Considering the emerging use of internet-based psychotherapy during the COVID-19 pandemic, information on videoconference-based EMDR (eEMDR) would be beneficial for many therapists. In this study, 23 therapists from the EMDR-Institute in Germany provided information about their experiences with eEMDR in a questionnaire-based survey. Information on the effectiveness and the course of 102 eEMDR sessions was recorded. Results showed the potential of eEMDR as an effective and viable method. The decrease in the subjective unit of disturbance (SUD), which is an important indicator of treatment outcome, was found to be at a similar level compared to that of previous EMDR studies that were not administered in eEMDR format. The most important predictor of the SUD decrease was the type of bilateral stimulation used in eEMDR sessions. Eye movements resulted in significantly greater SUD reductions than tapping. Perceived disadvantages and impediments for the implementation of eEMDR were mainly of bureaucratic and technical concerns. In addition, about one-third of the therapists stated that some patients were not willing to engage in eEMDR. In our study, eEMDR proved to be a practically applicable therapy method and therefore, therapists can consider using eEMDR. These findings will hopefully encourage EMDR therapists and their patients to use eEMDR due to its effectiveness and viability as an online treatment approach.Research on the effectiveness and applicability of eye movement desensitization and reprocessing (EMDR)  Mental health consequences of the COVID-19 pandemic suggest a high demand for psychological support during this unprecedented situation e.g., . At the Eye movement desensitization and reprocessing (EMDR) as well as trauma-focused Cognitive-behavioral therapy (CBT) are the primary choice for treatment of PTSD . EMDR isvia videoconference is feasible and appropriate ; however, they do not provide general conclusions about the effectiveness of eEMDR in an outpatient treatment setting. In times of COVID-19 and beyond, improvements in the provision of eEMDR and information on its effectiveness in treating PTSD especially in routine outpatient care are strongly required.A recently published review showed a lack of available evidence on effects of internet-based EMDR . Furtherropriate . As of nropriate . In the nce SUD; , signifiTo address this research gap, we studied the effectiveness of eEMDR on patients with PTSD and other diagnoses within the framework of an explorative approach. For the study purpose, we consulted therapists from the EMDR-Institute in Germany regarding their experiences with eEMDR sessions during the COVID-19 pandemic. Our study focused on obtaining information regarding the effectiveness of eEMDR sessions in a standard outpatient routine treatment, i.e., ratings of the EMDR process and the SUD decrease in patients reported during an eEMDR session. Notably, the SUD decrease can be seen as an important tool for therapists to assess the treatment process . Potentivia email, and 32 (8%) of them responded and returned filled-in questionnaires. Therapists were mainly female (81.3%) and more than half of them (65.6%) were aged between 51 and 65years. They had a mean professional experience of 19.77years  and a mean EMDR experience of 10.72years . Therapists were mainly trained in administering CBT (56.3%) and depth psychology (40.6%). Some therapists (12.5%) indicated they have already had experience with online-based psychotherapy before the COVID-19 pandemic.Four hundred EMDR-therapists were addressed M=41, SD=12.09). On average, patients had received 4.84 eEMDR sessions . Regarding their mental health, roughly 50% of the patient cohort fulfilled criteria of one diagnosis, while the other half of the patients had multiple comorbid diagnosis  and aged between 18 and 68years  and eventually, 102 protocols were available for evaluation. SD=19.75, range=30–100) minutes.Therapists were requested to return eEMDR session protocols of at least three different patients. Nine therapists did not return protocols because they had not yet conducted eEMDR. However, they provided information on generic questions like perceived barriers to online therapy, alternatives to eEMDR in COVID-19 pandemic, and experience with online therapy in general. The remaining 23 therapists on average returned 4.44 eEMDR session protocols , their experiences with online-based psychotherapy  and provided description of the technical as well as organizational measures they implemented to administer eEMDR .Therapists reported sociodemographic data, information about their professional background , technical aspects of the eEMDR session , and ratings concerning the quality of the eEMDR session . Moreover, on qualitative items, the therapists reported their opinion on advantages and disadvantages of eEMDR as well as adjustments and improvements necessary for the further use of eEMDR.via e-mail to therapists from the EMDR network in Germany. The e-mail informed therapists about the purpose and all procedures of the study as well as data protection issues. Most of the questionnaires were returned via postal mail; only a few were returned via e-mail. All study procedures were approved by the ethics committee of Ulm University.Questionnaires were sent Data were prepared and analyzed with IBM SPSS statistics (version 26.0.0.0) and R (version 1.3.1093). Our aim was to relate patient and therapist characteristics to quantified session effectiveness. Decrease in SUD ratings was analyzed for associations with potentially influential variables using Spearman’s rank correlation coefficients and Pearson’s correlation coefficients where applicable. Group comparisons with potential predictors of SUD decrease were computed with Kruskal-Wallis tests. Bonferroni-Holm corrections were conducted to correct the results for multiple testing. A multiple linear regression analysis was computed to examine the combined relevance of various predictors of SUD decrease. Qualitative statements about therapists’ attitudes towards eEMDR were collected as free text. The statements were reviewed, and frequency statistics were derived based on superior categories. This classification was crosschecked by one of the other authors (VT).In most eEMDR sessions, patients used computers or laptops (86.8%) followed by smartphones (18.4%). Likewise, therapists conducted the sessions mainly on computers or laptops (95.7%), followed by smartphones (8.7%), and tablets (8.7%). Some therapists also switched between different technical devices. Occasionally, they used additional equipment, i.e., headsets (30.4%), telephones (17.4%), and additional cameras (8.7%).Eye movement desensitization and reprocessing is a treatment algorithm developed by Shapiro that uses 8 phases and 3 temporal focusing steps to process stressful material. EMD is a modified EMDR protocol that focuses on a single high charge memory. EMDr is another slightly differently modified EMDR protocol that focuses on a single memory and allows only a limited range of associations in the processing phase . The stavia eye movements and in 36.3% of the sessions via tapping. In 7.8% of the sessions, both modes of bilateral stimulation were combined. When using eye movements for bilateral stimulation, supportive tools  were often (51.9%) used to evoke eye movements. Less often (31.5%), therapists guided the patients’ eyes with their fingers, while acoustical guidance (9.3%) and instructing the patients to switch their gaze between room corners (5.6%) were rarely used.In 52.9% of the sessions, bilateral stimulation was administered In most of the sessions, patient and therapist agreed on raising the hand as a stop signal (43.8%), while in 20.8% of the sessions, patient and therapist agreed on saying “stop,” and in 15.6% of the sessions, both signals were used concomitantly. Rarely, other stop gestures  were agreed.good or very good (Mdn=5 very good), and no session has been considered very bad as compared to face-to-face EMDR.In roughly 90% of the sessions, the therapists rated the overall impression of eEMDR sessions as very good . Similarly, when asked more specifically about the impression of the eEMDR process, therapists also submitted very good ratings. M=73.1% from the beginning to the end of an eEMDR session =8.82, p=0.116. The SUD decrease was lower in sessions with patients who have already had more eEMDR sessions, r=−0.23, p=0.049.Correlational analyses indicated significant associations neither between the therapists’ general professional experience and the relative SUD decrease, χ2(2)=14.11, p<0.001. As displayed in post hoc tests showed, eye movements have been more effective in reducing the SUD (Mdn =1) than tapping (Mdn =0.68).The relative SUD decrease differed significantly depending on the mode of bilateral stimulation, F=3.93, p=0.006, R2=18.1%. The analyses corroborated the relevance of the mode of bilateral stimulation as a significant predictor of the relative SUD decrease. However, the therapists’ general work experience and EMDR experience were not of relevance , while there was no difference between eye movements and combined eye movements and tapping (pHolm=0.166) and tapping and combined eye movements and tapping (pHolm=0.649).Linear Therapists most frequently reported that unstable internet connection either on patient or therapist side to be a barrier to implement eEMDR (37.5%). Several therapists also indicated that their patients refused online-based therapy (31.3%). Some therapists considered lack of personal contact (28.1%) and missing withdrawal options for the patients in their flat (21.9%) as barriers. 6.3% of the therapists considered their own media and technology skills or their patients’ cognitive or sensory limitations as barriers. Interestingly, no therapist indicated high impulsivity of patients as a barrier for online therapy.Furthermore, therapists were asked to report advantages and disadvantages of eEMDR sessions compared to face-to-face EMDR sessions as free text. Qualitative statements about advantages and disadvantages were reviewed and combined to superior categories. Results and frequencies of reported disadvantages can be seen in Difficulties with internet connection and dissociating patients were frequent answers concerning perceived disadvantages of eEMDR sessions. We therefore took a closer look at the actual connectivity issues and occurrence of dissociation during eEMDR sessions. Results showed that in 13.7% of the sessions, the connection was interrupted. Nevertheless, a SUD decrease of 68.3% could be accomplished in those sessions, and half of those sessions could be ended completely. Dissociations occurred in patients in 11.8% of the sessions. In those sessions, the mean relative SUD decrease was 53%, and one-third of those sessions could be ended as complete sessions.not to offer eEMDR post pandemic. Qualitative statements indicated, the reasons against and in favor of continuing to offer eEMDR coincided with its perceived disadvantages and advantages. Besides, some therapists suggested eEMDR could be inappropriate for certain patients, including patients with dissociation and severe mental disease status. With appropriate patient groups, however, therapists stated they consider continuing to offer eEMDR beyond the COVID-19 pandemic.Vast majority (87%) of the therapists stated, they would continue offering eEMDR beyond the COVID-19 pandemic. Conversely, 13% of the therapists considered In roughly half of the session protocols, the therapists provided information about their ideas and wishes for adjustments and improvements with respect to the future use of eEMDR beyond the COVID-19 pandemic. Answers were clustered into four categories as shown in good or very good. In this study, the most important indicator of effectiveness was the extent to which eEMDR enabled to reduce the patient-rated SUD from beginning to end of the session. According to Responses from therapists and the patients’ relative SUD decrease suggest eEMDR sessions can be an efficient and practically applicable alternative for face-to-face EMDR. Throughout eEMDR sessions examined in this study, therapist ratings of adherence, confrontation, grounding, and process were consistently positive, and the quality of more than 90% of the sessions was rated as Importantly, the SUD decrease was independent from the therapists’ professional experience or their EMDR experience. The question whether therapists’ experience influences treatment outcome has been debated since the origins of psychotherapy  and findvia eye movements enabled significantly stronger decrease in the patients’ SUD than the use of tapping or a combination of eye movements and tapping. These results were also corroborated in a regression analysis when controlling the therapists’ professional and EMDR experience. With regard to the debate on the effectiveness of different modes of bilateral stimulation . However, technology and connectivity issues are concerns that can be resolved through further network expansion throughout the country  and also by providing adequate technical equipment. According to the therapists, one of the advantages of eEMDR compared to face-to-face EMDR is that the patients would better focus on and engage in the therapy process. Some therapists also observed positive implications of the patients’ domestic surroundings . The barriers mentioned by therapists were not of therapeutic concerns, but rather related to the bureaucratic and technical framework of eEMDR. Importantly, more than 80% of the therapists participating in our study indicated they consider offering eEMDR beyond the COVID-19 pandemic, and this underscores the potential of eEMDR.The present study is of explorative nature and it focused on the outcome from a single eEMDR session instead of an entire EMDR therapy. In addition, there was no comparison group to compare the effects of eEMDR to a waitlist-control group, face-to-face EMDR, or other internet-delivered therapy methods. Although this was not the intention of our exploratory study, randomized clinical trials are clearly warranted to ascertain the effectiveness of eEMDR as compared to its face-to-face version and other therapy approaches.via videoconference but also to a lack of time and capacity considering the strenuous workload for therapists due to the pandemic situation.Notably, only 8% of the contacted therapists decided to participate, thus representing a selective and potentially biased subgroup of all therapists. The low response rate may be attributed to the restraint to offer EMDR The questionnaire asked therapists regarding their attitude and experiences with eEMDR. There could be therapist bias due to social desirability, especially regarding effectiveness measures as they may have chosen not to report on those eEMDR sessions which were assessed as ineffective. However, in the sessions where the standard protocol was applied, nearly half of the sessions ended incompletely, thus dispelling the presumption that therapists selectively returned protocols of only effective sessions. Furthermore, patient-rated SUD values might be also biased by social desirability.Future research could assess the experiences and compliance of patients participating in eEMDR, as this will provide additional insights for improving effectiveness of eEMDR. Furthermore, studies could investigate which patients benefit most from eEMDR formats and conversely, which diagnoses, or patient characteristics contraindicate the use of eEMDR.via videoconference (This study was one of the first to address the effectiveness of eEMDR by asking EMDR-therapists about their experiences with this treatment format. Our results show eEMDR as an effective and viable alternative to face-to-face EMDR. Especially the high SUD decrease in eEMDR sessions, an important indicator of treatment outcome was very promising. Thus, the results help to dispel doubts regarding the feasibility and appropriateness of EMDR nference .Therapists can consider conducting eEMDR even beyond the COVID-19 pandemic situation, as perceived impediments and disadvantages were mainly related to the bureaucratic and technical framework of eEMDR. However, with certain adjustments to the framework it is feasible to overcome these barriers. Thus, eEMDR has the potential to not only be a temporary solution during the pandemic but to become an integral part of everyday therapy.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/ participants or patients/participants legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.VT conceptualized the study, obtained ethical approval, and recruited therapists. CM wrote the manuscript under supervision of VT and AB, and performed statisical analysis together with LM under supervision of AB. AH provided expertise and feedback and recruited therapists. ML provided data. SV and RR edited the manuscript. I-TK supervised the study, revised and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


E. The charge density distribution in the vicinity of the graphene monolayer that is caused by E was determined using the pseudopotential density-functional theory approach. Different geometries were considered. The centroid of this extra density induced by an external electric field was determined as E, and its dependence on E has been obtained. The thus determined We present a detailed first-principles investigation of the response of a free-standing graphene sheet to an external perpendicular static electric field  The numerous properties of graphene have been intensively investigated after its experimental realization. Thousands of papers on this material were published. However, there still remains a simple unanswered question regarding the way in which the induced charge density is distributed in the vicinity of a graphene monolayer when an external electric field is applied to the graphene sheet. This topic was addressed, to some degree, by considering the problem of screening of the electric field induced by point charges in graphite ,2,3,4,5.z axis as pointing in the direction perpendicular to the carbon atoms basal plane) that is a “real position” of a solid surface for many phenomena occurring there. It determines a “physical” position of a metal surface when an external perturbation is applied. This problem was widely studied in the case of metal surfaces. In general, this “real” surface position is different from the spatial localization of the top atomic layer or a geometrical crystal edge, staying towards the vacuum side [The perpendicular charge distribution was studied by considering two- and multi-layer graphene films ,12,13, tuum side ,16,17,18It is usually assumed for a quasi two-dimensional (2D) system that the excess charge is confined within an infinitesimally thin 2D layer ,19. CertKnowledge of the position of the center of mass of the induced charge density is important in many fields of surface science. Thus, it determines the reference plane for the image-potential felt by an external charge placed in front of a surface. If this charge is an excited electron with energy below the vacuum level, it can be trapped by this image potential in a state belonging to an infinite Rydberg -like series ,24. The In the previous work devoted to the IPSs in graphene, it was assumed ,26 that redicted  to existUp to now, IPSs for a free-standing graphene were not studied experimentally. On the other hand, numerous measurements were performed on the graphene supported on various metallic or semiconducting substrates. Usually, the interface distance between the graphene sheet and the surface atomic layer is such that the conventional single Rydberg series of a whole system is observed. Thus, in the graphene/metal systems where the graphene atomic layer is placed closer to the substrate, only a single series of IPSs was observed ,34,35,36For a description of the IPSs in the graphene/substrate systems, a number of potentials have been developed. Indeed, an accurate description of IPSs is a challenge since the conventional density-functional theory (DFT) calculations do not accurately account for a correct long-range interaction in front of solid surfaces. One of the approaches consists of constructing the nonlocal van der Waals functional . Althougthe Ref. , the fitications ,26.Our goal in this work is to determine the The rest of this paper is organized as follows. In The band structure of a graphene monolayer in the absence and presence of an external electric field of varying intensity was obtained within the LDA by solving the Kohn-Sham equations employing a home-made band structure computer code . We usedx-y plane as shown in z-dependence is defined by a Gaussian with a decay length of 1 a.u. This extra charge was placed at a distance of 10 Å from the graphene plane. In order to ensure the neutrality of the system, the charge +z variation of the extra potential added to the system is schematically shown in The self-consistent procedure was realized by considering a repeated-slab geometry with the lateral lattice constant of 2.424 Å. The external electric field applied in the direction perpendicular to the graphene plane has no translation symmetry. In order to implement it in the repeated-slab geometry, we added to the Hamiltonian a term corresponding to the extra charge In other sets of calculations, we considered a geometry when the external electric field is applied from both sides of the graphene sheet as shown in The electronic structure of graphene around the Fermi level at zero external electric field is presented in E. x-y plane for the values of E ranging from −0.4 to 0.5 V/Å. In order to perform a comparison, E. One can see that its shape deviates qualitatively from the total valence density depicted by the green solid line. This can be understood, since the total density is dominated by the E. In general, we observe that at larger E, the shapes of E amplitude, the variations in E, defined asE. In the former case, E, the size of the Fermi surface shrinks and the possible calculation oscillations increase. For comparison, in the insert of E in a free-standing Al(111) monolayer. Since, for Al, the Fermi surface is large because there are three valence electrons, the deviation from the linear behavior is small.We have examined the way in which the induced charge density profile onolayer . A similonolayer . We expeWhen we apply an external electric field to the graphene sheet from both sides according to the scheme depicted in Clearly, in the symmetric case b, the cagen atom .Es reported in In the case of the geometry described in In our numerical calculations devoted to IPSs, the perpendicular lattice constant was increased up to 80 Å which allowed us to obtain convergent energies for the six lowest-energy members of the series. As it was mentioned previously, IPSs cannot be properly described with the use of conventional DFT calculations since the long-ranged image-potential on the vacuum side is not reproduced correctly. Additionally, the tight-binding methods are not inherently desired for its description ,50. Index and y coordinates, since The parameters x-y plane constructed with z larger than z only. For comparison, in z larger than n is increased this difference is gradually reduced. With In the Ref. . One canthe Ref.  to 1.58 z and z is large, let us account for the interaction between the charge A with real charges Az is large, for a point charge A, it can be considered as a point charge located at n. However, it may not be good for The approach described above for construction of a hybrid “LDA+image−tail” potential is an adoption of the conventional image-potential picture employed for solid surfaces . It can ocalized  around 2Based on this picture, we constructed a hybrid “LDA+image−tail” potential for which ce state ,54 obtaice state . HoweverWe believe, the measurements of energies of free-standing graphene will provide important information on its screening properties. It may contribute to establishing a detailed picture of what is going on there due to an external perturbation. So far, all the experiments on IPSs were performed on supported graphene. Sensitivity of the image-potential states to the environment where graphene was kept was significant. Our findings point out that they can also provide important information about free-standing graphene screening properties.In this theoretical study we have reported the detailed charge density distribution produced in free-standing graphene by external static electric fields with three geometries. The image-plane position was established. Surprisingly, it is rather large, located at 1.048 Å outside the carbon atom plane. Using this information, we constructed a new potential felt by an electron excited to the image-potential states. We checked several kinds of such a potential, demonstrating sensitivity of the energies of the lowest image-potential states to the details of this potential. It would be of interest to obtain the experimental information, such as from the photoemission spectroscopy, on the image-potential state energies for free-standing graphene. We believe that the experimentally determined image-potential energies will be extremely helpful for development of a more detailed picture for the graphene potential and how it reacts to the external perturbation on the atomic scale.Our data on  systems . Such pographene ,57,58,59graphene ,65,66,67graphene , borophegraphene , MXenes graphene ,71,72, agraphene .


Understanding the structural behavior of graphene flake, which is the structural unit of bulk crumpled graphene, is of high importance, especially when it is in contact with the other types of atoms. In the present work, crumpled graphene is considered as storage media for two types of nanoclusters—nickel and hydrogen. Crumpled graphene consists of crumpled graphene flakes bonded by weak van der Waals forces and can be considered an excellent container for different atoms. Molecular dynamics simulation is used to study the behavior of the graphene flake filled with the nickel nanocluster or hydrogen molecules. The simulation results reveal that graphene flake can be considered a perfect container for metal nanocluster since graphene can easily cover it. Hydrogen molecules can be stored on graphene flake at 77 K, however, the amount of hydrogen is low. Thus, additional treatment is required to increase the amount of stored hydrogen. Remarkably, the size dependence of the structural behavior of the graphene flake filled with both nickel and hydrogen atoms is found. The size of the filling cluster should be chosen in comparison with the specific surface area of graphene flake. The transformation of nanoscale structural elements into three-dimensional 3D) complex architecture is currently an important task of materials science. Since the discovery of fullerenes in 1985, a lot of new carbon structures have been proposed. Carbon polymorphs can be used to obtain nanostructures with unique mechanical and physical properties applicable in nanoelectronics, energy storage devices, sensors, supercapacitors, Li-ion batteries, etc. D complex,5,6,7.Graphene is capable of enhancing the performance, functionality as well as durability of many applications. The one-atom thin structure can serve as the platform for other materials especially since the graphene layer can be bent or crumpled. Extensive studies have been carried out in recent decades to investigate the crumpling behavior of thin sheets like graphene, by both theoretical and experimental methods ,17,18,19It is known that coupling metal nanoclusters with carbon materials can efficiently promote catalysis and electrocatalytic activities ,24 or prThe other important advantages of such structures are a large specific surface area (SSA) and, respectively, a high rate of gas adsorption, which makes it possible to predict their use in hydrogen storage ,51,52,53In the present work, two types of nanofillers for crumpled graphene flake were considered—Ni and hydrogen nanoclusters. Thus, the idea of using crumpled graphene flake for the storage of different atoms using Ni and hydrogen clusters as the example was realized. It is important to understand the dynamics of interaction between single graphene flake and a metal or non-metal nanocluster before the study of storage in three-dimensional crumpled graphene. The behavior of the hybrid structure is studied by atomistic simulation at different temperatures. For the carbon–nickel system, room temperature (300 K) and temperature close to the melting point of Ni (1000 K) were chosen. However, for the carbon–hydrogen system, the temperature of liquid nitrogen (77 K) and room temperature were chosen. The dynamics of the structure were studied during exposure for 20 ps.N = 21; 38; 47; 66; 78) inside the flake was considered  it is called “flake” rather than “nanoribbon”. This GF was filled with nanoclusters of two types: (i) nickel nanocluster and (ii) hydrogen nanocluster.To study the interaction between graphene flake (GF) and atomic nanocluster, an initial structure composed of the GF of one size . Equations of motion for the atoms were integrated numerically using the fourth-order Verlet method with the time step of 0.2 fs. The Nose–Hoover thermostat was used to control the system temperature. The periodic boundary conditions were applied in all directions, however, the simulation box is much bigger than the size of GF filled with nanocluster. The adaptive intermolecular reactive empirical bond order potential (AIREBO)  was usedGraphene flake with the nanocluster was exposed at 300 K and 1000 K for 20 ps to study the dynamics of nanocluster inside graphene flake. Previously , it was ab-initio simulation [To describe the interatomic interaction between Ni–Ni and Ni–C, Morse interatomic potential was used with the parameters or Ni–Ni ; and De mulation ,60.Graphene flake with hydrogen nanocluster was exposed at 77 K and 300 K for 20 ps. The temperature of 77 K was chosen since it was previously shown that better sorption of hydrogen can be found at 77 K . The temTo describe the interatomic interaction between C and H, AIREBO interatomic potential was used.It should be mentioned that the initially obtained structure contains atomic hydrogen, however, H atoms transform to Hngle see b. As it to 3.2 Å . HydrogeAt first, the evolution of the potential energy of the system during the crumpling process for graphene flake filled within spherical the Ni nanocluster was analyzed. In a is two times higher than for the biggest one which means that the time required to attach the nanocluster by the graphene flake is longer. For nanocluster NiThe longest time of stabilization at 300 K was found for NiAt higher temperature (1000 K), again a strong correlation between equilibration time and the size of the nanocluster was found. For GF with NiIn ioned in . Ni atomt = 1.5 ps one covalent bond appeared between two edges. Nanocluster disturbed by the temperature and several atoms attach to the graphene plane (state II). This leads to the spreading of the Ni atoms over the graphene plane (state III). Simultaneously, such a small nanocluster allowed the graphene flake to easily bend and the structural unit transforms to the bi-layer graphene with Ni atoms spread inside (state IV). Several more covalent bonds appeared on the side edges of GF. At 300 K, not many covalent bonds between the edges of GF can appear, since the temperature is far from the melting temperature of graphene [At first, consider GF with Nigraphene ,63.Increase in the exposure temperature to 1000 K facilitates the transformation. At 300 K, equilibration time is equal to 5 ps, while at 1000 K, it is equal to 3.5 ps. Subsquently, the structure came to a stable state and no further changes were observed despite slight thermal fluctuations. At 1000 K, more carbon atoms on the edges of GF found neighbors since the structure is disturbed by thermal fluctuations.For structures with NiA graphene flake with NiOne of the important applications of such structural elements is the fabrication of composites. In eratures ,42,43,44In At 77 K a, the eqAt 77 K, the time of equilibration was the longest (3 ps) for hydrogen clusters consist of 77, 66, and 38 atoms, and the shortest (1.5 ps) is for 21 and 47 atoms. At that temperature, the size of the cluster plays quite an important role which is connected with the number of sites for hydrogen on the side of GF. All hydrogen molecules and atoms can easily find sites on graphene for an initial number of atoms less than 47, while for bigger clusters, the number of sites is not enough and some molecules and atoms will move outside GF or seek better sits near GF.At 300 K, the main point is the process of dehydrogenation which is quite quick at such a high temperature, and for clusters with 38–78 atoms, about 1.5 ps is enough for all molecules to detach the side of GF and move outside. The long time of equilibration for cluster HTo understand the dynamics of the hydrogen sorption/desorption, snapshots of the structural units altogether with their potential energy are presented in At 300 K, hydrogen molecules and atoms have no chance to form even a weak bond to graphene. At 300 K, all hydrogen atoms move outside GF which is quite understandable. Numerous theoretical and experimental works confirm the sorption of molecular hydrogen on carbon nanostructures ,64,65,66A large number of hydrogen atoms almost completely fills the cavity of the GF. At 77 K, hydrogen atoms are easily converted into hydrogen molecules and under the action of chemical attraction or van der Waals forces, attach to the walls of the graphene flake. Mainly, hydrogen, which is located in the center of the flake under the influence of temperature, tends to leave the graphene cavity. Therefore, in structures with 66 and 78 hydrogen atoms, the stabilization of the structure takes longer (3 ps), in contrast to small hydrogen clusters, such as 21 and 47 (1.5 ps).For a bigger nanocluster, energy curves almost coincide for two temperatures, since hydrogen molecules at 77 K can find the places for sorption just after the first steps. In this case, hydrogen atoms are placed near graphene and link it as far as the van der Waals radius of the molecule reaches. Thus, the hydrogen adsorption capacities depend considerably on the initial size of the hydrogen cluster. The SSA of the GF should be big enough for a chosen number of H atoms and HIn As well as for a metal nanocluster, the three-dimensional structure of crumpled graphene filled with hydrogen is presented in Molecular dynamics simulation is used to study the dynamics of graphene flakes filled with the nanoclusters of two different types: metal and non-metal. The obtained results can shed the light on understanding the possibility of using crumpled graphene as a storage media from the point of single flake behavior.It was found that cavities of crumpled graphene (the structure consists of crumpled graphene flakes) can be used as containers for metal nanoclusters—for instance, for Ni. A nanocluster consisting of 21 to 78 Ni atoms was considered. A graphene flake can easily cover nanocluster, however, the dynamics of the interaction strongly depend on the nanocluster size. Small nanoclusters can be easily bent by rigid graphene flake, while the biggest conserve the shape. Such a structure, composed of Ni nanoclusters and graphene flakes, can be further used to obtain composite material with improved mechanical properties.The problem of hydrogen storage has been of high importance for decades and the application of carbon nanostructures from this point of view also looks promising. Crumpled graphene has a high specific surface area, light weight and a lot of pores and cavities which can be filled with hydrogen. It was shown that at 77 K, hydrogen molecules and atoms can be absorbed by both sides of graphene flakes. However, a single flake cannot store enough hydrogen for practical application. However, when graphene flakes composed in another structure and with additional treatment like hydrostatic compression ,47, it c


MC density was higher in the fibrotic areas of lung tissue compared with spared alveolar areas or in controls. Female gender, low diffusion capacity for carbon monoxide, diffuse alveolar damage, and smoking were associated with a low MC density. MC densities of fibrotic areas had declined significantly in five subjects in whom both SLB in the stable phase and autopsy after an acute exacerbation of IPF had been performed. There were no correlations of MC densities with survival time or future acute exacerbations. The MC density in fibrotic areas was associated with several clinical parameters. An acute exacerbation of IPF was associated with a significant decline in MC counts. Further investigations will be needed to clarify the role of these cells in IPF and in the pathogenesis of acute exacerbation as this may help to identify some potential targets for medical treatment for this serious disease.Mast cells (MCs) are known to be involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF), although their role in acute exacerbations of IPF has not been investigated. The aims of the study were to evaluate the numbers of MCs in fibrotic and non-fibrotic areas of lung tissue specimens of idiopathic pulmonary fibrosis (IPF) patients with or without an acute exacerbation of IPF, and to correlate the MC density with clinical parameters. MCs of IPF patients were quantified from surgical lung biopsy (SLB) specimens (The online version contains supplementary material available at 10.1007/s10753-021-01582-0. Idiopathic pulmonary fibrosis (IPF) is a severe interstitial lung disease of unknown cause, in which the phenomenon of acute exacerbation (AE) is responsible for significant mortality , 2. The Mast cells (MCs) are immune cells whose significance in pulmonary diseases has been best characterized in allergic asthma; the role of MCs in other respiratory diseases is less clear , 5. PeriA higher number of MCs have been observed in IPF compared with other interstitial lung diseases or normal controls , 9–15. MAlthough MCs appear to be involved in the pathogenesis of pulmonary fibrosis, the role of these cells in the development of AE-IPF has not been investigated. There were two aims of this study: (1) to evaluate the numbers of MCs in fibrotic and non-fibrotic areas of lung tissue specimens of IPF patients with and without an AE-IPF, and (2) to correlate the MC density with clinical parameters such as age, gender, pulmonary function test results, and smoking status. We also wanted to investigate whether the MC count was associated with the risk for mortality or the occurrence of AE-IPF.n = 21) were identified either by applying the current criteria for AE-IPF in those subjects in whom lung tissue material had not been obtained during the episode of AE-IPF (n = 13), or on the basis of the surgical lung biopsy material, i.e., diffuse alveolar damage (DAD) in parallel with usual interstitial pneumonia (UIP) indicative of AE-IPF (n = 8) [The study cohort consists of 47 IPF patients treated in Oulu University Hospital or Oulaskangas Hospital in Northern Finland between 1991 and 2019. All study subjects had undergone a surgical lung biopsy procedure for diagnostic purposes. The lung tissue specimens taken at autopsy were also studied from seven subjects out of 47. Non-diseased lung tissue material from 12 non-smokers who had undergone lung cancer surgery served as controls  results examined at a time near the biopsy date, antifibrotic drug use, medical treatment preceding AE-IPF in patients with DAD in a biopsy or autopsy specimen, and smoking data were collected systematically from the medical records. Patients with less than 5 pack-years of smoking history were regarded as non-smokers. Age and follow-up time were calculated by using the biopsy procedure date and death, transplantation or last follow-up date (11/8/2020). Death dates were collected from death certificates housed in the national registry of Statistics Finland.Formalin-fixed and paraffin-embedded lung specimens were cut into 3.5 μm sections, de-paraffinized in xylene and rehydrated in a descending ethanol series. The staining was performed by using Dako REAL EnVision Detection System . Microwave-stimulated antigen retrieval was performed in Tris–EDTA, pH9, and endogenous peroxidase was neutralized with peroxidase blocking solution (Dako). After the specimens had been incubated with a primary monoclonal mouse anti-human mast cell tryptase antibody  diluted 1:200 in antibody diluent (Dako) for 30 min at room temperature, a biotinylated secondary HRP rabbit/mouse antibody (Dako) was added. The color was developed with 3,3′-diaminobenzidine  and counterstaining was performed with hematoxylin. As a negative control, the primary antibody was replaced by mouse isotype control . The stained sections were digitized with Leica-Aperio AT2 .2 areas in fibrotic areas of lung tissue in IPF and in normal alveolar areas of control subjects. Due to the small amount of fibrotic areas in surgical lung biopsy samples of two subjects, five, and eight 0.16-mm2 areas were assessed instead of ten. In addition, three 0.16 square millimeter areas of non-fibrotic alveoli were assessed from specimens of 33 IPF patients, in whom there was enough preserved lung tissue to permit this analysis, i.e., in study subjects with IPF, the numbers of mast cells in both fibrotic and non-fibrotic areas were counted. The MC density of fibrotic areas was the value used when the correlations with clinical parameters were calculated. J.S. calculated the MCs from all lung tissue specimens. Two other investigators (S.L. and M.K.), calculated the mast cell densities of 20 different study subjects. In addition, MC densities of honeycombing areas, fibroblast foci and areas of dense fibrosis were calculated from 12 representative samples (S.L. and M.K.).Digitized lung tissue specimens were examined by using virtual microscopy software . The MCs were visualized maximally at 40 × magnification and were enumerated from ten randomly selected 0.16-mmt-test or paired samples t-test to compare means when appropriate. The intraclass correlation coefficient was determined to evaluate the interrater reliability of MC counts. Survival analysis was performed by using Kaplan–Meier curves, and risk for earlier death or earlier episode of AE-IPF was evaluated by using Cox regression model. Medians and quartiles of MC densities were utilized to determine the cutoff values for Kaplan–Meier and Cox regression analyses. We included MC densities of SLB samples, not autopsy samples, in the Kaplan–Meier and Cox regression analyses.IBM SPSS Statistics for Windows, Version 27.0  was used to perform statistical analysis, and Origin(Pro), Version 2019b , was utilized for preparing the graphs. Means and standard deviations were calculated for parameters that were normally distributed. Medians and interquartile ranges were determined for parameters that were not normally distributed. We used independent samples N = 1), autopsy (N = 5) or both in autopsy and lung biopsy specimens (N = 2).The characteristics of the study subjects are shown in Table 2 (SD 140) in the area of dense fibrosis, 530 MCs/mm2 (SD 110) in the honeycombing area, and 160 MCs/mm2 (SD 40) in fibroblast foci. Figure The MC densities in different types of lung tissue specimens are shown in Table 2 SD 40 in thep = 0.041) and current smokers had a lower MC density than ex- or non-smokers (p = 0.023). There was no association with forced vital capacity (FVC) and MC density, but in contrast, a high diffusion capacity for carbon monoxide (DLCO) was linked with a high MC density (p = 0.010). Patients with IPF had a higher MC density in their fibrotic lung tissue areas when compared with normal lung tissue derived from controls (p < 0.001), while the difference between non-fibrotic areas in IPF and controls was not statistically significant. We found no correlation of a future episode of AE-IPF with MC density in lung tissue biopsies taken in the stable phase of the disease.The MC densities in the fibrotic areas of lung tissue correlated with several clinical parameters, as shown in Table p < 0.001)  had a lower MC density than patients without AE at the time of biopsy  (1) Table , Fig. 2.p = 0.175). MC densities in tissue specimens obtained at diagnosis in the stable phase of the disease, without clinical or histological evidence of AE-IPF at the time of biopsy date, were not correlated with the time to the first future episode of AE-IPF .The correlation between the survival time and MC density was not statistically significant, although there was a slight trend towards a shorter survival and low MC density (319 MCs or less per square millimeter) ) with a high MC density , 13, 15,The discrepancies between the results mentioned above might be partly due to the differences in MC staining techniques: Andersson et al. found the association of a high MC density with low FEV1% predicted only with MCs double positive for both chymase and tryptase . ShimborIn addition to the differences in MC staining techniques, inconsistent results concerning MC densities and their correlations with clinical parameters might also be related to the methods used to calculate and choose the tissue areas for MC quantification. The determination of one MC density value for the whole area of tissue sample does not take into account the varying proportions of fibrosis and spared alveolar tissue in different tissue specimens, a phenomenon typically present in IPF where fibrotic areas of lung tissue alternate with less-affected parenchyma . ConsistWe could demonstrate a significant decline in the MC density of five patients who had undergone a lung biopsy for diagnostic purposes in the stable phase of disease and in whom an autopsy had been performed after death caused by an AE-IPF. No similar findings have been reported earlier, because there are very few studies involving multiple lung tissue specimens obtained from the same individual in different stages of pulmonary fibrosis.To our knowledge, there are no previously published studies investigating lung MC density in AE-IPF patients. Animal models have suggested MCs and their mediators have a role in the pathogenesis of acute respiratory distress syndrome (ARDS), a phenomenon in which DAD is present similarly to AE-IPF −30. HoweMCs are known to be activated by several different mechanisms, e.g., immune receptors, bacteria and their products, viruses, cytokines and inflammatory mediators, endogenic peptides, and physical stimuli . MCs areIn the study by Overed-Sayer et al., nintedanib, but not pirfenidone, inhibited fibroblast-mediated MC survival in vitro and this finding was confirmed in the rat bleomysin model . In our We were not able to find a statistically significant association of low MC density with short survival time, although a trend in this direction could be observed. It should be noted that we correlated survival time with MC density determined from patients’ SLB samples, of which only 3 were obtained during AE-IPF with a relatively low MC density. It was not reasonable to correlate MC cell densities of autopsy samples with survival time due to the lack of follow-up time of these patients. One may speculate that if we had had SLB specimens obtained slightly before death from a larger number of IPF patients, then it is possible that a correlation of low MC density and short survival time might have been found.Overall, we managed to gather a rather comprehensive lung biopsy material from IPF patients, namely lung tissue specimens from 47 patients with additional autopsy material from 7 patients; as far as we are aware, this is the largest histological material on IPF patients from which an MC quantification has been performed. In earlier studies, the study material has mainly included tissue specimens from fewer than 20 IPF patients , 13, 16,To conclude, the MC density had significantly declined in subjects with an AE-IPF indicating that these cells may play variable roles in different stages of the disease. Altogether, the role of inflammation, MCs and other immune cells should be studied further in order to gain a more profound understanding of the pathobiology of IPF as this may make it possible to identify new targets for medical treatment for this serious disease.Supplementary file1 (PDF 163 KB)Below is the link to the electronic supplementary material.


Therefore, it is necessary to design high-performance gas sensors for toxic gas detection. In this sense, graphene-based materials are promising for use as toxic gas sensors. In addition to experimental investigations, first-principle methods have enabled graphene-based sensor design to progress by leaps and bounds. This review presents a detailed analysis of graphene-based toxic gas sensors by using first-principle methods. The modifications made to graphene, such as decorated, defective, and doped to improve the detection of NOx, SOx, and CO toxic gases are revised and analyzed. In general, graphene decorated with transition metals, defective graphene, and doped graphene have a higher sensibility toward the toxic gases than pristine graphene. This review shows the relevance of using first-principle studies for the design of novel and efficient toxic gas sensors. The theoretical results obtained to date can greatly help experimental groups to design novel and efficient graphene-based toxic gas sensors.Detecting and monitoring air-polluting gases such as carbon monoxide (CO), nitrogen oxides (NO The conversion of energy from one form to another many times affects the air composition in several ways. It is well-known that fossil fuels have been powering industrial development and the amenities of modern life that we enjoy. However, the combustion of fossil fuels contributes to a great extent to composition variations of the atmosphere, and this is mainly due to harmful gas emissions.x), and sulfur oxides (SOx), among others. In this context, health expenditures have increased due to air pollution, which is mainly associated with the rapid industrialization of many countries. Consequently, the disruption of ecological balance and serious public health issues caused by harmful gases are raising global concerns . Ad. Ad2 andgraphene . The comadsorbed . Finallyadsorbed . The comadsorbed .Many theoretical studies have been conducted on the use of doped graphene as toxic gas sensors. The results evidence that doped graphene sheets are good candidate materials as gas sensors. To experimentally confirm some of the above-mentioned theoretical predictions, various doped graphene materials have been synthesized and evaluated as toxic gas sensors ,107,108.x, and SOx toxic gases were revised and analyzed in detail. Based on this review, we concluded the following:(a)The interaction between toxic gases and pristine graphene is weak, which reduces the sensitivity and selectivity of pristine graphene toward the toxic gases.(b)The pristine graphene decorated with transition metals is a promising material for use in a toxic gas sensor. However, up to now these types of studies are still scarce; therefore, more theoretical studies on the sensitivity and selectivity of pristine graphene decorated with transition metals toward the toxic gases should be carried out.(c)It was observed that graphene with single-vacancy defects interacts stronger with the toxic gases compared to pristine graphene. Therefore, it is a promising material for use in toxic gas sensors. In addition to point defects, line or multivacancy defects should be investigated at the DFT level, to enrich graphene functionalities.(d)Bilayer and multilayer graphene exhibit higher different dimensionalities than single-layer graphene, which can increase the number of possible defect types, namely, point defects, line defects, and so on. At the theoretical level, more attention should be paid to understanding stable bilayer and multilayer graphene with randomly distributed defects.(e)A large number of theoretical studies have addressed the use of doped graphene as a toxic gas sensor. The evidence indicates that doped-graphene sheets are good candidate materials. However, up to date, DFT studies on the selectivity of doped graphene toward the toxic gases are limited. Therefore, more theoretical studies on the selectivity of doped graphene toward the toxic gases should be carried out. In addition, feasible approaches to facilitate the desorption of toxic gas on the doped graphene surface should be investigated.(f)The pyridinic-type N-doped graphene and doped vacancy-defected graphene are good materials for use in toxic gases sensors. However, more DFT-based studies on pyridinic-type N-doped graphene and doped vacancy-defected graphene as toxic gas sensors are needed.(g)The reasons for the difference of adsorption energy obtained by using different functionals  in the calculation methods should be compared and analyzed.(h)This review shows the importance of theoretical studies for the design of novel and efficient toxic gas sensors. The theoretical results obtained up to now can help and motivate experimental groups to design novel and efficient graphene-based toxic gas sensors.This review presents a detailed and critical analysis of current progress of graphene-based toxic gas sensors using first-principle methods. Following the development of graphene as a gas sensor, it has gained considerable interest from both a theoretical and a technological viewpoint. Therefore, modifications made to graphene to improve the detection of CO, NO


Presentation at academic conferences is an important marker of research productivity. However, not all accepted abstracts progress to full publication, and there is anecdotal evidence suggesting an imbalance in sex and ethnicity amongst presenters. There is a lack of data evaluating the outcome of prize presentation sessions at academic surgical conferences in the UK. This study aimed to analyse the outcomes and demographics from presentations at prize sessions at two prestigious UK surgical conferences.This retrospective observational study compared data on all Moynihan (Association of Surgeons of Great Britain and Ireland) and Patey  prize presentations from 2000 to 2020. The primary outcome was rate of publication. Secondary outcomes included demographic differences in sex and ethnicity, publication according to prize outcome, academic affiliation, time to publication, and journal impact factor.P = 0.019).Some 442 accepted abstracts were identified over the 21-year period, with 71.0% from the Moynihan sessions and 79.3% from the Patey sessions leading to full publications, with a median time to publication of 448 days (IQR 179–859) in journals with relatively high impact factors . Of the 442 prize presenters, 85 (19.2%) were female. The majority of the presenters were White males , followed by Asian males . However, there was a continuously increasing overall trend of female presenters from 2000 to 2020 (Publication rates from the two prize sessions were high, with presenters publishing in journals with high impact factors. There, however, was a disparity in sex and ethnicity amongst presenters. Academic surgical conferences provide a forum to showcase up-to-date surgical research through education, discussion and presentation of new work. Participation at conferences is an important marker of academic productivity. Although abstracts submitted to conferences are reviewed by a panel of scientific experts prior to acceptance, they are not subject to the same rigorous peer-review process of a journal publication. Whilst desirable, full publication is not always achievable and studies have reported that less than 50% of abstracts presented at surgical conferences progress to a full publication –4.There is anecdotal evidence to suggest that imbalances in sex and ethnicity exist amongst presenters at conferences –8, whichhttps://www.asgbi.org.uk) annual congress and the Surgical Research Society  annual meeting are two of the most prestigious academic surgical conferences in the UK and Republic of Ireland. The highest ranked abstracts submitted to these annual conferences are shortlisted for presentation at the Moynihan and Patey prize sessions, respectively, with one presenter being awarded each prize.The Association of Surgeons of Great Britain and Ireland  and NamSor Version 2 diasporaBatch (https://www.namsor.com/)” application programming interfaces (API), respectively. Ethnicity was coded into four main ethnic groups  as defined by the Office for National Statistics . The sex and ethnicity APIs are automated linguistic matching software programmes used routinely to identify sex and ethnicity and limit observer bias or discrimination [Sex  and ethnmination , 12. ThrRussell Group (https://russellgroup.ac.uk/) or Ivy League (https://ivyleague.com/) universities. Outside the UK and USA, according to the Times Higher Education World University Rankings 2021 (https://www.timeshighereducation.com/), the top three universities within the Republic of Ireland  and international universities ranked within the top 100 institutions worldwide were also considered prestigious. Presentations were categorised as published if a PubMed citable original article was identified in addition to the published abstract presentation that was authored by the presenter/first author and contained data presented at the conference. Time to publication was calculated in days from the date of conference presentation to the date of first online publication. Where article publications occurred prior to conference presentation the time to publication was recorded as 0 days. Journal impact factors were derived from the 2019 Journal Citation Reports published by Clarivate in 2020 . The journals where these studies were published were subclassified into surgical, medical, basic science/translational and miscellaneous based on their attributions on Scimago Journal and country rank (SJR).Shortlisted presentations were assigned as nominees or winners depending on the outcome at each conference. Academic affiliation was categorised according to whether the presenter/first author was linked to a university and if so, whether the university was considered prestigious. Prestigious universities were classed as either 2) and Mann–Whitney U tests were used to compare categorical and continuous variables as appropriate. Univariate regression analysis with the likelihood ratio test was used to compare time trends by year and presented using 3-year rolling averages. Comparisons were made between prizes (Moynihan and Patey), winners and nominees, and by sex and ethnicity. All analyses were conducted using Stata Statistical Software v16.1  with significance set at the 95% level and P < 0.05 considered significant.Data normality was assessed by visualising distribution plots, and descriptive statistics were used to report demographics including percentage frequencies, medians and interquartile ranges (IQR). Fisher’s exact, Chi-square . Amongst the presenters, 61.1% were White, followed by 29.0% Asian, 0.5% Black, and 9.5% Other/Arab. Of the 442 presenters, 316 (71.5%) had affiliations with a prestigious university, 83 (18.8%) with a non-prestigious university, and 43 (9.7%) did not specify a university affiliation. Throughout 2000 to 2020, there was an increasing proportion of presenters having an affiliation to a prestigious university  at the ASGBI Moynihan and 304 (68.8%) at the SRS Patey prize sessions. The general characteristics of the presenters are summarised in Table P = 0.057, Table P = 0.468). The 98 full-text publications generated from the Moynihan prize group were published in journals with a median impact factor of 5.33 (IQR 3.36–5.79), whereas the 241 publications from the Patey prize group were accepted in journals with a median impact factor of 4.55 .The majority of the research output presented at both Moynihan and Patey prize sessions led to publications , with 71.0% from the Moynihan and 79.3% from the Patey prize sessions resulting in full-text publications (p < 0.001). The journals where these studies were published were surgical (38.6%), medical (23.3%), basic science/translational (33.3%) and miscellaneous (4.7%), with differences found in the journal type between the Moynihan and Patey prize sessions (P = 0.017), which mirrors the types of research presented  and 30.1% were on clinical research. Between the two prize sessions, presentations from the Patey prize sessions were more likely to be laboratory-based, 79.9% vs 47.8% . Prize winners were more likely to publish in journals with a higher impact factor  .There was a significant disparity in sex amongst the Moynihan and Patey prize presenters, with 80.8% of the overall prize presenters being male Table . BetweenP = 0.019). Throughout the 21-year period, there was a steadily increasing trend of non-White presenters across both conferences  were White men, followed by Asian men 112, 25.3%), as shown in Fig. %, as sho7.8% wereP = 0.027) Table p = 0.058).Of the abstracts presented at both prize sessions, an overwhelming majority of the senior authors were male and White (88.0%) with a significant disparity noted in female senior authors ((14.0%, This study found that the abstracts presented at the ASGBI Moynihan and SRS Patey prize sessions resulted in a high rate of publication following presentation, and with an acceptable time to publication. Nearly 71% of abstracts from the ASGBI Moynihan and 79% from the SRS Patey prize sessions led to publications. The median time from presentation to publication was 448 days, reflecting the length of the peer review and publication process . PresentInterestingly, there was significant disparity in sex across both prize sessions, with male presenters forming the majority at 80%. This was consistent in both the Moynihan and Patey prize sessions. However, there was a steadily increasing trend of female presenters, rising from 15% in 2000 to 33% in 2020. Additionally, ethnicity subgroup analysis highlighted that the majority of presenters were White, followed by Asians. Across both prize sessions over the 21-year period, only one Black female, one Black male and 16 Asian female presenters were recorded out of a total of 442 presenters. The significant sex and ethnicity differences persisted even amongst senior authors. A focus should be placed on encouraging more women and ethnic minority groups to consider academic surgery as a potential career choice early on in their training. Additionally, educating medical students on study design, statistics and the range of possibilities that a career in academia can bring is paramount to fostering interest. Finally, embedding mentorship and flexibility in the different pathways into academic surgery might encourage future aspiring trainees to consider this as a career option.A Cochrane review  of over Academic surgery is historically perceived to be one of the more difficult career options for women , 15. A rAlthough the issue of ethnic diversity in surgery has received more coverage in the USA , 24, the.Analysis of the Moynihan and Patey prize sessions at the ASGBI and SRS conferences, respectively, may not be representative of the remaining sessions at the conferences. However, the two prestigious prize sessions are important in highlighting the best of surgical research. Additionally, prestigious prize sessions provide an opportunity to showcase the breadth and excellence of surgical research across the UK and Republic of Ireland. “Gender” API and ethnicity software programmes were used for the names of presenters and senior authors and could be subject to some erroneous findings. However, both systems have been validated in multiple studies , 12. ForPrestigious prize sessions highlight important and impactful academic research in surgery. Associated publication rates were high, with prize winners publishing in journals with high impact factors. The significant disparity in sex and ethnicity of both presenters and senior authors, across both prize sessions, reflects the current state of academic surgery. More needs to be done, to address this imbalance and encourage diversity and representation across the echelons of academic surgery.


For the first time, this study investigated the extraction of bioactive substances with different polarities from immature honey pomelo fruit (IPF), a by-product of pomelo planting processing that causes resource waste and environmental pollution, using novel continuous phase transition extraction technology (CPTE). The results showed that CPTE was suitable for extracting essential oil, naringin, and pectin in sequence according to their polarities. The naringin extraction process was optimized by the response surface methodology, resulting in an extract ratio up to 99.47%. Moreover, the pectin extracted from IPF by CPTE showed better quality compared to commercial counterparts, as evidenced by lower protein and ash contents and higher white value. Together, these results suggested that CPTE could be a promising technology to improve the application value of IPF. For instance, the extracted bioactive components can be utilized as nutraceutical food ingredients. The scientific insights from this study will contribute to the development of functional food ingredients and comprehensive utilization of farming by-products.The immature honey pomelo fruit (IPF) is a huge agro-industrial by-product generated during pomelo planting. Although IPF is rich in nutrients, more than 95% of IPF is discarded annually, which causes resource waste and a serious environmental problem. Here, we report a novel continuous phase transition extraction technology (CPTE) to improve the comprehensive utilization of IPF by sequentially generating high value products and solve pollution problems related to their disposal. First, essential oil was successively extracted by CPTE at a yield of 1.12 ± 0.36%, in which 43 species were identified. Second, naringin extraction parameters were optimized using the response surface methodology (RSM), resulting in a maximum extraction rate of 99.47 ± 0.15%. Finally, pectin was extracted at a yield of 20.23 ± 0.66%, which is similar to the contents of commercial pectin. In conclusion, this study suggested that IPF was an excellent potential substrate for the production of value-added components by CPTE. Citrus grandis (L.) Osbeck), a member of the citrus family, is widely cultivated in Southeast Asia and some other Asian countries [Pomelo  and were stored at room temperature. The composition of the IPF  was analyzed according to methods adapted from previous research ,19,20,21HPLC-grade acetonitrile and hexane were purchased from Fisher Scientific . n-butane (99.99%) was obtained from Shenzheng Shenyan Gas Co., Ltd. . Galacturonic acid and the naringin standard were purchased from Aladdin Co., Ltd. . Anhydrous methanol, phosphoric acid, and petroleum ether were available from Guangzhou Chemical Reagent Factory .IPF was mainly produced in April and May each year, so the IPF samples were collected in both April and May. Two samples were washed twice with tap water, then cut in half and dried in oven at 35 °C until its weight remained constant. The dried IPF samples were stored at room temperature for further analysis.The essential oil content was determined by the Soxhlet extraction method . The pecPrior to extraction, the IPF was ground into a 40 mesh powder. The essential oil was extracted under n-butane using CPTE equipment. The extraction was performed under 0.5 MPa for 60 min at 50 °C. The solvent was concentrated at 70 °C for 70 min.The extracted essential oil was then analyzed with a gas chromatography mass spectrometer (GC-MS)  equipped with a DB-5 capillary column  following a published method . The temIn order to improve the extraction rate of naringin, all extraction experiments were performed in accordance with a central composite design (CCD) with 3 factors and 3 levels based on preliminary experiments. These factors were extraction temperature, time, and raw material granularity (RMG), respectively. The factors and independent variables are listed in The remaining IPF after essential oil extraction was used as the starting material for naringin extraction. Anhydrous methanol was used extract naringin under 0.3 MPa. The solvent was concentrated at 75 °C. The extracted naringin and the naringin remaining in IPF dregs after extraction were analysis by HPLC, using the parameters described above. The naringin extraction rate was calculated using the following equation:The remaining IPF dregs after naringin extraction were used as the starting materials for pectin extraction. Extractions were conducted under continuous deionized water (Ph = 1.5) under 0.3 MPa at 90 °C for 60 min. The solvent was concentrated by vacuum evaporation at 90 °C for 60 min. Pectin concentrate was desalinated using a small electrodialysis desalination unit . Subsequently, 2 volumes of 95% ethanol were added to the sample and incubated for 4 h under 4 °C to precipitate pectin. The pectin was separated by high speed centrifugation, then washed three times with 95% ethanol followed by freeze-drying to obtain the final product. The pectin yield was calculated using the following equation:The extracted pectin was analyzed for the determination of the content of galacturonic acid, moisture content, ash content, degree of esterification (DE), protein content, and total sugar content, according to methods adapted from previous researches ,19,20,21t-test was performed to test the mean difference between two groups, whereas analysis of variance (ANOVA) followed by Duncan’s multiple range test was used for the comparison of differences among three or more groups. p < 0.05 was used as the threshold for determining significance.All assays were performed in triplicate, and the results are expressed as the mean ± standard deviation (SD). Student’s p < 0.05). This is in agreement with previous research showing that flavonoid content is the highest during the immature period [p < 0.05). As the naringin content in April was twice as high as in May, we selected the IPF collected in April for the further study, even though the pectin content was slightly lower.In order to determine which sample should be used for further analysis, the content  of naringin and pectin of IPF produced in April and May were quantified A. We foue period . HoweverThen we determined the levels of essential oil and other components in IPF harvested in April. As shown in Essential oil, as a volatile oil, is a general term for oily liquids in plants which have an aromatic odor. Many studies have shown that pomelo peel essential oil presents numerous biological activities, including antibacterial, anti-inflammatory, as well as swelling and pain relief effects ,27. The D-limonene is a common substance in citrus essential oils and a major contributor to citrus aromas. It is considered as a marker substance in essential oils, such as pomelo, orange peel, and bergamot , and is In a previous study, a microwave irradiation treatment was conducted to extract essential oil from pomelo, and it performed better than hydrodistillation in terms of the yield of essential oil (1.88 ± 0.06% yield) . In anotThe IPF residue after essential oil extraction was used as the starting material for naringin extraction, and the naringin extraction rates obtained under different conditions are listed in p = 0.0003 < 0.001) (p > 0.05), indicating that the error of the test is small, and the non-test factor has little influence on the result. The goodness of fit (R2 = 0.9646) illustrated that the model has a high degree of fit and reflects real experimental results.ANOVA analysis revealed that the polynomial model was statistically significant (< 0.001) . The p-vThe relationship between independent variables and their effects on the extraction rate of naringin were determined using a three-dimensional response surface curve generated by modeling .We found that all parameters tested had significant effects on the extraction process. Noticeably, ANOVA analysis revealed that the extraction time is the most important factor affecting the extraction rate, followed by temperature. As the temperature rose, the extraction rate of naringin also increased, particularly at the beginning of the extraction process. In addition, our results indicated that the solubility of naringin was greatly affected by temperature. Moreover, an increased temperature could reduce the viscosity of the solvent and improve solute vapor pressure , leadingThe biological activities of naringin have been shown in many studies. In the medical and food industries, naringin is the subject of significant demand as a pharmaceutical component and food additive. The traditional extraction methods for naringin still have the disadvantages of low purity, low extraction rate, and environmental pollution. According to our analysis using Design-Expert software, the optimum conditions for maximum naringin extraction rate were 76.4 °C for 3.7 h at 39.1 mesh RMG. The predicted extraction rate was 99.32%. We then verified this result by extracting the naringin under the optimum conditions and determining the corresponding extraction rate. For our implementation, the conditions were adjusted to 75 °C for 4 h extraction time at 40 mesh RMG. The test was performed in triplicate and the actual extraction rate was 99.47 ± 0.15%, which was very close to the predicted value for the optimal conditions.Next, we compared the extracted sample and commercially available standards by HPLC. As shown in After naringin extracting, the IPF dregs remaining were used as the starting material for further pectin extraction. We found that the IPF dregs contained 44.02 ± 1.30% pectin, which was 3.12 times higher than that of the original proportion. This indicated that the extraction of essential oil and naringin from the IPF during the process led to the increase of the percentage of pectin remaining in IPF dregs, which would promote the effective extraction of pectin .After the extraction process described earlier, pectin was obtained from IPF dregs with a yield of 20.23 ± 0.66%. Data in In this study, IPF has been used to produce value-added components  via a novel CPTE technology. The naringin extraction process was optimized using RSM, and the extract ratio of naringin was up to 99.47%. The quality of extracted IPE pectin, characterized by protein content, ash content, and white values, were much better than commercial pectin. Together, these results indicate that CPTE is an efficient and eco-friendly extraction technology that provides a significant advancement in the development and utilization of IPF and offers new insights pertaining to the comprehensive utilization of this agro-industrial by-product.


To broaden the operating range of photoreceptors, mammalian rod photoresponse recovery is accelerated mainly by two calcium sensor proteins: recoverin, modulating the lifetime of activated rhodopsin, and guanylate cyclase-activating proteins (GCAPs), regulating the cGMP synthesis. We found that decreasing rod intracellular Ca2+ concentration accelerates the flash response recovery and increases the basal PDE6 activity (βdark) maximally by ~ 30% when recording local electroretinography across the rod outer segment layer from GCAPs−/− recoverin−/− mice. Our modeling shows that a similar elevation in βdark can fully explain the observed acceleration of flash response recovery in low Ca2+. Additionally, a reduction of the free Ca2+ in GCAPs−/− recoverin−/− rods shifted the inhibition constants of competitive PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) against the thermally activated and light-activated forms of PDE6 to opposite directions, indicating a complex interaction between IBMX, PDE6, and calcium. The discovered regulation of PDE6 is a previously unknown mechanism in the Ca2+-mediated modulation of rod light sensitivity.Phosphodiesterase-6 (PDE6) is a key protein in the G-protein cascade converting photon information to bioelectrical signals in vertebrate photoreceptor cells. Here, we demonstrate that PDE6 is regulated by calcium, contrary to the common view that PDE1 is the unique PDE class whose activity is modulated by intracellular Ca These molecules hydrolyze cyclic AMP and cyclic GMP, second messenger molecules involved in a vast number of signaling pathways, including gene regulation, cell proliferation and differentiation, metabolism, immune function, memory, and visual transduction3. PDEs, together with the cAMP and cGMP synthetizing cyclases, regulate the intracellular cyclic nucleotide concentrations. Therefore, the modulation of the activity of different PDEs is among the key therapeutic targets in humans and animals5.Cyclic nucleotide phosphodiesterases (PDEs) regulate numerous processes essential in health and disease12-fold range6. This requires efficient light adaptation mechanisms in various levels of visual perception, which in the photoreceptor level are mostly mediated by controlling intracellular Ca2+ concentration. Here we investigate calcium-dependent modulation of PDE6, an enzyme that hydrolyzes cGMP and mediates the conversion of photon absorption to an electrical signal in the phototransduction cascade, taking place in the outer segments of vertebrate photoreceptor cells. In rod photoreceptors, photon-activated rhodopsin sequentially activates several G-proteins, transducins. The activated transducins bind to PDE6 molecules and dislocate the inhibitory PDE6γ-subunits covering the catalytic sites. This converts PDE6 molecules from their basally active form, where the enzyme activity is set by spontaneous thermal activations, to light-activated form, which hydrolyzes cGMP at nearly diffusion-limited rate7. PDE6 activation causes a rapid drop in the cGMP concentration, leading to the closure of cyclic nucleotide-gated (CNG) channels in the rod outer segment plasma membrane, reduction of the circulating “light-sensitive” current, and hyperpolarization of the cell .Our visual system can function in different illuminations covering a 10+/Ca2+K+ (NCKX) exchangers located in the outer segment plasma membrane leads to a rapid decrease in the cytoplasmic calcium concentration. With strong light stimuli, the intracellular calcium level can drop by more than tenfold in mouse rods10. The decrease in Ca2+ activates fast light adaptation mechanisms through calcium sensor proteins and accelerates the photoresponse recovery. Guanylate cyclase-activating proteins (GCAPs) augments the cGMP synthesis by the activation of guanylate cyclase11. Recoverin controls the rhodopsin kinase affinity to rhodopsin and modulates the lifetime of light-activated rhodopsin14 and possibly light-activated PDE616. Calmodulin can modulate the affinity of cGMP to CNG channels17, but this mechanism has been regarded as negligible for mammalian rod light adaptation18. Further, we, together with others, demonstrated that in addition to GCAPs and recoverin, there exists an unknown calcium-mediated light adaptation mechanism in mouse rods16.The closure of CNG channels upon light absorption diminishes the inflow of calcium into the photoreceptor cells, while the continuing extrusion of calcium by the Na−/− recoverin−/− background. Using the cGMP clamp paradigm19 and the modeling of dim flash responses, we found that a drop in calcium concentration can cause ~ 30% increase in the basal PDE6 activity. In addition, we found that the decrease in the extracellular calcium concentration modulates the inhibition efficacy of the non-selective competitive inhibitor, 3-isobutyl-1-methylxanthine (IBMX), for PDE6, and surprisingly, this modulation shifts the inhibition constants against the light-activated and basally activated forms of PDE6 to opposite directions. This suggests a complex interaction between IBMX, PDE6 subunits, and calcium.In this study, we recorded local electroretinography across the rod outer segment layer (LERG-OS) from isolated mouse retinas with microelectrodes. We demonstrate that lowering of the extracellular calcium concentration reduces the light-sensitivity of rods by accelerating the photoresponse recovery in GCAPsThe use and handling of the animals were in accordance with the Finnish Act on the Protection of Animals Used for Scientific and Educational Purposes (497/2013) and the Government Decree on the Protection of Animals Used for Scientific and Educational Purposes (564/2013). The study was carried out in compliance with the ARRIVE guidelines.−/− and recoverin−/− mice11 kindly provided by Dr. Jeannie Chen , were used in this study. The mice were in 12/12 h dark/light cycle, and they were let to dark-adapt overnight before the experiment. The mice were euthanized by CO2 inhalation and cervical dislocation. The eyes were enucleated, and a small incision was made along the equator of the eyes. The eyes were bisected by enlarging the incision with micro scissors, and the isolated eyecups were placed to cooled nutrition solution. One eyecup was stored at + 7 °C in the nutrition solution in a light-tight container to be used later on the same day. The retina was removed from the eyecup with forceps and micro scissors under a microscope, and the whole retina was placed in the recording chamber inside a light-protective Faraday cage. All procedures were performed under dim red light.GCAPs recoverin double knockout (DKO) mice of both sexes, derived from GCAPs19. The retina was placed on a filter paper photoreceptor side upwards. Local electroretinography was recorded across the rod outer segment layer (LERG-OS). The recording electrode (tip diameter 2–5 µm) was passed into the depth of ~ 25 µm in the retina, and the reference electrode , a cell-permeant competitive PDE6 inhibitor, was added to the nutrition solution in concentrations of 5, 10, 20, and 40 µM. All chemicals were purchased from Merck Group / Sigma-Aldrich .During the experiments, the nutrition solution filled the open chamber, and the laminar flow of the solution perfused the photoreceptor side of the retina at a constant rate (~ 3 ml/min). The composition of the nutrition solution was (mM): Na2+ concentration to a very low level. In the low Ca2+ solution, the total calcium concentration was adjusted to 100 µM , and free calcium concentration of the solution was set to ~ 20 nM with 3.4 mM EGTA, calculated with the “EGTA calculator”23. The pH of this solution was adjusted to 7.5 with NaOH.The calcium-dependent mechanisms in rod photoreceptors were manipulated by decreasing the CaThe recording chamber was placed on top of a heat exchanger, the temperature of which was controlled with a water circulating heating bath . All recordings were conducted at 37 ± 1 °C. The temperature in the recording chamber was continuously monitored with a calibrated thermistor . The perfusing solution was connected to the signal ground with a 4.7 µF capacitor to reduce high-frequency noise.max = 532 nm; Lumileds, Amsterdam, Netherlands). The homogeneity of the full field stimulus beam on the retina was verified with a camera-based beam profiler . 1 ms light flashes were used to stimulate the retina. The absolute light intensity incident on the retina was measured with a calibrated photodiode . The amounts of rhodopsin photoisomerizations in rods (R*rod−1 or R*rod−1 s−1) caused by the stimuli were calculated based on the rod outer segments dimensions , the LED emission spectrum, the photodiode spectral sensitivity curve, and the pigment template24 as described in25.Light stimulation was accomplished with a LED  controlled feedback from the recorded LERG-OS voltage signal to the light source was used in the cGMP clamp procedure to keep the recorded signal constant by adjusting the background light level after the introduction of PDE inhibitor IBMX to the retina. The closed-loop light control was accomplished digitally in LabVIEW software.c = 500 Hz (8-pole Bessel filter) and afterward digitally filtered with fc = 100 Hz.Data acquisition and LED controls were handled with a data acquisition card  and custom-made LabVIEW software. The recorded DC signals  were sampled at 1000 Hz with a voltage resolution of 15 nV. The signals were first low-pass filtered with a cut-off frequency of f−/− recoverin−/− mouse flash responses was investigated in 10 isolated retinas. The responses were recorded while perfusing the isolated retina with normal Ca2+ solution (containing 1 mM Ca2+) and then with low Ca2+ solution (containing ~ 20 nM free Ca2+). In 5 experiments, the low Ca2+ solution was changed back to normal Ca2+ solution and the flash responses were recorded again. Complete response families covering the whole operation range of rods were collected in each solution for all but one retina. Additionally, in 4 experiments, response families were collected only in low Ca2+ solution . One to twenty-one responses were averaged with each flash strength depending on the response amplitude and noise level. The derived parameter values  and in low Ca2+ solution (n = 7 retinas) as described in19. The cGMP clamp experiments in normal and low Ca2+ were conducted with separate retinas. All experiments lasted less than 2.5 h. The statistical significance of the calcium-induced inhibition constant modulation against light-activated PDE6 by calcium was tested using a two-tailed unpaired t-test with unequal variances. Each retina was considered as one observation. The basal PDE6 activity and the inhibition constant against basally activated PDE6 were determined by pooling the data from each retina and calculating the mean value and the 95% confidence limits or the standard error of regression for the parameters.The inhibition constants against light-activated and basally activated PDE6, and the basal PDE6 activity with cGMP clamp were determined in normal Ca19, while the methods for determining the phosphodiesterase inhibitor inhibition constants are published in26. Here we give a summary of the utilized phototransduction models and the core ideas behind the methods.The phototransduction models used in this study and the theoretical background of cGMP clamp have been described in detail in28.In the rod photoreceptor cytoplasm without any stimulating light, the rate of cGMP synthesis by guanylate cyclase, The PDE6 activity can be decreased by introducing a phosphodiesterase inhibitor to the rod cytoplasm. A competitive PDE6 inhibitor decreases the catalytic activity of both basally activated PDE6 and light-activated PDE6 with inhibition constants mentclass2pt{minim−1 s−1)19:PDE6 inhibitors reduce the rate by which the PDE6 enzyme hydrolyzes cGMP. When a competitive inhibitor of PDE6 is introduced into the photoreceptor cell, it will reduce the rate of light-induced cGMP hydrolysis according to an equation26:The ratio of amplification constants without and in the presence of inhibitor yields a linear equation that can be used to determine the inhibition constant for PDE6 inhibitors against light-activated PDE6, −/− mice, the guanylate cyclase-activating proteins have been knocked out, and the cGMP synthesis rate by guanylate cyclase, Introduction of PDE6 inhibitor reduces the basal PDE6 activity from For a competitive PDE6 inhibitor, Eq.  gives10\8). Thus, 26:The relative change in the circulating current in rods, and thus the extracellular voltage across the rod outer segment layer, is proportional to the relative change in the intracellular cGMP concentration to the power of the Hill’s coefficient for CNG channels 8. The change in the cGMP concentration after a flash stimulus can be described by the differential equation that takes into account the synthesis of cGMP by guanylate cyclase and the hydrolysis of cGMP by both basal and light-activated PDE6 activity,r(t) depends on the relative change in the cytoplasmic cGMP concentration in the following way:−/− recoverin−/− mice40. Additionally, cell-to-cell variations are averaged out in the mass potential ERG signal, which reduces the deviation between experiments.Additionally, the LERG-OS flash response waveform mentclasspt{minimatial Eq.  numericaA), the lifetimes of activated rhodopsin α), and the Hill’s coefficient for CNG channels  play only a minor role. This approximation holds when When modeling photoresponses, the values for several free parameters need to be estimated: The amplification constant of phototransduction (t in Eq. ), while t in Eq. ) and theion, Eq.  simplifi19. The analysis presumes that the modeled response is sub-saturated and that the basal PDE6 activity is lower than 6 s−1. Here, we use Eq.  retina, in normal Ca2+ conditions  and after the extracellular calcium concentration was dropped to ~ 20 nM (red traces). The low extracellular Ca2+ is expected to drive the intracellular calcium in rods well below that during rod saturating light (near 20 nM10). Hence, the low Ca2+ treatment can be expected to drive the intracellular calcium sensor proteins out of their physiological operation range and to uncover any remaining component of calcium-mediated light adaptation, as found in16. Despite the absence of GCAPs and recoverin, the lowering of Ca2+ was still able to accelerate flash response recovery without affecting the activation phase , we compared the early phase of flash responses recorded in normal and in low Ca2+ conditions by fitting the flash response model to the first 34 ms of the response leading edge . This r2+ conditions. The −2. Figure 2+ in19, where the optimal fit to population-averaged response families was achieved with rhodopsin and PDE6 lifetimes of 28 ms and 196 ms, respectively, and with an amplification constant of 18.9 s−2 . The comparison of these analyses suggests that neither rhodopsin lifetime, PDE6 lifetime, nor phototransduction amplification constant change significantly in DKO mouse rods due to the reduction in intracellular Ca2+ concentration.Figure 2+, rhodopsin lifetime was fixed to the value determined by modeling, 2+ and in low Ca2+ solution. For 5 out of the 10 retinas, control recordings were conducted in normal Ca2+ solution also after the low Ca2+ treatment. We observed no substantial differences between the parameter values obtained before the introduction and after the washout of low Ca2+ solution.To analyze the key parameters of the dataset in normal and low Cages (Eq. ) separatmentclass2pt{minim2+ shown in Fig. 47. In DKO mouse retinas, the proteins regulating guanylate cyclase activity, GCAPs, are knocked out, and therefore, no calcium-mediated modulation of guanylate cyclase activity should remain. Hence, a potential source for the low Ca2+ induced acceleration of flash response kinetics might be the increased basal PDE6 activity. To test this hypothesis, we conducted cGMP clamp experiments with DKO mouse retinas in normal and in low Ca2+ conditions.Since the acceleration of photoresponse shut-off in low CaThe cGMP clamp experiments were conducted by recording the LERG-OS signal while illuminating the retina with closed-loop PID-controlled background light, which received its feedback from the recorded LERG-OS signal. One example of cGMP clamp runs is shown in Fig. 2+ conditions. Figure 26. The inhibition constant against light-activated PDE6, 2+ without IBMX (red traces) and with 40 µM IBMX (blue traces). Figure 2+  and in low Ca2+ (red circles). The amplification constant decreases in both solutions as predicted by Eq. (2+ but only 7.6 ± 0.5 µM in low Ca2+ (n = 7). This difference was statistically significant (t(9) = 3.52, P = 0.007, two-tailed unpaired t-test with unequal variances).In order to determine the basal PDE6 activity using the cGMP clamp paradigm, we needed to know the potency of IBMX in inhibiting PDE6 in its light-activated and basally active form both in normal and in low Caons (Eq. ). Figured by Eq. . Surprisrmax. The LERG-OS saturation amplitude before the addition of the inhibitor, rmax,control, was determined from the plateau level of LERG-OS response to a strong stimulus before the cGMP clamp procedure. The growth of the saturation amplitude was determined from the maximal increase in the LERG-OS voltage after the turn-off of the background light in the cGMP clamp  and in amp Fig. .2+  and in low Ca2+ (red dots). The downregulation of cGMP increase induced by high IBMX concentrations can be seen as the nonlinear behavior in Fig. 2+ and 49.0 µM  in low Ca2+. As described earlier, the 2+ solution  and 5.7 ± 0.1 s−1 in low Ca2+ solution .cGMP clamp experiments were conducted on 9 retinas in normal Can in Eq. . The Hil2+ induced desensitization of responses and acceleration of response shut-off kinetics, we modeled dim flash responses with a phototransduction model . Figureentclass1pt{minima2+-dependent regulation of phototransduction in mammalian rods. Our results support two major conclusions: (1) The basal PDE6 activity is not constant as previously thought but depends on calcium concentration. Low levels of calcium, mimicking the behavior of intracellular calcium in strong lights, increase the basal PDE6 activity. (2) Calcium modulates the PDE6 inhibition constant of the non-specific PDE inhibitor IBMX, revealing an interaction between Ca2+ and PDE6.In this study, we investigated the role of PDE6 in Ca16. The phototransduction machinery of mammalian rods has two well-known negative feedback mechanisms mediated by the calcium sensor proteins GCAPs and recoverin13. Here we show that calcium can modulate rod flash responses even in GCAPs−/− recoverin−/− background. In our experimental approach, we lowered the extracellular free Ca2+ to around 20 nM. The Na+/Ca2+K+ exchange mechanism in the outer segment plasma membrane drives the intracellular Ca2+ concentration down towards the equilibrium value 2+ conditions, the recovery of GCAPs−/− recoverin−/− mouse flash responses accelerated, leading to a decrease both in the fractional amplitudes and integration times of dim flash responses.In a previous study, we demonstrated that fast light adaptation in mouse rods is completely mediated by calcium ions and that at least one unknown negative feedback mechanism must be involved in mouse rod phototransduction2+ might be caused by modulation of several mechanisms: shortening of the lifetime of light-activated rhodopsin or PDE6, dynamic enhancement of guanylate cyclase activity, or increase in the basal PDE6 activity8. Since we made our investigations on the GCAPs−/− recoverin−/− mice, the acceleration of response deactivation cannot be mediated by calcium-dependent modulation of guanylate cyclase by GCAPs11, lifetime of rhodopsin14, or PDE616 by recoverin. Additionally, switching between normal and low Ca2+ did not affect the dominant time constant of saturated flash response recovery, reflecting the lifetime of light-activated PDE644. As Ca2+ did not modulate the dominant time constant, 2+-induced acceleration of flash response kinetics was apparent in the recovery phase, while the early phase remained unchanged. Hence, we conclude that low Ca2+ did not affect phototransduction amplification. A third known calcium sensor protein, calmodulin, modulates the affinity of cGMP to CNG-channels in rods17. The 50. Lowering the extracellular Ca2+ to ~ 20 nM should decrease the intracellular Ca2+ well below this value and drive calmodulin modulation out of its functional range, and thus, dynamic calmodulin-mediated cGMP affinity modulation during flash responses is unlikely in low Ca2+ solution. The low Ca2+-induced steady-state change in the affinity of cGMP to CNG-channels should manifest as modulation of absolute response amplitudes but would affect neither flash response amplitudes normalized with the saturation amplitude . Rod phototransduction, with PDE6 as a key effector enzyme, is probably the best-known G-protein mediated signaling cascade in vertebrates, and PDE6 function and inhibition can be investigated electrophysiologically in live photoreceptors.Cyclic nucleotide phosphodiesterases have a central role in a multitude of cellular signaling mechanisms, and therefore, specific inhibition of different PDE isoforms would have a great functional impact and therapeutic utility  and that this interaction would be modified by Ca2+ without significant effect on the catalytic activity of PDE6. Antagonistic interaction between the γ-subunit and IBMX would manifest as apparently larger inhibition constants determined in the presence of γ-subunits compared to those determined in the absence of γ-subunits, i.e. in trypsin-activated PDE6 . In order to explain the low Ca2+-induced modulation of KI values, the ability of the γ-subunit to hinder PDE6 inhibition by IBMX should weaken in the light-activated state and intensify in the basally activated state in low Ca2+. However, clarification of this mechanism requires further studies.Most phosphodiesterases share highly conserved amino acid sequences in their catalytic domains, suggesting high similarity in the hydrolytic sites between different PDEs78. If the basal PDE6 activity was caused by the subunit containing the high-affinity transducin-binding site (without or with the binding of transducin) and the light-activated cGMP hydrolytic activity was produced by the subunit with the low-affinity transducin-binding site, the inhibition of these subunits by IBMX could differ. This might explain the different KI against basally and light-activated PDE6 in low Ca2+.Our finding may also relate to a recent result suggesting that substantial PDE6 activation requires the binding of two transducins to one PDE6 dimer, the binding of the first transducin to the high-affinity transducin-binding site causing negligible cGMP hydrolytic activity while binding of the second transducin to the low-affinity transducin-binding site brings about the full PDE6 activity