High harmonic spectroscopy gives access to molecular structure with Angström resolution. Such information is encoded in the destructive interferences occurring between the harmonic emissions from the different parts of the molecule. By solving the time-dependent Schrödinger equation, either numerically or with the molecular strong-field approximation, we show that the electron dynamics in the emission process generally results in a strong spectral smoothing of the interferences, blurring the structural information. However we identify specific generation conditions where they are unaffected. These findings have important consequences for molecular imaging and orbital tomography using high harmonic spectroscopy. In the recombination process, a burst of extreme ultraviolet photons is emitted, encoding a wealth of information on the target. First, the recombination dipole moment imprints the structure of the molecular orbital involved in the emission, under the form of structural interferences3 equivalent to Cohen-Fano interferences in the photo-ionization dipole4. The accurate spectral position and shape of the signature of these interferences in the HHG spectrum (amplitude and phase) is the required information for performing molecular imaging3 and orbital quantum tomography7. Second, any dynamics occurring in the core during the EWP continuum excursion strongly affects the recombination. For instance, tunnel ionization from different valence orbitals results in interfering channels in the recombination10.High Harmonic Spectroscopy (HHS) is a powerful technique in which the process of High Harmonic Generation (HHG) is used to probe the structure and dynamics of the generating medium with Angström and attosecond resolution. In HHG, an Electron Wave-Packet (EWP) is prepared by tunnel ionization of the atomic/molecular target gas, it is then accelerated by the strong laser field and finally driven back to the core13. This was generally performed by recording the dependence on the laser parameters , since it was widely accepted that structural interferences do not depend on them14. This appears clearly in the Quantitative ReScattering theory (QRS)15 where the harmonic dipole is expressed as the product of the returning EWP, containing all the laser parameters, and the target-specific field-free recombination dipole, only dependent on the EWP energy and recollision direction. Such a factorization has been very successful in predicting and explaining resonant features in the harmonic intensity, e.g., in16. It was further confirmed by analytic calculations in the case of a model potential17.However, it is crucial to disentangle the different effects in order to access this rich informationdynamics on the structural interference occurring in the harmonic emission from diatomic molecules. Based on Time-dependent Schrödinger Equation (TDSE) calculations resolving the two shortest EWP trajectories for each harmonic, we show that the value of the laser field at recombination plays a crucial role on the shape of the two-center destructive interference. In most cases, the intensity minimum and the associated phase-jump are spread over a broad spectral range resulting in a very smooth behavior, and the phase-jumps for the short and long EWP trajectories are in opposite directions. Furthermore, we identify very peculiar situations for which this phase-jump is very sharp, providing ideal conditions for performing molecular imaging and quantum tomography. Using the Strong-Field Approximation (SFA) approach18modified for molecules20, we explain these features by the modification of the saddle-point trajectories induced by the fast phase variation of the recombination dipole. Our findings thus question the strict separation between continuum dynamics and recombination advocated, e.g., in QRS, with strong implications for HHS.Here, we study the signatures of the electron 22. Note that we carried out preliminary 2D computations that are in overall agreement with our 1D results, eventhough they present extra features, that we plan to address in a forthcoming paper. Our model system represents a diatomic molecule with fixed nuclei, under the single-active electron approximation.We chose to perform our study on a one dimensional (1D) system that contains the essential physics of two-center interference in HHG. This has two advantages: (i) we can easily achieve numerous computations both within the TDSE and the SFA framework, and implement analytical developments; (ii) we avoid additional effects like Coulomb refocusing or EWP spreading that was previously invoked to explain phase smoothing effectsR is the internuclear distance. This is valid for molecules with “heavy” nuclei that exhibit a strong two-center character, such as CO224. In order to study the R-dependence of the structural interferences, the regularization parameter a is adapted to maintain the same ground state energy (−Ip = −0.567 a.u = −15.43 eV) at each considered value of R. The electric field E(t), of amplitude EL, has a sine-square envelop lasting two optical cycles with frequency ωL corresponding to a 800-nm Ti:sapphire laser. The carrier -envelope phase is set equal to zero in order to obtain the contributions of only one set of short and long trajectories in the HHG spectrum. The t → ω Fourier transform of the acceleration25 provides the harmonic field. We calibrate the latter with the one calculated for a reference “atom” . However, we observe in Fig. R values, the intensity minimum and associated phase-jump are spread over a large spectral range; (ii) the phase-jumps for the short and long trajectories are in opposite directions for high values of R; (iii) the shape of the interference strongly depends on R. In particular for the long trajectory, at a critical internuclear distance Rc ≈ 1.55 a.u., the interference becomes extremely destructive at harmonic 41 (H41) with a very steep π phase-jump. For smaller R values, the interference becomes smooth again, but with a change of sign of the phase-jump that now resembles that of the short trajectory. Indeed, the interference is now positioned in the cutoff region (>H41) where the two trajectories coalesce.Figure exact scattering waves of the potential V(x) and found an excellent agreement only at the critical distance Rc and for the long trajectory.Within the QRS, the calibrated emission should be directly proportional to the molecular recombination dipole matrix element. We computed it with the numerically 28. We retrieved the emission times of the harmonics for which the destructive interferences occur and found that, at the particular internuclear distance Rc, it corresponds to an almost zero instantaneous electric field for the long trajectory. Furthermore, larger (lower) values of R lead to recollision with a negative (positive) electric field for the long trajectories while it is always positive for the short trajectories. Thus, the sign and shape of the phase-jump seem to be strongly correlated to the instantaneous value of the electric field when the harmonics are emitted.In order to shed light on the above features, we performed a time-frequency analysis of the harmonic dipole using Gabor transforms as inIL from 2 to 4 × 1014 W.cm−2 at fixed R = 1.4 a.u. and show the results in Fig. IL , the phase-jump behaves similarly when varying R at fixed IL. We found a critical intensity Ic = 3.24 × 1014 W.cm−2 for which we observe the inversion of direction with a very sharp πphase-jump. Here again, this value directly corresponds to an instantaneous electric field ≈ 0 when H49 is emitted.To further examine the influence of the electric field, we varied the peak laser intensity 20, the results of which are reported in Fig. 29. A major advantage of the SFA lies in its semi-analytical form. It allows for a semi-classical analysis by searching the saddle-points of the action that determine the main quantum orbits/trajectories contributing to HHG30. Thus, we performed a Taylor expansion of the stationary solutions for the molecular SFA around the ones for the reference atom to get physical insight into the electron dynamics. Following20, we approximate the symmetric electronic ground state of a homonuclear diatomic molecule by the sum of two symmetric atomic orbitals centered on each nucleus. The PW-recombination and -ionization dipole matrix elements become proportional to cos(pR/2), with pR/2) in terms of exp[±ipR/2], the action reads:A is the vector potential associated with the electric field. The first two terms are identical to the atomic SFA while the last two are induced by the fast phase variations of the molecular ionization and recombination dipoles. The saddle-point equations associated with this modified action20 allow to determine the stationary solutions for the momentum pstat, the ionization and recombination times tstat respectively. Compared to their atomic equivalents, we found that pstat is unchanged to first order in the molecular case, while the times tstat differ by:R/2 (+R/2) for j or k equal 1 or 2, respectively. As the former is classically forbidden, the change in ionization time is purely imaginary, while the latter, classically allowed, is purely real.We carried out the same study within the molecular SFA framework, derived followingζ is a constant and 27. One clearly sees that in addition to the “bare” recombination dipole (first term in Eq. (pR/2) and depending on the electric field at recombination time t. We have checked numerically that ζ, which originates from the rigorous handling of the prefactors arising from the saddle-point approximations within SFA, is almost constant and small compared to the other term in the bracket27. It explains why the discontinuous π-jump is observed at an almost but not exactly zero electric field at recombination time.The saddle-point harmonic dipole can then be factorized in the spirit of the QRS but with a modified recombination dipole, which reads to first order:pR/2), is zero, except when the bracket in Eq. (E(t)  and which value and sign drives the direction and the smoothness of the phase-jump. Thus, only when E(t) ≈ 0 at emission time of H49 of the long trajectory in the conditions of Fig. Therefore, (i) t in Eq.  is also entclass1pt{minimaζ in Eq. , sum of two symmetric atomic orbitals centered at ±R/2, and an antisymmetric state σu (field-free first excited state), difference of the same two atomic orbitals32. This decomposition is a very good approximation even at short distance R27.One should note that, if we neglect ζ in Eq. , we recoc(t) depends linearly on the instantaneous electric field E(t), i.e., c(t) = χE(t), and we checked, by computing the exact dressed ground-state33, that this is still valid for fields strength ≈1014 W.cm−2. Thus, the modified recombination dipole associated with Φ0 within the PW approximation, becomes:Within the time-dependent perturbation theory, 35.In practice, the influence of the electron dynamics on the structural interference could be observed by following the phase difference between the short/long trajectory contributions when varying the laser intensity, e.g., by recording the Quantum Path Interferences in the total harmonic dipoleIn summary, we have shown that the electron dynamics in the HHG process may blur the two-center interferences encoding the molecular orbital structure. The value and sign of the driving laser field at recombination become critical in spreading the intensity minimum and associated phase-jump over a large spectral range and inducing opposite phase-jumps for the short and long trajectories. Except in specific situations, our study demonstrates that the harmonic emission is not entirely proportional to the field-free recombination dipole moment. This raises questions since such factorization is central in HHS, e.g., in the QRS theory. This should be taken into account when comparing the recombination dipole extracted from HHG to the photoionization dipole, or using it for molecular imaging and quantum tomography. Our conclusions could be generalized to other types of structures occurring in the recombination dipole, like shape or autoionization resonances. If narrow enough, the corresponding fast phase variations could modify the electron dynamics and result in a smoother behavior in the harmonic emission. Further studies are in progress to uncover all the implications of the reported findings.


Drugs may cause liver injury in a few susceptible individuals, but the molecular events that lead to this idiosyncratic, largely dose-independent and non-predictable drug-induced liver injury (DILI) are mostly unknown, since animal models to explore the pathogenetic mechanisms of human idiosyncratic DILI are not yet reliable. More is known about molecular events in connection with the intrinsic dose-dependent DILI, since animal studies can clarify some questions of the underlying pathogenetic conditions. Molecular risk factors of idiosyncratic DILI include drug lipophilicity, high daily dose, high metabolic rate, and drug–host interactions, in addition to genetic factors that may initiate liver injury. Nevertheless, many pathogenetic challenges at the molecular level remain unknown. Uncertainties also relate to possible molecular diagnostic biomarkers, and whether these could assist RUCAM  in establishing causality for suspected drugs in DILI cases. Clearly, much more research is still warranted to elucidate molecular pathogenetic events in DILI which may help to establish diagnostic biomarkers. The aim of this Special Issue is to provide a broad updated overview on these molecular related hepatotoxicity features with their challenges and highlights and to encourage more molecular research in DILI. Experts in the field contributed their views to this emerging and fascinating topic of molecular aspects. Since some topics are still controversial, we expect and appreciate lively discussions.International Journal of Molecular Sciences to address the progress and current standing in the vast field of liver injury. A total of 10 reviews and original articles [Hallmarks of clinical liver injury will be presented in the Special Issue “Molecular Research on Drug Induced Liver Injury” published in the articles ,7,8,9,10The Special Issue opens with the review article of Iorga et al. , who expIn the second article, Teschke et al.  expand tSubsequently, McEuen et al.  considerIn the next clinically oriented review article, Sebode et al.  present N-acetyl moiety. Three cyanide adducts of KCZ were identified in mouse and human microsomal incubates as “flags” to trigger additional toxicity study. The oxidation of piperazine to iminium is likely the bioactivating step of toxicity. Therefore, this metabolomics approach contributes to our understanding of KCZ liver injury and asks to what extent these findings are transferable to clinical conditions of liver injury in patients under antifungal therapy with KCZ. Based on studies in humans and animals, Kim et al.  exploredAnother clinical issue supported by experimental toxicity studies was addressed by Goda et al. . They maTurning now to the next clinical topic supported by in vitro studies, Hirasawa et al.  focused Using two different experimental liver injury models in mice, Yamamotoya et al.  studied Experimental hepatotoxicity due to the anti-tuberculosis drug rifampicin was induced in mice, which led Kim et al.  to inves4-induced hepatic fibrosis. As a derivative of the hepatoprotective oleanolic acid originally from Ligustrum lucidum, the synthetic Oxy-Di-AO has a protective effect and prevented the development of CCl4-induced liver fibrosis when given intraperitoneally daily for 9 weeks. The protective effect was verified by measurements of ALT  and AST (aspartate transaminase) and by histological and histochemical analyses. After single-dose oral administration, pharmacokinetic studies showed that time to reach peak concentration of Oxy-Di-AO was 12 h, associated with an elimination half-life of 2.19 h. The LD50 value of Oxy-Di-OA exceeded 2000 mg/kg via intraperitoneal injection in mice. Whether Oxy-Di-AO is useful in clinical hepatology remains to be established.In another experimental study carried out in rats, Xiang et al.  reportedAs guest editors of this Special Issue with overall 10 contributions, we thank all experts who devoted their time and energy to writing their papers. For each submitted paper, the editorial team chose at least three experts in the field as external and independent reviewers whose recommendations were highly appreciated and substantially improved the quality of the articles as they were finally accepted for publication. These publications covered the broad field of molecular aspects of liver injury by drugs and also illustrate how crucial it is to assess pathogenetic events. It was a pleasure for us to work together with the authors and such a competent editorial team to get this exciting job done. Thank you again to all who contributed and assisted to get this Special Issue published.


Antimicrobial resistance (AMR) is a critical public health issue that involves interrelationships between people, animals, and the environment. Traditionally, interdisciplinary efforts to mitigate AMR in the food chain have involved public health, human and veterinary medicine, and agriculture stakeholders. Our objective was to identify a more diverse range of stakeholders, beyond those traditionally engaged in AMR mitigation efforts, via diagramming both proximal and distal factors impacting, or impacted by, use and resistance along the Canadian food chain.We identified multiple stakeholders that are not traditionally engaged by public health when working to mitigate AMR in the food chain, including those working broadly in the area of food  and health , as well as in domains as diverse as law, politics, demography, education, and social innovation. These findings can help researchers and policymakers who work on issues related to AMR in the food chain to move beyond engaging the ‘traditional’ agri-food stakeholders , to also engage those from the wider domains identified here, as potential stakeholders in their AMR mitigation efforts. Antimicrobial resistance (AMR) is a critical public health issue causing increased morbidity and mortality worldwide , 2. AntiCampylobacter and Salmonella, AMR is a complex issue involving interrelationships between people, animals, and the environment, and AMU in food-producing animals  is a recognized contributor to resistant human infections [In pathogenic bacteria transmitted via food, such as fections , 8. Thusfections –12.In addition to the direct relationship between AMU and AMR in animals and humans, it is important to assess the role of broader, systemic drivers. To-date, such assessments have evaluated the contribution of governance and corruption to AMR in a variety of pathogens including those transmitted by food ; explore® PLE Plus for Macintosh , and was drawn using a systems dynamics model format, which has been used for other public health issues to depict the underlying set of complex factors and interrelationships that drive the issue [We diagrammed factors related to AMU and AMR in the Canadian food chain, drawing on our own expertise in foodborne disease, AMR, and the Canadian food system, and informed by both group model building  and expehe issue , 19. We Salmonella Heidelberg in the province of Québec was high, and tracked well against the level of resistance found in Salmonella Heidelberg isolated from retail chicken samples collected from Québec. Upon receiving these data in 2005, Québec hatcheries responded and voluntarily stopped using ceftiofur to treat and prevent E. coli omphalitis in chicks, and in 2006 levels of resistance in both S. Heidelberg infections in humans and S. Heidelberg isolated from retail chicken decreased. However, in 2007, Québec hatcheries returned to partial use of ceftiofur to treat and prevent E. coli omphalitis, and resistance in human infections and retail chicken began to increase. By 2013, levels of resistance had returned to the 2003 levels.We then applied a case scenario to our draft model: the voluntary withdrawal of ceftiofur  in chicUsing this case scenario, we further refined our model by examining factors that may have: (1) been positively or negatively impacted when the voluntary withdrawal was in place; 2) been impacted had the withdrawal continued, either with or without any other changes in the underlying system of factors; or (3) contributed to the withdrawal being non-sustainable, that is, that might have driven hatcheries to start using ceftiofur again. For example, during the full withdrawal period we considered: did producers see a re-emergence of disease within flocks, and did this arise from the fact they were still producing chickens in the same manner as before, with no other changes in the broader production environment ? We used our knowledge of the industry and production practices to hypothesize about wider forces and added these factors to the model. We also examined the potential implications of the 2014 voluntary ban by the Canadian poultry industry, that “eliminates the preventative use of Category I antibiotics in Canadian chicken production” . Through been impWe then used the model to identify potential stakeholders by determining the individuals and organizations who act upon the different factors in the model. We classified stakeholders by whether or not they had been commonly engaged by public health practitioners in mitigating and reducing AMU and AMR in the Canadian food chain Table . InteresGenerating a conceptual model of the factors underlying AMR in foodborne pathogens was a useful process that allowed us to identify stakeholders who are not traditionally engaged by public health, to mitigate AMR in the food system. In addition to identifying stakeholders beyond the traditional agri-food partners, our model also identified that traditional methods for stakeholder engagement, that focus on engaging organizations, may be missing key stakeholders for whom formal or organized entities do not exist. Future efforts to engage a broader range of stakeholders are needed, in ways that allow for dialogue and exploration of how they act within and impact the system, in order to identify multi-pronged and sustainable approaches to mitigate and reduce AMR and its impacts across humans, animals, and the environment. Specifically, such dialogue is necessary to: (1) identify additional relevant model factors from domains like land use management, the media, access to health care and services; (2) explore other case scenarios to further identify factors that hold the current system ‘in place’ ; (3) co-define potential courses of action, including roles and key influencers; and (4) explore potential ramifications, both intended and unintended, of possible actions. The ideal expansion of this work would involve revising the model to include all additional relevant factors and inter-relationships, and assigning quantities and functions to all factors and relationships, respectively, so that predictive modelling can occur. To achieve this, expert engagement with the range of stakeholders identified here is needed, to determine where quantitative data exist, and where approaches like expert elicitation are needed to specify such a model in the absence of empirical evidence.We recognize that this work is subject to several limitations, most notably that it relies on our own expertise. However, this should not diminish the utility of the findings as a first depiction of the diversity of factors related to AMR in the Canadian food chain context, that allows us to identify additional domain experts with whom to engage in future model expansion and validation. Our approach was guided by expert elicitation, a method particularly useful for addressing questions that are difficult to answer via any other means , 22–25, Neisseria gonorrhea; [We also recognize that taking a higher level systems view of AMR, as we have done here, necessitates ignoring nuance and detail, and reduces entire disciplines to a summary arrow or label in our model. This is another reason that domain experts across the factors identified here must now be engaged to ensure model completeness and accuracy, particularly given that we recognize our perspective is biased towards public health, foodborne disease, and veterinary medicine and livestock-associated factors. As well, to manage the size of this model, we focused on AMR in foodborne pathogens; however, micro-organisms transmitted via different pathways interact to shape the resistance landscape, and it will be important to explore how AMR in non-foodborne pathogens  may linnorrhea; ).


Here, we present an electrochemical sensor based on gold electrodes modified with calix[4]arene functionalized with carboxypiperidino groups at the upper rim. It has been demonstrated that these groups are involved in a complex formation with dopamine (DA) on the surface of gold electrodes. The supramolecular complex calix[4]arene–DA created on the gold electrode surface has been characterized electrochemically and the measuring conditions have been optimized. The presented sensor displayed a detection limit in the pM range. The DA determination was performed successfully in the presence of ascorbic acid, uric acid and selected neurotransmitters. Dopamine is a very important biomolecule belonging to the catecholamine neurotransmitters family. A basal level of dopamine (DA) is 1 nM and 10 nM in human serum and in the brain, respectively ,3. For eIn the analytical methods currently available for DA determination in physiological fluids, the separation step is involved in chromatography, enzyme immunoassay and mass spectrometry ,6,7. AllThe electroactivity of the catecholamine neurotransmitters family is often used as the base for their direct determination. Electrochemical methods  attract scientific interest because of their simplicity and sensitivity. The devices used in such types of analysis are relatively cheap and easily miniaturized . The weaThe modification of electrode surfaces with enzymes, such as with laccase and/or nanomaterials, could overcome these problems ,12,13. HHere, we propose calix[4]arene as the sensing element in the electrochemical sensors destined for dopamine determination. The calixarenes belong to cavity-shaped cyclic compounds which are broadly applied in a supramolecular recognition process as host molecules ,18. A nu−11 to 1.0 × 10−6 M was achieved arene as a sensing element for dopamine determination . This seachieved . The weaIn the present work, we describe the sensor based on gold electrode modified with calix[4]arene derivatives functionalized with mercaptoalkyl groups at the lower rim, and carboxypiperidino groups on the upper rim. In this sensor our strategy for analytical signal generation and improving selectivity is based on the formation of an electroactive calix[4]arene–DA supramolecular complex located at the surface of the gold electrode. Its electroactivity originates from dopamine. Applying this strategy, we eliminate the need to add the redox marker to the sample solution. The presented sensor is designed for the determination of dopamine in the presence of a significant concentration of another neurotransmitter, as well as ascorbic and uric acid.N-(4-carboxypiperidino)methyl]-25,27-bis(3-mercapto-propoxy)-26, 28-dihydroxycalix[4]arene (HSCX-COOH) was synthesized by a previously reported procedure arene derivatives are applied very often as a sensitive element in sensors designed to detect neutral or ionic analytes ,28. ReceIn the present paper, we have introduced a sensor based on the HSCX-COOH as the host molecule for the detection of DA in the presence of a high concentration of remaining neurotransmitters. Modification of the gold electrode was done according to the scheme presented in N-4-carboxypiperidino groups forming the supramolecular HSCX-COOH–DA complex. The presence of DA as a component of this complex determines it redox activity, and because of that, it could fulfill the role of transducer. Additionally, the presence of the negatively charged carboxylic group should repulse negative charged interfering compounds, such as AA or UA.The first step of modification relies on the immobilization of the HSCX-COOH substituted in the bottom rim, with mercaptoalkyl groups added to the surface of the gold electrode through Au–S bonds. In the next step, the pinholes between HSCX-COOH molecules on the Au surface were filled with MET. The presence of the HSCX-COOH/MET layer on the gold electrode surface was confirmed by CV, done according to the procedure described in our previous publication . We haveo-quinone (DAQ) (reaction 1). The next step involves the irreversible closure of the ring via deprotonation of the amine side chain to leucodopaminechrome (LDAC) (reaction 2). LDAC is then oxidized to dopaminechrome (DAC) (reaction 3). Most researchers have not been interested in reaction 3, but rather, only reaction 1 -25,27,-bis(3-mercaptopropoxy)26,28-dihydroxycalix[4]arene, dedicated to determining DA in the presence of the physiological concentration of AA and UA, as well as EP and NEP. The sensor discussed demonstrated a very good linear dynamic range, from 2.0 to10.0 pM, and a detection limit of 1.4 pM. One of its main advantages is the possibility to determine DA in the presence of other neurotransmitters, AA and UA. This is very important for future sensor applications in physiological samples control. The presented electrochemical sensor is very simple to prepare , easy to use, and could well be miniaturized. In this sensor, the analytical signal generation is based on the electroactivity of the calix[4]arene–dopamine supramolecular complex. We have introduced a sensor based on gold electrode modified with 5,17-bis[


Treatment decision for coronary artery disease (CAD) is based on both morphological and functional information. Image fusion of coronary computed tomography angiography (CCTA) and three-dimensional echocardiography (3DE) could combine morphology and function into a single image to facilitate diagnosis. Three semiautomatic feature-based methods for CCTA/3DE registration were implemented and applied on CAD patients. Methods were verified and compared using landmarks manually identified by a cardiologist. All methods were found feasible for CCTA/3DE fusion. Due to the risks associated with invasive treatment procedures, treatment of functionally insignificant stenoses should be avoided. In the FAME study, disregarding measurements of fractional flow reserve during PCI treatment increased the risk of death or myocardial infarction within 2 years by 50%.,Morphological information can be noninvasively obtained from coronary computed tomography angiography (CCTA) as high-resolution three-dimensional (3-D) images of the coronary artery tree and stenoses.,To allocate stenotic segments to functional defects, morphological and functional information is traditionally viewed side-by-side and integrated mentally, a time-consuming and error-prone process. Side-by-side interpretation is further complicated by large individual variation of the coronary artery tree anatomy and myocardial distribution territories.Image fusion of CCTA and SPECT myocardial perfusion images enables more confident assessment of the functional significance of most stenoses than viewing images side-by-side.,Fusion of CCTA and 3DE has recently been shown to be feasible.22.1We enrolled 16 consecutive patients who were scheduled to undergo CCTA at Karolinska University Hospital  by their treating physician to rule out CAD. All patients consented to undergoing 3DE. The study was approved by the local ethics committee . Five patients were excluded due to poor image quality. The median age of included patients was 61 years, ranging from 17 to 74 years. One patient was female and 10 were male.2.2One 3-D volume from CCTA and one four-dimensional (4-D) volume (3-D + time) from 3DE were acquired for each patient. Ten patients underwent 3DE within 1 hour of CCTA. The remaining patient underwent 3DE 48 days later.All CCTA examinations were performed on a 64-channel detector CT scanner  with a prospectively ECG-triggered scan protocol. The tube potential was 120 kVp, tube current 450 to 650 mA depending on body mass index, rotation time 350 ms, and detector collimation All 3DE examinations were performed by an experienced sonographer using a Vivid E9 ultrasound system  with a 4-D matrix-array transducer (4V-D) with 1.67-MHz center frequency. Datasets were stored digitally for offline analysis using EchoPac . 4-D (3-D + time) volumes were stitched from subvolumes obtained by ECG gating from four to six consecutive cardiac cycles during one breathhold. Acquisition parameter ranges were 60 deg to 65 deg sector angle, 11 to 16 cm depth of field, and 29 to 52 volumes per second. The probe was positioned for standard transthoracic apical four-chamber view with the patient in left lateral decubitus position. No medication was given. The average patient heart rates during 3DE acquisition ranged from 39 to 79 beats per minute.2.3Semiautomatic segmentation was performed in both CCTA and 3DE volumes using the real-time contour tracking library.2.4The 3DE volume was registered to the CCTA volume using three alternative methods: landmark distance minimization based on Procrustes alignment,The segmented landmarks were mitral valve centers Landmark distance minimization: The mean squared distances between corresponding landmarks from the two volumes was minimized, i.e., rigid Procrustes analysis without scaling or reflection.Endocardium ICP alignment: The segmented endocardial surfaces from CCTA and 3DE, 642 points each, were aligned using the ICP method: the two surfaces were initially registered using the landmark distance minimization described above. Each point on the 3DE surface was associated to the closest point on the CCTA surface. A new transform was obtained by minimizing the mean squared distance of these 642 point pairs. Points were reassociated and a new transform obtained until the process converged.Three-chamber alignment: The three-chamber views were aligned by the unique rigid transformation such that 1.Mitral valve centers of both volumes coincided, i.e., 2.The long axes, as defined from mitral valve center to endocardial apex, coincided, i.e., the points 3.The three-chamber planes, as defined by mitral valve centers, apices and LVOT, coincided, i.e., the points Registration with the described methods required no manual intervention, other than the semiautomatic segmentation. All methods were implemented in C++ using the Visualization Toolkit.2.5Anatomical accuracy of the registration methods was quantitatively evaluated based on anatomical landmarks present in both volumes. An experienced cardiologist used a custom multiplanar reformatting visualization software to identify these LV anatomical landmarks. Note that the landmarks identified through this validation procedure are marked manually and are distinct from those detected in the semiautomatic segmentation step.The procedure performed for each volume was as follows:The software presented three approximate long-axis and one short-axis view simultaneously. The user adjusted these views to obtain an optimally aligned three-chamber view. The aforementioned landmark identification workflow was performed first for each 3DE volume in a random order, then for each CCTA volume in a random order. For the 4-D volumes from 3DE, the cardiologist viewed the entire cine loop but marked the landmarks only in the time frame of cardiac phase matching the CCTA volume. The LV endocardial apex in a volume was taken as the mean of the three apical landmarks identified in different views and the mitral annular center position as the mean of the 24 mitral annulus landmarks.Anatomical accuracy was quantified in each fused volume as the intermodality distances between corresponding anatomical landmarks . Apex diFurthermore, two intermodality angles were calculated to quantify LV rotation: long-axis angle and transverse-plane angle . The lonFinally, two additional measures were used to assess endocardial surface similarity: the mean point-to-point distance was obtained by averaging the distances from each point on the segmented 3DE endocardial surface to the CCTA surface. Dice’s coefficient was obtained by considering the intersection between the three-chamber view, as identified by a cardiologist in the CCTA volume using the visualization software described above and the two segmented endocardial surfaces. The two intersections, the 3DE three-chamber LV area The Wilcoxon signed-rank test was used to assess whether the quality measures vary between the registration methods presented. Tests were performed for each quality measure between each pair of registration methods for a total of 2.6Variations in the manual initialization of segmentation could affect the final registration. To evaluate this effect, segmentation was repeated on the data of four patients. For each of these patients, both the CCTA and 3DE volumes segmentation was repeated once by the original operator and once by another operator. Segmentation was performed as described in Sec. Using the three-chamber alignment method, as described in Sec. The landmark distances used were apex distance Interobserver variability for 3DE segmentation as well as inter- and intraobserver variability for CCTA segmentation was quantified analogously.2.7For one of the included patients, the registration was visualized by displaying the coronary artery tree, obtained from CCTA, with the instantaneous longitudinal strain of the myocardium, obtained from 3DE.3-D speckle tracking, including 3-D block matching method, was utilized to track and quantify myocardial wall motion in the 3DE volume. Instantaneous longitudinal strain was estimated using EchoPac 4D AutoLVQ . The estimated strain values at 337 points  in the myocardium at each time frame were exported. In the CCTA volume, the coronary artery tree was segmented by an experienced radiologist using Advantage Workstation.3Statistical analysis on the quality measures presented in 4This study aimed at comparing and validating three methods for automatic CCTA/3DE registration: landmark distance minimization, endocardium ICP alignment, and alignment of three-chamber views. All three registration methods showed good performance with regard to landmark distances, as seen in Differences in actual shape are unavoidable due to the difference in the nature of CCTA and 3DE. In CCTA, the patient was medicated to decrease the heart rate, which likely reduced LV volume throughout the heart cycle,,Cardiac image fusion systems combining CCTA with PET or SPECT have been shown to contribute to facilitated decision on revascularization strategy compared with side-by-side analysisPatients with complex CAD and multivessel disease are frequently referred to revascularization procedures, although studies have shown that stenoses found on their angiograms often are functionally insignificant.A benefit of using 3DE as functional modality in cardiac image fusion instead of SPECT or PET is the lack of additional ionizing radiation. Still, the individual radiation exposure needs to be minimized and the risk versus benefit of each imaging examination must be taken into account. Over the last years, CCTA has emerged as a promising noninvasive alternative to invasive coronary angiography due to improved spatial resolution and lower radiation dose. The conventional way of analyzing coronary artery anatomy is by curved multiplanar 2-D slices in a 3-D CCTA volume. 3-D techniques alone have demonstrated insufficient performance in the evaluation of stenosisA limitation of this study is the small group of patients enrolled and only one female patient was included. This was partly due to lack of female patients with suspected CAD undergoing CCTA on clinical indication in our clinic. However, this study aimed at comparing and demonstrating the potential of different registration methods for automatic CCTA/3DE registration. For this purpose, the included patient group was considered to properly represent a variety of cardiac anatomies present in a patient population with known or suspected CAD. The clinical effectiveness of the proposed fusion technique still needs to be determined in larger studies of both female and male patient groups.,,The registration methods presented depended on proper segmentation since the registration is based on alignment of either landmarks or surfaces determined by the segmentation. These segmentation algorithms have been validated in previous studies with credible results.,Myocardial strain assessment has been shown to be useful in the detection of hemodynamically significant stenosis, in particular, when performing stress echocardiography.Traditionally, fusion of morphological and functional information is performed by mental integration that relies on models allocating myocardial segments to specific coronary arteries.The registration of CCTA/3DE data was performed in a single time frame, 35% or 75% of the R-R interval. The CCTA was performed in these time frames since the heart is moving at least at end systole  and middiastole  in patients with heart rates below 80 beats per minute.5Three methods for registration of CCTA and 3DE volumes were implemented and compared on data from patients with suspected CAD. All methods provided adequate registration and performed well with regard to landmark distances. Landmark distance minimization and alignment of the three-chamber planes performed better with regard to LV rotation than minimization of distances between endocardial surfaces (ICP). However, clinical studies must be performed to ascertain whether or not CCTA/3DE image fusion could be clinically viable and provide incremental value in CAD treatment decision.


We determined the presence of 13 PFAAs in 293 beef muscle and liver samples collected in 22 cities covering northern, southern, and eastern Xinjiang using liquid chromatography, coupled with tandem mass spectrometry. Overall, the average values for PFAAs were relatively low compared with previous studies. Liver presented higher mean levels of total PFAAs at 1.632 ng/g, which was over 60-fold higher than in muscle (0.026 ng/g). Among the PFAAs analyzed, medium-chain compounds were dominant, accounting for more than 70% of the total. Perfluorooctane sulfonate (PFOS) was highly prevalent in the liver with the highest mean concentration (0.617 ng/g) and detection frequency (80%). When comparing the three regions of Xinjiang, we found differences in PFAA profiles, with the northern region showing the highest levels. Furthermore, the average daily intake and hazard ratios of PFOS and perfluorooctanoic acid varied by region, urban/rural environment, gender, ethnicity, and age. The highest risk value of 13 PFAAs was estimated to be 0.837 × 10 Perfluoroalkyl acids (PFAAs) are an anthropogenic, emerging class of persistent organic pollutants (POPs). In general, these compounds are composed of a fully fluorinated carbon chain with an acidic terminal group, such as a carboxylic, phosphonic, or sulfonic acid moiety, with the strong chemical bonds of C-F conferring their unique amphiphilicity, persistence, and bioaccumulation properties ,2,3. ConLarge quantities of PFAAs have been manufactured and used since the 1950s, representing more than 60 years of existence. The 3M Company declared a phase-out of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) (the two most frequently used PFAAs) and their long-chain homologs in 2000 due to their widespread contamination and potential risks to the environment and to humans, and then phased-in a series of shorter-chain alternatives with relatively low toxicity -octane sulfonate . Tetrabutylammonium hydrogen sulfate  and tert-butyl methyl ether  were supplied by Aladdin . Methanol  and acetonitrile  were from Fisher Scientific . Ammonium acetate  was supplied by Sigma-Aldrich . Sodium carbonate anhydrous  and sodium hydrogen carbonate  were purchased from J&K . Oasis HLB  was provided by Waters . All experimental water used in this study was ultrapure water (>18.2 MΩ/cm) prepared in the laboratory.All the analytical standards used in present study were obtained from Wellington Laboratories . Native standards were: perfluorobutanoic acid , perfluoropentanoic acid , perfluorohexanoic acid , perfluoroheptanoic acid , perfluorooctanoic acid , perfluorononanoic acid , perfluorodecanoic acid , perfluoroundecanoic acid , perfluorododecanoic acid , perfluorotridecanoic acid , perfluorobutane sulfonate , perfluorohexane sulfonate , and perfluorooctane sulfonate . Labeled standards used were: perfluoro-n-[1,2,3,4-2O, 1/9, v/v). After that, it was dried for 30 min under a vacuum and eluted with 7 mL MeOH. Throughout the treatment process, the velocity of flow was 1–2 drops per second. The obtained eluent was then blown to near dryness by nitrogen and reconstituted in 1 mL of mobile phase . The reconstituted extract was filtered into an autosampler vial using a 0.22 μm nylon syringe filter prior to the final HPLC-MS/MS analysis. Eventually, the concentration of dry weight was converted to wet weight through the ratio of dry and wet weights.The target compounds were extracted from muscle and liver by an ion-pairing method published previously ,25,26, w18 80A column . The mobile phase consisted of 10 mM NH4OAc aqueous solution (A) and 100% ACN (B). During the process of gradient elution, the initial stage was 60/40 A/B followed by 9 min ramped to 10/90 A/B and held for 3 min. Ultimately, it was reconditioned for 8 min of stabilization time using the initial conditions (60/40 A/B). Detection was performed with an Agilent 6410 triple-quadrupole mass spectrometer that operated in negative ion multiple reaction monitoring (MRM) mode. Further details, such as monitored MS/MS transitions, retention time (RT), and collision energy (CE) are given in the In this study, HPLC-MS/MS analysis was performed for 13 target PFAAs. A total of 10 μL sample was injected into an Agilent 1200 series HPLC system  equipped with an automatic degasser, a quaternary pump, and a thermostated column compartment (maintained at 40 °C). Separation of target compounds was achieved on an Zorbax Eclipse Plus C13C4-PFOA, 13C4-PFOS) into all samples at the beginning of extraction. Therefore, errors such as matrix interferences, extraction and/or MS acquisition discrepancies could be quantified and corrected. A 10-point matrix-matched calibration curve with concentration levels from 0.01–20 ng/mL was prepared using blank matrix extract serial dilutions for each analyte, and all correlation coefficients (R2) were better than 0.990. The limits of detection (LODs) for each substance were estimated as the concentration with a signal-to-noise ratio (S/N) of 3, and the limits of quantification (LOQs) were defined as the lowest calibration standard with S/N higher than 10. In addition, recovery tests for all PFAAs at three spiking levels  to blank matrices were performed. The LODs for muscle varied in a range of 0.012–0.06 ng/g; mean recoveries were between 73.8 and 115.4%. For liver, the LODs ranged from 0.003 to 0.036 ng/g, and mean recoveries were between 84.2 and 110.0%. Relative standard deviations (RSDs) repeated for six extractions were lower than 10%, indicating that the precision of the method was good. More detailed information  of the 13 measured PFAAs and 2 internal standards provided in Quantification was performed by adding the isotopically labeled standards  was injected every 10 samples to eliminate memory effects. To monitor contamination of reagents and glassware, procedural blanks were injected every 20 samples. New PP centrifuge tubes were used in all experiments and all materials with polytetrafluorethylene (PTFE) were removed from the experimental stations. All test containers were alternately rinsed twice with ultrapure water and MeOH before use. The instrument was rinsed overnight before use, and the ion source was cleaned regularly. All experimental results were deducted from blank experimental data.To elucidate the contribution of beef muscle and liver consumption to human exposure to PFAAs, we evaluated the daily intakes of PFOS and PFOA  and the possible health risks caused by them. Muscle consumption data were obtained from a survey on dietary structure analysis in Xinjiang , conductmuscle/liver) divided by the average body weight (BW), as shown in Equation (1). The hazard ratios (HR) for PFOA or PFOS were calculated with their respective mean concentrations in the samples (CPFOS/PFOA) multiplied by DI  and divided by their reference dose  [The daily intake (DI) is the average daily consumption for muscle and liver per unit body weight, ng/kg/day, and the calculation is the sum of daily consumption values of muscle and liver (CD bw/day) , which aThe estimated formulas were as follows:The aggregated data of 13 measured PFAAs are presented in For muscle samples, only PFOS, PFOA, PFHpA, and PFUdA were above their respective LODs, and all the detection frequencies were lower than 50%. However, nearly all target compounds were found in liver except for PFHxS (under its LOD) . A discoWe investigated variations in PFAA levels among northern, southern, and eastern Xinjiang, and these are displayed in Similar PFAA profiles were observed in the same food items a. PFOA aA summary of recent studies (2008–2014) of individual compounds in animal muscle and liver is given in When comparing PFAA contaminations among different animal matrices, higher levels were observed in beef muscle, whereas beef liver had lower levels than chicken and pork . DiffereLarge spatial variations of PFAAs levels are observed among countries. For muscle samples, those collected from Italy showed the highest concentrations (beef meat: 2.11 ng/g) ; for livSimilar to human exposure pathways, cattle may be exposed to PFAAs by ingesting food, drinking water, and inhaling air/dust. It has been found that PFOS and PFOA can be transferred from soil to crop plants  ,45, and The differences between PFAA levels in commercial feed and home-raised livestock may exist, due to living and eating habits causing different exposures to specific sources of these compounds, but there is no evidence for this yet. However, levels of PFAAs in eggs produced from differently raised chickens have been investigated , and homPFOS: 0.039 ng/kg bw/day, ADIPFOA: 0.034 ng/kg bw/day; HRPFOS: 0.257 × 10−3, HRPFOA: 0.023 × 10−3)  [PFOS: 0.021 ng/kg bw/day, ADIPFOA: 0.028 ng/kg bw/day) and the lowest  in the elderly (> 70 years). Diet composition is an important factor for intake, and higher consumption of dairy products and fish/shellfish constitutes the main source of exposure in young children (1–6 years) in Korea [In this survey, we made a hypothesis that beef muscle was the only consumed animal meat and beef liver was the only viscera intake. The health risks were assessed using the total meat/liver consumption data, and in this case, the estimated HR would be the highest exposure levels. PFOS and PFOA exposures though muscle and liver were examined for five parameters including region, urban/rural environment, gender, ethnicity, and age, and the results are shown in  × 10−3) . These A bw/day) ,44. It iPFOA; however, liver mostly contributed to HRPFOS (PFAAs) was initially calculated and is presented in PFAAs were calculated using PFOS as the reference, which is only a rough estimation. It is worth mentioning that the bioaccumulation potential and toxicity of PFAAs increases with the chain length [PFAAs was calculated to be 0.837 × 10−3 (northern Xinjiang)  b, which The pollution status and health risk assessment of various PFAAs in animal meat samples of Xinjiang were systematically investigated for the first time. In summary, PFAA contamination was widespread in cattle samples, especially in liver, although the pollution levels were lower than in most other studies. PFOS, which is overwhelmingly observed in liver, and PFOA, were the dominant contaminants. In terms of regional variation, total PFAAs decreased in the following order: north > south > east. In terms of structure, they decreased in the following order: medium-chain > long-chain > short-chain. The results of source analysis showed that many factors could cause PFAA contamination, and feeding modes have a great influence on exposure. Remarkably, the risk ratios were much lower than 1 in all cases and suggest that no imminent damage to the health of the Xinjiang population via beef intake exists. Nevertheless, more categories of food are expected to be analyzed in Xinjiang, with the aim of comprehensively estimating PFAA exposure in humans, and several uncertainties  should be taken into account. A wider age range, including toddlers, children, adolescents, and adults, should be considered to monitor and protect the health of different age brackets. Additionally, further studies are expected to investigate PFAAs found in the serum and plasma of humans, because their levels could better represent the degree of contamination, especially in the Xinjiang population.


The exposure pathways of perfluoroalkyl acids (PFAAs) to humans are still not clear because of the complex living environment, and few studies have simultaneously investigated the bioaccumulative behaviour of different PFAAs in humans. In this study, serum, dust, duplicate diet, and other matrices were collected around a manufacturing plant in China, and homologous series of PFAAs were analysed. PFAA levels in dust and serum of local residents in this area were considerably higher than those in non-polluted area. Although dietary intake was the major exposure pathway in the present study, dust ingestion played an important role in this case. Serum PFAAs in local residents was significantly correlated with dust PFAAs levels in their living or working microenvironment. Serum PFAAs and dust PFAAs were significantly higher in family members of occupational workers (FM) than in ordinary residents (OR) (p < 0.01). After a careful analysis of the PFAAs exposure pathway, a potential pathway in addition to direct dust ingestion was suggested: PFAAs might transferred from occupational worker's clothes to dinners via cooking processes. The bioaccumulative potential of PFHxS and PFOS were higher than other PFAAs, which suggested a substantial difference between the bioaccumulative ability of perfluorinated sulfonic acids and perfluorinated carboxylic acids. Perfluoroalkyl acids (PFAAs) have been extensively used as surfactants, lubricants, stain- and water-resistant coatings, and fire-resistant foams, due to their unique properties233PFAAs have been detected in human blood worldwide, and in certain cases, their levels were even higher than those of other persistent organic pollutants9111314161518Our previous study investigated the transportation of PFAAs from the manufacturing facility to the surrounding environment and elevated perfluorohexanesulfonate (PFHxS), PFOS, and PFOA levels in soil and biota samples were found within three kilometres of the plantA fluorochemical manufacturing plant located in the Hubei province of China was selected as the sampling area. The main synthesis method for PFAAs in this plant is by the electrochemical fluorination process. The sampling procedure was performed in December 2011 and 2012. Fifty-eight local residents who lived around this plant within a distance of 3 km were recruited in the present study. The gender, age, weight, occupation, dietary habits, and other information were collected by questionnaires; the demographic information of studied population is listed in Furthermore, to investigate the exposure pathways of PFAAs to residents around the manufacturing facility, relevant exposure matrices , and duplicate diet) from their living environment were also collected. Dust and TSP samples were collected from the plant and the corresponding participants' residential environment. Duplicate diet and drinking water samples were directly collected from the table and tap from the home of participants. In addition, the work clothes of workers and local residents were also collected. A total of 141 samples, including serum (n = 58), dust (n = 38), TSP (n = 15), drinking water (n = 6), duplicate diet (n = 9) and clothes (n = 15), were collected in this study. The detailed information of these samples  is listed in Eleven PFAAs (perfluoro-n-pentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), PFOA, perfluorononanoic acid (PFNA), perluorodecanoic acid (PFDA), perfluorododecanoic acid (PFDoDA), perfluoroundecanoic acid (PFUnDA), salts of perfluorobutanesulfonate (PFBS), PFHxS, and PFOS were determined by HPLC-ESI-MS/MS. Sample pretreatment, instrumental analysis, quality assurance, and quality control of the analytical method are based on our previous work2223foodC represents concentration of PFAAs in food (ng/g) and waterC stands for concentration of PFAAs in drinking water (ng/mL). According to the questionnaires, the average body weight of local residents was 60 kg (dust) was calculated through the following equation: DIdust = Cdust × Adust/body weight, where Cdust is the concentration of PFAAs in house dust, Adust is the average amount of the daily dust ingestion, and the mean gastrointestinal uptake of dust is set at 50 mg/day for adults according to the US EPA exposure factors handbookTSP = CTSP*Vair/body weight, where CTSP is the concentrations of PFAAs in TSP, Vair is the daily air volume inhaled by adults and was set at 16.0 m315The daily dietary intake of PFAAs is estimated by the sum of PFAAs in the ingested diet and water. Diet and drinking water consumption are set at 2500 g and 1200 ml per day, respectively. Thus, the daily dietary intake of PFAAs is calculated as as 60 kg . The nonE) and the change of PFAAs levels could be described as k is the first-order rate constant for PFAAs elimination per day . According to previous studies, dV of PFOA and PFOS were set at 170 and 230 mL/kg bw, respectively28pC is the PFAA concentrations in serum. If the serum PFAAs reached steady-state conditions, dpC/dt = 0. Thus, the former equation could be simplified as Furthermore, based on a simplified one compartment pharmacokinetic model, we estimated the total estimated daily intake (TEDI) of humans by serum PFAA levels. The model was developed by Andersen et al. and Thompson et al.283031PFPeA, PFHxA, PFNA, PFDA, PFDoDA and PFUnDA had low detection rates in all samples, and therefore, they were not included for further data analysis and discussion. PFBS, PFHxS, PFOS and PFOA were detected in all serum samples in local residents while PFHpA had a detection frequency of 67%. The average serum PFBS, PFHxS, PFOS, PFHpA, and PFOA concentrations were 22.7, 138, 445, 2.85 and 50.6 ng/mL, respectively. The detailed characteristics of PFAAs in resident demographic subgroups by gender and age (≤45 and >45 years) were provided in PFAAs in drinking waters were relatively low , the mai363740dust for occupational workers reached 1679 ng/day/kg bw, and the DIdust of PFOS and PFOA were 604 and 759 ng/day/kg body, respectively. These values were almost three orders of magnitude higher than those estimated in previous studies18dust of PFOS, PFOA, and PFHxS of local residents was 0.991, 0.295, and 0.057 ng/day/kg body, respectively. PFAAs exposure via TSP inhalation was lower than dietary intake and dust ingestion in local residents . In contrast to TSP, dust PFAA levels were significantly higher in the FM group (6775 ng/g) than in the OR group (429 ng/g) (p < 0.01). It was surprising that the high PFAA levels in dust seems not play a role in elevating the PFAA levels in TSP via resuspension in this study. It might because that the large noise of the TSP sampler kept human activities  away during TSP sampling process. High PFAA levels in dust samples collected from FM indicated that occupational workers elevated the PFAAs levels in the living environment. PFAAs levels in the duplicate diet were considerably higher in the FM group than in the OR group, PFOS level in duplicate diet from FM group was 1.41 ng/g ww, while it was only 0.077 ng/g ww in the duplicate diet from OR group. However, the food material of both groups was mainly purchased from the local market, and the drinking water was supplied by municipal water systems, so there was no special food source for the FM group according to questionnaires. Through our observation and further investigation, most of the occupational workers did not change their clothes after work. We collected clothes of both workers and residents, used distilled water to wash them, and then analysed PFAAs in the wash-off water , with the GM of 218275 and 4402 ng/g, respectively  of FM was higher than that of OR, which was consistent with the PFAAs body burden. Dietary intake was the major exposure pathway in the present study, which was consistent with previous studies151819dust of PFOS and PFOA accounted for 5.2% and 20.0% of the total daily intake (TDI) in the FM group, and 6.0% and 8.0% in OR group, respectively. Compared to that DIdust accounted for less than 2% of daily intake in the Chinese population12The total PFAAs daily intake  and two groups of serum data (FM and OR group) in this study. Thus, we cited a set of unpublished data (data only for the reviewer), the serum PFAAs levels of occupational workers from different departments in this plant in 2011 , to get more paired data of dust and serum samples and make a better significance of fit. The result indicated that logarithm serum PFAA concentrations were significantly correlated with the corresponding logarithm dust PFAA concentrations (p < 0.05), which suggested that PFAAs body burden was closely related to dust ingestion in this setting .The PFAAs profiles changed largely between the dust and serum samples of local residents. PFSAs accounted for 68.2% of the total PFAAs in dust samples, which then increased to 87.4% in serum samples in the present study . The resSerum to the proportion of PFAADust was calculated to illustrate the different accumulation potential of the studied PFAAs, and this value was defined as the potential bioaccumulative index (PBI) in later discussions. The PBI of five PFAAs were ranked as follows: PFHxS (7.41) > PFOS (3.90) > PFOA (0.359) > PFBS (0.057) > PFHpA (0.051). The PBIPFHpA and PBIPFBS exhibited a low bioaccumulation potential in the present study, and it was consistent with Conder et al.'s report that PFCAs with less than seven fluorinated carbons might not accumulate in biotaPFHxS and PBIPFOS were nearly 130 and 68.4 times higher than PBIPFBS, respectively. Olsen et al. reported that half-lives of PFHxS and PFOS were 107 and 70 times of PFBS, respectively, which were highly consistent with the PBI results in this study33833The ratio of the proportion of PFAASupplementary information


This highlights the possible role of phosphoproteins in the temporal aspects of striatal signal transduction.In reward learning, the integration of NMDA-dependent calcium and dopamine by striatal projection neurons leads to potentiation of corticostriatal synapses through CaMKII/PP1 signaling. In order to elicit the CaMKII/PP1-dependent response, the calcium and dopamine inputs should arrive in temporal proximity and must follow a specific  order. However, little is known about the cellular mechanism which enforces these temporal constraints on the signal integration. In this computational study, we propose that these temporal requirements emerge as a result of the coordinated signaling via two striatal phosphoproteins, DARPP-32 and ARPP-21. Specifically, DARPP-32-mediated signaling could implement an input-interval dependent gating function, via transient PP1 inhibition, thus enforcing the requirement for temporal proximity. Furthermore, ARPP-21 signaling could impose the additional input-order requirement of calcium and dopamine, due to its Ca A response towards an environmental stimulus could be reinforced if it elicits a reward. On the subcellular level, the environmental stimulus and the reward signal lead to a transient increase in striatal calcium- and dopamine-signaling, respectively. The integration of calcium and dopamine signals, which is important for reward-learning, could elicit a downstream response only if they are close in time and arrive in correct order . This study proposes that the requirement for the input signals to be temporally close and in correct order could emerge due to the coordinated signaling via two striatal phosphoproteins, DARPP-32 and ARPP-21. The DARPP-32 signaling implements an input-interval dependent gating function and ARPP-21 implements an input-order dependent threshold-like function. Thus, a molecular mechanism has been presented here which could explain the emergence of important temporal aspects of subcellular signal integration in reward-learning. Reinforcement learning plays an important role in building the learned-behavioral repertoire of an organism. It operates by updating the salience of an environmental cue or a cue-response association which has elicited a reward in the past. Basal ganglia are critical for reward learning and the input nucleus, striatum, is the locus of integration for the environmental and the reinforcement signals . The env2+-Calmodulin-dependent kinase II (CaMKII) and Protein-Phosphatase 1 (PP1) signaling plays an important role in this process [The postsynaptic integration of NMDAR-mediated calcium and dopamine-dependent D1R signaling leads to the potentiation of corticostriatal synapses on D1R-expressing MSNs, thus resulting in reward-learning –9. Ca2+- process ,11. HoweDopamine and cAMP-regulated Phosphoprotein 32kDa) could play an important role in the dopamine-dependent gating of the calcium signaling, which aligns with previous experimental observations [AMP-Regulated Phosphoprotein 21kDa), has the potential to introduce the input-order dependency into the integration process. ARPP-21, upon dopamine-dependent phosphorylation, has the ability to bind with Ca2+/calmodulin, thus affecting the calcium signaling [In this computational study, we investigated the integration of calcium and dopamine signals by D1R-expressing MSNs to explain the emergence of the aforementioned temporal constraints, using quantitative kinetic modeling. Our results suggest that DARPP-32  with a maximum timestep of 0.01s. This model assumes a well-mixed approximation in a single compartment. Thus, no spatial or molecular heterogeneity has been taken into account.If a reaction is an irreversible reaction then the backward rate constant, kThe topology of the signaling network is built using biochemical reactions which have experimentally known interpretations for the signaling pathways. While it is possible that there are additional reactions and pathways in the actual system, we selected a subset which is consistent with the known striatal literature and pertinent to the currently-studied behavior. In cases where biochemical details were incomplete, we assumed simple mass action reactions that could account for the known molecular dependencies. A detailed description of the signaling network is provided below. After fixing the topology, the free variables left in the system are the reaction kinetic parameters and starting amount for different proteins. The parameters have been constrained using various types of neuronal and, in many cases, MSN-specific data. A list of target phenotypes used to constrain the model is also described below. The model SBML file could be downloaded from BioModels database (Model Id: MODEL1603270000) [It is known that the integration of transient calcium and dopamine signals leads to synaptic potentiation of the corticostriatal synapses and this process is mediated by CaMKII activity ,9,17. Ca2+/calmodulin. The transition of calmodulin to Ca2+/calmodulin is modeled as a sequential two-step reaction. Each step results in the binding of calcium to one of the calcium-binding domains in calmodulin. The parameters for each of the steps are factored assuming a double binding-site Adair-Klotz’s model fitted to the known calcium calmodulin binding data [The sequence of calcium-triggered signaling events modeled in this study is shown in ing data . At eaching data .2+/calmodulin target is CaMKII. Under the basal condition, the inactive CaMKII is known to be enriched in the F-actin rich cytoplasmic compartment [In our model, an important Capartment . On the partment . In our partment . These o2+/calmodulin [2+/calmodulin [The activation of CaMKII leads to its autophosphorylation thereby further increasing its affinity to Calmodulin . The autlmodulin . We modelmodulin . Additiolmodulin , which ilmodulin ,26,27. TThe active CaMKII leads to the phosphorylation of its substrates and many of these substrates are dephosphorylated by PP1 ,11. To colf G-proteins [olf further predominantly activates Adenylyl Cyclase type V (AC5) which leads to an increased level of cAMP [The sequence of dopamine-triggered signaling events modeled in this study is shown in proteins ,30. The  of cAMP ,31. The  of cAMP –37. Thusolf-dependent AC5 activation. The parameters for the cAMP degradation by phosphodiesterases (PDEs) have also been refined in the current version of the model to match the known PDE kinetics observed in live cells [Under the basal condition the cAMP levels are expected to be low in the D1R expressing MSNs due to the low basal receptor activity. The basal cAMP level estimated for different types of neurons with low AC activity is around 30–90 nM ,39. In sve cells . It is ave cells . The eleve cells . Similarve cells . Furtherve cells . These MPKA activation in MSNs leads to the phosphorylation of DARPP-32 at its Thr-34 residue and this turns DARPP-32 into a potent inhibitor of protein phosphatase PP1 . DARPP-3As described in the “Results” sections, our results indicate that the DARPP-32-mediated dopamine signaling alone is insufficient to explain the emergence of certain temporal constraints of the calcium-dopamine integration  dopamine and (2) calcium. The dopamine input represents increased extracellular dopamine concentration due to the burst activity of dopaminergic neurons in response to an unexpected reward . Such a As described above, the integration of striatal calcium and dopamine inputs leads to the phosphorylation of downstream CaMKII/PP1 substrates in MSNs. This, however, requires that the inputs should fulfill certain temporal requirements . SpecifiTo investigate the integration of dopamine and calcium inputs by the subcellular signaling processes in the D1R expressing MSNs, we developed a kinetic model of the standard striatal signaling network, We first looked at how dopamine could affect the calcium triggered CaMKII signaling. To this end, we measured the difference between the CaMKII/PP1 substrate phosphorylation produced by a transient calcium input , where tcalcium and tdopamine are the start time of calcium and dopamine transients, respectively. A small absolute value of Δt indicates that the calcium and dopamine inputs are close in time. A positive value of Δt means that the calcium input is followed by the dopamine input and vice-versa. This relation between the substrate response and Δt could shed light on whether the input-interval and input-order constraints exist in this signaling network. If the substrate response is higher for smaller absolute values of Δt compared to large Δt values then this suggests the existence of an input-interval constraint. In other words, calcium and dopamine should be close in time for effective downstream activation. Similarly, if substrate response is higher for positive values of Δt compared to negative values then this indicates the existence of input-order constraint, i.e. the calcium input should be followed by the dopamine input for effective downstream response.Using the aforementioned signaling network, we then investigated how the substrate phosphorylation depends on the temporal relation between calcium and dopamine inputs. The interval between the inputs is referred as Δt, such that Δt =  and Ser-55 mutant of ARPP-21 (A21S55A) shows that loss-of-function mutation in ARPP-21 abolishes the input-order interval by significantly affecting the response in the negative Δt region, As shown above, the addition of ARPP-21 into the signaling network could introduce the input-order constraint by significantly reducing the downstream response for the negative Δt regime, We looked at how active CaMKII and PP1 levels are affected for different Δt values to understand the relation between substrate phosphorylation and Δt for this updated signaling network, 2+/calmodulin [2+/calmodulin sequestration by ARPP-21 thus reducing the CaMKII activation. To ascertain this possibility we first considered the case of Δt = -2s as an example, 2+/calmodulin. A significant part of the Ca2+/calmodulin binds to the pARPP-21 produced by the preceding dopamine, 2+/calmodulin available for CaMKII activation, 2+/calmodulin leads to a lower CaMKII activation. Thus, the sequestration of Ca2+/calmodulin by pARPP-21, thereby reducing its availability, indeed explains the low CaMKII activation in the negative Δt regime.We then investigated how the ARPP-21 could reduce the CaMKII activation in the negative Δt regime, i.e. only when dopamine input precedes the calcium input. It is known that dopamine leads to the phosphorylation of Ser-55 residue on ARPP-21 and this phosphorylation turns ARPP-21 into an excellent binding partner of Calmodulin . This co2+/calmodulin by pARPP-21 is also reduced, 2+/calmodulin available for CaMKII activation, 2+/calmodulin by ARPP-21 is negligible, 2+/calmodulin available for CaMKII activation is not reduced, As the interval between dopamine and calcium decreases in the negative Δt region, e.g. Δt = -1s, the sequestration of CaAs highlighted in the previous section, the dopamine-dependent phosphorylation of ARPP-21 results in the dampening of a subsequent calcium-triggered CaMKII activation. In a two-trial scenario, this could mean that the ARPP-21 which is phosphorylated in the first trial may affect the CaMKII activation in the second trial. To test this cross-talk between successive trials we considered the calcium-dopamine integration in a two-trial scenario. The two trials are separated in time by an inter-trial interval (ITI). Each of the trials is represented by a pair of calcium and dopamine inputs with Δt = 1s.2+/calmodulin-dependent downstream response in an ITI dependent fashion.We first tested a scenario with an ITI = 10s. In this scenario, the substrate response produced by the first trial was similar to the response observed in a single trial case (as in the previous sections), ITI = t is the area under the curve for substrate phosphorylation in the second trial which is separated by the first trial with an ITI = t seconds. ITI = 100 is the activation area for second trial with an ITI = 100s. The trials with ITI = 100s are considered as well separated trials because with this ITI the second trial response is not affected by the first trial. Thus, if REF = 1, then the response in the second trial is fully eliminated due to the inhibitory effect of the first trial and if REF = 0, then there is no effect of the first trial on the response of the second trial. On the other hand, a negative value for REF indicates a potentiating effect of the first trial on the second trial.We define a metric called refractoriness (REF) to quantify the ITI dependent effect of the first trial on the response of the second trial. REF is defined as follows:The simulations suggest that the REF decreases as a function of ITI, i.e. REF is higher for lower ITI values and it decreases as ITI increases It could be possible that in a more complex scenario  the downstream response may integrate over the trials thereby overcoming the refractoriness imposed by pARPP-21. To test this possibility, we implemented a 10 trial scenario with ITI = 10s. Similar to the two trial scenario, each trial consists of 10 calcium pulses at 10 Hz followed by a dopamine input with Δt = 1s. The simulation for this 10 trial scenario suggests that there appears to be a small build of substrate response over the trials, Amplitude of the striatal dopamine represents the reward prediction error and it could vary depending on the difference between the expected and the acquired reward . In a siThe quantitative model built in this study is quite detailed and contains several parameters. Many of the parameters which might be important are not directly measured for the system of interest. They are rather optimized to fit experimental observations/phenotypes which emerge from the interactions of multiple parameters. However, it is important that the biological phenomena and predictions in the study should be robust with respect to changes in parameters around the selected point in the parameter space. Here, the parameters of interest are the kinetic rate constants of individual reactions and the starting amount of various conserved species. As mentioned above, the model output appears to be quite robust with respect to different parameter perturbation, The top sensitive-parameters list for the positive Δt region includes several DARPP-32 related parameters, In the case of negative Δt, the top sensitive parameters include the total amount of ARPP-21 and the kinetic parameter for ARPP-21 phosphorylation by PKA, 2+/calmodulin. It is also interesting to note that the total amount of calmodulin appears to be a sensitive parameter for both positive and negative Δt region. This could be because calmodulin has several interactions with the DARPP-32, ARPP-21 and CaMKII mediated signaling. The combined effects of calmodulin on the DARPP-32 and CaMKII significantly affect the gating potential of DARPP-32 signaling. Moreover, the core ARPP-21 mediated signaling relies on the calmodulin sequestration/competition thus adding further to the sensitivity of the model/predictions towards calmodulin.Additionally, the total amounts of CaMKII and PP2B also appear in the list of sensitive parameters in this case. This becomes clear by considering that both CaMKII and PP2B are also the downstream targets of calmodulin similar to ARPP-21 and changes in their total amounts could affect the level of binding between phospho-ARPP-21 and CaThe subcellular integration of the NMDA-dependent calcium and dopamine by D1R-expressing MSNs is believed to be an important biological process for reward learning. In this kinetic modeling study, we propose a novel mechanism which could explain the emergence of the observed input-interval and input-order constraints on the integration of calcium and dopamine. These temporal constraints could emerge as a result of the coordinated signaling through two striatally enriched phosphoproteins, namely DARPP-32 and ARPP-21, In our simulations, dopamine acts as a gate for the calcium-triggered CaMKII/PP1 signaling and this aligns with known observations ,9,17. ThThe transient nature of both CaMKII activation and PP1 inhibition limits the possible time-interval (Δt) between the respective calcium and dopamine inputs, which could be synergistically integrated. Moreover, the PP1 inhibition kinetics should be fast enough to match the temporal characteristics of CaMKII activation. In other words, the PP1 inhibition should occur before the transient CaMKII activation fades away if these two were to interact. This requires particularly fast kinetics for the cAMP/PKA signaling axis which is already known to be the case in striatal neurons . Recent 2+/calmodulin, thus reducing the available Ca2+/calmodulin for CaMKII activation. Similar to the DARPP-32 mediated phenomenon, this ARPP-21 mediated effect also demands fast phosphorylation kinetics.Even though the transient DARPP-32-dependent effect could implement the observed input-interval constraint, according to our results, DARPP-32 signaling alone may not distinguish between the order of calcium and dopamine incidence. Moreover, the molecular identity of the substance which could make the striatal CaMKII/PP1 signaling sensitive to the temporal order of calcium and dopamine is not clear. However, as presented above, ARPP-21 has the potential to implement an input-order dependent filtering mechanism by acting as a competitor to CaMKII. This could enable the striatal signaling machinery to distinguish between the incidence order of calcium and dopamine. When ARPP-21 is phosphorylated by a preceding dopamine it turns into a potent sequestering agent for subsequent calcium-triggered Ca2+/calmodulin. This competition stipulates that the amount of calmodulin be lower than the collective amount of its possible binding partners, i.e. Ca2+/calmodulin is a limiting resource. If Ca2+/calmodulin is not a limited resource then pARPP-21 and CaMKII do not have to compete with each other, thereby reducing the inhibitory effect of ARPP-21 on CaMKII activation. This in turn will eliminate the input-order sensitivity of the calcium-dopamine integration. Even though the absolute amount of calmodulin could be high in many cell types, various studies suggest that free calmodulin is indeed a limiting resource [2+/calmodulin seems to exist also in MSNs [As aforementioned, the ARPP-21 mediated mechanism rests on the idea that ARPP-21, upon phosphorylation, competes with CaMKII for the available Caresource –69. The resource . Moreove in MSNs  thus sup2+/calmodulin is also responsible for the activation of PP2B which dephosphorylates DARPP-32 Thr34. However, unlike CaMKII, pARPP-21 does not have any significant effect on the level of PP2B activation, for transient input signals, in our signaling model because the affinity of Ca2+/calmodulin towards PP2B is assumed to be very high . Thus, the current study does not predict any significant ARPP-21 mediated effect on DARPP-32 dephosphorylation within the assumed parameter space for transient input signals. However, this does not rule out such a regulation. Rather this could be due to the insufficiency of data to tune this aspect of the current signaling model.In addition to CaMKII, Car range; ) compareEven though the role of striatal ARPP-21 is not clear, it seems to be involved in addiction and motivational behavior ,72. The Apart from imposing the input-order constraint on the calcium-dopamine integration, our simulations also suggest that ARPP-21 could impose an inter-trial refractoriness as a function of the ITI in a multi-trial conditioning scenario. This could imply that an MSN, or its dendritic segment, which is potentiated in the recent past would resist a further potentiation within a refractory period. This could mean that multiple trials with shorter ITIs may not be completely independent to each other. As the ITI increases the trials start to become decoupled. Such, an inverse relation between conditioned response and ITI is well known for classical conditioning ,74. Alth2+/calmodulin stimulated Adenylyl cyclase, which exhibits calcium-dependent priming on the G-protein activation [2+/calmodulin stimulated Adenylyl cyclase, Adenylyl cyclase type I (AC1), has also been suggested to be involved in the emergence of striatal input-interval and input-order constraints [s (Golf) G-protein and Ca2+/calmodulin and is known to act as a coincidence detector for these two signals [olf and does not display any calcium-dependent synergy [This study highlights the possible role of phosphoproteins in controlling the temporal aspects of striatal signal transduction. Furthermore, we also attempt to illustrate the idea that different aspects of a single phenomenon  could be distributed among parallel signaling modules (DARPP-32 and ARPP-21) and do not necessarily have to be implemented by a single signaling node. One such single-signaling node which is believed to be responsible for similar temporal constraints in other systems, like Aplysia serotonin response, is Cativation ,76. A sistraints . AC1 is  signals . Even th signals ,79. Duri signals ,79,80. A synergy . It coulOn the other hand, the DARPP-32/ARPP-12 dependent mechanism proposed here is quite generalizable to various systems consisting of different AC isoforms. Even though we focused on the potential role of DARPP-32 and ARPP-21, they might just be part of a much wider signaling mechanism to produce the temporal constraints on striatal calcium-dopamine integration. For example, the activation of other signaling processes, like PKA, have also been observed to display the input-interval and input-order constraints . Thus, fIt is interesting to note that there are brain regions other than the striatum, e.g. the amygdaloid complex and the bed nucleus of the stria terminalis (BNST), where the co-expression of DARPP-32 and ARPP-21 is known to exist ,85–87. BS1 Text(PDF)Click here for additional data file.


The precipitates were characterized by chemical and thermogravimetric analyses, x-ray powder diffraction, infrared spectroscopy, polarized light microscopy, and by their fluorescent properties. The precipitation boundary was established tindallometrically and microscopically. On the basis of these measurements, the stability conditions, structural parameters, and solubility of the tetragonal polymorph of NH4[UO2PO4]·3H2O were determined. This compound shows a close structural relationship with H3O[UO2PO4]·3H2O (space group P4/ncc) and alkali uranyl(2+)phosphates polyhydrates M[UO2PO4]·nH2O . The unit-cell dimensions determined for NH4UO2PO4·3H2O are: a=b=7.02 Å, c=18.08 Å (P4/ncc). The thermodynamic solubility product constant, Ks=a(NH4+)×a(UO22+)×a(PO43−), for NH4UO2PO4·3H2O was determined: log Ks= −26.50±0.09. The Ks values of M[UO2PO4]·n H2O  calculated from previously published experimental data by using correct stability constants of uranyl(2+)phosphate complexes are:Ks=−22.61±0.08 for M=Na;log Ks= −23.92±0.12 for M=K;log Ks= −24.13±0.19 for M=Rb;log Ks= −23.80±0.20 for M=Cs; andlog Ks= −24.74±0.10 for M=NH4,log showing that NH4UO2PO4·3H2O is less soluble than corresponding alkali uranyl(2+)phosphates.Precipitates formed in the system UO At pH>9 mixtures of amorphous NH4UO2PO4·3H2O (prevailing solid phase) and an undefined compound (designated by X in NHnge pH⩽2 . Its cry4UO2PO4·3H2O(s) . The ionic concentration product, Ks=c(NH4+)×c(UO22+)×c(PO43−), expressed in greater detail form isc(UO2)soln and c(NH4)soln are the total concentrations of uranyl and ammonium species in the solution, respectively. K13, K12, and K1 are the association constants of phosphoric acid  are presented in Ks (I = 0) has at 298 K an average value of −26.50±0.09 phosphate tetrahydrate as compared to that of alkali cations ·3H2O reveals a close structural relationship with the series M[UO2PO4]·nH2O . The crystal structure of M[UO2PO4]·nH2O  ions. Uranium exhibits an octahedral coordination. The PO4 tetrahedron acts as a monodentate bridging group; each PO4 group is coordinated to four UO22+ ions. A striking structural feature is the arrangement of the water molecules. The size of the hydrogen, alkali and ammonium ionic species in particular compounds affects the content of the crystalline water in the unit-cell.The x-ray powder pattern of NHO4]·3H2O ) reveals4[UO2PO4]·3H2O , K(ri= 1.33 Å), NH4(ri= 1.43 Å), and Rb(ri= 1.47 Å).The unit-cell parameters of alkali uranyl(2+)phosphates are calculated ,22 from O4]·3H2O . Calcula NH4, Rb , correlaThe results of this work along with the recalculations of previously published experimental results ,4,14 giv


AbstractSenticaudata. The tacitly assumed status of truth implied in some molecular higher phylogenies is called in to question.A response is given to criticisms in a recent paper of the validity of the amphipod suborder  Senticaudata was established by Senticaudata is recognised by the WoRMS online database and is cited by most taxonomic papers reporting on taxa in the group. The paper has been cited in at least 115 publications (Google Scholar). It has recently been included in a new phylogenetic classification of the Amphipoda . In this family, uropods 1 and 2 are tending towards lanceolate rather than ferrulate and uropod 3 is clearly lanceolate, a character state that does not occur in the Senticaudata. The third family, the Calliopiidae (of which they quote numerous examples) does indeed exhibit the senticaudate character state and we classified them as Senticaudata  are sister taxa to Bathyporeia, (Gammaroidea); Salentinella (Bogidiellidae) is a sister taxon to Haustorius (Haustoriidae); Stegocephalus  is a sister taxon to Antatelson (Stenothoidae) and Syrrhoe (Synopiidae) is a sister to Bactrurus/Crangonyx (Crangonyctidae). During the three centuries that scientists have turned their attention to amphipod relationships, none of these associations have ever before been suggested. Whereas our concept of the Senticaudata is not in any way revolutionary, some of the relationships suggested by It is not clear why the “other” amphipods are considered by ood tree , fig. 2,Molecular studies of higher phylogeny cannot be considered as the truth. Much has still to be learned about molecular evolution. Genes can also be homoplastic. It would be statistically improbable that any given base pair substitution, occurring by random mutation in a neutral gene, could have occurred only once in evolutionary history. It is likely that homoplasy is common in the genotype as well as in the phenotype, even where neutral genes are concerned. Molecular approaches have been shown to give meaningful results at population level and may perhaps reflect real relationships at species level. However, it appears that at higher taxonomic levels the effectiveness of current molecular methods decreases, so that higher phylogenetic relationships are not reliable. This can be seen in the study by PageBreak