SEM/TEM and XPS analysis indicated that the carbonaceous materials were mostly coated on the TiO2 surface and clearly revealed that the Cu species exhibited multivalence states, existing as CuOx (0≤x≤2). The optimal catalyst showed a 56-fold enhanced hydrogen evolution rate compared with that of the pure C/TiO2 catalyst. Further, an intensive multiple electron transfer effect originating from CuOx and the carbonaceous materials is proposed to be responsible for the elevated photoactivity. CuOx species serve as electron donors facilitating charge carrier transfer and proton reduction sites. The carbonaceous materials function as the “bridge” that transfers the electrons of TiO2 to the CuOx species, which provides a new route for electron transfer and reinforces the effect of CuOx as a co-catalyst. In this study, the CuOx and C co-modified TiO2 catalyst was prepared with multiple electron transport pathways and enhanced hydrogen production evolution, which provides a deep understanding for the design of co-catalyst-based photocatalysts.Titanium dioxide nanoparticles co-modified with CuO Hencsistance , 6. Howeications . It is a2, increasing the hydrogen evolution rate significantly. Noble metals can accept electrons from a semiconductor due to its high work function, which facilitates electron-hole separation. Their high-activity surfaces can act as active sites for hydrogen evolution. However, noble metals are hardly employed for practical applications due to their scarcity and valuableness.Co-catalysts –10 have 2 evolution. Among them, copper is one of the most promising candidates [2 modified with Cu and other ions (iron(III), nickel(II) and chromium(III) ions), revealing that metal ions can serve as both electron acceptors and donors based on a double-beam photoacoustic spectroscopy measurement. Characterization experiments and theoretical calculations confirmed this conclusion [2 in which transition metal ions can function as a trap for excited electrons to decrease recombination. The authors believed that Cu(I) can be formed by the photoexcited electron reduction of Cu(II), and Cu(II)-grafted samples are capable of reducing O2 to O2∙-. The multivalent states of Cu are regarded as the active species for the photoreaction. Our group fabricated Cu(II) nanodot doped nanosheet TiO2 in a previous report [2 evolution increased 25-fold due to the multiple charge-transfer pathways. Here, we introduced another species co-modified with Cu and managed to raise the hydrogen quantity.As an alternative, transition metals  –15 have ndidates –18. Ohnondidates  preparednclusion . In addinclusion  proposeds report , in whic2 evolution rate was observed. The authors revealed that graphene can facilitate electron transfer by accepting photoelectrons as well as functioning as active sites for the photocatalytic reaction. A similar mechanism is also applicable to CNT-TiO2 composites [Carbonaceous materials, such as carbon nanotubes (CNTs)  and grapmposites .2 co-modified with transition metals and nonmetal carbonaceous materials is a new route to facilitate the charge carrier separation and increase photoactivity. Zhang et al. [2/graphene photocatalyst for efficient phenol degradation, in which graphene provides a new electron transfer route and enhances the co-catalyst effect for superoxide radical generation.TiOg et al.  preparedx-C/TiO2 catalyst via a facile hydrolysis and photo-reduction method for photocatalytic hydrogen generation. By controlling the amount of water, the titanium precursor can be hydrolyzed completely. Thus, the carbonaceous materials originated from the calcination process without an external carbon precursor. The formation of carbonaceous materials and Cu species were systematically explored. The results demonstrated that carbonaceous materials serve as electron conductors to enhance the co-catalyst effect of CuOx, inducing multiple electron transfer pathways for photocatalytic hydrogen generation.Inspired by the above achievements, we have prepared a foam-like CuO4), copper chloride (CuCl2.2H2O), ethanol (C2H5OH), and glycerol (C3H8O3) were purchased from Sinopharm Chemical Reagent Co., Ltd, China. All reagents were analytical grade and used without further purification. Deionized water was used for all experiments.Tetrabutyl titanate (Ti(OBu)2 (CT) photocatalysts were synthesized by a facile sol-gel method. Typically, 5.5 mL of deionized water was added to 25 mL of tetrabutyl titanate (Ti(OBu)4) under magnetic stirring for 48 h. The mixture was filtered by a Buchaer funnel and dried in an oven at 80°C overnight. The prepared catalyst was denoted as CT. Then, CT samples were calcined at 350°C, 400°C, 500°C and 550°C for 2 h, respectively. The corresponding dark brown powders were denoted as CTR .C/TiOx-C/TiO2 was prepared according to a previously published photo-reduction method[2 aqueous solution (0.01mol/L). The mixture was placed to a photocatalytic quartz reactor which was bubbled with Ar for 0.5 h and then irradiated with a 300 W xenon lamp for 2 h under magnetic stirring. After extensive washing, the precipitate was dried at 80°C overnight and then calcined at 350°C with a heating rate of 5°C min-1 for 2 h. The prepared powders were denoted as CuOx-CTR , the Cu/Ti molecule ratio of which is 1%.CuOon method. A certaThe structural characterization of the as-synthesized materials was performed by X-ray diffraction , and their morphology of them was observed by a scanning electron microscope , transmission electron microscope , and high-resolution TEM, as illustrated in a previous work . MolybdeTo explore the charge carriers, photoluminescence emission spectra were measured at room temperature with a fluorescence spectrophotometer  using a 325 nm wavelength laser as the excitation source. In addition, all photoelectrochemical measurements  were conducted with a three-electrode workstation .2 was detected every half hour by a gas chromatograph  equipped with a thermal conductivity detector using Ar as carrier gas. Another gas chromatograph with a flame ionization detector  was used to determine the evolution of CO and CO2.The photocatalytic activity experiment was performed in a top-irradiation jacketed quartz photoreactor with water flowing through to maintain a constant temperature (30 ± 1°C). In a typical reaction, 50 mg of photocatalyst was dispersed in 100 mL of 5 v% glycerol with magnetic stirring. The catalyst suspension was first degassed with an Ar stream for an hour. Subsequently, the solution inside the reactor was irradiated by a 300 W xenon lamp . The generated Hx-CT400. As shown in 2 (JCPDS 21–1272) [x modification, the spectrum exhibits a similar peak shape, suggesting that the addition of Cu species did not change the crystal form. Moreover, the average grain size of CT400 was estimated to be approximately 9 nm by using Jade 6.0 and Scherrer’s formula [X-ray powder diffraction was used to analyze the crystal structures of CT, CT400 and CuO21–1272) . After C formula  from the2 catalyst shows an approximately 5% weight loss when heated to 100°C, suggesting water evaporation from the catalyst surface [2 at the calculated temperature of 400°C. From the DSC curves, it is worth noting that a peak of an exothermic reaction appeared at approximately 352°C indicating a crystal transition of the catalyst from the amorphous to the anatase phase [To investigate the thermostability and components of the CT catalyst, thermogravimetric analysis was conducted. As it demonstrated in  surface . When itse phase . Crystal2 and CuOx-C/TiO2 catalysts were characterized by SEM and TEM. Fig A in S1 File shows the SEM images of CT . The sample is composed of many aggregated nanosized sphere particles. These spheres result from the hydrolysis of TBOT. To further observe the microstructure, the corresponding TEM images are presented in Fig B in S1 File. The nanosized sphere particles have a distribution in average size of 200 to 300 nm, with smooth edges. From the HR-TEM , layered amorphous structure on surfaces of the spheres are detected, which could be identified as carbon materials. It is noted that no lattice fringes were found in the catalyst, which suggests that the catalyst remains amorphous. The results are in accordance with the previous XRD pattern.The microstructures of the C/TiO2, respectively, as seen in After heat treatment for 2 h, CT400 shows a similar SEM image with CT in Fig C in x-CT400 is presented in Fig D in x-C/TiO2 clearly indicate 0.352 nm  [2.The SEM structure of CuO21–1272)  and 0.2321–1272) . The EDXx-CT400, both the catalysts exhibit type-IV patterns with H2 hysteresis, ascribed to a typical mesoporous structure [2 cluster was first formed during the hydrolysis of TBOT as the nucleus and then grown into larger particles with the framework of confined small mesopores [x-CT400 are summarized in tructure . After eesopores . The Brux-CT400. In 3/2 and Ti 2p1/2 states with a gap of 5.7 eV, which suggests the presence of the typical Ti4+ state [2 sample [1/2 state is at 464.6 eV), the CT400 shows a slight 0.2 eV shift to higher binding energy. The result may suggest the surface interaction between the carbonaceous species with the TiO2 surface. The Ti 2p binding energy of CuOx-CT400 shifts to lower binding energy in contrast with CT400, which indicates charge transfer between the Cu species and C/TiO2 [3/2 for CuOx-CT400 =2+, Cu+, and Cu0, respectively.With this calculation, the values of α’ are 1850.8 eV, 1849 eV and 1851.4 eV corresponding to Cux-CT400. Three evident peaks at 284.8, 286.3, and 288.7 eV are detected for CT400. The C1s peak at 284.8 eV originated from sp2 hybridized carbon [2 lattice, other carbon may exist as the carbonaceous species formed at the surface of TiO2 to serve as a conductor. After loading the Cu species, the CuOx-C/TiO2 catalyst also exhibits the similar peak shape but a small peak at 282.5 eV, which is close to the Ti-C bond [d carbon . The pead carbon , 38. Thei-C bond  and shif2, the absorption edge of CT350 obviously shifts to the visible light region, which can be attributed to the photo absorption property of the carbonaceous materials absorbed on the TiO2 surface [2 decreased significantly with increasing calcination temperature. CT550 exhibits a similar spectrum to the pure TiO2 lattice. This result proves that carbonaceous species were gradually removed due to the higher temperature, which lead to the decreased absorption of visible light. Notably, the spectra of CT350 exhibit DRS spectra similar to those reported in the literature [2 surface. Based on these results, it can be speculated that carbonaceous materials in the CT catalyst mainly adsorb on the TiO2 surface, which is in accordance with the XPS analysis. As shown in 2 [UV-vis diffuse reflectance spectroscopy (DRS) was performed on CT350, CT400, CT500, and CT550 to determine the light harvesting, and the results are shown in terature , in whicown in 2 , which cown in 2 .x, which indicates that CuOx favors the transfer of photo-induced electrons and restrains the recombination of photo-induced electrons and holes [To explore the charge carrier transfer and recombination, photoluminescence emission spectra were employed. As shown in nd holes .x-CT400 catalysts. The experiment was conducted to reveal the photoexcited electron transfer. As x-CT400 catalyst exhibits higher photocurrent up to 11.3 μA compared to CT400 with 4.4 μA. Generally, the higher transient photocurrent indicates more electron transfer [x-CT400 catalyst shows an evident spectrum trail when the light is off. The phenomenon is ascribed to complex photoelectron transfer involving the valence state change of Cu species.transfer . It is wx-CT400. Anode slope and corrosion current (j) are two major parameters of Tafel characterization. The anode slopes are 1.77 and 2.40 and the corrosion currents are 1.79 and 7.01 μA/cm2 for CT400 and CuOx-CT400, respectively. The larger anode slope and j values suggest the faster photoelectron transfer [x-CT400, compared with CT400.transfer  of CuOx-ct (the reactivity resistance), Rs (the solution resistance), Cdl (double-layer capacitance), and Zw (Warburg impedance), as shown in ct indicates the resistance of the charge carrier transfer. Notably, Rct values for CT400 (122 ± 1.69 Ω/cm2) decreased to 80.6 ± 1.4 Ω/cm2 for CuOx-CT400. The result indicates that CuOx modification significantly contributes to making the photoexcited charge carrier transfer more favorable and faster [To further test the photoexcited charge carries transfer resistance of the catalyst, electrochemical impedance spectroscopy (EIS)  methods d faster .2 evolution amount follows the order of CuOx-CT400 > CuOx-CT350 > CuOx-CT500 > CuOx-CT550 > CuOx-anatase > CT400. The CuOx-CT400 catalyst exhibits a high hydrogen generation rate of 433.3 μmol/h, which reaches 56 times that of CT400 (7.7 μmol/h). CuOx-anatase was prepared for comparison since it is difficult to prepare CuOx-TiO2 without carbon according to this method (the precursor Ti(OBu)4 was both a carbon source and a titanium source). The H2 evolution efficiency with CuOx-anatase is 216.7 μmol/h, about half that of with CuOx-CT400. The calcination temperature plays an important role for the H2 generation of CuOx-CTR catalysts. Generally, the higher temperature can result in higher crystallinity and lower carbonaceous material content of the catalyst. However, 400°C is the optimal temperature indicating that crystallinity is not the main factor for the photoactivity. It can be speculated that carbon content and crystalline type are associated with photocatalytic H2 generation.x-CT400 is presented in 2 generation is always high in the first four cycles and shows a little decrease in the 5th run.The cycle experiment of the hydrogen evolution of CuO2 and CO production of CT400 and CuOx-CT400 are shown in Fig F in 2 and CO production rate was observed between CT400 and CuOx-CT400, which is consistent with the H2 evolution rate.The COx-CT samples is important for the elevated photoactivity. However, the CT400 catalyst without Cu modification exhibited low photocatalytic efficiency. It seems reasonable to deduce that carbon materials could act as good electron conductors instead of proton reduction sites. Moreover, Joo et al. [2 catalysts and concluded that electrically conductive carbon could only facilitate charge carries separation but made no direct contribution to the hydrogen formation. Further, in the synthesized materials, CuOx species can be crucial co-catalysts which serve as both electron acceptor and surface redox reaction sites. Therefore, the carbonaceous material is speculated to act as an electron-bridge providing a new electron transfer channel from TiO2 to the CuOx species.The results of the photoactivity experiment reveal that the carbon coated on the CuOo et al.  preparedx modified C/TiO2 for photocatalytic H2 evolution featuring a synergistic effect of CuOx and carbon is presented in 2 surface through Ti-O-C bonds and CuOx species can graft on both carbon materials and the TiO2 surface. Electrons were photoexcited to the conduction band (CB) of TiO2 with holes staying in the valence band (VB) under UV-visible light irradiation (Eqs x species to form Cu(I) or Cu(0), which results in efficient electron-hole separation  [Accordingly, the hydrogen evolution rate is significantly boosted by the synergistic effect of CuO4, etc.)  by the p2 surface modified with CuOx has been prepared via a simple hydrolysis and photo-reduction method. With systematic structure characterization, carbonaceous materials were observed to be absorbed onto the surface of TiO2 nanoparticles. The Cu species existed as CuOx with multivalence states grafted both on the TiO2 surface and the carbonaceous materials. With CuOx functioning as a cocatalyst, the CuOx-CTR exhibited high activities in photocatalytic hydrogen evolution. The photocatalytic hydrogen generation rate of the CuOx-CT400 catalyst (433.3 μmol/h) is 56 times as high as that of CT400 (7.7 μmol/h). This significant increase is attributed to the synergistic effect between CuOx and the carbon species. CuOx grafted on the surface of the catalyst accepts photoexcited electrons inhibiting electron-hole recombination and serving as proton reduction sites. Meanwhile, carbonaceous materials function as good conductors by transferring electrons to the CuOx, which significantly enhances the CuOx co-catalyst effect. This work demonstrates a simple method to prepare a Cu and C co-modified TiO2 catalyst with high photocatalytic performance and proposes the synergistic effect between copper and carbon, which presents a significant step in determining the photoexcited electron transfer mechanism.Carbon coated on a TiOS1 FileTable A in S1 File. Comparison of various TiO2-based photocatalysts. Fig A in S1 File. SEM images of CT. Fig B in S1 File. TEM (a–c) and HRTEM (d) images of CT. Fig C in S1 File. SEM images of CT400. Fig D in S1 File. SEM images of CuOx-CT400. Fig E in S1 File. Elemental mapping of CuOx-CT400 (a-d) for C, Cu, O, and Ti. Fig F in S1 File. CO2 (a) and CO (b) production of CT400 and CuOx-CT400. Additionally, the raw data for figures has been uploaded to Figshare (http://dx.doi.org/10.6084/m9.figshare.7951724).(DOCX)Click here for additional data file.


Neisseria gonorrhoeae include screening for and treating asymptomatic infections in high-prevalence populations in whom antimicrobial drug–resistant infections have typically emerged. We argue that antimicrobial resistance in these groups is driven by a combination of dense sexual network connectivity and antimicrobial drug exposure . Sexual network connectivity sustains a high-equilibrium prevalence of N. gonorrhoeae and increases likelihood of reinfection, whereas antimicrobial drug exposure results in selection pressure for reinfecting N. gonorrhoeae strains to acquire antimicrobial resistance genes from commensal pharyngeal or rectal flora. We propose study designs to test this hypothesis.Contemporary strategies to curtail the emergence of antimicrobial resistance in  Neisseria gonorrhoeae has led to fears that gonorrhea may soon become untreatable   in en (MSW) .N. gonorrhoeae with resistance to all classes of antimicrobial drugs tested   may temporarily reduce N. gonorrhoeae prevalence in this setting but produce selection pressure for N. gonorrhoeae to acquire AMR.We focus on the emergence of N. gonorrhoeae . N. gonorrhoeae and C. trachomatis screening. In the absence of a global screen-and-treat strategy that leads to N. gonorrhoeae extinction, a typical local screen-and-treat approach induces a temporary decline in N. gonorrhoeae prevalence. Without altering the underlying determinant of high N. gonorrhoeae prevalence (network connectivity), N. gonorrhoeae tends to return to its high-equilibrium prevalence. Moreover, this strategy also increases the prevalence of the genes that encode AMR in N. gonorrhoeae. On the basis of studies using macrolides for other indications, ≈90% of patients treated with ceftriaxone and azithromycin, the currently recommended therapy for N. gonorrhoeae infection, would be expected to acquire macrolide resistance that can persist for up to 4 years in commensal pharyngeal and colonic bacteria  and consequently are prone to outbreaks of various bacterial, viral, and parasitic diseases , establishing the validity of the connectivity–AMR hypothesis in general and the place of screening in high-prevalence populations specifically is imperative. Part of the answer lies in accurately describing the mechanisms underpinning AMR in n of AMR  (18). AlN. gonorrhoeae screening and treatment (vs. no screening and limiting therapy to patients with symptomatic N. gonorrhoeae infection) on several parameters: prevalence of N. gonorrhoeae infection; susceptibility to other STIs, including HIV; effect on adaptive immunity to N. gonorrhoeae; effect on individual and population resistome and microbiome; and emergence of AMR. Researchers could increase the probability of reducing N. gonorrhoeae prevalence in these studies by including aggressive contact tracing strategies, such as by using sexual networking apps. A practical challenge would be the large cohort size required to demonstrate a difference in the probability of AMR between the screening and no-screening arms of the study because AMR emergence is a rare event. Nonetheless, establishing whether N. gonorrhoeae screening reduces infection prevalence in dense networks and at what cost to the resistome  would be informative. If screening is found to have little or no effect on N. gonorrhoeae prevalence but a large effect on the population resistome, it may cause a reevaluation of screening policies. Researchers could also assess the significance of altered resistomes to AMR in N. gonorrhoeae in vitro by assessing whether N. gonorrhoeae is able to acquire AMR via transformation with DNA extracts from posttreatment microbiomes. Such studies could also provide the probabilities of resistome alteration following specific therapies . These data could then be used to construct more realistic models of AMR induction in N. gonorrhoeae –producing bacteria in a hospital in Spain was best described by an increase in ESBL production in a range of bacteria  rather than epidemics of single species or clones (N. gonorrhoeae AMR in sex workers, for example, has been linked to extensive antimicrobial drug use (Although we have focused our discussion on the connectivity–AMR thesis in MSM, similar considerations would also apply to other high connectivity populations. The emergence of


Background: Polycystic ovary syndrome (PCOS) is associated with an adverse cardiovascular risk profile including a prothrombotic state. Exenatide has been shown to be effective at improving insulin sensitivity and weight loss in PCOS; therefore this study was undertaken to assess its effects on weight, endothelial function, inflammatory markers, and fibrin structure/function in overweight/obese women with PCOS.Methods: Thirty overweight/obese anovulatory women with all 3 Rotterdam criteria received exenatide 5 mcg bd for 4 weeks then 10 mcg bd for 12 weeks. The primary outcome was change in weight; secondary outcomes were changes in endothelial function [Reactive Hyperemia-Peripheral Arterial Tonometry (RH-PAT)], serum endothelial markers , change in inflammation (hsCRP), and alteration in clot structure and function .Results: Twenty patients completed the study. Exenatide reduced weight 111.8 ± 4.8 to 108.6 ± 4.6 kg p = 0.003. Serum endothelial markers changed with a reduction in ICAM-1 (247.2 ± 12.9 to 231.3 ± 11.5 ng/ml p = 0.02), p-selectin (101.1 ± 8.2 to 87.4 ± 6.6 ng/ml p = 0.01), and e-selectin (38.5 ± 3.3 to 33.6 ± 2.6 ng/ml p = 0.03), without an overt change in endothelial function. Inflammation improved , there was a reduction in clot function  but not clot structure.Conclusion: Exenatide caused a 3% reduction in weight, improved serum markers of endothelial function, inflammation, and clot function reflecting an improvement in cardiovascular risk indices in these women with PCOS. This suggests exenatide could be an effective treatment for obese women with PCOS.Clinical Trial Registration: ISRCTN81902209. Polycystic ovary syndrome (PCOS) is a common condition affecting 10% of women of reproductive age  has consistently been shown to be greater in PCOS than controls , 15. PCOAbnormal clot structure and function contributes to the prothrombotic state; clots composed of a compact structure with thin fibers and small pores are associated with early and more severe cardiovascular disease . Clot stExenatide, an incretin mimetic, is a glucagon like polypeptide (GLP-1) analog used in the treatment of type 2 diabetes . It has We therefore hypothesized that exenatide would exert a direct effect on endothelial function, inflammatory markers and blood clot structure and function in overweight/obese women with PCOS.An open labeled study was undertaken using exenatide 5 micrograms twice daily for 1 month followed by exenatide 10 μg twice daily for 3 months. The diagnosis of PCOS was based on all three diagnostic criteria of the Rotterdam consensus being present for each patient to reduce the heterogeneity of the diagnosis, namely clinical, and biochemical evidence of hyperandrogenemia , oligomenorrhoea or amenorrhea, and polycystic ovaries on transvaginal ultrasound. Non-classical 21-hydroxylase deficiency, hyperprolactinaemia, Cushing's disease, and androgen-secreting tumors were excluded by appropriate tests. Subjects had no concurrent illness, were not on any prescription or over-the-counter medication that was likely to affect insulin sensitivity, lipids, or ovarian function including hormonal contraceptives for the preceding 6 months. Subjects were not planning to conceive and were using barrier contraception. Subjects were advised not to change their lifestyle, including physical activity, or dietary habits, during the study period.Thirty patients (mean age 27 ± 4 years) who fulfilled both inclusion and exclusion criteria were given exenatide. Compliance with treatment was calculated by determining the amount of exenatide in the pens returned at the next clinic visit (2 and 4 months).All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Leeds East Local Research Ethics committee.Clinical and fasting biochemical assessments were performed at baseline and at the end of the 4-month period. The primary endpoint of the study was a change in weight, and the secondary endpoints were changes in endothelial function, inflammation, and clot structure.g for 15 min at 4°C, and the aliquots stored at −20°C. Serum testosterone was measured by liquid chromatography/mass spectroscopy on an Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer . Sex hormone binding globulin (SHBG) was measured by an immunometric assay with fluorescence detection on the DPC Immulite 2000 analyzer using the manufacturer's recommended protocol (upper limit of the reference range 2.0 nmol/l). The FAI was calculated as the total testosterone × 100/SHBG. Total cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL-C) levels were measured enzymatically using a Synchron LX20 analyzer . Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald equation. Serum insulin was assayed using a competitive chemiluminescent immunoassay performed on the DPC Immulite 2000 analyzer . The analytical sensitivity of the insulin assay was 2 μU/ml, the coefficient of variation was 6%, and there was no stated cross-reactivity with proinsulin. Plasma glucose was measured using a Synchron LX 20 analyzer (Beckman-Coulter), using the manufacturer's recommended protocol. The coefficient of variation for the assay was 1.2% at a mean glucose value of 5.3 mmol/L during the study period. The insulin resistance was calculated using the homeostasis model assessment for insulin resistance (HOMA) method [HOMA-IR = (insulin x glucose)/22.5]. Alanine aminotransferase was measured on the Unicel® DxC 80 analyser .Fasting venous blood was collected into serum gel and fluoride oxalate tubes. Samples were separated by centrifugation at 2,000 × The serum concentrations of serum intercellular adhesion molecule 1 (ICAM-1), serum vascular cell adhesion molecule 1 (sVCAM-1), and sE-selectin were measured by commercially available quantitative ELISA  according to the manufacturer's instructions.Reactive hyperemia peripheral arterial tonometry (RH-PAT) is a non-invasive technique to assess peripheral microvascular endothelial function by measuring changes in digital pulse volume during reactive hyperemia. The RH-PAT data were analyzed by a computer in an operator-independent manner as previously described . The RH-−1 Tris, 100 mmol−1 NaCl pH 7.4 (75 μl). Activation mix (50 μl) containing 0.045 U ml−1 human thrombin , and 22.5 mmol−1 calcium in assay buffer was added to each column with a multi-channel pipette at 10-s intervals. Plates were shaken and read at 340 nm every 12 s for 60 min in an ELx-808 IU ultramicroplate reader .Clot structure and fibrinolysis measurements were conducted using turbidity and lysis measurements as previously described . In brie−1 tissue plasminogen activator (tPA) (75 μl) . A number of parameters were subsequently analyzed including final turbidity (FT), time from initiation of clot formation to 50% lysis (LT1) and time from start of clot formation to 50% lysis (LT2).For lysis measurements, the same procedure was applied, but with the addition of 333 ng mlThe sample size was based on the study of Elkind-Hirsch et al treating women with PCOS on the effect of exenatide on weight loss in women with PCOS . Using nt-test for biochemical data and clinical observations. The Wilcoxon signed rank test was applied to biochemical data that violated the assumptions of normality when tested using the Kolmogorov–Smirnov test, and associations used Pearson's correlation or Spearman's correlation as appropriate. Statistical analysis was performed using SPSS for Windows NT, version 18.0 . Data are reported as mean ± SEM.A comparison between the groups at baseline and follow up was carried out using the paired Twenty patients aged 26 ± 4 years completed the study. One patient became pregnant during the study, and six patients withdrew due to side effects . Two patients were withdrawn due to poor compliance during the first month and one patient was lost to follow up. The pregnancy was followed to term with no maternal or fetal complications.2 [3 women were overweight (BMI 26–29) whilst 27 were obese (BMI 30 or greater)] Systolic blood pressure was 119.7 ± 2.2 mmHg and diastolic was 71.5 ± 1.6 mmHg.The mean weight of the women at baseline was 110.0 ± 3.4 kg with BMI 40.4 ± 1.3 kg/mp = 0.003) and BMI 41.0 ± 1.9 to 39.8 ± 1.8 kg/m2 (p = 0.005). There was no effect on waist circumference but there was a reduction in hip circumference. Blood pressure was unaffected by the exenatide treatment ]; however, the surrogate measures of endothelial function improved with a reduction in ICAM-1 (247.2 ± 12.9 to 231.3 ± 11.5 ng/ml p = 0.02), p-selectin (101.1 ± 8.2 to 87.4 ± 6.6 ng/ml p = 0.01), and e-selectin (38.5 ± 3.3 to 33.6 ± 2.6 ng/ml p = 0.03). There was no change in VCAM-1.Endothelial function determined by RH-PAT was unchanged [RH-PAT .For clot formation there was a reduction in time from full clot formation to 50% lysis (LT) from 2,987 ± 494 to 1,926 ± 321 s r = −0.59 p = 0.02), change in p-selectin and hip circumference (r = 0.61 p = 0.009) and change in CRP and insulin/HOMA (r = −0.57 p = 0.02 r = −0.49 p = 0.047). There was also a correlation between change in LT and systolic blood pressure .There was a correlation between a change in ICAM and triglycerides  compared to other studies. The study was too short to determine changes in menstrual frequency though increased menstrual frequency with exenatide has been shown by others  which may be relevant as an increase in LT has been shown to be associated with cardiovascular risk ; these fAn improvement in inflammation was seen in our study with a significant reduction in both CRP and serum triglycerides. This is in accord with reports of exenatide use in patients with type 2 diabetes , 44, butIn contrast to a previous study with exenatide our study showed there was no reduction in total testosterone or HOMA  that mayThe weakness of this study is the high drop-out rate which largely was due to side effects of the medication (namely nausea and vomiting), however the withdrawal rate was similar to that reported in a previous study  of 30%. In summary, this study has shown that exenatide is effective in improving the parameters of adverse cardiovascular risk in obese women with PCOS. There was no change of endothelial function by direct measurement that may indicate that surrogate markers do not reflect endothelial change in PCOS.The datasets generated for this study are available on request to the corresponding author.AD performed the study. RV performed and interpreted the serum endothelial markers. A-MC performed the radiology. RA supervised the clotting studies. TS, EK, and SA supervised the study. All authors contributed to writing 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.


It has been suggested that C3 glomerulonephritis (C3GN) and atypical hemolytic-uremic syndrome , two rare entities caused by complement alternative pathway dysregulation share overlapping genetic origin and can be triggered by infections.CFI gene (complement factor I). These two variations are segregated with disease in the core family member of this patient. Plasma CFI levels of the patient and family members were all in normal range. We considered that these two variations only impair CFI function rather than its quantity in the serum.We report a case of concomitant C3GN and thrombotic microangiopathy (TMA) after pulmonary infection in a young male receiving kidney transplantation. Genetic assessment revealed two missense variations in compound heterozygous form in Our case supports that C3GN and TMA shared overlapping genetic variations and might be triggered by infection in genetically susceptible patients after kidney transplantation. The etiology of which remains elusive, however, some studies have suggested that genetic mutations leading to complement dysregulation play a critical role , 2. Like pattern . The com and C3b . The delCFI gene, while his father and mother who are carriers of only one variant were asymptomatic.Herein, we report a rare case of a young male with a diagnosis of concomitant C3GN and TMA in the renal allograft that harbored two variants of compound heterozygous form in 9/L. Forty days prior to this admission, a renal allograft biopsy was performed. He was managed with intravenous antibiotics and immunosuppression enhancement by increasing the dosage of mycophenolate mofetil. His Scr level decreased initially with a nadir of 1.58 mg/dl, but elevated progressively with increased proteinuria (11.38 g/24 h). He denied family history of any kidney diseases or inheritable illnesses. A repeat kidney biopsy was performed in our hospital. Written informed consent to publish this case was obtained from this patient.A 32-year-old male was referred to our hospital for elevated level of serum creatinine (Scr) 3.71 mg/dl) and proteinuria (3+) following a previous deceased cardiac donor-derived KT due to an unidentified cause of end-stage renal disease (ESRD). Laboratory workups and results of diagnostic procedures performed are summarized in Table  mg/dl anLight microscopy showed multiple periodic acid-Sciff stain (PAS)-positive materials in the capillary lumens Fig. . CapillaImmunofluorescence study indicated prominent C3 positivity (3+) along the basement membrane and in the mesangium Fig.  in all tElectron microscopy examination of 2 glomeruli showed widespread foot process effacement and electron-dense deposits in the subendothelial and subepithelial spaces Fig. . FurtherRetrospective review of the 1st biopsy slides (X.F.) indicated similar light microscopy findings Fig. . Immunof. We found two rare missense variants in compound heterozygous form, c.848A > G (p.Asp283Gly) and c.1339C > T (p.Pro447Ser) in the CFI gene (NM_000204.3) in the patient while his father and mother were found to harbor only the c.848A > G and c.1339C > T respectively   were perely Fig. . Both paOur patient was treated with 2 sessions of plasma exchange, but no clinical improvement was achieved as indicated by persistent nephrotic-range proteinuria and progressive elevation of Scr. After approximately one-year follow-up, this patient was in dialysis.CFI variation the most likely underlying etiology. Second, the compound heterozygosity of the two rare CFI gene variants may related with diseases in the patient while the patient’s father and mother, who harbors only one of the CFI gene variant, were phenotypically normal, indicating that the disease is autosomal-recessive . Serum CFI concentrations of all family members were normal, indicating that CFI functional impairment rather than quantitative deficiency of CFI protein was probably the underlying cause.There are 2 unique aspects of this particular case. First, C3GN and TMA, both rare diseases, were present simultaneously in this patient. Moreover, the possibility that TMA in this patient was caused by allograft rejection or calcineurin-inhibitor toxicity could be excluded histologically and clinically, making genetic CFI gene mutations in this series, 5 CFI gene mutations have been previously reported in aHUS patients. Some investigators even have proposed that aHUS and C3 glomerulopathy should be lumped together as specific disease entities of the alternative complement pathway [It has now been increasingly appreciated that mutations in genes coding for complement regulators are largely responsible for inheritable or acquired C3G and TMA , 2, 6. I pathway . In anot pathway .CFI gene missense variant. The c.848A > G (p.D283G) in his father results in substitution of the highly conserved Asp residue by Gly at the position 283 in low-density lipoprotein receptor domain class A of the CFI protein, which is identified as an important calcium-binding site in this domain . As far as we are aware, these 2 variants of CFI gene have not been reported in patients with C3 glomerulopathy or TMA.Genetic analyses of our patient and his family members were also informative. Both of his parents were phenotypically normal with a heterozygous ain Fig. . CFI genquantity , 10. TheIt is being increasingly recognized that a host of patients with complement factor gene variations only develop diseases or experience exacerbation after various triggers, one important factor of which is infections . A classSellier-Leclercet al [CFI gene mutation rapidly evolved to ESRD and plasma therapy had only modest efficacy in aHUS patients with complement regulator gene mutations. In fact, the efficacy of plasma exchange for C3 glomerulopathy is dubious as considered by most experts [Our patient failed to respond to plasma exchange therapy which is currently a main-stay treatment modality in areas without access to eculizumab, a humanized monoclonal antibody against terminal portion of the complement cascade . Sellierrcet al  reported experts . Althoug experts  and C3 g experts , this drCFI gene that presumably resulted in CFI functional deficiency. Our case supports that C3GN and TMA shared overlapping genetic variations and might be triggered by infection in genetically susceptible patients.In conclusion, we report an interesting case of C3GN and TMA of a transplanted kidney after pulmonary infection in a young male with two variations in the


Metabolomics has started to embrace computational approaches for chemical interpretation of large data sets. Yet, metabolite annotation remains a key challenge. Recently, molecular networking and MS2LDA emerged as molecular mining tools that find molecular families and substructures in mass spectrometry fragmentation data. Moreover, in silico annotation tools obtain and rank candidate molecules for fragmentation spectra. Ideally, all structural information obtained and inferred from these computational tools could be combined to increase the resulting chemical insight one can obtain from a data set. However, integration is currently hampered as each tool has its own output format and efficient matching of data across these tools is lacking. Here, we introduce MolNetEnhancer, a workflow that combines the outputs from molecular networking, MS2LDA, in silico annotation tools (such as Network Annotation Propagation or DEREPLICATOR), and the automated chemical classification through ClassyFire to provide a more comprehensive chemical overview of metabolomics data whilst at the same time illuminating structural details for each fragmentation spectrum. We present examples from four plant and bacterial case studies and show how MolNetEnhancer enables the chemical annotation, visualization, and discovery of the subtle substructural diversity within molecular families. We conclude that MolNetEnhancer is a useful tool that greatly assists the metabolomics researcher in deciphering the metabolome through combination of multiple independent in silico pipelines. Metabolomics has matured into a research field generating increasing amounts of metabolome profiles of complex metabolite mixtures aiming to provide biochemical insights. Mass spectrometry has become the workhorse of metabolomics and typical untargeted experiments currently result in qualitative and semiquantitative information on several thousands of molecular ions across tens to hundreds of samples. Technical advances in the last decade have allowed researchers to fragment increasing amounts of mass peaks that result in mass fragmentation spectra (MS/MS or MS2). Metabolite annotation and identification tools have benefited from these advances as now more MS2 spectra per sample can be queried in reference libraries in order to find candidate structures or submitted to in silico tools that propose a putative structure ,6,7,8,9.Despite these tremendous advances, a key challenge remaining for metabolomics researchers is to biochemically interpret large-scale untargeted metabolomics studies due to the complexity of the metabolomes represented by mass fragmentation spectra to which actual chemical structures need to be assigned, and for which reference spectra are not available. In biological samples, many metabolites share molecular substructures and form structurally related molecular families (MFs) of various chemical classes, which has inspired metabolome mining tools exploiting these biochemical relationships. Based on the assumption that structurally similar molecules  generate similar mass spectrometry fragmentation spectra, one can group analogs by comparing their fragmentation spectra resulting in the construction of molecular families. To do this on a larger scale, computational tools have been developed such as molecular networking (MN) . HoweverIndeed, since the molecular networking approach was proposed in 2012 , numerouThe outputted structural information for each mass feature can be mapped on a molecular network, for example, to show for which mass features library matches or in silico predicted structural matches are available. The recently introduced Network Annotation Propagation (NAP) also exploits the network topology to rerank candidate structure lists based on neighboring matches within molecular families . FurtherTo assess whether molecular families are of particular interest for your research question, knowing their chemical class may provide sufficient information. The recently proposed ClassyFire tool  takes moTaken together, all these recent developments enable the discovery of relations between millions of spectra and the listing of candidate structures from various spectral libraries or alternatively from compound libraries using in silico approaches.Euphorbia [Whilst each of those tools produce useful structural information, their combined application has been hampered by the use of different file formats, platforms, and the challenge to match molecular features across the outputs of these tools. We postulate that whilst each tool provides complementary insights, their combined use allows an increased level of biochemical interpretation, i.e., the sum becomes greater than the individual parts. Furthermore, it would be practically advantageous to combine all these results in one place. We have previously described the integration of Mass2Motifs and chemical classifications with molecular networks to assess the chemical diversity within a subset of species of the plant genus uphorbia  and the uphorbia . HoweverEuphorbia and Rhamnaceae plants. Illustrated by four case studies, we demonstrate how our integrative workflow discovers dozens of MFs in large-scale metabolomics studies of these plant and bacterial extracts. Moreover, discovered MFs can be divided into subfamilies using the mapped MS2LDA results. Structural annotation of Mass2Motifs is facilitated by having chemical and structural annotations at hand, for example by recognizing substructures in peptidic molecules. We conclude that our workflow provides chemical refinement of metabolomics results beyond spectral matches through large-scale MF and substructure discovery and annotation by integrating outputs of various tools in one place allowing for enhanced visualization. This also guides the metabolomics researcher in prioritizing MFs to explore and in structurally annotating molecules.Here, we introduce MolNetEnhancer a software package available in Python and R that unites the output of many of the above-mentioned metabolome mining and annotation tools  independent of what dataset it processes, thus making the algorithm accessible in an easy-to-use format to the community . MolNetEhttps://gnps.ucsd.edu).Perform mass spectral molecular networking analysis through the Global Natural Products Social Molecular Networking platform  and DEREPLICATOR through the GNPS platform. Alternatively, other in silico tools for putative chemical structural annotation  ,4 can alhttp://ms2lda.org).Perform unsupervised substructure discovery using MS2LDA  and (ii)  b. Manualskeleton ,42. Furtxylation b. Chemics2Motifs c.Euphorbia diterpenoid skeletons of the jatrophane, deoxy tigliane, or ingenane ester type are found within all Euphorbia subgeneric clades, whereas nicotinoyl sidechain modifications are unique to subgenus Esula  and N,N-dimethyl-pyrrolosamine (lomaiviticin) which have overlapping fragment ions and are therefore characterized by the same Mass2Motif. With a frequency of more than 70 throughout the entire molecular network , the amino sugar Mass2Motif can be used as a handle to identify known and potential novel natural products throughout network. Indeed, the Mass2Motif was found in all members of the Rosamicin MF  nodes. In total, using the MolNetEnhancer workflow we could annotate 32 peptidic motifs of which we could link 11 to peptides known to be produced by Xenorhabdus/Photorhabdus strains whilst the other 21 Mass2Motifs represent substructures not yet elucidated. The peptidic nature of these Mass2Motifs was assessed by recognition of typical fragment ion patterns as seen for known peptides as well as doubly charged precursor ions that are often a sign of peptides in these extracts.Annotations included Mass2Motifs that form peptidic substructures related to well-known nortides ,53. We cnortides  which thde class . The corXenorhabdus and eight variants have been described in detail with variants A and B present in peptidic databases [With the help of the integrative display of DEREPLICATOR and VarQuest annotation results, we could also annotate two xenoamicin-related peptidic MFs a,b. Xenoatabases . Xenoamiin silico approaches. The smaller MF of 22 nodes consisted of doubly-charged precursor ions where no ring-related Mass2Motifs were assigned. Some members like xenoamicin A appeared in both MFs as singly and doubly charged precursor ions; the differences in motif distributions between the two MFs indicates that the initial charge has an impact on the fragmentation pathways and thus the acquired spectra given that we know the ring A is part of xenoamicin A.We could also find additional MFs and singletons in which the xenoamicin ring or tail Mass2Motif was present, pointing to related peptidic molecules not linked through the modified cosine score. Further inspection with help of VarQuest annotations strengthened these annotations as VarQuest annotated modified amino acids in both rings  and the in silico tools together.Altogether, this example highlights how the MolNetEnhancer approach facilitates fragmentation based metabolomics analysis workflows by increasing the “structural resolution”, the discovery of more xenoamicin variants than previously described, and highlighting previously unseen connections between MFs and molecules. Furthermore, the integrative approach enabled straightforward annotation of Mass2Motifs found in the xenoamicin MF by using the VarQuest fragment ion annotations as guide for Mass2Motif feature annotation. Both Mass2Motif and VarQuest results strengthened each other since when predicted amino acid changes occurred in the peptidic ring, the corresponding ring-related Mass2Motif was absent, and vice versa—made possible by combining the outputs of several in silico tools, thus increasing structural information retrieved and enhancing biological interpretation. Here, we have chosen a representative number of in silico tools covering mining, annotation, and chemical annotation to provide the user with different chemical insights. Although we used DEREPLICATOR and NAP to exemplify in silico annotation tools here, MolNetEnhancer is platform independent, meaning that chemical structures retrieved from any in silico annotation platform could be used given the molecular feature identities correspond across all molecular mining and annotation tools.Although significant advances have been made in molecular mining workflows, chemical annotation as well as classification tools ,14,15,16Particularly in natural products research, the rapid annotation of known  as well as unknown specialized metabolites from complex metabolic mixtures hinders interpretation in an ecological, agricultural or pharmaceutical context. Many specialized metabolites from natural sources are used as pharmaceuticals , in agriEuphorbia, we were able to retrieve chemical structural information of previously described pharmaceutically highly valuable diterpenoid skeletons corresponding to an annotation level 3 according to the Metabolomics Standard Initiative’s reporting standards [Salinispora and Streptomyces bacterial extracts, MolNetEnhancer aided the annotation of a previously unreported tryptophan-based MF, and a xenoamycin-related MF in the Gammaproteobacteria of the genus Xenorhabdus and Photorhabdus could be studied in more detail than in previous studies.In the plant genus tandards . The useIt is of utmost importance to note that results retrieved from MolNetEnhancer summarize results retrieved from third-party software and manual inspection and validation of all structural hypotheses remain essential. However, MolNetEnhancer significantly aids the manual inspection and validation process conducted by the expert, by making substructural as well as chemical class information readily available and visible within one data resource. As exemplified in the case studies, MolNetEnhancer can for example help in prioritizing molecular families within a molecular network, which consists of many hundreds to thousands of molecular features, be it by highlighting different chemical classes of interest or molecular families, for which only very few structural hypotheses could be retrieved, potentially highlighting novel chemistry.in silico structure prediction [in silico annotation tools will improve our confidence in in silico spectral annotation [Limitations introduced through data acquisition on different mass spectrometric instrument types do also apply to MolNetEnhancer. Acquiring data on different instruments can cause different MS2 fragmentation patterns, thus in some cases leading to different structural hypotheses through library matching or ediction . Also, tnotation .These limitations highlight the importance of good practices during data acquisition and processing to minimize the time spent analyzing mass spectrometry artefacts and improving the confidence in any downstream annotations. Here, the use of feature-based molecular networking could also help to focus the analysis on those molecular features that are very likely molecular ions —and it hp-values, and false discovery rates to assess confidence in the annotations. Using MolNetEnhancer, it is now also possible to explore the consistency in peptidic annotations within MFs, along with their associated Mass2Motifs, which also assist in improving confidence in the annotations, as we have shown for the xenoamicin MFs in the nematode symbiont bacteria where the majority of the MFs were annotated with xenoamicin variants.Apart from limitations caused by experimental conditions, analysis bias can be introduced for structural predictions based on chemical structures available in public databases, which are still limited especially for particular compound classes. This is particularly true for the chemical class annotations provided through ClassyFire, which rely on collecting correct or structurally closely related candidate structures from compound databases. The chemical annotation score was implemented to guide the researcher in assessing how consistent the chemical annotations are and for how many molecular features at least one candidate structure is found. The peptidic annotations by DEREPLICATOR and VarQuest come with scores, m/z in most of their mass fragment peaks. Incorporation of amino acid-related mass differences as features for MS2LDA could be a route to also discover Mass2Motifs for noncyclic peptides. As it is, cyclic peptides do often contain one or more Mass2Motifs and peptides containing positively charged amino acids such as lysine and leucine have this structural information represented by Mass2Motifs. Furthermore, many Mass2Motifs are currently still unannotated, which hampers fast structural analysis. To partially solve this bottleneck, MotifDB (www.ms2lda.org/motifdb) was recently introduced [One limitation of the use of MS2LDA on the bacterial datasets is that most noncyclic peptidic molecular families do not share any motifs as typically analogues differ by modifications such as methylation or hydroxylation causing a shift in troduced  and the Despite the limitations discussed above, MolNetEnhancer assists in metabolite annotations by its combined analysis of chemical class annotations, structural annotations, and Mass2Motif annotations. If these annotations support each other, as for example for the actinomycin MF in the marine sediment bacteria, there is more confidence that these in silico annotations will indeed be correct. It is noteworthy that the modularity of MolNetEnhancer allows for complementary sources of structural information to be added on in future. We showed that MolNetEnhancer is a practical tool to annotate the chemical space of complex metabolic mixtures using a panel of complementary in silico annotation tools for mass spectrometry based metabolomics experiments. Although we have highlighted the use of MolNetEnhancer using two plant and bacterial datasets, MolNetEnhancer is sample type-independent and may be used for any mass spectrometry-based metabolomics experiment, where chemical structural annotation and interpretation is of interest. Future work will focus on making the complete MolNetEnhancer workflow available within the GNPS platform in order to further increase its user friendliness. Currently, the chemical classification workflow is available to run within the GNPS framework directly outputting an annotated network (see URL in code availability MolNetEnhancer is a powerful tool to accelerate chemical structural annotation within complex metabolic mixtures through the combined use of mass spectral molecular networking, substructure discovery, in silico annotation as well as chemical classifications provided by ClassyFire. The MolNetEnhancer workflow is presented both as an open source Python module and R package, allowing easy access and usability by the community as well as the possibility for customization and further development by integration into future collaborative modular tools and by integration of other existing or future metabolome mining and annotation tools. Whilst its use was showcased using natural product examples, we expect that MolNetEnhancer will also enhance biological and chemical interpretations in other scientific fields such as clinical and environmental metabolomics.Publicly available mass spectrometry fragmentation data sets from four studies were used for this study. Details on how samples and data were collected can be found in the original studies ,28,48,50Data from case studies 1 & 2 illustrating MolNetEnhancer applied to feature-based molecular networking are publicly accessible through the links listed below.Euphorbia study—combined analysis of 43 Euphorbia plant extractsCase study 1: https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=c9f09d31a24c475e87a0a11f6e8889e7MASSIVE: MSV000081082 https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=26326c233918419f8dc80e8af984cdaeGNPS Molecular Networking job: https://proteomics2.ucsd.edu/ProteoSAFe/status.jsp?task=2cfddd3b8b1e469181a13e7d3a867a6f and https://proteomics2.ucsd.edu/ProteoSAFe/status.jsp?task=184a80db74334668ae1d0c0f852cb77cGNPS NAP jobs: http://ms2lda.org/basicviz/summary/390MS2LDA experiment: Case study 2: Rhamnaceae study—combined analysis of 71 Rhamnaceae plant extractshttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=36f154d1c3844d31b9732fbaa72e9284MASSIVE: MSV000081805 https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=e9e02c0ba3db473a9b1ddd36da72859bGNPS Molecular Networking job: https://proteomics2.ucsd.edu/ProteoSAFe/status.jsp?task=6b515b235e0e4c76ba539524c8b4c6d8GNPS NAP job: http://ms2lda.org/basicviz/summary/566MS2LDA experiment: https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=de2d18fd91804785bce8c225cc94a44MASSIVE: MSV000082678 https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=b817262cb6114e7295fee4f73b22a3adGNPS Molecular Networking job: https://proteomics2.ucsd.edu/ProteoSAFe/status.jsp?task=c4bb6b8be9e14bdebe87c6ef3abe11f6GNPS NAP job: http://ms2lda.org/basicviz/summary/907MS2LDA experiment: GNPS example study used in Jupyter notebook to show MolNetEnhancer based on feature-based molecular networking—subset of American Gut Project:Data from case studies 3 & 4 illustrating MolNetEnhancer applied to classical molecular networking are publicly accessible through the links listed below.Salinospora and 26 Streptomyces bacterial strain extractshttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=9277186021274990a5e646874a435c0dhttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=a507232a787243a5afd69a6c6fa1e508MASSIVE: MSV000078836, MSV000078839 http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=c36f90ba29fe44c18e96db802de0c6b9GNPS Molecular Networking job: https://proteomics2.ucsd.edu/ProteoSAFe/status.jsp?task=60925078e0c148cbaba3593569e983d6GNPS NAP job: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=0ad6535e34d449788f297e712f43068aGNPS DEREPLICATOR 0.005 job: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=e494a63be6d34747a4b8cdfb838ef96eGNPS DEREPLICATOR 0.05 job: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=f1f00c1c20ba4f61ad471d340066df76GNPS VARQUEST 0.005 job: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=f5ffcc8f63ab4e6f96a97caabc11048bGNPS VARQUEST 0.05 job: http://ms2lda.org/basicviz/summary/551MS2LDA annotation experiment: http://ms2lda.org/basicviz/summary/912MS2LDA MolNetEnhancer workflow experiment: Case study 3: Marine-sediment bacteria study—combined analysis of 120 Xenorhabdus and 5 Photorhabdus bacterial strain extractsCase study 4: Nematode symbionts study—combined analysis of 25 https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=dcc30b777c344d668a5626d01f26c9a0MASSIVE: MSV000081063 https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=aaff4721951b4d92b54ecbd2fe4b9b4fGNPS Molecular Networking job: http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=677f076eb04b4518958ca8cd56b4c753GNPS NAP job: http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=338b422483d1432e82afd1bf848f1292GNPS DEREPLICATOR 0.005 job: http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=83bca3c45665470891d41ead275dcae7GNPS DEREPLICATOR 0.05 job: http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=20cfb9af4a244feea102aa9c9da2651cGNPS VARQUEST 0.005 job: http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=a4ffda169823476a9b1e81616aeccbdaGNPS VARQUEST 0.05 job: http://ms2lda.org/basicviz/summary/570MS2LDA annotation experiment: http://ms2lda.org/basicviz/summary/917MS2LDA MolNetEnhancer workflow experiment: https://gnps.ucsd.edu/ProteoSAFe/result.jsp?task=7c4b25d21a6348df9a6942d3071a4b1f&view=advanced_viewMASSIVE: MSV000081098 https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=b76dd5a123e54a7eb42765499f9163a5GNPS Molecular Networking job: https://proteomics2.ucsd.edu/ProteoSAFe/status.jsp?task=cb63770fe307410492468f62f9edb8f3GNPS NAP job: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=4d971b8162644e869a68faa35f01b915VarQuest job: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=c62d3283752f4f98b1720d0a6d1ee65bDEREPLICATOR job: http://ms2lda.org/basicviz/summary/909MS2LDA experiment: GNPS example study used in Jupyter notebook to show MolNetEnhancer based on classical molecular networking—drug metabolism in set of sputum samples:https://github.com/madeleineernst/RMolNetEnhancer and the MolNetEnhancer package in Python including Jupyter notebooks with an exemplary analysis workflow for mapping Mass2Motifs and chemical class annotations onto classical and feature-based molecular networks is publicly accessible at https://github.com/madeleineernst/pyMolNetEnhancer. A beta version of the MolNetEnhancer workflow is also available from within GNPS: https://gnps.ucsd.edu/ProteoSAFe/index.jsp?params=%7B%22workflow%22:%22MOLNETENHANCER%22%7D. This currently outputs the chemical class annotated molecular network by user provided task ids to the individual jobs run within GNPS.The MolNetEnhancer package in R including Jupyter notebooks with an exemplary analysis workflow for mapping Mass2Motifs and chemical class annotations onto classical and feature-based molecular networks is publicly accessible at Tutorials to get familiar with individual tools from which the output is combined with MolNetEnhancer can be found here.GNPS molecular networking: https://ccms-ucsd.github.io/GNPSDocumentation/networkingDEREPLICATOR/VarQuest: https://ccms-ucsd.github.io/GNPSDocumentation/dereplicatorNetwork annotation propagation: https://ccms-ucsd.github.io/GNPSDocumentation/napClassyFire: http://classyfire.wishartlab.comMS2LDA: https://ccms-ucsd.github.io/GNPSDocumentation/ms2lda/http://ms2lda.org/user_guideMolNetEnhancer workflow tutorials in both R and Python can be found here: https://github.com/madeleineernst/pyMolNetEnhancerhttps://github.com/madeleineernst/RMolNetEnhancer


In this review, molecular brushes and other macromolecular architectures bearing a bottlebrush segment where the main chain is synthesized by ring opening metathesis polymerization (ROMP) mediated by Mo or Ru metal complexes are considered. A brief review of metathesis and ROMP is presented in order to understand the problems and the solutions provided through the years. The synthetic strategies towards bottlebrush copolymers are demonstrated and each one discussed separately. The initiators/catalysts for the synthesis of the backbone with ROMP are discussed. Syntheses of molecular brushes are presented. The most interesting properties of the bottlebrushes are detailed. Finally, the applications studied by different groups are presented. In 2005, Yves Chauvin, Robert H. Grubbs and Richard R. Schrock shared the Nobel award in chemistry on their work regarding the development of the metathesis method in organic synthesis . MetatheROMP is a special type of polymerization, where the unsaturated ring of a cyclic olefin opens and the number of unsaturated bonds is retained in the polymer backbone. This is contradictory with the classic polymerization of alkenes, where the main chain consists of single carbon–carbon bonds . The dri4/Et3Al, MoCl5/Et3Al, WCl6/EtAlCl2/EtOH, Re2O7/Al2O3, RuCl3/polar solvents and many more 2/tricyclohexylphosphine (PCy3)/(trimethylsilyl)diazomethane system. The poly(norbornene)-g-poly(ε-caprolactone) bottlebrush copolymers were synthesized employing “graft from” and “graft through” methods. The results were not very encouraging, since low degrees of polymerization were achieved using the “graft through” method [2(PPh3)2CHCHCPh2], was used with little success . Th. Th33]. G3’ from G2 , because the reaction is quite easy experimentally. G3 is air and thermally quite stable and exhibits great functional group tolerance compared to the other carbenes. High molecular weight polymacromonomers have been synthesized with a variety of side chains. Usually, the synthesized polymacromonomers have quite low molecular weight distributions, so model bottlebrushes can be studied. Short polymerization times (<1 h) are common and no byproducts are produced. The living character of the catalyst is exploited in more complex bottlebrushes, like (multi-)block brushes, stars with bottlebrush arms, modifications at the end of the main chain, etc. Summarizing, G3 is commercially available or easy to prepare from G2. The polymerization is fast and well-controlled. Well-characterized (model) molecular brushes bearing a variety of functional groups can be synthesized. Put simply: ‘easy’, fast and good results. The incorporation of Grubbs’ third generation catalyst (and the pyridine derivative) in the “grafting through” toolbox in 2009 ,36,37,38Even though more than three small monomers undergo ROMP, only three have been employed for the synthesis of the main chain of molecular brushes: (a) cyclobutene leading to 1,4-butadiene main chain (8 articles), (b) oxanorbornene (22 articles) and (c) norbornene (>160 articles) . The comUsually, the preparation of the macromonomers requires much more time than the synthesis of the bottlebrush itself. There are two main routes to prepare macromonomers:(a) α-monotelechelic macromonomers. The macromonomer is synthesized from an initiator which bears a norbornene (NBE) moiety . There a(b) ω-monotelechelic macromonomers. There are two ways to synthesize these macromonomers: (i) termination of the polymerization with a suitable norbornene derivative, which attaches the norbornene moiety at the end of the polymeric chain, e.g. termination of anionic polymerization of styrene with an NBE acyl chloride , (ii) prDepending on the side reactions the α- or the ω- route is chosen. The quality of the macromonomers is essential for the preparation of model molecular brushes . If teleG1 catalyst, no significant amount of macromonomer was left unreacted (<1%) 2/PCy3/(trimethylsilyl)diazomethane as the catalytic system for “graft from” and “graft through” ROMPs [G2). Xie’s bottlebrushes exhibited narrow polydispersity (1.2) for DP = 5, but larger ones (1.7–1.8) for DP > 25 [The first attempts to incorporate PCL as side chains in molecular brushes was made in 1998 by Jerome and his team, using [RuClh” ROMPs ,32. Desp DP > 25 . b-PCL [b-PCL [co-PCL [co-PCL [co-PCL, PE-co-PCL-co-PEO, PE-co-PCl-co-PEO-co-PS [co-ClCL) side chains have also been synthesized and Cl was used for side chain functionalization [Diblock molecular brushes A that hab-PCL  and PE-bL [b-PCL . Molecul [co-PCL , PEO-co- [co-PCL , PE-co-PEO-co-PS  have beeEO-co-PS ,44,150. EO-co-PS  and PEO/EO-co-PS . MoleculEO-co-PS . Core–shEO-co-PS . Bottleblization . In 2012, Qiao and his team synthesized a cylindrical bottlebrush polypseudorotaxane with PCL and α-cyclodextrins . This intBA), poly(n-butyl acrylate) (PnBA) and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) are some of the most common side chains, but bottlebrushes for special applications demanding exquisite side chains have also been prepared. A total of 47 papers involving poly(acrylate) and poly(methacrylate) side chains has been published.Poly(acrylate) and poly(methacrylate) linear polymers have found significant applications in the modern society. Molecular brushes bearing such side chains have been synthesized and their properties studied by several scientific teams. Poly(methyl methacrylate) (PMMA), poly(t-butyl acrylate)  were achieved for the bottlebrushes . In 2006b-PtBA [b-PAA [b-PnBA [b-PtBA [pHS-b-(PpHS/PhMI) [b-(PpHS/PhMI) [b-PtBA [b-PAA [b-PnBA [nBA [tBA [tBA/PDMS [tBA/PS [co-PMMA as side chains have been synthesized [Molecular brushes have been synthesized by “graft through” and “graft from” methods. All generations of Grubbs’ catalysts have been used with a variety of macromonomers. Diblock molecular brushes A that hab-PtBA ,157, PI-A [b-PAA , PDLLA-b [b-PnBA , PS-b-Pt [b-PtBA , PTFpHS-HS/PhMI) , P(fluorHS/PhMI) ,115, PEO [b-PtBA ,71,97, P [b-PnBA  have bee [b-PnBA  and PnBAnBA [nBA  side chanBA [tBA ,158, PMMnBA [tBA  and PDMAnBA [tBA  chains pnBA [tBA , PtBA/PDtBA/PDMS , PtBA/PS [tBA/PS  have beethesized . Core–shthesized . More cothesized ,73.b-PtBA nanoparticles exhibited rigidified cylindrical shape and narrowly-dispersed size as revealed by AFM [tBA bottlebrushes with two side chains per monomeric unit by AFM [b-PnBA brush block copolymers by SAXS. AFM also revealed large cylindrical micellar structures of 200-nm wide and several mms long [tBA-b-PEO brush block copolymer. They reported an exceptionally large volume of highly ordered arrays on the order of millimeters, which is 109 times larger than that of typical self-assembled linear block copolymers [tBA-b-PEO, with varying side chains lengths was conducted by the same team [tBA and PDMS/PtBA molecular brushes and studied by Johnson and his team [The morphology of the bottlebrushes has been studied by many researchers. Wooley and her team observed PMMA ellipsoidal shapes with variable sizes by Atomic Force Microscopy (AFM) measurements. Aggregation of the polymer gave interesting surface nanosized irregular patterns . Core-crd by AFM . Fontaint by AFM . Grubbs mms long . Watkinspolymers . A detaiame team . Janus bhis team . Qiao anhis team . PMMA-Fuhis team . Hong, Xhis team . Direct his team .nBA bottlebrushes, which were reversibly cross-linked [Ohe and his team incorporated complex bottlebrush architectures with PMA chains to accomplish targeted tumor imaging ,73. Kasis-linked .In this section molecular brushes that bear side chains other than the aforementioned are listed. Some of the papers have already been mentioned since the molecular brushes contained PEO, PS, P(D)LLA, PCL, P(meth)acrylates too. They are mentioned here to provide useful information to the synthetic chemist mainly.G3’ or by “grafting from” the backbone. Morphologic properties have been investigated in every paper by TEM, AFM, SEM, WAXD, etc. Their thermal properties have been studied as well. Light harvesting, optoelectronics, membrane separation and nanodielectric materials are some of the applications these polymers could be efficient to.Thiophene-containing molecular brushes have appeared in 6 papers ,170,171.Bottlebrushes containing PDMS side chains have been synthesized in 6 cases ,142,172.Molecular brushes with N-phenyl maleimide side chains have been synthesized by Wooley and her colleagues in four cases ,114,115.b-PHIC, PLLA-b-PHIC-b-PLLA and PHIC-b-PLLA-b-PHIC have been synthesized by our team [b-PHIC side chains [Poly(hexyl isocyanate) and poly(4-phenylbutyl isocyanate) have been incorporated in molecular brushes exhibiting photonic crystal properties . Chiral our team  and a coe chains . Their oBottlebrushes with NBE side chains have been synthesized using Mo catalysts by Nomura and his coworkers ,29,41. Tb-PS diblock [Poly(2-vinylpyridine)/PS pseudo-alternating  and poly diblock  moleculaOne article referring to bottlebrushes with poly(vinyl acetate) side chains has been published by Matson and his colleagues .Molecular brushes with poly(N-isopropylacrylamide) (PNIPAAM) side chains has been mentioned in the literature in one case .Bottlebrushes with poly(isobutylene) side chains have been synthesized recently . The relPoly(phenyl isocyanide) side chains have been grown from the norbornene backbone by Wu and his team. “Grafting through” the appropriate macromonomers was proven impossible as the authors mention. The bottlebrushes exhibited worm-like morphology .G1 [In 2001, Allcock and his colleagues “grafted through” polyphosphazene macromonomers. Their main purpose was to copolymerize the macromonomer with small monomers, but molecular brushes were also prepared using G1 .G3 provided the means towards controlled polymerization reactions leading to well-characterized bottlebrushes with narrow polydispersities. This feature enabled the design and synthesis of advanced structures containing bottlebrush segments for special applications.In the last decade, polymer brushes prepared by ROMP of NBE macromonomers have acknowledged great interest from various research teams and more than 100 papers have been published. Since polymerizing macromonomers by the “graft through” method is the best way to synthesize molecular brushes, Grubbs’ third generation catalyst provided the required tool to polymerize macromonomers bearing a variety of functional groups in minimum reaction time. The living character of Drug and gene delivery, tissue engineering scaffolding and tumor imaging are some of the fields where molecular brushes have been studied. Materials for photonic crystals, liquid crystals, batteries, solar cells, lithography have also been synthesized and studied.


Due to their multiple advantages, piezoceramic materials have been widely used in structural health monitoring (SHM). Piezoceramic patch-based smart aggregate (SA) and spherical piezoceramic-based smart aggregate (SSA) have been developed for damage detection of concrete structures. However, the stress waves generated by these two types of transducers are limited by their geometry and are unsuitable for use in two-dimensional concrete structures . In this paper, a novel embeddable tubular smart aggregate (TSA) based on a piezoceramic tube was designed, fabricated and tested for use in two-dimensional (2D) structures. Due to its special geometry, radially uniform stress waves can be generated, and thus the TSA is suitable for damage detection in planar structures. The suitability of the transducer for use in structural health monitoring was investigated by characterizing the ability of the transducer to transmit and measure stress waves. Three experiments, including impedance analysis, time of arrival analysis and sweep frequency analysis, were conducted to test the proposed TSA. The experimental results show that the proposed TSA is suitable for monitoring the health condition of two-dimensional concrete structures. Structural health monitoring ,2,3 and Piezoelectric-based transducers are commonly used for the structural health monitoring of concrete structures. Due to their rapid response times, low cost and availability in different geometries, piezoelectric transducers are a staple of many practical projects ,22,23,24To resolve the aforementioned problems, smart aggregates (SAs) were proposed by Song et al. . In the More recently, a spherical smart aggregate (SSA) was proposed by Kong et al. ,45. In tTo make the shapes of the generated stress wave more compatible with 2D structures, a novel tubular smart aggregate (TSA) was developed in this research. The actuating and sensing core in the TSA is a piezoceramic tube, which can generate a radially expanding stress wave along the plane of slab-like concrete structures while minimizing reflections at the two surfaces. This paper is organized as follows: The fabrication of the TSA is detailed in The proposed TSA was composed of a piezoceramic tube as the core. The inner diameter, the height and the thickness of the piezoceramic tube were 16 mm, 25 mm and 2 mm, respectively. After soldering the wire electrode, a thin layer of epoxy resin was coated over the piezoceramic tube to enhance the strength of the piezoceramic materials and provide a waterproofing layer. Finally, a liquid electrical tape was coated over the entire assembly to provide electrical insulation and further improve the waterproofing. The entire electrical insulation and waterproofing process is shown in Due to the fragility of piezoceramic material, care must be taken during the casting of concrete to prevent damaging the transducers. Therefore, to ensure the durability of the proposed TSA, the piezoceramic tube was encased within a layer of ultra-high-performance concrete (UHPC), which formed a high strength protective layer around the piezoceramic tube. UHPC is a suitable choice due to its high strength compared with normal concrete .To form the UHPC layer, a two-part mold which includes a wooden pedestal and a polyvinyl chloride tube was fabricated. The tube was cut along the length to form two half tubes, which were then bonded together by epoxy resin. Alternatively, if larger scale production is considered, the mold can be fabricated by a three-dimensional (3D) printer. After the mold was prepared, the electrically insulated and waterproofed piezoceramic tube was suspended in the mold by a fine copper wire. After the UHPC was poured into the mold, the completed TSA could be removed from the mold after 28 days of curing. The diameter and the height of the TSA was 25 mm and 40 mm, respectively .The functionality of the TSA was tested by embedding it in a hexahedral concrete specimen. The specimen was 280 mm in length, 280 mm in width, and 200 mm in height. The detailed parameters of the concrete for casting the experimental specimen are listed in Three experiments were conducted to study the characteristics of the proposed TSA. In the first experiment, an impedance analyzer  was used to analyze the natural frequencies of the proposed TSA . Based oThe second experiment aimed to demonstrate the advantage of the proposed TSA over conventional SAs  and SSAsImpedance analysis was performed to study the natural frequencies of the TSA and the operational frequency ranges. As shown in To characterize the abilities of the TSA to generate and receive stress waves in 2D structures, a time of arrival analysis was conducted. The results of the second experiment are shown in From LT is the velocity of the longitudinal waves, ∆L is the distance between the transmitter and the receiver and ∆t denotes the time difference between the actuating signal and the corresponding receiving signal, as shown in According to prior research ,47, the In this way, the velocity of each test can be computed. The results are listed in The received time-domain signal responses of the surface bonded unidimensional SA when using the TSA as the actuator are shown in Meanwhile, the proposed TSA can also clearly receive signals from all directions within the plane. As shown in In this research, a new type of smart aggregate (SA) using a piezoceramic tube is proposed. Compared to the conventional piezoceramic patch-based SAs and the recently reported piezoceramic SSAs, this tubular smart aggregate (TSA) is more suitable for actuating and receiving stress waves in two-dimensional concrete structures. Three experiments were conducted to characterize the TSA. In the first experiment, the natural frequencies of TSA were analyzed to determine the suitable operating frequencies. The unique feature of the TSA in generating and receiving radially uniform stress waves was tested in the second experiment. Finally, the ability of the TSA to implement active sensing was tested by transmitting and receiving swept sine stress waves with the surrounding SAs. The experimental results indicate the feasibility of the proposed TSA in the damage detection of two-dimensional concrete structures. In the authors’ future work, a numerical simulation will be conducted to further study the characteristics of the proposed TSA, including the optimal height of the TSA needed to induce the appropriate stress waves in two-dimensional concrete structures and the optimal thickness of the protective layer outside the TSA. In addition, the performance of the TSA for different applications of damage detection and structural health monitoring will be investigated.


Dopamine is involved in numerous neurological processes, and its deficiency has been implicated in Parkinson’s disease, whose patients suffer from severe sleep disorders. Destruction of nigrostriatal dopaminergic neurons or dorsal striatum disrupts the sleep–wake cycle. However, whether striatal dopamine levels correlate with vigilance states still remains to be elucidated. Here, we employed an intensity-based genetically encoded dopamine indicator, dLight1.1, to track striatal dopamine levels across the spontaneous sleep–wake cycle and the dopaminergic response to external stimuli. We found that the striatal dLight1.1 signal was at its highest during wakefulness, lower during non-rapid eye movement (non-REM or NREM) sleep, and lowest during REM sleep. Moreover, the striatal dLight1.1 signal increased significantly during NREM sleep-to-wake transitions, while it decreased during wake-to-NREM sleep transitions. Furthermore, different external stimuli, such as sudden door-opening of the home cage or cage-change to a new environment, caused striatal dopamine release, whereas an unexpected auditory tone did not. Finally, despite both modafinil and caffeine being wake-promoting agents that increased wakefulness, modafinil increased striatal dopamine levels while caffeine did not. Taken together, our findings demonstrated that striatal dopamine levels correlated with the spontaneous sleep–wake cycle and responded to specific external stimuli as well as the stimulant modafinil. Recent 1R with a circularly permuted GFP (cpGFP) and permits the tracking of dopamine levels by detecting cpGFP fluorescence without activating D1Rs signaling cascades downstream.Classical analytical approaches such as intracerebral microdialysis and electro-chemical voltammetry have been used for the quantitative measurement of extracellular dopamine concentrations, but they provide poor temporal resolution. Using intracerebral microdialysis with a 2-min temporal resolution, a previous study found that dopamine concentrations in the nucleus accumbens (NAc) and prefrontal cortex (PFC) are higher during both the awake state and REM sleep compared to non-REM (NREM) sleep in rats . Anotherin vivo studies. We detected the dLight1.1 fluorescent signals using fiber photometry, while simultaneously collecting polysomnographic recordings in freely behaving mice after environmental or pharmacological manipulations. We found that striatal dopamine levels were at their highest during wakefulness, lower during NREM sleep, and lowest during REM sleep. We also revealed that striatal dopamine levels were correlated with sleep-state transitions. Furthermore, dopamine levels were enhanced in the striatum following the sudden opening of the home-cage door but did not respond to a high-frequency auditory stimulus whether asleep or awake. Moving the mice from their home cage to a new cage also caused striatal dopamine release. Finally, the wake-promoting agent modafinil, but not caffeine, induced the release of striatal dopamine. Taken together, our results provided strong evidence that striatal dopamine levels correlated with wakefulness and could respond to defined stimuli and stimulants.In our current study, we employed an optimized variant of this dopamine sensor called dLight1.1, which is suitable for This study was carried out in accordance with the principles of China Regulations on the Administration of Laboratory Animals, the Decree NO.2 of National Science and Technology Commission of the People‘s Republic of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of Fudan University (permit number: 20140226-024).ad libitum. Every effort was made to minimize animal suffering, and the minimum number of animals required to generate reliable scientific data was used.Male, specific pathogen-free (SPF), inbred C57BL/6 mice (10–14 weeks old weighing 20–25 g) were obtained from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences . The mice were housed at a constant temperature (22 ± 0.5°C and humidity (55 ± 5%), under an automatically controlled 12/12 h light/dark cycle  . Food an12 genome copies per mL. Aliquots of virus were stored at -80°C until stereotaxic injection.The adeno-associated virus (AAV) plasmid pAAV-CAG-dLight1.1 was a gift from Lin Tian (Addgene plasmid # 111067) . A recomin situ for at least 5 min and then withdrawn slowly. After injections, the mice used for in vivo fiber photometry experiments were implanted with an optical fiber cannula , Newdoon, China) into the dorsal striatum. The fiber cannula was implanted 0.2 mm above the virus injection site. After injection and implantation, the mice were placed on a heating pad for post-operative recovery. Mice were housed for at least 2 weeks after injections for complete recovery and to allow viral expression prior to any experiments.Adult mice were anesthetized with pentobarbital  and 1% lidocaine hydrochloride . After shaving the fur on the head and sterilizing the skin with 75% ethanol, the mice were placed on a stereotaxic frame . The skull surface was cleaned with sterile saline on a sterilized cotton swab. Small craniotomy burr holes were made and 100 nL of the AAV-CAG-dLight1.1 virus was unilaterally microinjected through a fine glass pipette into the dorsal striatum (anteroposterior (AP): 0.80 mm, mediolateral (ML): +1.5 mm, dorsoventral (DV): -2.5 mm), according to the Allen Mouse Brain Atlas . The virAs previously described , the EEGAfter 2 weeks of post-operative recovery, each animal was connected to an EEG/EMG recording cable in a recording apparatus (transparent barrel) and habituated for 3 days before polysomnographic recordings were conducted. The uninterrupted, synchronous recordings of EEG and EMG were performed by means of a slip ring, which was designed to let the mice move freely. Cortical EEG and neck EMG signals were amplified and filtered , digitized at a sampling rate of 512 Hz, and recorded by a Power 1401 digitizer and Spike2 software . The Spike2 data were then converted to text format for the analysis of vigilance states using SleepSign software . After the experiment was completed, the EEG/EMG data were automatically classified off-line using 4-s epochs for wakefulness, REM sleep, and NREM sleep using SleepSign software according to standard criteria . These aFollowing the 2-week recovery period from the virus injection and implantation surgery, dLight1.1 fluorescence emission was recorded with a fiber photometry system  using methods similar to previous studies . The fibF/F by calculating ΔF/F = (F -Fmean)/Fmean, where Fmean is the average fluorescence in recording episode. We recorded data for 4–10 h per session and calculated the averaged ΔF/F during periods of wakefulness, NREM, and REM sleep. For the analysis sleep-state transitions, we identified each state transition and aligned ΔF/F with a ±60 s window before and after the switch point. For stimuli analysis, the photometry signal was aligned with a ±20 s window before and after the event onset. For the modafinil, caffeine, and cage-change experiments, we recorded signals for 6 h (from 1 h before to 5 h after the administration of drugs or the cage change) and calculated the averaged ΔF/F value pre- and post-treatment.Photometry data were analyzed by customized Matlab software  as described in our previous study . In brieTo examine whether striatal dopamine levels respond to external stimuli, a high-frequency auditory tonal stimulus  or a sudden door-opening stimulus was applied to mice during NREM sleep or wake periods as previously described . The louOne hour following the onset of the photometry recording, modafinil  was dissolved in sterile saline containing 10% DMSO and 2% (w/v) cremophor and administered intraperitoneally at doses of 45 and 90 mg/kg. Caffeine  was dissolved in sterile saline and given intraperitoneally at a dose of 15 mg/kg. Both drugs were prepared fresh, immediately before use.Histological verification of viral expression was performed as described previously . After at-tests were used for two-group comparisons and one-way analysis of variance (ANOVA) was used for multiple-group comparisons. A two-way ANOVA was used to analyze the experiments with modafinil and caffeine treatment. Following the ANOVA, Sidak or Bonferroni’s post hoc tests were used to make pairwise comparisons. All the statistical tests were two-tailed and P-values less than 0.05 were considered significant. All the statistical analyses were performed using Prism 7.0  and MATLAB R2016a software.Data are presented as the means ± SEM. Paired or unpaired Student’s n = 7 mice; F2,18 = 116.1, P < 0.01; post hoc Tukey test: wake vs. NREM P < 0.01, wake vs. REM P < 0.01, NREM vs. REM P < 0.01). Although the peak value of REM sleep is higher and the trough value is lower . These findings demonstrated that striatal dopamine levels not only varied across spontaneous sleep–wake states but also showed dynamic changes within wakefulness episodes. Next, we assessed the striatal dopamine levels during state transitions. We found that the striatal dLight1.1 signal increased significantly during NREM sleep-to-wake transitions , whereas it decreased during wake-to-NREM sleep transitions . However, there was no significant dLight1.1 fluorescence change during REM sleep-to-wake transitions and NREM-to-REM sleep transitions (P = 0.333 and P = 0.182). Since the diversity of animal behaviors depend on the duration of the wakefulness episode, we further examined whether striatal dopamine levels fluctuated with the duration of wakefulness episodes; we calculated the dLight1.1 signal at longer wake episodes (duration > 30 s) and brief wake episodes (duration < 30 s). Interestingly, the net growth of dLight1.1 fluorescence was significantly higher when mice were awake for longer periods than for brief wake periods . These results indicated that striatal dopamine levels at wake onset were correlated with the duration of the following wake episode. Taken together, these findings demonstrated that striatal dopamine levels were highest during wakefulness and that they fluctuate dynamically across spontaneous state transitions.To examine whether the striatal dopamine levels correlated with distinct vigilance states, we recorded striatal dLight1.1 fluorescent signals across spontaneous sleep–wake cycle. As shown in is lower , the meais lower . We calcP = 0.260 and P = 0.127). These results indicated that acute, short auditory tone stimuli did not elevate striatal dopamine release. Surprisingly, the striatal dLight1.1 signal ascended when the door of the mouse’s home cage was suddenly opened at the end of the trial session. This observation prompted us to systematically investigate whether unexpected door-opening during sleep or wake states induced striatal dLight1.1 signal changes. We discovered that the striatal dLight1.1 signal rapidly increased whenever the home-cage door was opened suddenly during the sleep period  and the wake period . In addition, the amplitude of the striatal dLight1.1 signal induced by sudden door-opening was higher than the amplitude during spontaneous awake periods. Although both auditory tone stimulation and door-opening were able to wake up sleeping mice, the door-opening stimulus elevated dopamine release while auditory stimulation did not. The behavioral paradigm door-opening test maybe mix with visual and olfactory stimuli. In order to explore whether visual and olfactory stimuli enhance striatal dopamine tone, predator odor TMT or light flash were employed. We used light flash at 1 Hz for 10 s when mice were sleeping or awaking , but failed to enhance during sleep . These results indicated that awareness of visual stimuli enhanced the striatal dopamine tone. Application of air or predator odor TMT didn’t enhance striatal dLight1.1 fluorescence  was employed as previously described . We expo awaking , and fouF2,4 = 7.284, P = 0.0464), However, when mice were tail handled followed by moved to new cage, the mean striatal dLight1.1 fluorescence was significantly higher for post 30 min and the following 90 min than baseline . These findings revealed that moving the mice to home cage or new cage induced wakefulness and enhanced striatal dopamine release which sustained for 30 min for mice returned to home cage and for at least 2 h in mice exposed to a new cage.Our previous study showed that dopamine receptors were necessary for arousal when mice were exposed to new environments . However2A receptors (A2ARs) . Moreover, the mean dLight1.1 signals for 2 h after administration of modafinil 90 mg/kg was 13.81% ± 1.55%, significantly higher than 6.83% ± 1.27% for modafinil 45 mg/kg, -0.86% ± 0.55% for caffeine 15 mg/kg, and -0.02% ± 0.40% for vehicle. The dLight signal for modafinil at 45 mg/kg was higher than that for caffeine 15 mg/kg or vehicle, but there was no statistical significance between caffeine 15 mg/kg and vehicle . Taken together, these findings indicated that modafinil increased striatal dopamine levels but caffeine did not.Stimulants such as caffeine and modafinil are universally used to stay awake and to boost mental performance. Our previous studies have shown that caffeine promoted wakefulness via adenosine A (A2ARs) , whereasand D2Rs . A2ARs wstriatum , 2013. hstriatum , consiststriatum . Adminisstriatum . For betUsing a dopamine sensor and simultaneous polysomnographic recordings, we demonstrated that striatal dopamine levels were highest during wakefulness and dopamine fluctuations correlated with spontaneous sleep–wake transitions. Furthermore, we revealed that some external salient stimuli and certain wake-promoting stimulants elicited striatal dopamine release. These findings provide strong evidence that dopamine in the dorsal striatum is important for wakefulness under baseline conditions, induced by cage change or wake-promoting drug modafinil but not caffeine.2R/A2AR-containing neurons, mimicking the action of dopamine on D2Rs, promotes wakefulness (2+)  . Moreoveess (2+) . Recent ess (2+) . Using iess (2+) . These p1R/D2R inactivation. Our previous study revealed that the genetic deletion of D2Rs destabilizes the wake stage and shortens the duration of wakefulness episodes  reduce the duration of wake episodes in mice following being moved to a new cage. The plasma corticosterone levels are elevated after cage change, suggesting that cage change or new environment induces an elevating arousal level  with low affinity  and the odafinil . In addiodafinil . Moreoveil (2ARs . A2ARs astriatum . Previoucaffeine . Chemogecaffeine . The ext the SNc . The dif the SNc . In facty for PD . Our stuy for PD . Hence, Pharmacological, genetic, and clinical studies have demonstrated that striatal dopamine is involved in numerous behavioral and psychological processes that operate on the basis of wakefulness, including motor behaviors, attention, motivation, reward, and feeding. Dysregulation of nigrostriatal dopamine results in severe neurological disorders such as PD and Huntington’s disease with diversified sleep disturbances. Our study demonstrated that striatal dopamine levels fluctuated across the spontaneous sleep–wake cycle and responded to external stimuli and wake-promoting stimulants. By understanding the dynamics of striatal dopamine under various conditions, our findings provide insight into the role of striatal dopamine in sleep regulation and suggest a potential treatment alternative for sleep disturbances in PD.All datasets generated for this study are included in the manuscript and/or the HD, JW, Y-FY, W-MQ, and Z-LH: conceptualization. HD and Y-FY: methodology, investigation, and formal analysis. HD, Y-FY, YS, W-MQ, and Z-LH: writing original draft. HD, JW, Y-FY, YS, W-MQ, and Z-LH: revised the manuscript. W-MQ and Z-LH: supervision and funding acquisition.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.