercross-linked polymers (HCPs) are microporous organic materials with a high specific surface area (SSA) –2. The pThe porosity and functionality of HCPs is highly dependent on the core structural monomers –15. Howe2  calcd for C40H40O6, 639.3; found: 639.4.Sodium hydride  was added portionwise to a solution of phenyl β-D-glucopyranoside  in DMF (5.0 mL) over 40 min under nitrogen atmosphere in an ice bath. After being stirred at room temperature, benzyl bromide  was added to the mixture. The resulting mixture was stirred for 4 h at room temperature and then ice water was added to quench the reaction. The suspension was extracted with ethyl acetate (2 × 50 mL). The combined organic layer was washed with water (3 × 50 mL) and dried with anhydrous sodium sulfate. After removing the solvent under reduced pressure, the residue was chromatographed on silica gel to give 1H NMR  δ (ppm) 7.38–7.19 , 4.92 , 4.71 , 4.66–4.52 , 3.87–3.70 , 3.61 , 3.26 , 1.38–1.26 ; 13C NMR  δ (ppm) 138.8, 138.7, 138.5, 128.5, 128.1, 128.0, 127.8, 127.7, 127.6, 99.2, 80.6, 80.3, 75.5, 74.9, 72.9, 72.2, 68.0, 54.7, 18.1; MS (MALDI–TOF) m/z: [M + Na] calcd for C28H32O5, 471.2; found: 471.2.Sodium hydride  was added to a solution of methyl α-L-rhamnopyranoside  in DMF (5.0 mL) in an ice bath over 40 min under a nitrogen atmosphere. Then benzyl bromide  was added and the reaction mixture was stirred at room temperature for 4 h. After completion (TLC and carbonation), the organic layer was extracted twice with ethyl acetate (50 mL). The combined organic layer was washed three times with water and dried with anhydrous sodium sulfate. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to give a colorless solid foam . SugPOP-1). Anhydrous FeCl3  was added to a stirred solution of Sug-1  and FDA  in 10 mL dry DCE under a nitrogen atmosphere. After the solution was well mixed, the resulting mixture was heated to 45 °C for 5 h and 85 °C for 19 h. The obtained precipitate was washed three times with methanol and THF, respectively. The residue was further purified by Soxhlet extraction with methanol and THF for 24 h each, then dried under reduced pressure at 50 °C for 24 h to give Sug-1 as a brown powder .The representative synthesis procedure was as follows  was added to a freshly prepared Tollens’ reagent solution (15 mL). The reaction mixture was stirred at 45 °C in the dark for 24 h. The obtained product was filtered and washed with water and ethanol and then dried under reduced pressure at 45 °C for 24 h to give AgNPs/SugPOP-1 composite as a brown solid (100 mg)./SugPOP-1 composite, the reduction of 4-NP was performed in a quartz cuvette  in the presence of sodium borohydride (NaBH4). 4-NP  and a freshly prepared aqueous NaBH4 solution  were added in the quartz cuvette. Then, the AgNPs/SugPOP-1 composite  suspended in deionized water was subsequently added to the above solution and the reaction progress monitored by UV–vis spectroscopy. The absorption spectra were measured at room temperature by recording absorbance from 244–600 nm within defined time intervals. The reduction reaction was conducted within minutes after the solution was prepared to minimize decomposition of NaBH4.To investigate the catalytic performance of the AgNPsFile 1Additional spectra.SugPOP-1–3; 13C CP/MAS NMR spectra of SugPOP-1 and SugPOP-2; TGA curves of polymers SugPOP-1–3 under nitrogen atmosphere; CO2 adsorption isotherms of SugPOP-1–3 with pressure up to 1.13 bar; X-ray diffraction patterns of SugPOP-1 and AgNPs/SugPOP-1 composite; TGA plot of the AgNPs/SugPOP-1 composite under air atmosphere; 1H NMR, 13C NMR, and MS spectra of new monomers are provided.IR spectra of the


HbNPs/AuE were characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) before and after immobilization of HbNPs. The HbNPs/AuE showed optimum response within 2.5 s at pH 6.5 in 0.1 M sodium phosphate buffer (PB) containing 100 μM H2O2 at 30°C, when operated at –0.2 V against Ag/AgCl. The HbNPs/AuE exhibited Vmax of 5.161 ± 0.1 μA cm−2 with apparent Michaelis-Menten constant (Km) of 0.1 ± 0.01 mM. The biosensor showed lower detection limit (1.0 μM), high sensitivity (129 ± 0.25 μA cm−2 mM−1) and wider linear range (1.0–1200 μM) for H2O2 as compared with earlier biosensors. The analytical recoveries of added H2O2 in serum (0.5 and 1.0 μM) were 97.77 and 98.01% respectively, within and between batch coefficients of variation (CV) were 3.16 and 3.36% respectively. There was a good correlation between sera H2O2 values obtained by standard enzymic colorimetric method and the present biosensor . The biosensor measured H2O2 level in sera of apparently healthy subjects and persons suffering from diabetes type II. The HbNPs/AuE lost 10% of its initial activity after 90 days of regular use, when stored dry at 4°C.The nanoparticles (NPs) of hemoglobin (Hb) were prepared by desolvation method and characterized by transmission electron microscopy (TEM), UV spectroscopy and Fourier-transform IR (FTIR) spectroscopy. An amperometric H Characterization of the cellular functions of H2O2 requires measurement of its concentration selectively in the presence of other oxygen metabolites with spatial and temporal fidelity in live cells . AuE offers many attractive features such as good conductor of electricity, low cost, low background current, and easy availability [2O2 biosensor based on covalent immobilization of HbNPs on to AuE, its characterization, evaluation, and application for amperometric determination of H2O2 in sera of diabetic patients.Hydrogen peroxide  , Hb/collet (NGP) , Hb/Ag set (NGP) , Hb/AuNP/Pt disk , Hb/coll/Pt disk , DNA–Hb//Pt disk . The varene (Fc) , self-asene (Fc) , graphenene (Fc) , HRP/polene (Fc) , electroene (Fc) , 3,3′,5,) [2NPs) , turnip ) [2NPs) , GPtNPs ) [2NPs) , PtRu/3D) [2NPs) , cytochrPANI)/Au . Howeverlability . The pre+, D-Glucose, uric acid, urea, glucose, ascorbic acid, potassium chloride, silica gel from SRL, Mumbai; sulphuric acid from Qualigens Fine Chemicals, Mumbai were used. Au wire (diameter: 2 mm) for the preparation of AuE was purchased from a local jewellery shop. Sera samples of apparently healthy subjects and persons suffering from diabetes type II were collected from local PGIMS hospital. All other chemicals were of analytical reagent (AR) grade. Double distilled water (DW) was used throughout the experimental studies.Hb, Tris buffer, NADPotentiostat/galvanostat  with a three electrode system consisting of Pt wire as an auxillary electrode, an Ag/AgCl electrode as a reference electrode and HbNPs immobilized on to polycrystalline Au as a working/enzyme electrode. SEM , UV spectrophotometer , X-ray diffractometer (XRD) , Fourier transform IR (FTIR) spectrometer  were used.g for 10 min at 4°C, followed by redispersion of HbNPs in 0.1 M phosphate buffer (PB) (pH 7.3) and sonication for 5 min. Moreover, it is assumed that α-NH2 group of cysteamine reacts with excess unreacted –CHO groups of glutaraldehyde cross-linked Hb molecules to form Schiff’s base. Thus, the glutaraldehyde cross-linked HbNPs get functionalized with –SH groups. These –SH functionalized HbNPs were stored at 4°C until use [The HbNPs were prepared by desolvation method. To the Hb solution (1 mg/ml), 5 ml of absolute ethanol was added dropwise at a rate of 0.1–0.2 ml/min under continuous stirring at a speed of 500 rpm. The desolvating agent encouraged the aggregation of protein molecules into small NPs by eliminating water molecules amongst them. In the same way, process was followed by addition of 1 ml of 1% glutaraldehyde solution into the reaction mixture. The mixture was kept under the similar stirring conditions at 4°C for 24 h to ensure complete cross-linking of HbNPs. Such a high concentration of glutaraldehyde is likely to provide intermolecular cross-linking of HbNPs molecules through Schiff’s base. Besides, above all the enzyme HbNPs thus formed were thiol functionalized by adding 0.02 g/ml cysteamine solution (0.02 g/ml) to HbNPs suspension under constant stirring for 5–6 h. Last, HbNPs were separated from the supernatant by centrifuging NPs suspension at 1200×ntil use .−1.HbNPs were characterized by recording their TEM images, UV and visible spectra and FTIR spectra. The size and shape of HbNPs were measured by a high-resolution transmission electron microscope, Zeiss EM 912, at an accelerating voltage of 120 kV. TEM samples of the HbNPs were prepared by placing the product solution on to the carbon-coated copper grids, allowing the solvent to evaporate in air. UV absorbance of HbNPs dispersed in 0.1 M sodium PB (pH:7.0) was recorded in the range from 200 to 600 nm at an interval of 50 nm, using UV spectrophotometer . FTIR spectra of the HbNPs were measured by FTIR spectrometer  using the standard KBr method over a range of 500–4000 cm2SO4 until the voltammogram characteristic of the clean polycrystalline Au was established. The polycrystalline AuE was placed in the HbNPs suspension under mild stirring at 4°C for 12 h to get covalent coupling between thiol-functionalized cross-linked HbNPs and polycrystalline AuE via Au-thiolated bond through dehydration process (Thiol groups on the surface of HbNPs provide a facile way for attachment of HbNPs on the surface of Au electrode (AuE). Before immobilization, the cleaned bare AuE was scanned over the 0–0.5 V range in freshly prepared 0.2 M H process . The HbN2O2 biosensor, HbNPs/Au, as working electrode, Ag/AgCl as reference electrode, and Pt wire as counter electrode were connected through potentiostat. Cyclic voltametry (CV) of working electrode was recorded in potentiostat–galvanostat at the potential range, –0.75 to –0.25 V in 25 ml of 0.1 M sodium PB (pH 6.5) containing 0.1 ml of 100 μM H2O2. The current (in mA) was measured at different times (in s).To construct the H2O2) concentration on biosensor response were studied. To determine the optimum pH, it was varied between 4.0 and 8.0 at an interval of 0.5 using the following buffer, each at a final concentration of 0.1 M: pH 4.0–5.0 sodium acetate buffers and pH 5.5–8 sodium PB. Similarly, to determine optimum temperature and incubation time, the reaction mixture was incubated at different temperatures (5–70°C) and time durations (1–10 s). The effect of H2O2 concentration on biosensor response was determined by varying the concentration of H2O2 in the range 1–1200 μM. The S.E.M. was calculated using the formula: S.E.M. = S.D./√n, where n is the of samples. Basically, it just tells the general variability of the points around their group means. The limit of detection (LOD) was calculated using the following formula: LOD =3σ/slope, where σ is S.D. The correlation coefficient (2R) was calculated by regression equation.To optimize the working conditions of the biosensor, effects of pH, incubation temperature, time of incubation, and substrate  prepared under optimal assay conditions of biosensor (Blood samples (1 ml each) were drawn from apparently healthy males and females (20 each), and persons with diagnosis of diabetes type II at Pt B.D.S. PGIMS, Rohtak Hospital and centrifuged at 2000 rpm for 10–15 min and their supernatant (serum) was collected. Hiosensor .−1. The strong absorption band at 1317 cm−1 may arise due to the stretching vibrations of C–N aromatic functional groups of protein. The medium absorption bands located at 1679 and 1553 cm−1 corresponds to carbonyl stretch and NH– stretch vibrations in the amide II linkage of the protein, respectively. The band at 1062 cm−1 indicates the presence of an amine group. In addition, there was a stretching located at 3497 cm−1, which could be assigned to the O H stretching vibrations, indicating the presence of hydroxyl groups. The FTIR spectrum of HbNPs showed distinct peaks at 1090, 1305, 1561, 1701, and 3488 cm−1  which controls the electron transfer kinetics of the redox probe at the electrode interface, while the linear part at lower frequencies corresponds to Warburg diffusion process. The Nyquist plot (3Fe(CN)6/K4Fe(CN)6. The RCT value for native Hb was 200 Ω which is lower than HbNPs/Au, i.e. 300 Ω. These results showed that increased RCT value of HbNPs/Au electrode was due to the immobilization of HbNPs on to Au surface. This increase in RCT is attributed to the fact that most of the biological molecules, including HbNPs, are poor electrical conductors at low frequencies  and cause hindrance to electron transfer.EIS provides useful information on impedance changes on the electrode surface during the fabrication process. It was carried out to investigate immobilization of HbNPs on to Au electrode. The diameter of the semicircle at higher frequencies corresponds to the electron transfer resistance  [p-ABSA/Pt disk (pH 7.0) [2NPs (5.0) [p-ABSA/Pt disk (25°C) [2O2 in the serum sample. Hence, all the subsequent electrochemical experiments were carried out in 0.1 M sodium PB, pH 6.5 at 30°C. There was a linear relationship between biosensor response and H2O2 concentration in the range 1–1200 μM, the response was constant after 1200 μM  , PtRu/3D (0.1 V) , Cytc/Ni (0.1 V) , GPtNPs  (0.1 V) .The bios (0.1 V)  but lowe (0.1 V) , Hb/coll (0.1 V) , Hb/Ag s (0.1 V) , Hb/AuNP(pH 7.0) , Hb/coll(pH 7.0) , DP-AuNP(pH 7.0) , GRCAPS/(pH 7.0) , ERGO/GC(pH 7.0) , TTP/SPC(pH 7.0) , PtRu/3D(pH 7.0)  and high(pH 7.0) , TMB/HRPPs (5.0)  and DNA–Ps (5.0) . The optPs (5.0) , Hb/AuNPk (25°C) , Hb/collk (25°C) , and lowk (25°C) , GRCAPS/k (25°C) . Figure  1200 μM d. The ap45.35 μM ). Smalle2O2 concentration in the range 1.0–1200 μM, which is better than earlier biosensors, 10–150 μM [2O2, showing the high sensitivity of biosensor. The average recoveries of H2O2 added in sera at levels of 0.5 and 1.0 μM were 97.77 and 98.01%, respectively (Supplementary Table S1), demonstrating the reliability of the present biosensor. The within and between batch coefficients of variation for determination of H2O2 in five sera samples on the same day and after 1 week of storage were 3.16 and 3.36%, respectively (Supplementary Table S2). These high precisions reveal the good reproducibility and consistency of the present method. The H2O2 level in sera of 20 apparently healthy subjects and persons suffering from diabetes type II, as measured by present biosensor were compared with those obtained by the standard Enzo kit method. There was a good correlation (2R =0.99) between these two methods  and the H0–150 μM , 5–30 μM0–150 μM , 0.21–310–150 μM , 1–25 μM0–150 μM   had practically no effect on biosensor response. This practically no interference could be attributed to functioning of biosensor at lower applied potential (–0.2 V). Hence, the present biosensor could be highly selective/specific for H2O2 content as measured by the present biosensor was in the range from 23.9 ± 0.04 to 45.6 ± 0.05 µM (n=20) for sera of apparently healthy persons, which is in normal established range and from 68.2 ± 0.05 to 143 ± 0.05 µM (n=20) for persons suffering from diabetes type II (Supplementary Table S3).The HThe HbNPs/Au electrode were tested every fifth day over a period of 90 days, while being stored dry at 4°C. 2O2. However, biosensor has some drawbacks such as lack of substrate specificity, requirement of expensive Au wire for construction of working electrode and cold temperature for its storage.The present biosensor could be advanced with respect to use in biomedicine or clinical settings by miniaturizing it into commercial/portable model, which could be used at the bedside of the patient. The miniaturization means designing of an electronic chip for current measuring device (potentiostat) and transformation of HbNPs/AuE into a microelectrode (like screen-printed electrode), which could be dipped into one drop of biological sample and an adopter to connect the miniaturized current device with the microelectrode. The current device needs to be battery operated and finally calibrated according to the concentration of H2O2 biosensor has resulted into its better analytical performance in terms of lower working potential (–0.2 V), which is similar to HRP/PAN-PNMThH (–0.2 V) [p-ABSA/Pt disk (0.1 V) [The covalent immobilization of HbNPs on to AuE in the construction of H(–0.2 V) , ERGO/GC(–0.2 V)  Hb/NGP ((–0.2 V) , but hig(–0.2 V)  Hb/Colla(–0.2 V) , Hb/Ag s(–0.2 V) , GRCAPS/(–0.2 V) , DNA–Hb/(–0.2 V) , and low(–0.2 V) , DP-AuNP(–0.2 V) , PtNPs/R(–0.2 V)  Hb/AuNPs (0.1 V) , PtRu/3D (0.1 V) , Cytc/Ni (0.1 V) , GPtNPs  (0.1 V) , LOD (0. (0.1 V) , 0.91 μM (0.1 V) , 0.4 μM  (0.1 V) , 0.37 μM (0.1 V) , 0.1 μM  (0.1 V) , 0.07 μM (0.1 V) , wider l (0.1 V) , 10–120  (0.1 V) , 5–30 μM (0.1 V) , 0.21–31 (0.1 V) , 1–25 μM (0.1 V) , rapid r


Abacavir and rilpivirine are alternative antiretroviral drugs for treatment-naïve HIV-infected patients. However, both drugs are only recommended for the patients who have pre-treatment HIV RNA <100,000 copies/mL. In resource-limited settings, pre-treatment HIV RNA is not routinely performed and not widely available. The aims of this study are to determine factors associated with pre-treatment HIV RNA <100,000 copies/mL and to construct a model to predict this outcome.HIV-infected adults enrolled in the TREAT Asia HIV Observational Database were eligible if they had an HIV RNA measurement documented at the time of ART initiation. The dataset was randomly split into a derivation data set (75% of patients) and a validation data set (25%). Factors associated with pre-treatment HIV RNA <100,000 copies/mL were evaluated by logistic regression adjusted for study site. A prediction model and prediction scores were created.3; and pre-treatment HIV RNA was 100,000  copies/mL. Factors associated with pre-treatment HIV RNA <100,000 copies/mL were age <30 years , body mass index >30 kg/m2 , anemia , CD4 count >350 cells/mm3 , total lymphocyte count >2000 cells/mm3 , and no prior AIDS-defining illness . Receiver-operator characteristic (ROC) analysis yielded area under the curve of 0.70 (95% CI 0.67–0.72) among derivation patients and 0.69 (95% CI 0.65–0.74) among validation patients. A cut off score >25 yielded the sensitivity of 46.7%, specificity of 79.1%, positive predictive value of 67.7%, and negative predictive value of 61.2% for prediction of pre-treatment HIV RNA <100,000 copies/mL among derivation patients.A total of 2592 patients were enrolled for the analysis. Median [interquartile range (IQR)] age was 35.8 (29.9–42.5) years; CD4 count was 147 (50–248) cells/mmA model prediction for pre-treatment HIV RNA <100,000 copies/mL produced an area under the ROC curve of 0.70. A larger sample size for prediction model development as well as for model validation is warranted. Antiretroviral therapy (ART) for the treatment of human immunodeficiency virus (HIV) infection has dramatically reduced HIV-associated morbidity and mortality and has transformed HIV infection into a manageable chronic condition , 2. FurtThe initial ARV regimen for a treatment-naïve HIV-infected patient generally consists of 2 nucleoside/nucleotide reverse transcriptase inhibitors, usually abacavir (ABC) plus lamivudine (3TC) or tenofovir disoproxil fumarate plus emtricitabine (TDF/FTC), plus a drug from 1 of 3 drug classes: an integrase strand transfer inhibitor, a non-nucleoside reverse transcriptase inhibitor (NNRTIs), or a boosted protease inhibitor , 5. ABC 3 .HIV-infected adults enrolled in TAHOD were eligible if they had an HIV RNA measurement documented at or around the time of ART initiation (pre-treatment HIV RNA). The window period of pre-treatment HIV RNA measurement was between 3 months prior to 1 day after the date of starting ART. ART was defined as a regimen containing ≥3 ARVs. Those exposed to mono or dual therapy prior to starting combination ART were excluded. Baseline was defined as the date of ART initiation. At baseline, co-variables included age, sex, HIV exposure, hepatitis B and C serology (ever positive), time since diagnosis of HIV infection, HIV subtype, and AIDS diagnosis prior to baseline. The window period of the following co-variables was between 3 months prior to 3 months after the date of ART initiation; body mass index (BMI), anemia , total lymphocyte count, CD4 count, CD8 count, CD4:CD8 ratio, and syphilis serology  age was 35.8 (29.9–42.5) years, 56.2% had heterosexual HIV exposure, median (IQR) BMI was 21.1 (19.0–23.4) kg/m>30 kg/m2 , anemia , total lymphocyte count >2000 cells/mm3 , and no prior AIDS-defining illness   1.10–1.80, p = 0.01], body mass index <30 years, +2.5 for BMI of 18.5–29.9 kg/m2 or +14.5 for BMI of >30 kg/m2, +7.0 for non-anemia, +17.0 for CD4 count >200 cells/mm3 or +5.5 for 100–199 cells/mm3, and +8.5 for no prior AIDS-defining illness. The possible maximum score was 50.5.Clinical prediction tool scores for pre-treatment HIV RNA <100,000 copies/mL are shown in Table AUROC analysis was 0.70 (95% CI 0.67–0.72) among the derivation patients Fig.  and 0.693, however these models did not perform better.A cut off total score >25 yielded sensitivity of 46.7 and 47.4%, specificity of 79.1 and 77.1%, positive predictive value of 67.7 and 64.2%, and negative predictive value of 61.2 and 63.0% for pre-treatment HIV RNA <100,000 copies/mL among the derivation patients and validation patients, respectively (Tables Plasma HIV RNA is one laboratory test used to stage HIV disease and to assist in the selection of ARV drug regimens , 5. If tTo our knowledge, this is the first study on prediction tool of pre-treatment HIV RNA <100,000 copies/mL in treatment-naïve HIV-infected patients that aims to facilitate the use of ABC and RPV as one of ARV in the first-line ART in resource-limited settings. We found some clinical and laboratory factors statistically significantly associated with pre-treatment HIV RNA <100,000 copies/mL. Our prediction tool of pre-treatment HIV RNA <100,000 copies/mL performed AUROC curve of 0.70. A cut off score >25 yielded the highest specificity of 79.0% for predicting pre-treatment HIV RNA <100,000 copies/mL.Few studies focus on the association between HIV RNA levels and HIV-related outcomes. The results from some previous studies showed that HIV RNA level is rarely directly associated with the type of opportunistic infection  or HIV d<30 years had higher odds of 1.4 of having pre-treatment HIV RNA <100,000 copies/mL compared to patients 41–50 years old. Furthermore, patients with baseline CD4 count 100–199 cells/mm3 had higher odds of 1.6 of having pre-treatment HIV RNA <100,000 copies/mL compared to patients with baseline CD4 count <100 cells/mm3. These factors might be easily applied in the assessment of patients in resource-limited settings because they are patients’ clinical characteristics and routine baseline laboratory investigations.We also found six independent factors associated with pre-treatment HIV RNA <100,000 copies/mL: age, BMI, anemia, CD4 count, total lymphocyte count, and prior AIDS-defining illness. For example, patients with age The AUROC curve is a single index for measuring the performance a test and can be used to estimate the discriminating power of a test. The AUROC of a ‘perfect’ test would be 1.00, that of a useless test, 0.50 , 17. TheThis study had some limitations. First, some patients must be excluded from the regression analysis and from the prediction tool due to missing data. Second, the performance of the model described by the AUROC of 0.70 might be associated with the small sample size of the study population among derivation and validation group.In conclusion, in situations where HIV RNA cannot be obtained prior to ART initiation due to high costs or limited availability, certain risk factors and models for predicting pre-treatment HIV RNA <100,000 copies/mL might be useful to predict pre-treatment HIV RNA and afford opportunities for ABC and RPV initiation among naïve HIV-infected patients. A larger sample size with greater data variety would be warranted for prediction model construction as well as for model validation. Pre-treatment HIV RNA should be performed before ABC and RPV initiation if it is available and affordable.


Pseudomonas stutzeri LH-42, a bacterium isolated from petroleum-contaminated soil in Liaoning Province, China. The bacterium contains a series of specific genes involved in the oxidation of organic sulfur compounds.We used Illumina HiSeq technology to sequence the whole genome of  Pseudomonas genus belongs to the gamma subgroup of Proteobacteria, and Pseudomonas stutzeri is a characterized heterotroph known to degrade several hydrocarbons , the components of which were as follows: 2.44 g/liter KH2PO4, 14.03 g/liter Na2HPO4·12H2O, 0.36 g/liter MgCl2·6H2O, 2.00 g/liter NH4Cl, 0.004 g/liter MnCl2·4H2O, 0.001 g/liter FeCl3·6H2O, 0.001 g/liter CaCl2, and 1.62 g/liter glycerol, and dibenzothiophene (DBT) was used as the sole electron donor (30°C at 170 rpm). The total genomic DNA of strain LH-42 was extracted using the TIANamp bacteria DNA kit , and the whole genome was sequenced using an Illumina HiSeq 2000 . The genome sequence assembly was performed using SOAPdenovo  were annotated as known proteins in the public database, 29%  were conserved hypothetical proteins, and 5% (220 CDSs) were hypothetical proteins.The draft genome of dsr), an adenylyl-sulfate reductase (APSR) gene, and a phosphoadenylyl-sulfate reductase (PAPSR) gene, but the DBT monooxygenase gene (dsz) was not detected, which indicated that the P. stutzeri LH-42 metabolized the organic sulfur in the Kodama metabolic pathway. A complete whole-genome analysis is in progress, with an expectation to reveal additional information on the catabolic potential of P. stutzeri LH-42.A number of genes potentially responsible for the oxidation of inorganic sulfur were identified: a dissimilatory sulfite reductase gene (NCKS00000000. The version described in this paper is the second version, NCKS02000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Background AZD4547 is a potent, oral, highly selective fibroblast growth factor receptor (FGFR) inhibitor in clinical development for treating tumours with a range of FGFR aberrations, including FGFR mutations, amplifications and fusions. Methods This open-label, Phase I, multicentre study (NCT01213160) evaluated the safety, pharmacokinetics, and preliminary antitumour efficacy (RECIST v1.1) of AZD4547 monotherapy in Japanese patients with advanced solid tumours. Part A was a dose-escalation part; Part B was a dose-expansion part in patients with FGFR-amplified tumours, confirmed by fluorescence in situ hybridization. Results Thirty patients enrolled in Part A , four in Part B (80 mg bid). No dose-limiting toxicities were observed and maximum tolerated dose was not determined. Most common adverse events  were: dysgeusia (50% of patients); stomatitis (41%); diarrhoea (38%); hyperphosphataemia (38%); dry mouth (35%). Common grade ≥3 AEs were nausea (12% of patients) and neutropenia (9%). No complete or partial responses were observed: 21/30 patients had stable disease ≥4 weeks in Part A, and 1/4 patients had stable disease ≥10 weeks in Part B. Following single and multiple dosing, absorption rate appeared moderate; peak plasma concentrations generally occurred 3–4 h post-dose, then declined biphasically with terminal half-life ~30 h. Steady state was reached by day 8. Compared with single dosing, plasma concentrations were, on average, 2.4- and 3.3- to 5.4-fold higher after qd and bid dosing, respectively. Conclusions AZD4547 was well tolerated in Japanese patients, with best response of stable disease ≥4 weeks. Fibroblast growth factor receptors (FGFRs) are transmembrane receptor tyrosine kinases with varied biological roles in regulating angiogenesis, cell proliferation, differentiation, migration and survival. Altered FGFR signalling has the potential to drive mitogenic, invasive, anti-apoptotic and pro-angiogenic cells and has been increasingly implicated in a range of solid tumour types, including breast cancer (BC), high-grade bladder cancer, non-small-cell lung cancer (NSCLC) and gastric cancer (GC), as well as haematological malignancies. Of the five known FGFRs found in humans, FGFR1–4 are characterized by extracellular immunoglobulin-like and intracellular tyrosine kinase domains, whereas the atypical FGFR5  lacks the cytoplasmic tyrosine kinase domain; consequently, its role is less understood. There are several mechanisms underlying the misregulation of FGFRs in neoplastic disease, including activating mutations in FGFRs , FGFR gen = 1; bladder cancer, n = 1; BC, n = 1)  and ECG) and ophthalmic assessments were also conducted.Blood samples for PK analysis were collected pre-dose and at defined intervals up to 96 h following single dosing, and up to 24 h following multiple dosing. Urine samples were also collected during the 24 h after multiple dosing in order to perform urinary PK assessments. For multiple dosing, the 80 mg bid dosing level was evaluated using combined data from patients in both Part A and Part B, as FGFR amplification status was unlikely to have a significant impact on PK. Concentrations of AZD4547 in human plasma and urine were determined using a validated high-performance liquid chromatography–tandem mass spectrometry method at PRA International . PK parameters were analysed by standard non-compartmental methods using WinNonlin software .Tumour assessments were performed according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) at baseline, on day 21 of the first treatment cycle, then every 6 weeks after the start of treatment for 12 weeks, and thereafter every 12 weeks (±1 week) until discontinuation of study treatment or withdrawal of consent.No formal hypothesis-led statistical analysis was performed. Safety, tolerability, PK data and efficacy were summarized using descriptive statistics. Analysis sets for safety and efficacy contained all patients who received ≥1 dose of AZD4547. PK analysis included all patients who provided blood samples.n = 16; female, n = 14) and four in Part B . All patients received at least one dose of AZD4547 and were evaluable for safety, PK, and efficacy analyses. A summary of patient characteristics is given in Table Between 5 November 2010 and 22 November 2012, 30 Japanese patients were enrolled in Part A of this study . Based on the emerging safety profile, the safety review committee authorized the initiation of schedule two, a once-daily dose, in a fourth cohort 160 mg qd). However, based on emerging data from the study in Western patients [0 mg qd. No DLTs were observed across any of the four cohorts examined and given the decision not to titrate beyond the once-daily schedule of 160 mg qd, the MTD was not determined for Japanese patients in this study. Instead, the recommended dose of 80 mg bid for assessment in Part B was determined based on safety data from Part A of this study alongside the data in Western patients [n = 3; 160 mg qd, n = 6), and death in 1/4 patients . All patients  in Part B discontinued AZD4547 following disease progression.All patients had discontinued the study by the data cut-off date (16 August 2013). The most common reasons for discontinuation in Part A were disease progression in 14/30 patients (46.7%), AEs in 9/30 patients , the most common CTCAE grade ≥3 AEs were neutropenia in three patients (8.8%), nausea in two (5.8%), and decreased appetite in three (8.8%). Three SAEs were experienced by two patients (6.7%) in Part A and one patient (25.0%) in Part B. All patients with SAEs required hospitalization. Only one SAE, decreased appetite and nausea, was deemed to be causally related to AZD4547 treatment . Two further SAEs, stomatitis  and decreased appetite , were not considered to be causally related to treatment with AZD4547. One death occurred during the study period (160 mg qd) following disease progression; the study investigators concluded that this death was not causally related to AZD4547 treatment.Overall, 32/34 patients (94.1%) experienced at least one AE following AZD4547 treatment (Table Nine patients (30.0%) in Part A reported dose interruptions following AEs, and 13/30 patients (43.3%) experienced dose reductions, of whom 12 (40.0%) had reductions following AEs, most commonly detachment of retinal pigment epithelium or other retinal disorders, as well as hyperphosphataemia and dizziness. One patient (25.0%) in Part B had a dose interruption as a result of an AE of decreased appetite, and two patients (50.0%) had dose reductions after reporting AEs of retinal detachment, nausea, and hypoglycaemia. The mean actual treatment duration was 80.1 days in Part A and 36.0 days in Part B. The mean relative dose intensity was 87% in Part A and 83.5% in Part B.In Part A, 9/30 patients (30.0%) had an AE leading to discontinuation of the study drug, and these AEs were considered causally related to the study drug by the investigator. None of the patients in Part B had AEs leading to discontinuation. Retinal events led to study-drug discontinuation in 7/34 patients (20.5%) and all patients recovered.Treatment-related increases in blood phosphate levels were observed in 11 patients (36.7%) in Part A and two patients (50.0%) in Part B, with median change in phosphate levels from baseline ranging from –0.16 to 0.79 mmol/L in the 80 mg bid cohort (combined from Parts A and B) to –0.29 to 0.72 mmol/L in the 160 mg qd cohort. Time to onset ranged from 9 to 24 days. All except one patient received treatment with fosrenol in accordance with the management guidelines for hyperphosphataemia and recovered. No clinically relevant changes in vital or physical signs were observed. One patient (120 mg bid) with a normal ECG at baseline experienced an abnormal ECG with AZD4547 treatment; however, this was not considered to be clinically relevant. Three patients experienced a decrease in left ventricular ejection fraction (LVEF) of ≥10 percentage points and three patients experienced an absolute LVEF value of <55%; however, no patients were reported to have fulfilled both criteria simultaneously and, consequently, these changes were not considered to be clinically relevant. Grade 1 and 2 decreases in platelet counts were observed in 13/30 patients (43.3%), and only in the 160 mg qd cohort. All other clinical laboratory observations were comparable between dosing levels. A trend in mean-value increase for transaminases and blood creatinine was observed, which consisted mainly of a one-grade shift.max) ranged from 2.9 to 4.0 h across the dose levels of 40–160 mg. After reaching maximum plasma concentration (Cmax), AZD4547 concentrations declined biphasically, with a mean terminal half-life  ranging from 22.4 (±7.21) to 33.5 (±7.49) h. The ratio of the area under the plasma concentration–time curve from time 0 to infinity (AUC) to that from time 0 to time of last measurable concentration (AUC0–t) was >0.87, indicating that the sampling scheme used had reliably captured the plasma concentration–time profiles. The percentage coefficient of variation (CV%) values for Cmax and AUC were 54.0–142% and 53.3–117%, respectively, across the dosing levels. Dose-normalized Cmax and AUC values were 9.58–21.4 ng/mL and 0.61–1.35 h · ng/mL, respectively, for the dosing levels tested. Mean (±SD) oral clearance (CL/F) ranged from 57.8 (±27.3) to 116 (±77.0) L/h and was independent of dose across the dosing range of 80–160 mg.Following single dosing, AZD4547 plasma levels were quantifiable across all investigated dosing levels. The mean plasma concentration–time profiles for single and multiple dosing are shown in Fig. ss,max was 23.0–65.9%, and the CV% range for AUCss was 10.5–61.0%. Dose-normalized values for Css,max and AUCss were 1.60–3.41 ng/mL and 11.4–29.7 h · ng/mL across the different multiple dosing levels. Median tmax ranged from 2.9 to 4.0 h post-dose, in line with the data from single dosing. Mean (±SD) CLss/F ranged from 37.9 (±19.9) to 87.9 (±9.37) L/h, which were lower than the CL/F values for single doses. The mean (±SD) accumulation ratio  was 3.34 (±1.98), 4.87 (±2.42), and 5.34 (±5.43), respectively, for 40 mg, 80 mg, and 120 mg bid dosing. The mean (±SD) value of RAC (ratio of multiple-dose AUCss to single-dose AUC0–24h) for the 160 mg qd cohort was 2.42 (±1.56). The mean (±SD) values of temporal change  were 1.37 (±1.01), 1.75 (±0.86), 1.61 (±1.41) and 1.37 (±0.88), respectively, for 40 mg, 80 mg, 120 mg bid and 160 mg qd dosing.AZD4547 plasma levels were quantifiable across all levels and time points for multiple dosing, and steady state (ss) was reached by day 8. The CV% range for CR) values were variable at 3.23 (±0.58), 1.25 (±0.43), 2.32 (±0.63) and 1.38 (±0.59) L/h, respectively, for 40 mg, 80 mg, 120 mg bid and 160 mg qd dosing, but showed no dose-dependency trend.Urinary PK data were available for 27 patients . The mean (±SD) steady-state fraction of the AZD4547 dose excreted in urine (fe) was 3.92% (±0.828%), 3.66% (±1.25%), 4.13% (±1.68%) and 3.78% (±2.48%) for 40 mg, 80 mg, 120 mg bid and 160 mg qd dosing, respectively, suggesting that urinary excretion of AZD4547 is dose independent. Mean (±SD) steady-state renal clearance  in Part A, with one patient  continuing to experience stable disease at 10 weeks in Part B. Except for one patient in Part A with a non-evaluable response, all remaining patients showed disease progression. Post-baseline target lesion measurements were available in 25/30 patients in Part A and all patients in Part B; the median percentage change in the sum of the diameters was 5.9% (range –6.8% to 48.9%) in Part A and 3.0% (range –16.3% to 23.4%) in Part B.Complete responses and partial responses, according to RECIST v1.1, were not observed; however, stable disease (≥4 weeks’ duration) was observed in 21 patients . Consistent with data from Western patients, the most common AEs in our study were gastrointestinal disorders, dryness, hyperphosphataemia, and eye disorders and included diarrhoea, stomatitis, dry mouth and skin, nausea, dysgeusia, and detachment of retinal pigment epithelium , 34, 35.1/2λz. Dose-normalized PK parameters for single and multiple doses were similar across all dosing levels; however, the small number of patients in each cohort and the variability between plasma concentration–time plots make dose proportionality difficult to establish. Tc tended to be close to or slightly higher than unity, suggesting that there were no notable time-dependent changes in PK upon multiple dosing. A relatively small proportion of AZD4547 was excreted in the urine unchanged (3.8–4.1% of the dose), suggesting that urinary excretion may be a minor route of AZD4547 elimination if AZD4547 absorption is good in humans. The results reported here are the first published PK data for AZD4547 in any patient population, and it would therefore be interesting to compare our PK findings with subsequent PK data that may emerge from the ongoing clinical development of AZD4547.The PK findings were generally consistent between AZD4547 single and multiple dosing. Following bid dosing in three cohorts  and qd dosing in one cohort (160 mg), steady state was achieved by day 8, and the accumulation ratio was consistent with the prediction from single-dose tThe best response following AZD4547 treatment in this study was stable disease for ≥4 weeks in 70% of patients, with one BC patient experiencing stable disease at 10 weeks. Previous efficacy data from the Western population showed a best response of PR (80 mg bid) for ≥12 weeks in one patient with FGFR1-amplified squamous NSCLC ; stable FGFR2 or FGFR3 translocations, and tumour types were reported as glioblastoma, UC, and endometrial cancer. Phase II studies of FGFR inhibitors are ongoing in different tumour types harbouring FGFR gene alterations. These include assessment of AZD4547 at the RD of 80 mg bid in FGFR2-amplified GC and FGFR1-amplified BC  [Efficacy data from clinical studies of other selective FGFR inhibitors have also been reported. In a Phase I study of BGJ398 (Novartis), PRs were observed in 2/17 evaluable patients with lung squamous cell carcinoma, with durations of 8 and 3 months . Publish1795768) , 41, BGJ1795768) , and JNJ1795768) . Selecti1795768) , 44.Further investigation is required to establish the treatment settings in which this new class of drugs can provide the most meaningful clinical benefit. The initial expectation was that selection of patients by screening for FGFR amplification may identify responsive patients; however, patients with FGFR gene amplification have responded inconsistently to FGFR inhibitors , 45. A kSeveral non-selective, multi-targeted TKIs have been licensed that can act as FGFR inhibitors, including pazopanib, lenvatinib, ponatinib, regorafenib, and nintedanib. In addition to FGFRs, these compounds have activity against a wide range of targets, including VEGFR1–3 and platelet-derived growth factor , 48, andAZD4547 was well tolerated in this Phase I study and no DLTs were reported. Based on safety data from Part A, and taking into account previous data in Western patients , the rec


BPD patients had lower P3 amplitudes and longer N2 latencies than HC, independent of WM load . Impulsiveness was not correlated with N2 latency or P3 amplitude, and no correlations were found between N2 latency or P3 amplitude and affect intensity scores in any WM load . In conclusion, the lower P3 amplitudes and longer N2 latencies in BPD patients suggested that they might have some dysfunction of neural activities in sub-processing in WM, while impulsiveness and negative affect might not have a close relationship with these deficits.Whereas some studies have demonstrated impaired working memory (WM) among patients with borderline personality disorder (BPD), these findings have not been consistent. Furthermore, there is a lack of neurophysiological evidence about WM function in patients with BPD. The goal of this study was to examine WM function in patients with BPD by using event-related potentials (ERPs). An additional goal was to explore whether characteristics of BPD  are associated with WM impairment. A modified version of the N-back task (0- and 2-back) was used to measure WM. ERPs were recorded in 22 BPD patients and 21 age-, handedness-, and sex-matched healthy controls (HCs) while they performed the WM task. The results revealed that there were no significant group differences for behavioral variables (reaction time and accuracy rate) or for latencies and amplitudes of P1 and N1 (all  Borderline personality disorder (BPD) is a serious mental disorder that is characterized by a pervasive pattern of instabilities in affect regulation, impulse control, interpersonal relationships, and self-image  measure electrical brain activity that is time-locked to sensory or cognitive events. ERPs elucidate the time course of ongoing brain activity during WM tasks and reflect the spatiotemporal sequence of cortical information processing  criteria . All subjects in the control group were interviewed by two professional psychiatrists to exclude All subjects had normal or corrected-to-normal vision. This study was carried out in accordance with the recommendations of “ethics committee of Central South University”. The protocol was approved by the ethics committee of  Central South University. Every subject signed an informed consent form in accordance with the Declaration of Helsinki. In the case of BPD patients, this consent form was also signed by a well-informed relative.The CES-D was used to assess depressive levels of participants. This scale consists of 20 items, each of which is assigned a value from 1 to 4. Four items are positively worded and reverse-scored. The total score is computed by summing the 20 items, such that the range of scores is 20–80  to 4 . Items are summed to determine the overall impulsiveness score, with higher scores indicating greater impulsivity  to 5 (very often true). There are five questions for each type of trauma, scored 5–25, with an additional three questions to assess minimization/denial .As participants completed the N-back task, continuous EEG signals were acquired with a 64 channel electroencephalographeic system . Electrodes were placed by using a 10/20 extended QuikCap system . References were placed at a vertex by default and off-line re-referenced to averaged mastoids. Impedanc values were kept lower than 5 KΩ for all electrodes. Horizontal electrooculograms (EOGs) were recorded with electrodes placed on the bilateral external canthi. Vertical EOGs were recorded from electrodes placed above and below the left eye. EEG data were sampled at 1000 Hz and analyzed offline with a 30-Hz low pass filter. Trials with undesired eye movements and eye blink artifacts were removed from analysis by a semiautomatic and manual block rejection procedure.The continuous EEG was subsequently segmented beginning at 200 ms before stimulus onset and lasting for 800 ms. The baseline for ERP analysis was 100 ms before appearance of the target stimuli. Individual segments were excluded if the absolute voltage of each channel exceeded 100 μV. In each subject, artifact-free trials were averaged for each task (0-back/2-back) to obtain the corresponding ERP waves. Subjects with less than 30 epochs for each task were excluded. All analyses were performed by using Scan 4.3 and Curry 7.0 .Three electrode positions  were chosen for statistical analyses of N2 and P3. Occipital electrodes  were selected for P1 and N1 because these components are usually maximal at these electrodes. ERP waves were analyzed in terms of peak latency and baseline-to-peak amplitude, as determined by visual inspection. Latency ranges for potentials were designated as follows: 50–150 ms for P1, 140–200 ms for N1, 200–400 ms for N2, and 250–500 ms for P3.t-test. Reaction time and accuracy data were analyzed by 2 (group: BPD vs. control) × 2 (WM load: 0-back vs. 2-back) repeated-measures ANOVA with WM load as a within-subject factor and group as a between-subject factor. ERP components (latency and amplitude) were analyzed by 2 ) × 2 (WM load: 0-back vs. 2-back) × 3  repeated-measures ANOVA with WM load and electrodes as within-subject factors, and with group as a between-subject factor. Greenhouse–Geisser was used to correct compound symmetry violations in the ANOVAs. Correlations between psychological measures (impulsiveness and different affect intensity) and ERP components were calculated by Pearson’s correlation. Where appropriate, Cohen’s d and η2 were calculated as indices of effect size.We used SPSS 17.0 to analyze all data. Demographic data of participants were analyzed by using a Chi-Square or t = 3.94, p < 0.001) and depression  were obtained for BPD patients compared to HCs. BPD patients had higher scores on CTQ subscales of emotional abuse , emotional neglect , and physical neglect , as well as on the SAIS subscale of negative intensity .Clinical data are reported in Table F = 0.001, p = 0.979), no significant effects of WM load  = 0.79, p = 0.380), and no significant Group × WM load interaction effects  = 0.22, p = 0.644). However, WM load had a significant effect on accuracy  = 312.38, p < 0.001, η2 = 0.88). Low WM load had a higher accuracy rate (97.7% ± 3.5%) than high WM load (60.9% ± 12.3%), but no significant group differences  = 0.21, p = 0.652) or significant main Group × WM load interactions  = 0.04, p = 0.848) were observed on accuracy.Mean reaction time and accuracy data for both groups under each condition are reported in Table Mean amplitudes and latencies of P1, N1, N2 and P3 for each WM load are shown for both groups in Table F = 18.38, p < 0.001, η2 = 0.31). P1 amplitude was more positive under low WM load (4.16 ± 3.15 μV) compared to high WM load (2.67 ± 2.56 μV). The effect of electrodes was also significant  = 14.06, p < 0.001, η2 = 0.26), the amplitude of Oz was the minimum (2.61 ± 2.49 μV). The group difference in P1 amplitude, interactions of WM load × Electrodes, WM load × Group, Electrodes × Group, and WM load × Group × Electrodes were not significant .The main effect of WM load in P1 amplitude was significant  = 10.52, p < 0.001, η2 = 0.20) and interaction of WM load × Electrodes  = 8.58, p < 0.001, η2 = 0.17). Further analysis revealed that O2 had a shorter latency (102.20 ± 14.25 ms) compared to O1  or Oz  under low WM load. Oz had a shorter latency (102.31 ± 20.22 ms) compared to O1  or O2  under high WM load. The group difference, main effect of WM load and remaining interactions were not significant .The repeated-measures ANOVA on P1 latency showed significant main effect of electrodes  = 27.03, p < 0.001, η2 = 0.40), electrodes  = 12.18, p < 0.001, η2 = 0.23). The interaction of WM load × Electrodes was also significant  = 5.47, p = 0.011, η2 = 0.12), post hoc analysis showed that the N1 amplitude was smallest in O1 under both low (−2.58 ± 3.47 μV) and high (−4.19 ± 3.39 μV) WM load. The group difference and the remaining interactions were not significant .The ANOVA conducted on N1 amplitude revealed significant main effects of WM load  = 4.66, p = 0.015, η2 = 0.10). The N1 latency of Oz (149.26 ± 23.31 ms) was shorter than the N1 latency of O1  or O2 , but there was no significant difference between O1 and O2 (p = 0.572). None of the group difference, main effect of WM load and remaining interactions were significant .Electrodes exhibited a main effect on N1 latency  = 30.54, p < 0.001, η2 = 0.43) and electrodes  = 10.47, p = 0.001, η2 = 0.20). The interaction of WM load × Electrodes  = 12.12, p < 0.001, η2 = 0.23) was also significant, N2 amplitude was smaller for Pz (−3.35 ± 4.35 μV) than for Fz  or Cz  under low load, while the amplitude of Fz (−3.13 ± 2.77 μV) was larger than that of Pz  or Cz  under high WM load. The group difference and other interactions were not significant .There were significant main effects of WM load  = 4.33, p = 0.044, η2 = 0.10). BPD patients had longer latencies (257.34 ± 20.03 ms) than HC (244.66 ± 19.91 ms). The main effect of electrodes  = 23.97, p < 0.001, η2 = 0.37) and the Group × Electrode interaction  = 3.95, p = 0.034, η2 = 0.09) were also significant, indicating that BPD patients had a longer latency (249.64 ± 35.28 ms) than HC  in the Pz electrode only. The main effect of WM load and other interactions were not significant .A significant group difference was found in N2 latency  = 5.62, p = 0.023, η2 = 0.12), BPD patients had a smaller P3 amplitude (5.03 ± 2.61 μV) compared to controls (7.24 ± 3.46 μV). WM load  = 66.46, p < 0.001, η2 = 0.62), electrodes  = 41.71, p < 0.001, η2 = 0.50) and WM load × Electrodes interaction  = 20.68, p < 0.001, η2 = 0.33) all had significant effects. Post hoc analysis showed that P3 amplitude was significant larger for Pz (10.92 ± 5.18 μV) compared to Fz  or Cz  under low WM load, the P3 amplitude for Fz (2.85 ± 3.19 μV) was smaller than for Cz  or Pz  under high WM load. The remaining interactions were not significant .Significant group difference was found in P3 amplitude  = 6.98, p = 0.004, η2 = 0.15). Fz had a longer latency (372.37 ± 27.46 ms) than Cz  or Pz . Furthermore, Cz had a longer latency than Pz (p = 0.033). There was no significant group difference or main effect of WM load, as well as no significant interactions for WM load × Group, Electrodes × Group, WM load × Electrodes, or WM load × Electrodes × Group .Electrodes significantly affected P3 latency  or 2-back task .Pearson’s correlation analyses showed that N2 latency was not significantly correlated with P3 amplitude in the 0-back task (p > 0.05).Pearson’s correlation analyses showed that impulsiveness, as detected by the BIS, was not correlated with N2 latency under any WM load. There were no significant correlations between N2 latency and affect intensity scores, or between P3 amplitude and impulsiveness or affect intensity scores  and impulsiveness or negative affect intensity, we still could not conclude that the abnormalities of ERPs during WM tasks are independent of the symptoms of BPD, just as Fertuck et al.  concludeThis study has several limitations. First, although there is high temporal resolution, the poor spatial resolution of ERPs makes it difficult to identify which brain regions are related to the impairment of WM in BPD patients. Second, no patients had taken medicine in this cross-sectional study. Therefore, it is unclear whether medicine or psychological treatments would reduce impairment of WM. Further studies tracking the performance of BPD patients after taking medicine would help us to identify which impairment of WM are related to symptoms of BPD. Third, previous research suggests there are sex differences in BPD (Hoertel et al., YL was responsible for analyzing data and writing the manuscript. MZ designed the experiment and wrote the manuscript with YL. CX, XJ, XZ and SY were responsible for data collection. JY was responsible for designing the study and revising the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this work, experimental evidences were achieved by analyzing Mg doped high Al-content AlGaN alloys and Mg doped AlGaN SLs as well as MgGa δ doped AlGaN SLs. Mg acceptor activation energy was significantly reduced from 0.378 to 0.331 eV by using MgGa δ doping in SLs instead of traditional doping in alloys. This new process was confirmed to be able to realize high p-type doping in high Al-content AlGaN.P-type doping in high Al-content AlGaN alloys is a main challenge for realizing AlGaN-based deep ultraviolet optoelectronics devices. According to the first-principles calculations, Mg activation energy may be reduced so that a high hole concentration can be obtained by introducing nanoscale (AlN) A), the compensation by nitrogen vacancies, the increased hole scattering, and the limited acceptor solubility10A in AlxGa1−xN increases monotonically with increasing Al-content from 0.17 eV in GaN to 0.51 eV in AlN11−9) of Mg dopants can be activated in AlN at room temperature 11. Therefore, decreasing Mg acceptor activation energy is one of the most challenges in AlGaN-based DUV optoelectronic devices.High Al-content AlGaN alloys are ideal materials for deep ultraviolet (DUV)23468Great efforts have been devoted to improve p-type conduction in group-III nitrides131415161718191315161718192021Ga δ doping in Al0.6Ga0.4N alloys to increase the n-type carrier densitym/(GaN)n (m > n) SL could convert the valence-band maximum (VBM) from the crystal-field split-off hole to heavy hole band, leading to the increase of the transverse electric (TE) polarized light emission efficiency315/(GaN)1 SL substitution for Al0.83Ga0.17N disorder alloy was theoretically investigated by Zhong et al.A decreases if the NN Ga atom number increased and the Mg-centered tetrahedron volume decreased. In this way, the Mg acceptor activation energy can significantly be reduced to 0.26 eV, very close to that of GaN, in (AlN)5/(GaN)1 SL by MgGa δ-doping0.63Ga0.37N/Al0.51Ga0.49N SLsGa δ doping in (AlN)m/(GaN)n SLs to study Mg acceptor activation energy, aiming to find a proper way to minimize it in high Al-content AlGaN.Many works were concentrated on the doping in the superlattices (SLs)232425262728Ga δ doped AlGaN SLs were grown on c-plane sapphire substrates by using the radio-frequency plasma-assisted molecular beam epitaxy system . The growth details are shown in Ga δ doped AlGaN SLs with a cycle period of 15 s for 180 cycles. Although the cycle period of sample D5 and D6 was 15 s, the effective growth time in a cycle was still kept as 10 s and the total effective growth time was also 30 min. For sample D5, the growth process consisted of two loops as shown in 2 in area. Current versus voltage (I-V) characteristics were measured by using a semiconductor device analyzer .In this work, traditional Mg doped AlGaN alloys , Mg doped AlGaN SLs and Mg2O3 (0006) peaks were normalized at 41.700°. The full widths at half maximum (FWHM) of the Mg-doped AlGaN (0002) peaks were around 800 arcsec in alloys and 1000 arcsec in SLs. AlGaN peaks were fitted with Gauss model to get more accurate peak information. According to the alloy crystal parameter formula hklsinθ = nλ as well as hexagonal interplanar distance formula xGa1−xN thin films were determined by using standard crystal parameter cGaN of 0.5189 nm and cAIN of 0.4981 nm. In this way, the compositions of all samples from D1 to D6 were Al0.97Ga0.03N, Al0.75Ga0.25N, Al0.79Ga0.21N, Al0.53Ga0.47N, Al0.76Ga0.24N and Al0.47Ga0.53N, respectively. The compositions of (AlN)m/(GaN)n in D3 and D5 nearly matched to the designed value of 4:1, while those in D4 and D6 nearly approached to the designed 1:1.m/(GaN)n SLs were confirmed by HRTEM results. 4/(GaN)1 SLs, indicating that the monolayer SL structure was achieved and NN Ga atom number increased in sample D5. The one bisection spot might be caused by the dislocations. Hence sample D5 was measured to be (AlN)4/(GaN)1 superlattices, achieving the designed (AlN)m/(GaN)n SLs structure with increased NN Ga atom number.As XRD results reveal only the macro compositions of the AlGaN films, the (AlN)ρ) versus temperature (T). Since A) of Mg in AlxGa1−xN in sample D5 was fitted to be 0.331 eV as shown in A was calculated separately under positive and negative voltages and then made of an average. The EA for samples D1-D6 were 0.386, 0.378, 0.358, 0.344, 0.331, and 0.311 eV, respectively. The highest EA as 0.386 eV was in sample D1 and the lowest EA as 0.311 eV lied in sample D6, in match with calculated results that high EA in high Al component alloys, and low EA in low Al component SLs.A on Al composition in AlGaN with three doping methods. Obviously, the EA reaches the lower values in MgGa δ doped AlGaN SLs, the medium in Mg doped SLs and the higher in traditional Mg doped AlGaN alloys. As for Al content around 0.8, the EA decreases from 0.378 to 0.358, then to 0.331 eV by the three methods in turn as Mg doped AlGaN alloys, Mg doped SLs and MgGa δ doped SLs. Hence, Mg acceptor activation energy can be significantly reduced from 0.378 to 0.331 eV by using MgGa δ doping in (AlN)4/(GaN)1 SLs instead of traditional Mg doping in Al0.8Ga0.2N alloys. The difference between the theoretical value (0.26 eV) and the experimental one (0.331 eV) is attributed to two reasons: firstly, the macro function of Mg activation energy in Ga and Al was measured in this experiment while only the Mg activation energy in Ga was calculated to be 0.26 eV; secondly, the as-grown (AlN)4/(GaN)1 SLs were not perfect single crystal with dislocations which might affect the Mg activation energy. Therefore, we have experimentally proved the theoretical prediction that Mg acceptor activation energy can be significantly decreased in (AlN)m/(GaN)n SLGa δ doping in (AlN)4/(GaN)1 SLs instead of traditional Mg doping in Al0.8Ga0.2N alloys. Our experimental study verifies the prediction of the first-principles calculationsIn conclusion, we have systematically studied Mg doping in high Al-content AlGaN by using different doping methods. For high Al-content AlGaN, Mg acceptor activation energy can be significantly reduced from 0.378 to 0.331 eV by using MgHow to cite this article: Wang, X. et al. Experimental evidences for reducing Mg activation energy in high Al-content AlGaN alloy by MgGa δ doping in (AlN)m/(GaN)n superlattice. Sci. Rep.7, 44223; doi: 10.1038/srep44223 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Using ACE inhibitory screenings, two penta-peptides, KTCGY and KRIHF, were selected for KTCGY, KRIHF, and captopril were shown to have similar vasodilating effects against phenylephrine (PE)-induced tensions in rat endothelium-dependent thoracic aortic rings, however, KTCGYKTCGY (two-repeated KTCGY) and TCGYTCGY (two-repeated TCGY) were showed endothelium-independent vasodilating effects against PE-induced tensions. KTCGY, KRIHF (10 or 20 mg/kg), and captopril (10 mg/kg) were used to evaluate antihypertensive activity during 24-h after a single oral administration to spontaneously hypertensive rats (SHRs). The KTCGY and KRIHF showed significantly different and reduced the systolic blood pressure of SHRs compared to the blank.in vivo.These results suggest that KTCGY and KRIHF may contribute important roles in yam dioscorin for regulating blood pressure The online version of this article (doi:10.1186/s40529-014-0049-3) contains supplementary material, which is available to authorized users. Dioscorea spp., Dioscoreaceae) are an important tuber crop in Africa, Asia, and Middle and South America and are also a staple food in the Caribbean. Yam dioscorin and its peptic hydrolysates have been reported to exhibit several biological activities in vitro and in vivo , includu et al. ; Liu et u et al. ; Han et u et al. , 2014a]Dioscoreau et al. ; Lin et u et al. ; Liu et u et al. ), immunou et al. ; Liu et u et al. ), and enu et al. , [b]; Hou et al. ).50 = 0.4 μM) and ERKIKVYL (IC50 = 1.2 μM) were potent ACE inhibitors, but none of them were effective in spontaneously hypertensive rats (SHRs) to reduce the blood pressure. These potential ACE inhibitory peptides were further hydrolyzed by the rat’s gastrointestinal proteases and then lose their antihypertensive effects on SHR in vivo. We reported that yam dioscorin and its peptic hydrolysates exhibited ACE inhibitory activity  which may contribute important roles in yam dioscorin for regulating blood pressure in vivo.The untreated high blood pressure is considered to be the central factor in stroke which accounts approximately 33% deaths (Mark and Davis ). There u et al. ) and antu et al. ) using Sex vivo and in vivo experiments used in this study  were synthesized by Shanghai Hanhong Chemical Co., Ltd  or Shanghai Mocell Biotech Co., Ltd . The purity of each peptide was determined to be greater than 97% by performing HPLC chromatography and mass spectroscopy. Captopril was purchased from Calbiochem Co. . N-(3-[2-furyl]acryloyl]-Phe-Gly-G ly (FAPGG), ACE , dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. . Other chemicals and reagents were from Sigma Chemical Co. .The 23 peptides for ACE inhibitory activity screenings were synthesized by Mission Biotech. . Peptides for http://www.expasy.org/proteomics) to participate in a computer-aided simulation of pepsin hydrolysis (pH > 2) (http://web.expasy.org/peptide_mass/). Twenty-three peptides were selected for syntheses following the general rules which the peptide contained aromatic or branched-chain aliphatic amino acids closed to the COOH-terminus for ACE inhibitions  and dioscorin B (UniProtKB/TrEMBL:Q9M501) were selected from the website of the ExPASy SIB Bioinformatics Resource Portal , includblank/min). The decreased absorbance at 345 nm (ΔAsample/min) was recorded during 90 sec at room temperature. The ACE inhibition (%) was calculated as followed: [1 - (ΔAsample/min ÷ ΔAblank/min)] × 100.The ACE inhibitory activity was measured following the previous reports with some modifications . Each s2, 1 mM MgSO4, 27 mM NaHCO3, 1 mM KH2PO4, 10 mM glucose, pH 7.4, with 95% O2, 5% CO2) and gently dissected free of fat and connective tissue. The endothelium was removed by gently rubbing the intimal layer of the vessel with a wire. The isolated aortic rings, 3 to 4 mm in length with or without the endothelium, were fixed isometrically in organ chambers under passive tension (1.8 g) for 60 min. Changes in vascular tension were recorded with a polygraph  via a force displacement transducer . After equilibration, the near maximal concentration was induced by PE (0.3 μM). When the rings achieved a stable tension, the acetylcholine (3 μM) was added to the bath to assess endothelial integrity. The relaxing effects of KTCGY, KTCGYKTCGY, TCGYTCGY, KRIHF, and captopril on PE (0.3 μM) pre-contracted endothelium-intact or endothelium-denuded aortic rings were examined. When PE treatments reached a steady plateau (considered as 100%), KTCGY, KTCGYKTCGY, TCGYTCGY, KRIHF, and captopril were then tested at cumulative concentrations (0.1 to 100 μM) for their ability to induce aortic ring relaxations.Effects of four peptides and captopril on vascular tension were examined following the previous method . Male SExperimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Taipei Medical University . Male SHRs  were housed individually in steel cages kept at 24°C under a 12-h light–dark cycle, with free access to water and a standard mouse/rat chow . SHRs were randomly divided into 4 groups (N = 6 of each group), KTCGY or KRIHF at concentration of 10 mg/kg and 20 mg/kg was orally administered to the SHRs, and the SBP were measured after 0, 2, 4, 6, 8, and 24 h by using an indirect tail-cuff blood pressure meter . Distilled water (0.5 ml) was administered to the SHRs in the blank group. The captopril (10 mg/kg) was used as the positive control.t-test, and the P-value of less than 0.05 (*), 0.01 (**), and 0.001 (***) were recognized as different significantly. The statistical analysis was performed using the SigmaPlot software 10.0.Data were expressed as mean ± S.D. For animal experiments, the differences between the blank and the experimental group at the same time was analyzed using Student’s ex vivo and in vivo antihypertensive experiments. Table The deduced sequences of dioscorin A (UniProtKB/TrEMBL:Q9M519) were selected for computer-aided simulation of pepsin hydrolysis (pH > 2). There were sixty-four peptide fragments and seven amino acid residues  were released from dioscorin A in a computer-aided simulation hydrolysis  and TCGYTCGY (two-repeated TCGY) were also synthesized for s Figure B and C. P < 0.01) and 8-h (P < 0.01) for 10 mg/kg and at 2-h (P < 0.05), 4-h (P < 0.001), 6-h (P < 0.001), 8-h (P < 0.001) for 20 mg/kg. The average reduced SBP for 10 mg/kg at 6-h and 8-h was 24.17 and 11.93 mmHg, respectively; the average reduced SBP for 20 mg/kg at 2-h, 4-h, 6-h, and 8-h was 24.20, 26.24, 31.03, and 21.40 mmHg, respectively. The positive control of captopril (10 mg/kg) showed antihypertensive activity by lowing SBP and showed significantly different compared to the blank at 2-h (P < 0.001), 4-h (P < 0.01), 6-h (P < 0.001), 8-h (P < 0.01), and 24-h (P < 0.05). The average reduced SBP for captopril (10 mg/kg) at 2-h, 4-h, 6-h, 8-h, and 24-h was 26.85, 24.97, 29.82, 24, and 13.80 mmHg, respectively. It was noted that KTCGY at 20 mg/kg showed similar antihypertensive activity to captopril at 10 mg/kg. Figure P < 0.05), 4-h (P < 0.01), 6-h (P < 0.01) and 8-h (P < 0.01) for 10 mg/kg and at 2-h (P < 0.01), 4-h (P < 0.05), 6-h (P < 0.01), 8-h (P < 0.01) for 20 mg/kg. The KRIHF at dose of 10 and 20 mg/kg showed similar antihypertensive activities toward SHRs. The average reduced SBP for 10 mg/kg at 2-h, 4-h, 6-h, and 8-h was 17.30, 21.95, 21.47, and 16.65 mmHg, respectively; the average reduced SBP for 20 mg/kg at 2-h, 4-h, 6-h, and 8-h was 18.57, 19.64, 20.87, and 8.99 mmHg, respectively.The KTCGY or KRIHF (10 or 20 mg/kg) was orally administered once and the SBP was measured at 2-, 4-, 6-, 8-, and 24-h, and the captopril (10 mg/kg) was used for comparisons. Figure ex vivo and in vivo data showed KTCGY and KRIHF with vasodilating effects against PE-induced endothelium-intact aortic tensions and lowering SHR’s SBP which were derived from a computer-aided simulation of pepsin hydrolysis of the yam tuber dioscorin. The penta-peptide of KTCGY was positioned at amino acid residue 52 to residue 56 of dioscorin B protein , and the penta-peptide of KRIHF was positioned at amino acid residue 118 to residue 122 of dioscorin A protein  and dioscorin B protein . Conlan et al. . The pru et al. ). It seeex vivo and in vivo experiments. KTCGY and KRIHF showed endothelium-dependent aortic ring relaxations in PE-induced tensions  . Theref2+ influx  isolated from peptic hydrolysates of egg ovalbumin showed bradykinin B1 receptor antagonist and exhibited endothelium-independent canine mesenteric artery relaxations in prostaglandin F2α-induced tensions . Recenta et al. ), which g et al. ), and thi et al. ). The HRa et al. ). Therefin vivo SHR oral administration, both penta-peptides of KTCGY and KRIHF at doses of 10 and 20 mg/kg exhibited antihypertensive activities by lowering SBP, but not diastolic blood pressure (data not shown), among which KTCGY of 20 mg/kg exhibited the similar lowering SBP profile to captopril of 10 mg/kg after a single oral administration. However, the vaso-relaxing peptide with antihypertensive activity is not necessary for potent ACE inhibition. Therefore, it might loss some antihypertensive peptides from ACE inhibitory screenings in the present study. The RF di-peptide  and IHRi et al. ) isolatein vivo and will be beneficial for anti-hypertension in functional food preparations.The KTCGY and KRIHF show vasodilating effects and can reduce SHR’s systolic blood pressure which may contribute important roles in yam dioscorin for regulating blood pressure Figure S2. The computer-aided simulation of pepsin hydrolysis of yam dioscorin B (Q9M501). (DOCX 905 KB)Additional file 1: Figure S1.: The computer-aided simulation of pepsin hydrolysis of yam dioscorin A (Q9M519). Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Below are the links to the authors’ original submitted files for images.