Enterocytozoon hepatopenaei (EHP) causes hepatopancreatic microsporidiosis (HPM) in shrimp. It is probably endemic in Australasia and was first characterized and named from the giant or black tiger shrimp Penaeus monodon from Thailand in 2009. Later, it was also found to infect exotic Penaeus vannamei imported for cultivation in Asia. HPM is not normally associated with shrimp mortality, but information from shrimp farmers indicates that it is associated with significant growth retardation that is not clearly noticeable until 2–3 months of cultivation. In order to study modes of HPM transmission and to test possible control measures, a laboratory challenge model was needed that would mimic the mode of infection in shrimp ponds.E. hepatopenaei (EHP)-infected shrimp in closed, perforated plastic containers placed in aquaria together with free-swimming, uninfected shrimp. After a period of 14 days all the free-swimming shrimp tested positive by PCR (approximately 60% with heavy infections evident by 1-step PCR positive test results) and gave positive histological and in situ hybridization results for E. hepatopenaei (EHP) in the hepatopancreas.We describe successful transmission in a cohabitation model with natural E. hepatopenaei (EHP) has been developed and used to confirm that E. hepatopenaei (EHP) can be directly transmitted horizontally among shrimp via water. The model will facilitate studies on methods to prevent the E. hepatopenaei (EHP) transmission.A laboratory cohabitation model for studying  Penaeus monodon and P. vannamei in Thailand. These are Agmasoma penaei  at 37 °C for 30 min after which the sections were covered with the DIG-labelled probe (200 ng/slide) in hybridization buffer . A cover glass was added followed by incubation at 42 °C for at least 16 h. This was followed by high stringency washes and equilibration in buffer I , followed by incubation in buffer II [Buffer I containing 0.5% Blocking reagent ] for 30 min at RT. After the incubation, a solution containing alkaline phosphatase conjugated anti-digoxigenin (1:500) was added for 1 h. The unbound antibody was washed off twice using 1X buffer I before the sections were equilibrated in buffer III . At the same time, all of the samples of purported E. hepatopenaei (EHP)-infected shrimp gave positive results by the same test, showing either a single nested PCR amplicon of 176 bp (relatively light infections) or two amplicons (severe infections), one of 779 bp for the first-step PCR and one of 176 bp for the second, nested PCR step.All samples of 10 shrimp from all batches of naïve shrimp tested gave negative results by nested PCR analysis for E. hepatopenaei (EHP). In addition, none of the 6 specimens from day 14 gave positive in situ hybridization results for E. hepatopenaei (EHP). By contrast, all 6 shrimp samples taken from the E. hepatopenaei (EHP) cohabitation aquaria at day 14 were positive for E. hepatopenaei (EHP) by the nested PCR assay, 4 at the first PCR step (indicating severe infections) and 2 at the nested step only (indicating light infections)  none gave positive nested PCR assay results for E. hepatopenaei (EHP) developmental stages such as plasmodia and spores  gave positive hybridization results revealing that many of the HP tubule epithelial cells were infected  infections indicated by the PCR results, even though the samples showed no clear developmental stages such as plasmodia and spores by H&E staining in the single tissue sections examined for each specimen. Although tedious and time consuming, the in situ hybridization method provided a more reliable and accurate assessment of the severity of infection than did H&E staining.H&E stained sections of HP tissue from the PCR positive cohabitated shrimp specimens showed varying levels of infectivity. Some samples showed extensive histological signs corresponding to res Fig. , albeit ted Fig. . This coE. hepatopenaei (EHP)-infected hepatopancreatic tissue [E. hepatopenaei (EHP) in a non-destructive manner. It also allows for the testing of water or feed treatments for possible efficacy in protecting against E. hepatopenaei (EHP) infection via this natural infection route. Protection might result from treatments to kill spores or to interfere with their process of polar tube extrusion that must occur for the spore contents to enter host cells [E. hepatopenaei (EHP) in both directions, i.e., from shrimp to potential carriers and vice versa. At the same time, the previous method of oral challenge using infected HP tissue [E. hepatopenaei (EHP) inactivation by heating, chilling, freezing, etc.The successful cohabitation model described herein overcomes the disadvantage of the previous destructive, oral challenge model using c tissue . It allost cells . The cohP tissue  will stiThe main reason we developed this cohabitation method was that spores we had purified by density gradient centrifugation from hepatopancreatic tissue of infected juvenile shrimp following previously published methods  failed tE. hepatopenaei (EHP) since our cohabitation tests employed infected juvenile shrimp similar in age to those used for spore purification. On the other hand, it is also possible that the juvenile shrimp may produce two different types of spores, one for horizontal transmission and another for transfer to an alternative host. Clearly, much more work is needed on this emerging pathogen to elucidate its host range and life stages.Another possibility is that the spores produced at different host life stages may differ in ability to re-infect the same host. This phenomenon has been previously reported for other microsporidians  where spin situ hybridization probe specific for E. hepatopenaei (EHP) with cultivated P. vannamei sometimes reveal severe infections (approximately 75% hepatopancreatic tissue involvement) with few recognizable intracellular stages of plasmodial development and spore production, as assessed by histological analysis of tissue sections [in situ hybridization assays with the same tissue section were performed in parallel with the standard H&E analysis to better estimate the extent of infection. Nonetheless, the histopathological assay is a tedious and time-consuming process. We, therefore, recommend instead that PCR based on DNA extracts from homogenates of hepatopancreatic tissue of individual or pooled shrimp samples be used for initial E. hepatopenaei (EHP) diagnosis [E. hepatopenaei (EHP) is positively related to pathogen load, it is probably best to test for E. hepatopenaei (EHP) using quantitative PCR methods  transmission between shrimp and suspected carriers. In the meantime, the problems associated with the use of purified spores may be solved allowing for additional bioassays related to issues such as infectious spore dose.The bioassay for inducing laboratory infection is a primary requirement for studying any newly emerging pathogen and our co-habitation model is straightforward and simple. It can be applied to develop protective treatments for water or feed and to test


Antimicrobial resistance (AMR) is a global public health threat. Emergence of AMR occurs naturally, but can also be selected for by antimicrobial exposure in clinical and veterinary medicine. Despite growing worldwide attention to AMR, there are substantial limitations in our understanding of the burden, distribution and determinants of AMR at the population level. We highlight the importance of population-based approaches to assess the association between antimicrobial use and AMR in humans and animals. Such approaches are needed to improve our understanding of the development and spread of AMR in order to inform strategies for the prevention, detection and management of AMR, and to support the sustainable use of antimicrobials in healthcare. Antimicrobial resistance (AMR) – the ability of microbes to grow in the presence of a drug that would normally kill them or limit their growth  – is a mAntimicrobials have played a crucial role in reducing morbidity and mortality from infectious diseases. However, these healthcare gains are now under threat because of the emergence and spread of organisms that are resistant to antimicrobials , 5. In rThe development of AMR is primarily due to selective pressure on microorganisms – as a result of exposure to antimicrobials. There are several mechanisms by which organisms can adapt and become resistant to antimicrobials; these include the production of enzymes, alteration of target sites, alteration of metabolic pathways, alteration of outer membrane permeability and efflux pumps . ResistaThe first step to tackling AMR is to determine the burden and understand its determinants before implementing public health policies to contain it. Whilst the USA and many European countries have surveillance systems in place, these are mostly lacking in low-resource settings. Surveillance of AMR in these regions is limited by the lack of laboratory infrastructure for monitoring infections and detecting resistance. Smaller studies examining the prevalence of AMR in these regions are likely to be hospital-based, and potentially overestimate the prevalence of AMR . DevelopThe use of antimicrobials in clinical medicine has materially decreased the burden of infectious diseases and facilitated complex medical interventions such as organ transplantation and advanced surgery . AntimicStaphylococcus aureus (MRSA) such as clonal complex 398 (CC 398) [mecC MRSA in a wide range of host species [There is an intricate interplay between humans, animals and the environment in relation to the development and spread of AMR , 8, 25. (CC 398)  and mecC species , 30 has  species , 32 – hiEscherichia coli strains can spread from livestock to humans via food consumption [Salmonella Typhimurium DT104 bacterium and its resistance genes are distinct between animal and human populations with limited transmission between the two [The integration of genetic and epidemiological approaches, including targeted and whole-genome sequencing methods, has allowed the examination of the evolutionary origins of pathogens in both human and other animal populations. Analyses of sequence data suggest that a globally distributed MRSA lineage (ST5) may have originated after human-to-poultry transmission . Converssumption , 37, a rsumption . Similar the two . CollectDespite growing worldwide attention, there are substantial limitations in our understanding of the burden, distribution and determinants of AMR at the population level. A recent systematic review of studies assessing the observational evidence for an association between antimicrobial use and development of MRSA resistance found that individual study reporting was poor . This hiWith a lack of new therapeutic agents to target resistant bacterial infections, it is increasingly important to understand the factors that contribute to the emergence of AMR. While most preventative strategies emphasise the need to reduce antimicrobial misuse and overuse by health providers, the lack of advice regarding specific interventions that are likely to have an impact on reduction in AMR in the hospital and community setting limits the implementation of such guidance. Well-designed large-scale epidemiological studies are needed to understand the relationship between AMR and distinct patterns of antimicrobial prescribing and consumption, in order to inform specific guidelines for AMR stewardship in humans as well as agricultural treatment practices. Such research could also facilitate identification of high-risk populations  for AMR transmission, and provide specific guidance for prevention and treatment in these subgroups. There is a need to assess this association in both hospital and community settings, including the use of routine clinical data, electronic health records (EHRs) and concomitant pathogen surveillance, as well as capitalising on experimental/interventional designs . PotentiIn addition to limitations of population-based epidemiological studies of AMR, there is also a gap in individual-level data to assess individual patient outcomes in response to antimicrobial use . ExistinAs well as efforts to increase our understanding of the factors contributing to the emergence and spread of AMR, there are preventative strategies that could help to reduce its burden. Preventing the spread of infections in the first instance would reduce the need to use antimicrobials and, consequently, might moderate the development of resistant infections. There are a number of ways in which this might be achieved. Examples include immunisation, safe food preparation, infection control measures  and improved waste management to prevent the spread of resistant organisms in the hospital, community, agricultural and environmental settings. However, basic health interventions, such as good hygiene practices and adherence to antimicrobial stewardship, can be particularly difficult to achieve in some LMICs . FurtherPatterns of antimicrobial consumption are influenced by multiple factors including self-diagnosis and treatment with over-the-counter medications and local prescribing practices. The lack of point-of-care (POC) tests that are able to reliably distinguish bacterial from viral infections means that patients are frequently prescribed empirical antimicrobials whilst waiting for the results of microbiological tests . The dosIncreasing globalisation also contributes to the development and spread of AMR. Population movement facilitates the spread of resistant infections from areas of high to low prevalence. However, the risk of imported drug-resistant infections is difficult to quantify and better international surveillance is needed to detect and monitor these infections. Deployment of new technologies, such as next-generation sequencing, could help to better understand the origin of organisms, and the role of population movement in the spread of AMR. Furthermore, in LMICs that are undergoing rapid economic development, urbanisation has resulted in overcrowding and poor sanitation. Development may also result in a rise in migration from rural to urban areas. This not only increases exposure of susceptible populations to drug-resistant infections, but further contributes to the problem of overcrowding. Collectively, these factors associated with urbanisation increase the risk of the development and spread of AMR. Better control of the spread of AMR will rely on a multi-sectorial approach and the involvement of stakeholders on a global scale.Antimicrobial stewardship programmes (ASPs) involve a number of interventions to promote prudent antimicrobial use and reduce AMR, through evidence-based treatment regimens. EHR systems have recently been shown to provide a successful framework for ASPs . These pThe advancement of precision medicine will provide additional opportunities to reduce the emergence and spread of AMR. On the one hand, it will enable the delivery of more targeted therapy against the infective organism. On the other hand, it may help to predict to susceptibility to infection, severity of disease or response to treatment in the infected individual. The application of a precision medicine approach may also help to reduce unnecessary antimicrobial prescribing and thus slow the emergence and spread of resistance. However, this approach often requires the availability of accurate, rapid and affordable diagnostic tests if we are to derive the potential health care benefits.Finally, there is an urgent need to develop new therapeutic options for treating drug-resistant infections. Since 1987, no new class of antibiotics has been discovered and drug development has largely relied on structural changes to existing compounds , 54. ChaAMR is a global public health threat. Despite growing worldwide attention to AMR, there are substantial limitations in our understanding of the burden, distribution and determinants of AMR at the population level. The integration of a broad range of context-specific epidemiological study designs is needed to improve our understanding of the burden of AMR in populations, as well as the population-level factors influencing the development and spread of AMR . Togethe


Lactococcus garvieae. The bacteriocin has a broad inhibitory spectrum, inhibiting members of all the 19 species of Gram-positive bacteria tested. Unlike other bacteriocins from Gram-positive bacteria, garvicin KS inhibits Acinetobacter but not other Gram-negative bacteria. Garvicin KS was tested in combination with other antimicrobial agents. We demonstrated synergy with polymyxin B against Acinetobacter spp. and Escherichia coli, but not against Pseudomonas aeruginosa. Similar effects were seen with mixtures of nisin and polymyxin B. The synergistic mixtures of all three components caused rapid killing and full eradication of Acinetobacter spp. and E. coli. In addition, garvicin KS and nisin also acted synergistically against Staphylococcus aureus, indicating different in modes of action between the two bacteriocins. Both bacteriocins showed synergy with farnesol, and the combination of low concentrations of garvicin KS, nisin and farnesol caused rapid eradication of all the S. aureus strains tested. Its broad inhibitory spectrum, rapid killing, and synergy with other antimicrobials makes garvicin KS a promising antimicrobial.The increasing emergence of antibiotics resistance is of global concern. Finding novel antimicrobial agents and strategies based on synergistic combinations are essential to combat resistant bacteria. We evaluated the activity of garvicin KS, a new bacteriocin produced by  Infections caused by antibiotics resistant pathogens from both Gram-positive and Gram-negative bacteria have become of global concern. In addition, the slow discovery and development of new antibiotics is unable to catch up the rapid increasing of antibiotics resistance , 24. SeaThe best-studied of these bacteriocins is the lantibiotic nisin, which inhibits most Gram-positive bacteria but not Gram-negative , 30. HowLactococcus garvieae [We recently described garvicin KS, a bacteriocin produced by garvieae . We repoLactococci were grown in M17 broth  supplemented with 0.4% (w/v) glucose (GM17), Lactobacilli and Pediococci were grown in deMan, Rogosa and Sharpe (MRS)  medium at 30 °C. E. coli, Pseudomonas aeruginosa, and Acinetobacter strains were grown in Lysogeny broth  or Mueller Hinton Broth (MHB)  at 37 °C with shaking at 225 rpm. Other strains were grown in Brain Heart Infusion  broth at 30 °C.Lactococcus lactis (2.5% purity), polymyxin B sulfate and farnesol were from Sigma-Aldrich, Co. . Crude garvicin KS was prepared by following procedure. 1 L of GM17 broth was inoculated with 1% of an overnight culture of L. garvieae KS1546 and incubated at 30 °C for 8 h. The cells were removed by centrifugation at 12,000×g for 25 min and 258 g of ammonium sulfate was dissolved in the cell-free supernatant, and the mixture was left at 4 °C for 24 h. The crude bacteriocin was precipitated by centrifugation at 15,000×g for 30 min at 4 °C, dissolved in 20 mL water and kept in a boiling water bath for 10 min.Nisin from 50) refers to the concentration of antimicrobial agent causing 50% growth inhibition, the MIC50 was detected once the OD600 of testing strains reaches 0.4–0.5. Bacteriocin unit [L. lactis IL1403 in this assay. The checkerboard assays using mixture of antimicrobials were performed in microtiter plates as described above.Growth inhibition was determined in microtiter plates with 200 µL of growth medium in each well as described by Holo et al. . The minInteractions between antimicrobial agents were determined by using the fractional inhibition concentration (FIC). The FIC was calculated as follows: FIC = FICa + FICb + FICc, where the FICa means MIC of A in combination/MIC of A alone, FICb means MIC of B in combination/MIC of B, and FICc means MIC of C in combination/MIC of C alone. Effects were considered as synergistic if FIC was ≤0.5 for two components mixture  and ≤0.7Time-kill assays were performed using cultures grown overnight and then diluted 100× in MHB with different concentrations of antimicrobial agents. The assays were done in triplicate. Total viable count (TVC) was determined by plating on Mueller Hinton agar. The agar plates were incubated at 37 °C, and the TVC was estimated after 24 h incubation. Interactions between antimicrobial agents were interpreted as synergistic when they in mixture caused ≥2-log decrease in TVC compared to the antimicrobial agents alone .L. garvieae strain, is composed of three similar peptides [S. aureus strains, were sensitive (results not shown) tested. The MICs of garvicin KS against a representative selection of the strains are shown in Table Listeria monocytogenes, S. aureus, and vegetative cells of Bacillus cereus. The effect of garvicin KS on spores were not tested. The most sensitive bacteria were strains of the lactic acid bacteria Lactococcus and Enterococcus, with MIC ranging from 5 to 80 BU/mL. 5 BU/mL corresponds to a mixture of 10 nM of each of the pure peptides of garvicin KS [Garvicin KS, produced by peptides . Initialpeptides  on 250 ivicin KS . ConcentA. baumannii, A. iwoffii, and A. calcoaceticus (MIC = 2560 BU/mL). However, other Gram-negative bacteria, such as E. coli and P. aeruginosa, did not show any sensitivity to garvicin KS in this assay.Notably, garvicin KS was inhibitory to Staphylococcus aureus LMGT 3242 was the least sensitive Gram-positive bacterium tested in the microtiter assay, and in time-kill assays we were unable to fully prevent growth of this bacterium even with high concentration of garvicin KS. We therefore used this strain to investigate the effects of combining garvicin KS with other antimicrobials, including nisin, another bacteriocin with a wide inhibitory spectrum [Staphylococci [spectrum  and farnylococci . Using tS. aureus were also studied in time-kill assay. In the experiments, concentrations corresponding to the MIC . We therefore replaced farnesol with polymyxin B in the mixtures.A unique feature of garvicin KS is that it has antimicrobial effects on aumannii , and likA. baumannii and A. calcoaceticus strains tested were both sensitive to polymyxin B with a MIC of 0.63 µg/mL, while A. iwoffii with a MIC of 25 µg/mL is considered resistant [Acinetobacter strains tested, even the polymyxin B resistant A. iwoffii. Similar synergistic effects were seen between nisin and polymyxin B. The combinations of three antimicrobial agents also showed synergistic effects against all the Acinetobacter strains. Compared to the individual antimicrobials combining the three compounds caused about 10-fold and 15-fold MIC reductions for garvicin KS and polymyxin B, respectively . Both bacteriocins showed synergy with polymyxin B. A FIC of 0.4 was found for two- and three components mixture with polymyxin B . Both bacteriocins improved the killing rates compared to polymyxin B alone, but regrowth was seen. The mixture of all three components, however, caused eradication of the bacteria after 8 h exposure.Prompted by the results using  MIC = 0. µg/mL. B MIC = 0. µg/mL. BP. aeruginosa. The MIC for polymyxin B against the strain tested was 0.8 µg/mL. However, there were no synergy effects on their combinations (data not shown).We also tested synergy effects by using garvicin KS, nisin, and polymyxin B against Acinetobacter genus. However, the sensitivity towards garvicin KS varied more than 500-fold between the bacteria tested. Relatively high amounts of garvicin KS were required to kill many of the pathogenic species tested, and these bacteria were not completely killed and regrew after initial killing.Garvicin KS is a new bacteriocin comprised of three similar peptides of 32–34 amino acids . The bacCombining antimicrobial agents offers a potential for increasing antimicrobial treatment efficacy and for reducing resistance evolution, and the use of combination antimicrobial therapy is widely used in the treatment of serious infections . ImportaA. baumannii, but because surviving bacteria are frequently found most patients are recommended to receive combination therapy of polymyxin B with other agents active against A. baumannii [Importantly, garvicin KS showed synergy with polymyxin B, a drug of last resort in the combat infections by multidrug resistant Gram-negative bacteria but which is avoided due to its toxicity at relevant concentrations , 26. Moraumannii , 29. TheS. aureus. The synergy indicates that garvicin KS and nisin have different modes of action, which is different from classical antibiotics. Importantly, against S. aureus they were both synergistic with farnesol, a cheap and harmless compound which has been considered a promising adjuvant for antibiotics [S. aureus tested.Strong synergy between nisin and garvicin KS was also observed against ibiotics . MoreoveIn conclusion, garvicin KS is a promising antimicrobial agent. We have demonstrated activity against a wide variety of bacteria, including pathogenic species known to account for a large number nosocomial infections, often with multi-resistant strains. The mode of action of garvicin KS is unknown, but different from many other used antimicrobials. Furthermore, garvicin KS mixtures with other antimicrobial compounds can be highly efficient by improving killing kinetics and eradication hence lower resistance development.


Emitting dipole orientation is an important issue of emitting materials in organic light-emitting diodes for an increase of outcoupling efficiency of light. The origin of preferred orientation of emitting dipole of iridium-based heteroleptic phosphorescent dyes doped in organic layers is revealed by simulation of vacuum deposition using molecular dynamics along with quantum mechanical characterization of the phosphors. Consideration of both the electronic transitions in a molecular frame and the orientation of the molecules at the vacuum/molecular film interface allows quantitative analyses of the emitting dipole orientation depending on host molecules and dopant structures. Interactions between the phosphor and nearest host molecules on the surface, minimizing the non-bonded van der Waals and electrostatic interaction energies determines the molecular alignment during the vacuum deposition. Parallel alignment of the main cyclometalating ligands in the molecular complex due to host interactions rather than the ancillary ligand orienting to vacuum leads to the horizontal emitting dipole orientation. Iridium-based phosphors show high photoluminescence quantum yield in organic light-emitting diodes. Here, Moon et al. reveal the mechanism responsible for the preferred orientation of iridium complexes in an organic host and highlight the interaction between phosphor and host molecules at play. Therefore, understanding and control of molecular orientation in organic films have been a research topic with central importance in organic electronics and photonics, including the fields of liquid crystals9, organic field effect transistors10, and organic photovoltaics11. In organic light-emitting diodes, the molecular orientation of emitter embedded in the emissive layer has been an issue to enhance the outcoupling efficiency of light pursuing the horizontal alignment of the emitting dipole moment21.The orientation of molecules in molecular films dictates their electrical and optical properties such as charge mobility20. However, it was difficult to assert the reason why the spherical-shaped phosphors have a propensity toward preferred molecular alignment in the emissive layers. A few mechanisms have been proposed to explain the preferred molecular orientation of the Ir complexes doped in vacuum-deposited organic semiconducting layers: molecular aggregation of the dopants leading to randomizing their orientation by suppressing the intermolecular interaction between the dopant and host molecules22, strong intermolecular interactions between electro-positive sides of the dopant, and the electro-negative host molecules promoting parallel alignment of the N-heterocycles of Ir complexes by forming host-dopant–host pseudo-complex mainly participating in 3MLCT transition23, and π–π interactions between the dopant and host molecules on the organic surface bringing alignment of aliphatic ligands to the vacuum side24. Currently, it is not very clear which mechanism most comprehensively describes the origin of the preferred EDO of the heteroleptic iridium phosphors. Moreover, the models are too oversimplified to describe the EDO values quantitatively, which depend on structures of the phosphors and host molecules23. Therefore, the molecular configurations and the interactions responsible for the EDOs of Ir complexes should be established by atomic-scale simulation of the Ir complexes interacting with host molecules during film fabrication.Interestingly enough, it is only in recent years has attention turned to the orientation of emitting dipoles of iridium-based phosphors, the most verified light-emitting dyes with high photoluminescence quantum yield and variety of chromatic spectrum as doped in the emissive layers; probably because their iridium-centered spherical shape and the amorphous surrounding nature in the emissive layers are far from having strong molecular alignments. Recently, some heteroleptic Ir complexes exhibiting efficient electroluminescence in organic light-emitting diodes are reported to possess preferred horizontal emitting dipole orientations (EDOs)In this paper, we carefully examine the vacuum deposition process of phosphors on organic layers using a combination of molecular dynamics (MD) simulations and quantum mechanical analyses. The triplet EDO of heteroleptic Ir complexes doped in organic layers is studied with systematic variations of the molecular structures of both host and dopant. Theoretical prediction of EDO from simulated deposition process reveals excellent quantitative agreement with experimental observations, reproducing the anisotropic molecular orientations of heteroleptic Ir complexes in the emissive layers. In-depth analysis indicates that the molecular orientation originates from the coupling of the cyclometalated main ligand participating in the optical transition with neighbor host molecules rather than from the alignment of aliphatic ancillary ligand toward the vacuum. Close observation of the simulation results indicates that non-bonded interaction energy has a critical influence on the molecular orientation during the deposition.mx-, my-, and mz-axes) was determined by quantum mechanical calculations after optimization of molecular geometry. For iridium-based phosphors, spin-orbit-coupled time-dependent density functional theory (SOC-TDDFT) was employed for the calculation of the triplet TDM vectors for phosphorescence. Second, vacuum deposition of the emitting molecules on organic surfaces was simulated using MD. Finally, the TDM vectors in the molecular axis in each frame of MD were transformed to the vectors in the laboratory axis  by rotation matrix method  to the vertical transition dipole moment (TDMV) follows the trigonometric relationship:μ0 is the magnitude of the dipole moment and squares of the components indicate the intensity of the transition (emission intensity). The EDO describes an average fraction of the horizontal and vertical dipole moment of whole emitters embedded in the emissive layer. An ensemble average of the horizontal dipole moment gives the fraction of horizontal emitting dipole moment in the emissive layer (Θ) as a parameter of the EDO byThe simulation method for obtaining EDO of an emitter in the vacuum-deposited layer is schematically illustrated in Fig. hod Fig. . We deteDetails about the rotation matrix and the vector transformation are given in Methods section.25 Force field of OPLS_200526 and periodic boundary conditions were used for the MD simulations. Organic substrates were prepared by packing of 256 host molecules at 300 K and 1 atm. The simulated substrates have random molecular orientations and similar densities as the experimental results. Detailed steps of preparation of the substrates are given in Supplementary Fig. φC) were analyzed using Eq. 2tmd18, and Ir(dmppy-ph)2tmd19 possessing high Θ values were adopted to investigate the effect of the phosphor molecular structure. The molecular C2 symmetry axis toward the center of the ancillary ligand from the origin located at the Ir atom was set as mz, the orthogonal vector to mz normal to the molecular Ir-O-O plane was set as mx, and my was determined by a cross product of mz and mx in the dopants. The triplet TDM vectors of the three Ir complexes align along the direction of the iridium atom to the pyridine rings by 3MLCT as displayed in Fig. 2tmd, Ir2tmd, and Ir(dmppy-ph)2tmd were φM = 89°, θ = 156°], respectively, indicating that the substituents at the 4-position of the pyridine of the main ligands do not change the direction of triplet TDM vectors much.Chemical structures of the materials used in this study are depicted in Fig. N-carbazolyl)-1,1′-biphenyl (CBP) were selected as host materials to investigate the effect of ground state dipole and conjugation length of the host on the EDO. TSPO1 has large permanent dipole moment due to the polar phosphine oxide group and the asymmetric structure, while UGH-2 and CBP molecules have small ground state dipole moments compared to TSPO1 due to the symmetric structures and the less polar groups. On the other hand, CBP has longer conjugation length than UGH-2 and TSPO1, indicating that CBP has a larger polarizability than UGH-2.Diphenyl-4-triphenylsilyphenyl-phosphineoxide (TSPO1), 1,4-bis(triphenylsilyl)benzene (UGH-2), and 4,4′-bis2tmd and Ir(dmppy-ph)2tmd doped in TSPO1 layers have enhanced horizontal dipole orientation with the Θ values of 0.80 and 0.86, respectively .Experimentally, Ir(ppy)H and C2 axis of the dopants with time on the different hosts are displayed in Supplementary Fig. The simulated variations of the orientation of the TDMblue lines represent the probability density of TDMH 2tmd were 0.63, 0.72, and 0.74 on the UGH-2, CBP, and TSPO1 substrates, respectively. Ir2tmd and Ir(dmppy-ph)2tmd on TSPO1 substrates have the Θ values of 0.76 and 0.82, respectively. In addition, the simulation was performed for Ir(ppy)3, a homoleptic complex exhibiting isotropic EDO when doped in CBP as a refs 23. The distribution of the emitting dipole moment of Ir(ppy)3 was close to the random distribution with a simulated Θ value of 0.67 and random orientation of the C3 symmetry axis of the molecule . Higher Θ values are obtained when population of molecules possessing high TDMH is getting larger with the reduced population with low TDMH.Figure mentclass2pt{minimC2 axes of the phosphors on the organic layers is shown in Fig. 24. One expects that the distribution function follows blue line) if the orientation is random. We can extract several interesting results from Fig. C2 axis of Ir(ppy)2tmd is similar on the UGH-2, CBP, and TSPO1 layers with the average φC of 70°, but the EDO on UGH-2 host is different from the EDOs on other two hosts. Third, the orientation of the C2 axes of Ir2tmd and Ir(dmppy-ph)2tmd on TSPO1 are more random . Fourth, the dopant molecules with vertical TDM (red regions in the stacked histogram with 0 ≤ TDMH ≤ 0.3) have φC close to 90° for all the system, indicating that the ancillary ligands align parallel to the surface. All the results show that there is little correlation between the orientation of TDMs and alignment of the ancillary ligands.The orientation of the The size of the substrates turns out to be large enough to simulate the vacuum deposition of the phosphorescent dyes adequately, as confirmed by the similar results obtained on a larger substrate consisting of 1024 molecules 2tmd is much lower than Ir2tmd and Ir(dmppy-ph)2tmd. These results are the reverse direction from the prediction based on the model and clearly demonstrate, therefore, that alignment of aliphatic ligands to the vacuum side is not a necessary condition for the alignment of EDO in heteroleptic Ir complexes. The reason why it is not required can be understood from the following consideration.Alignment of aliphatic ligands of heteroleptic Ir complexes to vacuum model 3) is not required for preferred horizontal EDO either as shown in Fig.  is not rC2 axis is toward the ancillary ligand (dark blue arrows) and the TDM vector (red arrows) is approximately along the direction from the iridium center to one of the pyridine rings. The alignment of iridium-pyridines determines the orientations of TDM for Ir(ppy)2tmd, Ir2tmd, and Ir(dmppy-ph)2tmd. Figure C2 axis for the horizontal TDM and one configuration for the vertical TDM. Rotation of the C2 axis from the vertical to the horizontal direction can result in the horizontal TDM as long as the TDM is located on the horizontal plane (substrate) with an arbitrary orientation of the ancillary ligand. In other words, horizontal EDO is possible no matter which direction of the ancillary ligand aligns, either toward vacuum or film. On the other hand, the vertical TDM is obtained only when the pyridine rings are aligned perpendicular to the substrate. It accompanies horizontal alignment of the C2 axis (φC~90°) on the configuration. This consideration is consistent with the simulation results in Fig. The relationship between the orientation of molecules and emitting dipole moment can be easily figured out using schematic molecular orientations of a heteroleptic Ir complex shown in Fig.  Ir3′,5′,-mppy2tmdδ) of the phosphor and host molecules. Predicted solubility parameters of the molecules are 16.2 (UGH-2), 18.5 (CBP), 17.2 (TSPO1), 15.3 (Ir(ppy)3), 14.7 (Ir(ppy)2tmd), 13.8 2tmd), and 14.4 MPa1/2 (Ir(dmppy-ph)2tmd), respectively, calculated from OPLS_2005 NPT MD by the equation:27Ev is the internal energy change of vaporization, and Vm is the molar volume, respectively. In general, the differences in the solubility parameters (Δδ) between two components in a chemical mixture can be an indicator of the degree of miscibility, with smaller and larger values of Δδ indicating more and less miscible, respectively. In this work, the host and the phosphor Δδ are much less than 7 MPa1/2, suggesting that all the phosphors are miscible with the hosts28. However, Δδ’s between Ir(ppy)3 and the hosts are smaller than between Ir(ppy)2tmd and the hosts. Since the difference comes from the ppy and tmd groups, it follows that the tmd group is less miscible in the host substrates than the main ligand ppy group, which explains the orientation of the aliphatic ancillary ligand toward vacuum side for Ir(ppy)2tmd. On the other hand, the difference in the solubility parameters among Ir(ppy)2tmd, Ir2tmd, and Ir(dmppy-ph)2tmd comes from the difference in main ligands. The reduced solubility of Ir2tmd and Ir(dmppy-ph)2tmd indicates that both 3′,5′,4-mppy and dmppy-ph groups are less miscible to the host than ppy of Ir(ppy)2tmd and less preference to attachment to the substrate compared to the ppy group. Therefore, the orientations of the ancillary ligand of the two phosphors are more randomized during the deposition, consistent with the simulated distributions in Fig. The distributions of the ancillary ligand can be partly explained by analyzing the differences in Hildebrand solubility parameters (H = 0.1−0.5 for Ir(ppy)2tmd on the UGH-2 host and the energy increases with further increasing of TDMH, thereby resulting in rather a vertical EDO compared to random orientation because the population of TDMH is expected to be concentrated in the regions of low (large) non-bonded interaction energy. On the other hand, non-bonded interaction energies of Ir(ppy)2tmd on CBP and TSPO1 layers, and the energies of Ir2tmd and Ir(dmppy-ph)2tmd on TSPO1 are lowered as TDMH increases. As a result, molecular alignment with horizontal TDM is energetically preferred when they are deposited onto the organic semiconducting layers. Furthermore, much lower energies were obtained from Ir2tmd and Ir(dmppy-ph)2tmd than Ir(ppy)2tmd on the TSPO1 layer, indicating that the increased EDOs are also related to the stabilization by neighbor molecules. The calculated non-bonded interaction energy and the statistical results indicate that the host–dopant interaction plays a pivotal role in orienting heteroleptic Ir complexes and the force applies to the alignment of the iridium–pyridine bonds of the phosphors toward the horizontal direction.Non-bonded interaction energy is calculated as a summation of van der Waals and Coulomb interaction energies from the MD simulation to investigate if the intermolecular interaction between the phosphor and neighbor host molecules is responsible for the spontaneous molecular alignments of the phosphors on the surfaces. Figure 2tmd, TSPO1:Ir(ppy)2tmd, TSPO1:Ir2tmd, and TSPO1:Ir(dmppy-ph)2tmd systems decrease, whereas the energy in UGH-2:Ir(ppy)2tmd increases as the ratio of the horizontal transition dipole moment (TDMH) increases. For a polar host molecule of TSPO1, Coulomb energies of the phosphors are lowered as TDMH increases. The variation of vdW energy depending on the molecular orientation was 3–5 kcal/mol, which is larger than the variation of Coulomb energy of 0–1 kcal/mol. The results indicate that vdW interactions (dipole-induced dipole and induced dipole-induced dipole interactions) between the aromatic ligands and the nearest host molecules are the main mechanism contributing to the molecular alignment of the phosphors. The Coulomb interaction helps further alignments of the phosphors if polar host materials are employed. For instance, Ir(ppy)2tmd and Ir2tmd have a quadrupole composed of two dipoles from pyridines (δ+ charge) to Ir atom (2δ− charge). If there is a dipole in host molecule , dipole and quadrupole interaction [‒P = Oδ− and δ+H(pyridine)] anchors one phosphor molecule to two host molecules, leading to a rather horizontal orientation of iridium–pyridines bond of the phosphors, which is approximately parallel to the TDM. In contrast, if host molecule has the positive surface potential 3-Si-phenyl-Si-(Phenyl)3δ+ in UGH-2), there must be a repulsive force between pyridine of phosphors and host molecules so that pyridine ring must be pushed to vacuum. Dispersion force between the conjugated phenyl substituents of Ir(dmppy-ph)2tmd and nearest neighbors anchors the pyridines onto the surface as well and lowers the energy with the molecular long axis lying on the surface. Meanwhile, random EDO of Ir(ppy)3 is attributed to three intermolecular interaction sites, resulting in random orientation of the molecule.The type and magnitude of the non-bonded interactions are different for different phosphors and hosts, leading to different EDOs. The separated vdW and Coulomb energies as a function of the molecular orientation are depicted in Fig. 2tmd, TSPO1:Ir(ppy)2tmd, and TSPO1:Ir(dmppy-ph)2tmd, respectively. Large vibrations, rotations, and diffusions of Ir(ppy)2tmd on the surface of the UGH-2 layer without lowering the energy were observed in the trajectory shown in Fig. 2tmd faced toward an oxygen atom of TSPO1 with the −P = O··H(pyridine) distance around 0.4 nm at t = 2472 and 4560 ps, thereby the parallel alignment of the Ir-pyridines of Ir(ppy)2tmd to the surface. Larger quadrupole moment of Ir2tmd than that of Ir(ppy)2tmd increased the strength of quadrupole–dipole interaction and resulted in enhancement of fraction of the horizontal dipole compared to Ir(ppy)2tmd. The horizontal EDO of Ir(ppy)2tmd in CBP could be understood by the dipole inducement in carbazole groups of CBP when the positive pole of pyridine approaches, but the interaction strength for the iridium−pyridines alignment between Ir(ppy)2tmd-CBP is smaller than that between Ir(ppy)2tmd−TSPO1. Compared to the former cases, the picture of Ir(dmppy-ph)2tmd shown in Fig. 2tmd in Fig. Figure 2tmd has larger quadrupole moment than Ir(ppy)2tmd resulted in further molecular alignment for the horizontal emitting dipole moment. The increase of the dispersion force along the direction of TDM was also effective on control of the molecular orientation for the horizontal EDO with lowered non-bonded interaction energy.The origin of the molecular orientation and EDO of doped heteroleptic iridium complexes in vacuum-deposited organic layers is investigated using MD simulations and quantum mechanical analyses in direct comparison with experimental observation. Careful analyses of the simulation results revealed that molecular alignments of the phosphors are spontaneous by local electrostatic and van der Waals interaction with nearest host molecules interacting in a smaller scale than a molecule. The orientation of the TDM vector of the phosphors on the organic surfaces follows the direction of the ligand mainly participating in optical transition, such as pyridines in ppy, in the molecular alignment, whereas the alignment of ancillary ligand does not have a direct correlation with the EDO. Attractive interactions between pyridines of a phosphor and CBP (quadrupole-induced dipole interaction) or TSPO1 (quadrupole–dipole interaction) anchor the phosphor onto host molecules with the parallel iridium-pyridine alignment, thereby increasing the horizontal EDO. IrT1 to S0 were also calculated via SOC-TDDFT. The DFT and SOC-TDDFT calculations and the follow-up analyses were performed with Schrodinger Materials Science Suite25 along with the quantum chemical engine, Jaguar29. A TDM having the largest oscillator strength among the three degenerated states of T1 level  obtained from the density functional calculations was used in this study as a representative TDM. All MD simulations were performed by Desmond31, a MD engine implemented in the Schrodinger Materials Science Suite. Equilibration simulations prior to deposition were performed in NPT ensembles, where pressure and temperature were set constant via Nose–Hoover chain and Martyna–Tobias–Klein method, respectively. There were no explicit constraints to geometry and/or positions to any of the molecules that were introduced in the simulation box. The simulations were performed over NVIDIA general-purpose GPU cards (K80).Density functional theory (DFT) was used to obtain molecular geometries and electrostatic potentials of host and phosphors. Triplet TDMs of the same phosphors from α, β, and γ are defined as the clockwise rotations to laboratories axes of nx-, ny-, and nz-axes, respectively. Then, the rotation matrixes for α, β, and γ rotations are followings:Rotation matrix method was used for transformation of the TDM vector from the molecular coordinate to the laboratory coordinate. Rotation angles of αβγ rotations of the dopant molecules were extracted in every configurations of the MD simulation. A product of the three rotation matrixes gives a matrix representing the orientation of the dopant molecule bySequential Finally, the TDM vectors in the laboratory coordinate were obtained byf, indicates a probability of a variable X between X = a and b.Integration of a probability density function, To calculate the probability density function of fX) is obtained asIf we put The authors declare that all data supporting the findings of this study are available in the article and in Supplementary Information file. Additional information is available from the corresponding author upon request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


The detection of dopamine in a highly sensitive and selective manner is crucial for the early diagnosis of a number of neurological diseases/disorders. Here, a report on a new platform for the electrochemical detection of dopamine with a considerable accuracy that comprises a 3D porous graphene oxide (pGO)/gold nanoparticle (GNP)/pGO composite-modified indium tin oxide (ITO) is presented. The pGO was first synthesized and purified by ultrasonication and centrifugation, and it was then further functionalized on the surface of a GNP-immobilized ITO electrode. Remarkably, owing to the synergistic effects of the pGO and GNPs, the 3D pGO-GNP-pGO-modified ITO electrode showed a superior dopamine-detection performance compared with the other pGO- or GNP-modified ITO electrodes. The linear range of the newly developed sensing platform is from 0.1 μM to 30 μM with a limit of detection (LOD) of 1.28 μM, which is more precise than the other previously reported GO-functionalized electrodes. Moreover, the 3D pGO-GNP-pGO-modified ITO electrodes maintained their detection capability even in the presence of several interfering molecules . The proposed platform of the 3D pGO-GNP-pGO-modified ITO electrode could therefore serve as a competent candidate for the development of a dopamine-sensing platform that is potentially applicable for the early diagnosis of various neurological diseases/disorders. Dopamine is a part of the catecholamine family that is produced by the dopaminergic neurons in the brain . DopaminAmong the currently available methods  ,12,13, t2–graphene/GCE, and GO/GCE electrodes have been developed to enhance the performances of dopamine biosensors [To this end, numerous attempts have been made to overcome the selectivity and sensitivity issues by functionalizing the surface of the electrodes or integrating other types of conductive materials. Graphene, composed of pure carbon molecules that are arranged in a two-dimensional (2D) honeycomb structure, has been widely applied for various research areas, such as batteries, display panels, solar cells, and even biomedical applications ,20,21,22osensors ,26,27. Hosensors , was disosensors . DespiteThe ITO electrode was obtained from U.I.D. , and the gold nanoparticles with a diameter of 60 nm were purchased from BB Solutions . The single-layer graphene oxide (275 mg/L) that was dispersed in water was acquired from Graphene Supermarket , and polydimethylsiloxane (PDMS) was obtained from Dow Corning Corp., . Lastly, (3-Aminopropyl)triethoxysilane (APTES), Dulbecco’s phosphate-buffered saline (DPBS), cysteamine hydrochloride, dopamine, and the interfering materials used for the selectivity test including the AA and glucose (dextrose) were all purchased from Sigma Aldrich .To synthesize the pGO, the GO dissolved in the distilled water (DW) was first sonicated for 30 min to disperse the aggregated GO particles. Thereafter, 20 mL (200 μg/mL) of the sonicated GO solution was subjected to an ultrasonic homogenizer for 12 h using a sharp ultrasonic probe (diameter: 5 mm). Cooling units were not utilized to increase the temperature during the ultrasonication process. The GO solution was then distributed into 1.5 mL conical tubes and centrifuged for 10 min . The supernatant was collected and used for TEM imaging and electrochemical studies.2, 0.5 mm thickness) through a consecutive sonication process with a 1% Triton X-100 solution, DW, and ethanol. The electrodes then underwent a treatment of oxygen plasma for an effective attachment of the APTES, and this was followed by a 15 min coating with a 5% APTES solution. After the removal of the residual APTES solution with the ethanol, the electrodes were baked for 2 h in an oven at 80 °C for an improved annealing, resulting in stable covalent bond between the –OH groups of ITO electrode and silicon oxide groups. A chamber, however, was needed for the electrode to contain a small volume of the solution for the detection, and such a container was fabricated on the surface of the electrodes using a commercially available circular plastic chamber with a 1 cm diameter and PDMS as an adhesive. Subsequently, the gold nanoparticles were distributed into the chamber, and the electrodes were kept in the refrigerator (4 °C) overnight. The gold nanoparticles were then washed off with DW, and the electrodes were coated with the cysteamine hydrochloride and GO solution for 2 h and 1 h, respectively. As well-known, the thiol group of cysteamine could form stable Au-S bond on the surface of GNPs via self-assembly process, leaving free amine groups that are effective for the immobilization of GNPs. Since ITO substrates were functionalized with APTES for GNP immobilization prior to the cysteamine modification, both the ITO and GNP were positively-charged in the aqueous medium owing to the presence of free amine groups, which could attract negatively-charged pGO via electrostatic interactions. Lastly, the cysteamine hydrochloride and GO solutions were washed off with the DW after the coating process.The preparation of the working electrodes involved cleaning the ITO-coated glass , D = diffusion coefficient (m2/s), v = scan rate (V/s) and C = concentrations of analytes (mol/L).Cyclic voltammetry (CV) and amperometric detection were conducted using a DY2000 Series Multi-Channel Potentiostat (Digi-Ivy). While the ITO working electrodes were used for the dopamine-detection platform, the Ag/AgCl (1 M KCl) and a platinum wire were each used as the reference electrode and the counter electrode, respectively. For the CV, various dopamine-solution concentrations were prepared and placed in the chip chamber for the detection. The scan rate and the range are 0.05 V/s, and −0.1 to 0.6, respectively. Lastly, for the selectivity test, the amperometric detection was completed after 20 μL of the 10 μM dopamine, the AA, and glucose were added to the DPBS-filled electrode chamber. The sampling time, initial voltage and sensitivity are 0.05 s, 0.3 V, and 10N = 3). The significant differences were determined for p < 0.05.The height of the cathodic peaks (Ipc) in the cyclic voltammogram were used for the quantitative analysis. The data were analyzed using the computerized statistical program Origin 8 or Microsoft Excel 2013. Data are expressed as mean ± SE  and G (1580 cm−1) peaks were observed in both substrates in contrast to substrates A and C. Generally, the G band indicates the in-plain vibrations of SP2-bonded carbon atoms, while the D band represents the out-of-plane vibrations . The I (D/G) values of Substrate C and Substrate D were calculated as 0.98 and 0.96, respectively, indicating that the pGO structure is similar to that of the GO. Accordingly, the conclusion here states that, based on the TEM and SEM images and the Raman spectroscopic results, the pGO was successfully synthesized and modified on the surface of the ITO-GNP substrates, thereby elucidating its application possibility regarding electrochemical dopamine detection.After the confirmation of pGO structure, four different substrates: ITO electrode (Substrate A), gold nanoparticle-modified ITO electrode (Substrate B), pGO-modified ITO electrode (Substrate C), and ITO-pGO-GNPs-pGO were characterized by scanning electron microscopy and Raman spectroscopy. As shown in pa– Epc| values (peak-to-peak separation) are 0.47 V, 0.24 V, 0.176 V, and 0.066 V for Substrate A, Substrate B, Substrate C, and Substrate D, respectively, while the calculated Ipa/Ipc values are 1.22, 0.33, 0.96, and 1.03 for Substrate A, Substrate B, Substrate C, and Substrate D, respectively. Consequently, these values indicate that the redox reaction of dopamine on those electrodes was quasi-reversible. Of the four different substrates, however, the dopamine CV curve of Substrate D most resembles the ideal reversible redox reaction . The Ip values of Substrate D in the presence of the 10 μM of dopamine are respectively 42.8, 6.88, and 1.67 times higher than those of Substrate A, Substrate B, and Substrate C, implying that Substrate D is the most suitable candidate for the electrochemical detection of dopamine. Moreover, Substrate D even resulted in a highly enhanced reduction current in the presence of 1 μM of dopamine, while the other substrates either failed to detect the presence of dopamine, or nominally produced insignificant levels of electrochemical signals under the same condition.As previously mentioned, dopamine is vital to several human bodily functions, and numerous studies have reported effective ways to detect dopamine with a considerable accuracy. However, owing to the distinct nature of dopamine that reduces or oxidizes at specific electrical potentials, electrochemical tools have gained significant attention for their capability to detect dopamine in a simple, rapid, and sensitive manner. After the confirmation of pGO structure as discussed in 2 based on the voltammogram obtained using K3Fe(CN)6 as a redox couple and the Randles-Sevcik equation  showed the best performance in terms of dopamine detection, as confirmed in Remarkably, even the 0.1 μM of dopamine showed weak redox peaks that were distinguishable with the control group (DPBS without dopamine). The reduction and oxidation peaks on the cyclic voltammogram clearly increased with increasing the dopamine concentrations from 0.1 μM to 50 μM, as shown in Since dopamine generally co-exists with the other molecules in the blood plasma including the AA, UA, glucose, and protein, it is important to confirm the selectivity of the developed electrode toward dopamine detection. As an interfering molecule, AA and glucose were chosen for amperometric dopamine detection. As shown in Conclusively, the new material reported in this paper, 3D pGO-GNP-pGO, underwent two perceptible alterations in its surface property. First, the graphene oxide substrate was imbued with substantial porosity on its surface (pGO), and the porous surface was further incorporated with GNPs. The new material has proven its suitability for the electrochemical detection of dopamine, and this could be attributed to the changes on the substrate surface that were undertaken in this research, which eventually delivered two benefits: increased specificity and selectivity.First, the porous nature of the synthesized pGO demonstrated higher efficiency in dopamine detection than that of a typical GO sheet. The enhanced specificity was retained even in low concentrations of dopamine (10 μM). The reduction peaks that were obtained from the 3D pGO-GNP-pGO are 42.8, 6.88, and 1.67 times higher than those of the bare ITO, ITO-pGO, and ITO-GNP electrodes, respectively The superior specificity of the 3D pGO-GNP-pGO substrate for dopamine detection is still unclear, however, but it is reasonable to expect that the presence of many hydroxyl groups on the pGO plane might facilitate hydrogen-bonding formation between the dopamine molecule and the pGO. Furthermore, it could be postulated that the nanoholes on the pGO plane assisted the electron transfer from the dopamine molecules to the GNP surfaces, which could contribute to the enhancement of the electrochemical-redox signals. On the other hand, the ability of the pGO in the detection of dopamine was reportedly further improved by the incorporation of GNPs, which are commonly known to enhance electrochemical-signals. The selectivity of the 3D pGO-GNP-pGO toward the dopamine detection was further confirmed with amperometry analysis in the presence of other interfering molecules including 10 μM of the AA and glucose. However, further studies need to be conducted to thoroughly investigate the physicochemical properties of pGO, especially in terms of the interaction between the pGO and dopamine molecules.In conclusion, the 3D pGO-GNP-pGO is excellent for dopamine detection and could be utilized for the development of biosensors that are capable of detecting other types of neurotransmitters , and these might be helpful for the early diagnosis of various neurological diseases .


Lactobacillus spp. in female (sixteen-fold) versus male mice (two-fold). In addition, combined with alterations in gut microbiota, KS also significantly reduced body weight while maintaining normal gut homeostasis. Altogether, we first demonstrated a sex-dependent effect of KS on gut microbiota and highlighted that it may be used as a novel prebiotic for disease management.Keratan sulfate (KS) represents an important family of glycosaminoglycans that are critical in diverse physiological processes. Recently, accumulating evidence has provided a wealth of information on the bioactivity of KS, which established it as an attractive candidate for drug development. However, although KS has been widely explored, less attention has been given to its effect on gut microbiota. Therefore, given that gut microbiota plays a pivotal role in health homeostasis and disease pathogenesis, we investigated here in detail the effect of KS on gut microbiota by high-throughput sequencing. As revealed by heatmap and principal component analysis, the mice gut microbiota was readily altered at different taxonomic levels by intake of low (8 mg/kg) and high dosage (40 mg/kg) of KS. Interestingly, KS exerted a differing effect on male and female microbiota. Specifically, KS induced a much more drastic increase in the abundance of  N-acetylglucosamine (GlcNAc), KS stands out as the only type of GAGs that does not bear an uronic acid residue [N-linked to the asparagine resides in protein and is usually found in corneal and the epidermis of animal skin  [O-linked to the serine and threonine residues in protein and is much more often detected in cartilage [Keratan sulfate (KS) is a glycosaminoglycan (GAG) that is widely distributed in the extracellular matrices of animal tissues such as bone, cartilage and cornea ,2. As a  residue . Currentmodulin) ,3,4. In artilage ,2.As a functional macromolecule, KS has been comprehensively studied . For exaGut microbiota is a newly discovered “metaorgan” that has fundamental roles in almost every aspect of host physiology ,9,10. ReLactobacillus spp. and Bifidobacterium spp. [B. bifidum [Lactobacillus spp. in the gut of rats [Lactobacillus spp. In the present study, to verify this hypothesis, we analyzed the gut microbiota of Kunming mice after low-dose and high-dose of KS treatment by high-throughput sequencing. As expected, oral intake of KS induced a dramatic change in the composition of the gut microbiota in both male and female mice. However, interestingly, the prebiotic effect of KS was observed to be much more pronounced in female than in male mice. Collectively, our study indicates that KS can be used as a new prebiotic candidate and highlights that potential therapy should be tailored to the individual patient when KS is used as a prebiotic ingredient.Prebiotics are defined as microbiota accessible carbohydrate that could beneficially modulate gut microbiota by increasing the abundance of ium spp. ,26. Prev bifidum . This st of rats  and mice of rats . Given t of rats ,32, and  of rats ,2, we exLactobacillus spp. in Kunming mice. Our results rationalize the commercial utilization of KS as a novel prebiotic substrate and will benefit the development of KS as a new generation of marine drug.The gut microbiota is an extremely dense and diverse microbial community which plays a pivotal role in both health homeostasis and disease pathogenesis ,9,22. GiDuring the experiment, no significant clinical symptoms were observed and all mice remained in good health. To fully demonstrate the effect of KS on commensal bacteria, high-throughput sequencing covering the V3-V4 hypervariable regions of 16S rRNA was applied. The Good’s coverage (>99%) indicates a sufficient sequencing depth for all samples . After tTo validate this hypothesis, we constructed a Venn diagram based on shared OTUs and performed the principal component analysis (PCA) and 3D-principal coordinate analysis (3D-PCoA) . Served Previously, we have identified a similar sex-dependent effect of chondroitin sulfate and its oligosaccharides on gut microbiota . Here inGiven that KS changed the overall structure of intestinal microbiota, the bacterial populations in each group were then compared at the phylum level. As revealed by Heatmap, both female microbiota and male microbiota were dominated by Firmicutes, Bacteroidetes and Proteobacteria . HoweverBacteroidales S24-7 group norank, Alistipes spp., Lachnospiraceae NK4A136 group and Bacteroides spp.  producers, could significantly increase the abundance of SCFAs in the gut and regulate energy homeostasis by promoting satiety [The gut microbiota is composed of Gram-negative and Gram-positive bacteria and both of these microorganisms could trigger inflammation in the host once gut symbiosis is disturbed. Lipopolysaccharide (LPS) is an endotoxin that is only produced by Gram-negative bacteria, which, once entered the circulation system, can stimulate inflammation with the most potent capability ,60. Simi satiety . Toll-like receptors (TLRs) are a family of evolutionary conserved receptors which play a pivotal role in host defense against invading pathogens ,63. TLRsOur study has two limitations. First, as we pooled the DNA samples before sequencing, it is inevitable that information about inter-animal variations was lost. Although studies using pooled DNA samples for sequencing were still acceptable ,68, we aIn summary, we first demonstrate a prebiotic activity of KS from shark cartilage using in vivo models. And in accordance with the sex-specific impact of KS on gut microbiota, the prebiotic effect of this molecule was found to be more pronounced in female mice than in males. Our current results indicate that KS can be potentially used as a novel prebiotic candidate for disease management, which merits further investigation.KS was isolated and purified from chondroitin sulfate (CS) using the method adapted from Galeotti et al. . The CS Pasteurella pneumotro pica, Klebsiella pneumonia, Staphylococcus aureus, Streptococcus pnemoniae, β-hemolyticstre ptococcus, Pseudomonas aeruginosa, Helicobacter pylori, Pneumonia Virus of Mice, Reovirus type III, Minute Virus of Mice, Theiler’s Mouse Encephalomyelitis Virus, Mouse Adenovirus and Polyoma Virus. During the experimental session, all mice were housed in a controlled environment  in groups of three mice per cage, and kept with free access to the same batch of standard laboratory diet  and autoclaved fresh water. The diet is composed of 9.2% water, 22.1% casein, 5.28% lard, 9.32% cellulose, 24.08% corn starch, 23.9% sucrose, 1.24% calcium carbonate, 0.92% potassium citrate, 1.34% l-cystine, 0.95% vitamin mix, 0.95% mineral mix, and 0.72% methionine. After one-week adaptation, all animals were randomly assigned to six groups (n = 6 per group): Male control group (KSNM), male low-dosage group (KSLM), male high-dosage group (KSHM), female control group (KSNF), female low-dosage group (KSLF), female high-dosage group (KSHF). The control groups were given normal saline. The treated groups were given either a low-dosage (8 mg/kg) or a high-dosage (40 mg/kg) of KS by gavage. After six weeks of treatment, all mice were sacrificed by cervical dislocation. Blood samples were collected after a 12 h fasting period and centrifuged at 12,000× g for 20 min to pellet the blood cells. The serum lipopolysaccharide (LPS)-binding protein (LBP) levels were determined using a commercial ELISA kit  according to the manufacturer’s instructions. The cecal contents in each mouse were collected aseptically and stored at −80 °C before being analyzed.All animal experimental procedures used in the present study were approved by the Ethical Committee of Ocean University of China and complied with the National Guidelines for Experimental Animal Welfare  and the Guide for the Care and Use of Laboratory Animals . BrieflyThe metagenomic DNA from each cecal sample was extracted using a QIAamp DNA Stool Mini Kit  according to the manufacturer’s instructions. The DNA concentration was quantified using a Nanodrop ND-2000 UV-VIS spectrophotometer  and the quality of DNA was checked by gel electrophoresis. Based on different treatment protocols, the DNA samples in the same group were pooled at equimolar concentrations to generate six samples before being analyzed ,67,68. AQuality control of the raw sequencing data was performed by QIIME (version 1.17) using the criteria previously described . The Opep < 0.05 versus the control group.Data for the body weight and serum LBP levels are expressed as the mean ± standard deviation. Statistical analyses for the body weight and serum LBP levels were performed by one-way ANOVA and Bartlett’s test . All results were considered statistically significant at *


Molecular weights (molar masses), molecular weight distributions, dissociation constants and other interaction parameters are fundamental characteristics of proteins, nucleic acids, polysaccharides and glycoconjugates in solution. Sedimentation equilibrium analytical ultracentrifugation provides a powerful method with no supplementary immobilization, columns or membranes required. It is a particularly powerful tool when used in conjunction with its sister technique, namely sedimentation velocity. Here, we describe key approaches now available and their application to the characterization of antibodies, polysaccharides and glycoconjugates. We indicate how major complications, such as thermodynamic non-ideality, can now be routinely dealt with, thanks to a great extent to the extensive contribution of Professor Don Winzor over several decades of research. Sedimentation equilibrium (SE) analysis in the analytical ultracentrifuge was introduced by Svedberg and Fåhraeus  nearly aM , or equivalently the ‘molar mass’ (g/mol), is one of the most important parameters defining a macromolecule. SE in the analytical ultracentrifuge is a well-established method for obtaining the molecular weights of polymers . The situation has been worse for SE compared to sedimentation velocity because the former generally requires high concentrations to register sufficient optical signal for analysis. Both these drawbacks have now been dealt with.The molecular weight z-averages) and also oligomeric states of assemblies using the SEDFIT-MSTAR procedure, which does not have the requirement of the assumption of a model. Then, if there is a suggestion of an interaction  more advanced analysis of molecular weight distributions can be made, as recently reviewed (http://www.ncbi.nlm.nih.gov/pubmed/23377850).Analysis procedures start with the basic analysis of molecular weight averages (primarily the weight and http://www.ncbi.nlm.nih.gov/pubmed/23377850). These strategies include the global fitting of many SE signal profiles acquired at different loading concentrations, different rotor speeds and different data acquisition with models that create constraints through implicit mass conservation and different interaction models, yielding binding affinities and stoichiometries , became a relative measure of the size of macromolecules , although unless combined with measurement of the translational diffusion coefficient to eliminate the frictional/ shape contribution, these did not provide an absolute measure of molecular weight. The sedimentation equilibrium technique—wherein the sedimentation and diffusive forces come to equilibrium, leading to a steady-state concentration distribution not affected by frictional/shape considerations—established soon after by Svedberg and Fåhraeus  versus r are conventionally obtained using either UV/visible absorption optics  or Rayleigh interference/refractometric optics (any macromolecule). Distributions of c(r) versus r can also be obtained in principle using fluorescence optics. In older or specially adapted instrumentation, Schlieren (concentration gradient) optics directly gives dc(r)/dr versus r  and polydisperse systems, such as polysaccharides and many glycoconjugates. Because of thermodynamic non-ideality, the Mw returned will be an apparent value Mw,app. Thermodynamic non-ideality arises from co-exclusion and polyelectrolyte effects, which, in some cases, under conditions of high dilution are not significant, but otherwise need to be taken into account. These effects can be corrected for by measuring and extrapolating 1/Mw,app to zero concentration (c = 0), although for many systems  such effects are almost negligible at low concentration (∼0.5 mg/ml). SEDFIT-MSTAR Mw,app changes with local concentration c(r) in the ultracentrifuge cell and provides an estimate of the molecular weight distribution and Mz,app.The SEDFIT-MSTAR procedure is a model-independent SE analysis routine for obtaining primarily the weight-average molecular weight M* function . k is defined by:for sector shaped solution columns with ρ the solvent density (Fujita R the gas constant and T the absolute temperature. M*(r) has several useful properties, the most important being the M* extrapolated to the cell base (r = rb) = Mw,app, the apparent weight-average molecular weight for the whole distributionwhere y Fujita , ω the rc(r) is equal to the initial cell loading concentration, co . The SEDFIT-MSTAR algorithm evaluates the local or “point” apparent weight-average molar masses as a function of radial position, Mw,app(r), and at the hinge point (r = rhinge)the hinge point method: the “hinge point” in the radial distribution is the radial position at which the local concentration It is based on the co from the conservation of mass. SEDFIT-MSTAR also offers a “smart-smoothing” procedure for providing an accurate estimate for the meniscus concentration cm and baseline correction, and also yields an estimate of the overall molar mass distribution and the z-average molecular weight Mz,app. An example of the output from SEDFIT-MSTAR for a near-monodisperse immunoglobulin (Ig) G1 antibody is given in Fig. Mw,app(c) versus local concentration c. A comprehensive set of other examples for synthetic and real data systems are given in Schuck et al.  should be employed to minimize non-ideality effects. For a standard 12-mm path length the concentration of cells needs to be at least ∼0.5 mg/ml to give a sufficient fringe increment between cell meniscus and base (this is considerably higher than the lowest limit for sedimentation velocity experiments). The availability of cells with a 20-mm path length  makes it possible to use a concentration as a low as ∼0.3 mg/ml, which is usually sufficient for rendering thermodynamic non-ideality contributions insignificant in many  macromolecular systems.Mw ∼ Mw,app is still not valid, the conventional way of dealing with this situation is to perform a series of measurements at different loading concentration and extrapolate back to zero concentration where these effects tend to vanish. The form of the extrapolation can be linear (straight line) or non-linear . For obtaining Mw,app using procedures that do not involve an integration there is a simple relation relating Mw,app and Mw in dilution solution:B is the second thermodynamic virial coefficient (ml mol g−2). In more concentrated or non-ideal solutions additional virial terms may be necessary . Mw,app values evaluated according to Eq. Mw,app plotted versus loading concentration c yields the reciprocal of the true Mw from the intercept at c = 0. At higher concentrations, the extrapolation may not be linear (straight line), and an extra virial term in c2 may be required. Furthermore, for evaluations involving an integral transformation such as Eq. Mw, there may also be a speed-dependent enhancement of the non-ideality effects /2 with k defined by Eq. However, if working at these low loading concentrations the approximation mentclass2pt{minims Fujita , b leadiMw,app from Eq. Mw,app evaluated from Eq. Mw,app evaluation  on c(r)] obtained from SEDFIT-MSTAR, along with the corresponding sedimentation coefficient or sedimentation coefficient distribution from sedimentation velocity, may provide sufficient information about a particular system. such as its monodispersity, oligomeric state/state of aggregation, etc.. Alternatively, further processing may be required. If, on the basis of the dependence of Mw(r) on c(r)  and on the sedimentation velocity records there is a suggestion of a self-association or an interaction, then the c(r) versus r records can be further analysed to estimate interaction constants (section The  section . If it i section .g(s) versus s. Although this is still a very useful marker of heterogeneity, the sedimentation coefficient depends not only on molecular weight but (to some extent) on macromolecular shape, and so it is desirable to convert this to a molecular weight distribution, f(M) versus M.Although the SEDFIT-MSTAR algorithm can provide an approximate distribution, because of the lower speeds the resolution is quite poor. Conversely, sedimentation velocity—at higher rotor speeds—gives a much better resolution for a heterogeneous/ polydisperse system, although the distribution is (primarily) a sedimentation coefficient distribution, g(s) versus s profile to a molecular weight distribution, although his method was specifically aimed at random coil polymers. The “Extended” Fujita method  versus s distribution being converted should be obtained at the lowest concentration possible to minimize non-ideality. The s value used in Eqs. s value obtained at the same concentration as the distribution. By contrast the M value used to evaluate κs using Eq. always be the ideal value .The b value, then at least two plausible values at the possible extremes should be tried to give an idea of the effect on the measured distribution. An example is given in Fig. If there is uncertainty in the The c(r) versus r will not only depend on molecular weight M, but also on mass action parameters, such as the equilibrium association constant Ka, or the equivalent dissociation constant Kd . The distribution will also, as above, be influenced by thermodynamic non-ideality, soB is the second thermodynamic virial coefficient as described above. K represents either Kd or Ka, whichever is the more convenient:d (mol/l) = 1/Ka. For a monomer–dimer equilibrium, for example, correct to first order in concentration—and if virial terms higher than the second order are ignored and a binomial approximation to incorporate the contribution of Ka is accepted, then the relationship between the apparent molecular weight as estimated by SE and the total solute concentration c can be approximated by:M1 is the monomer molecular weight, B11 is the monomer–monomer second virial coefficient, c (g/ml) as above is the total solute concentration (of monomer and dimer) and the distribution of mass between these two species is given by the Law of Mass Action. The c(r) against r distribution (Eq. B or BM) and mass-interactive (Ka) terms can be floated in a single fit. As an example, we have chosen the application of this approach to a strong interaction of the A + B = AB type, the electron-transfer flavoprotein heterodimer ETF, where the association is between one polypeptide chain of M ∼ 29 kDa and another polypeptide chain of 34 kDa  was plotted against the local concentrations c(r) in the ultracentrifuge cell on the same axes for different loading concentrations. If the system is a genuine reversible equilibrium, then these data sets should overlay and fall on the same Mw,app(r) against c(r) curve (Roark and Yphantis c(r) against r data sets to Eq. a (or Kd)  interacting system, the concentration distribution in the ultracentrifuge cell, tion Eq. . One poption Eq. , which fmentclasspt{minimaTAR Fig. ; plots oKd) Fig. . This deKd) Fig. .Fig. 5SeBex. To do this, an estimate of the triaxial shape of the monomeric species is required  and CD48 (M = 28.7 kDa). A value for the second virial coefficient (B11) based on the dimensions from X-ray crystallography of 8.5 × 2.3 × 2.5 nm for the protein CD2 and 9.4 × 4.9 × 6.7 nm for the protein CD48 and the application of the software COVOL  yielded an average B11 = 1.8 × 10−4 ml mol g−2. Hence knowing this, from the experimental data, a value for Kd of ∼ (1.0 ± 0.3) × 10−4 mol l−1 was estimated, in good agreement with an estimate of ∼(7.5 ± 1.5) × 10−5 mol l−1 from surface plasmon resonance. In general, cases such as this, where the second virial term can be either computed or estimated, the use of software such as SEDPHAT  versus r obtained at different rotor speeds and temperature provides a simple and reliable way of securing a value for Kd and for understanding, including by “bootstrapping”, the likely levels of error present in the estimate made.The example shown in Fig. Sedimentation equilibrium, facilitated by modern computer analysis, continues to provide a vital, matrix-free tool for characterizing the molecular weight/oligomeric state, molecular weight distribution and interaction parameters for a wide range of macromolecular systems. Although the application of analysis procedures is becoming easier, attention to detail and awareness of the complications—most notably due to thermodynamic non-ideality—remains crucial, and for this researchers are indebted to the important underpinning and authoritative work provided by Professor Winzor and colleagues over many decades.


To study the effectiveness of exenatide with metformin and sequential treatment with exenatide and glargine added to metformin and their influence on insulin sensitivity and adipose distribution.  20 newly diagnosed obese type 2 diabetic patients were enrolled, and 2-month washout treatment of metformin, 6-month exenatide treatment, and 6-month glargine treatment were administrated sequentially accompanied with previous metformin. Glucolipid metabolic parameters were compared among groups. Adipose distribution was quantified with computerized tomography according to anatomy, dividing into visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), adding up to total adipose tissue (TAT).  The 6-month exenatide treatment dramatically ameliorated the glucose and lipid profile, improved insulin sensitivity, and mainly decreased VAT and also the ratio of VAT/SAT (RVS). The following 6-month glargine treatment increased VAT. The whole 12-month sequential treatment with exenatide and glargine added to metformin basically improved the insulin sensitivity and glucolipid control though VAT rebounded at the end, however without deteriorating the other parameters. ChiCTR-OOC-17013679. Exenatide is an ideal treatment for obese type 2 diabetic patients in the aspect of adipose tissue distribution. Sequential treatment of exenatide and glargine could be an alternative for low-income patients who cannot afford GLP-1 agonist for long time. This trial is registered with  Obesity is epidemic throughout the world. Obesity, especially visceral obesity, could increase the risk of type 2 diabetes, hypertension, dyslipidemia, and atherosclerosis, suggesting that obese patients with type 2 diabetes are at high risk for cardiovascular diseases . ChineseExenatide is a synthetic derivative of exendin-4, a peptide found in the saliva of the Gila monster . It is a2, and (4) HbA1c levels between 7 and 11% under 2-month treatment of metformin (daily dosage not less than 1500 mg). The exclusion criteria were as follows: (1) type 1 diabetes mellitus, (2) allergic history to incretin mimetics, (3) interstitial pneumonia, (4) severe renal or liver disease, (5) malignant tumors, and (6) history of acute coronary syndrome or stroke or acute pancreatitis within 3 months prior to enrollment.A total of 20 newly diagnosed obese type 2 diabetic patients (age 51.0 ± 10.5 years) were enrolled, including 9 males and 11 females. The inclusion criteria were as follows: (1) type 2 diabetes mellitus according to the criteria of the WHO of 1999, (2) Chinese adult aged 30~65, (3) BMI equal to or greater than 28 kg/mThe glycated hemoglobin (HbA1c) levels were measured with the HPLC method on a Bio-Rad Variant II glycosylated hemoglobin analyzer . Fasting insulin (FINS) and C-peptide (CP) were detected with the radioimmunoassay (RIA). Fasting/postprandial blood glucose and blood lipid profile including total cholesterol (TC), total triglyceride (TG), high-density lipoprotein-cholesterol (HDL-c), and low-density lipoprotein-cholesterol (LDL-c) were detected with the ADVIA 2400 Chemistry System  as routine clinical tests in the hospital.The design and procedure of the study were shown in β as (20 × fasting insulin (mU/L))/(fasting glucose (mmol/L) − 3.5).Data from the 20 patients at visit 1 and visit 2 and 15 patients at visit 3 were statistically compared. The insulin resistance index was calculated by homeostasis model assessment of insulin resistance (HOMA-IR) as (fasting insulin (mU/L) × fasting glucose (mmol/L))/22.5. Beta-cell secretion function was calculated by homeostasis model assessment of 3. And then the VAT/SAT ratio was calculated.Computerized tomography (CT) was performed with a Somatom Emotion 16 CT scanner  by one designated specialist. Patients were scanned above the level of S1 in a supine position. Briefly, 25 contiguous 5 mm thick slices  were acquired covering 125 mm above the level of S1. Images were reconstructed with Volume Viewer 3.1 software on an AW Workstation  . The musp < 0.05.All statistical analyses were carried out using the Statistical Package for Social Sciences . Results are expressed as mean ± standard deviation (SD). The mean values of variables are studied by analysis of variance (ANOVA). Pearson correlation was used to analyze the relation between abdominal fat and other clinical parameters. The level of significance was accepted as 2. Age is 51 ± 10.5 years.Data from the 20 patients showed that the baseline glycated hemoglobin (HbA1c) was 9.745 ± 1.50%, while the baseline fasting blood glucose was 13.07 ± 2.36 mmol/L. BMI was 29.77 ± 1.49 kg/mr = 0.813, 0.822, 0.665, 0.464, 0.484, 0.643, and 0.518, resp., p < 0.05). VAT was positively correlated with BMI, waist circumference, TG, LDL-c, HbA1c, FINS, CP, and HOMA-IR  and negatively correlated with HDL-c . In comparison, SAT was only positively correlated with BMI and waist circumference . The ratio of VAT/SAT was positively correlated with TG and FINS .To analyze the parameters influencing the adipose distribution pattern before any medical therapy, Pearson correlation was performed. Results indicate that TAT was positively correlated with BMI, waist circumference, LDL-c, HbA1c, FINS, CP, and HOMA-IR (p < 0.05).As shown in Figures As for the change of abdominal adipose tissue volume after the six-month exenatide treatment, VAT volumes decreased significantly, while SAT volumes and TAT volumes decreased slightly, without statistical significance. The ratio of VAT/SAT after the treatment was lower than that before. These results indicated that exenatide mainly functions on the visceral adiposity.After the 6-month exenatide treatment ended, glargine was used to take the place of exenatide. Glargine was started with 0.2 unit/kg and then modified according to the fasting blood glucose, adding two units every time when fasting glucose was higher than 7.0 mmol/L. At the end of the 6-month treatment, the final daily dosage of glargine was 23.01 ± 4.94 units or 0.27 ± 0.04 unit/kg, similar to that of previous reports .p value > 0.05). The glucose control level with exenatide and the following glargine treatment seemed to have no significant difference.Patients were called back to repeat the tests mentioned above in the three major visits, that is, at the end of the single metformin washout period, exenatide plus metformin period, and exenatide plus metformin period. At the end of the whole 12-month sequential treatment, though most people rebound a little in body weight and adipose tissue amount, nobody regains their body weight completely. Their blood glucose levels kept stable compared with those of exenatide treatment  receptor agonists, used as glucose-lowering drugs, can also reduce body weight by decreasing food intake and reversing hepatic steatosis . Accordigood and bad adipose tissue. It was reported that exenatide could improve β-cell function up to 3 years in patients with type 2 diabetes [β which represents the insulin secretion function had no significant change during the whole periods of the treatments. Exenatide and glargine seemed to affect more insulin sensitivity rather than insulin secretion. The change of sensitivity might be related partly to glucose control or adipose distribution.By comparing exenatide with glargine in their 6-month treatment combined with metformin, they had comparable glycemic and lipid control and their fasting glucose, HbA1c, TG, TC, FINS, and HOMA-IR were similarly lower than those of the pretreatment, making exenatide and glargine both reasonable methods for newly diagnosed obese type 2 diabetic patients, from the aspect of glucose/lipid control and insulin resistance. But from the aspect of adipose tissue distribution and visceral obesity, exenatide and glargine are different in the fact that exenatide decreased the visceral adiposity while glargine increased it . In consdiabetes . HoweverFor the low-income level, most Chinese patients cannot afford GLP-1 agonists for long term in consideration of their high price and no coverage by local medical insurance. Consequently, many patients had to switch to other remedies after several months of GLP-1 agonist treatment. According to our own unpublished survey in our hospital during the past years, most people could only stick to GLP-1 agonists for 3 to 12 months; then, they had to change to insulin, sulfonylureas, metformin, or DPP4 inhibitors, mostly for the economic reason. Therefore, our study aimed to simulate what is happening every day in our real world. In our observation, there were three patients that chose to pay for exenatide because it gave them confidence with fine blood glucose and body weight control. But because of their small number and higher-income level, the possible difference on their willingness and compliance to therapy might lead to unavoidable statistical confounding; therefore, we did not put these three patients into the comparison and statistics. But VAT volumes of these three patients also reached a plateau, making our sequential treatment with exenatide and glargine a cheaper alternative of long-term GLP-1 agonist, at least within 12 months. More clinical trials may be needed to confirm more affordable treatments.


Overall Abstract: Dopamine plays a central role in shaping circuitry within the brain, thus affecting learning and behavior. It also plays a central role in schizophrenia and addiction. This panel will examine the impact of dopaminergic signaling on specific circuits that may create special vulnerability for the emergence of comorbidity between schizophrenia and addiction. The talks will include two presentations in clinical samples using molecular and functional imaging and two presentations in animal models.Jared Van Snellenberg will discuss connectivity of striatal substructures to the rest of the brain in drug free patients with schizophrenia and their relationship to abnormal cortical D2 signaling and psychotic symptoms. He will present recent unpublished work, motivated by pre-clinical studies with a D2 receptor over-expressing mouse model, using simultaneous multi-slice (multiband) functional MR imaging in these patients. These results suggest that unmedicated patients have altered connectivity between specific basal ganglia subnuclei, consistent with the animal model.Nora Volkow will focus on the role of bidirectional interactions between dopamine reward system and prefrontal regions in the addicted brain with emphasis on the role of D2 receptor signaling in the striatum. She will discuss the functional impact of these interactions on reward/motivation and executive-function networks and will discuss the variables that influence D2 receptor function including genes, sleep and social stressors and how they interact with drug exposures to provide resilience or vulnerability to substance use disorders or schizophrenia. Finally, she will discuss how this knowledge can be used to tailor interventions to remediate or buffer neurocircuity dysfunction triggered by drugs and for prevention.Bita Moghaddam will present an animal model of behaviorally induced dysfunction in the mesocortical circuit by using a task where actions are consistently rewarded but probabilistically punished. Spike activity and local field potentials are recorded during this task simultaneously from VTA and mPFC, two reciprocally connected mesocortical regions. Under no risk of punishment, a synchronous interaction at multiple time scales between PFC and VTA dopamine neurons is observed. This synchrony collapsed as a function of punishment contingency during reward-seeking actions, with risk of punishment diminishing VTA-driven neural synchrony between the two regions. These data reveal a dynamic coding scheme in VTA-mPFC neural circuits in representing aversion-based modulation of rewarded actions. These data suggest that driving VTA dopamine neurons by drugs of abuse may reverse the diminished synchrony and serve as self-medication in comorbid conditions.Finally, Aurelio Galli will discuss the structural, functional, and behavioral insights into the dopamine dysfunction of comorbid conditions as modeled by a deletion of the SLC6A3 affecting the function of the human dopamine transporter (hDAT). Genetic variants in hDAT have been associated with neuropsychiatric disorders. An in-frame deletion in hDAT at N336 (∆N336) leads to abnormal DA homeostasis. He demonstrated these dysfunctions in brains of Drosophila melanogaster expressing hDAT ∆N336. Furthermore, these flies are hyperactive and display fear and impaired social interactions, traits associated with impaired DA neurotransmission. Insights from X-ray crystallography, electron paramagnetic resonance, molecular dynamic simulations, electrophysiology and behaviors describe how a genetic variation causes DA dysfunction resulting in combined behavioral alterations and psychostimulant use.