Music therapy has been found to be an effective intervention for persons with dementia (PWD) and their primary caregivers (PC), yet the implementation of musical strategies to improve daily care in the home environment requires further exploration. This study developed and examined a home-based music therapy (HBMT) work model that offers weekly joint music therapy sessions, and additional bi-weekly phone-counseling sessions with the PC. This was followed by an additional 12-week support period that included 3 therapy sessions and 3 phone counseling sessions once every other fortnight, so that the same type of session occurred at a frequency of once a month. Participants were five couples (PWD + spouse as PC) who live in their home. Findings based on the qualitative multiple case study research method showed the importance of the music therapist’s (MT) continuous support. The MT’s presence made it possible to address the needs of both spouses, separately and together, while maintaining the required balance. Moreover, the MT’s presence enabled better implementation of the musical strategies independently and this was maintained during the intervention and the support period. The number of persons with dementia (PWD) has increased parallel to the increase in life expectancy. Most live at home and are supported by a primary caregiver (PC) . SpousesMusic therapy has been found to be a positive and beneficial intervention for both PWD and for their spouses as PCs. In recent years, various studies engaged in developing musical strategies for PCs have been conducted so that they can employ these strategies independently and daily without the ongoing presence of a music therapist (MT) . These sThere is a large number of based-music interventions that are not conducted by a MT. However, studies in which the intervention included a MT’s presence indicate better implementation. A more extensive presence can be seen in Melhuish’s study  where a The purpose of the current study was to examine whether a work model that includes extensive support enables the independent implementation of music used in daily life and over time. Although the frequency of the support was gradually reduced to increase independence, we believe it is important to continue providing the couple with ongoing assistance.The current study employed principles of the qualitative multiple case study research method, which gathers a collection of specific cases and gleans insights using similarity or contrast . The stuThis study was approved by the ethics committee of the Department of Music at Bar-Ilan University (E.MUS.2018-7), and the PCs signed their consent to participate in the study together with their spouses. Pseudonyms were used, identifying details were omitted, and the remaining information was kept confidential.The participants were recruited through organizations in Israel that provide home services to PWD. An explanation about the proposed program was given to the study participants. Criteria for participation included: (1) An individual with a diagnosis of dementia who lives at home with a spouse as a PC. (2) The spouse expressed their willingness to be involved in therapy and/or to receive phone counseling. (3) The spouse had difficulty coping with caring for the PWD at home. (4) The participants spoke Hebrew or English.Five couples (a PWD + spouse as PC) participated in the study, ages 73–95. A diagnosis of dementia was given 3–7 years before the study began. Four spouses lived in assisted living and one couple lived at home. They were all assisted by a paid caregiver.According to the model, the MT met with each couple at their home over 26 weeks, including a follow-up period, as follows: The intervention of 10 joint weekly music therapy sessions with both spouses, and five phone counseling sessions, which were held concurrently with the caregiving spouse. This was followed by an additional 12-week support period that included three therapy sessions and three phone counseling sessions once every other fortnight, so that the same type of session occurred at a frequency of once a month. Approximately 4 weeks after the support period ended, a follow-up session was held. The intervention included singing, listening to music, dancing, or playing percussion instruments, according to the couple’s preferences. The musical strategies were personalized and tailored to each couple’s needs.All therapy sessions were recorded and transcribed. Transcriptions of the sessions included verbal and musical content and they enabled an inside look at what occurred in the intervention.All counseling sessions were recorded for documentation purposes. The talks were conducted in order to offer musical interventions and to examine whether these contributed to the daily care of the PWD. These sessions employed topics from the burden questionnaire (Zarit Burden Interview - ZBI)  for PCs Around a month after the intervention ended, a follow-up call was held with the spouse. This call examined whether the musical strategies had been implemented and how had the therapy impacted the daily life of the PWD and their spouse even after the intervention had ended.The journal included documentation of the treatment goals, preliminary planning of the therapy sessions, interpretation, feelings, and personal conclusions of the therapist-investigator that emerged during the intervention. Writing the journal led to an observation of the processes the therapist underwent with the clients and provided a necessary distance from the therapeutic process required as an investigator.The data was analyzed according to the cross-case synthesis method , which iFive main themes emerged from an analysis of the content of all the couples’ sessions and phone counseling.The study emphasized positive and joint musical work to facilitate moments of connection and quality time together for the couples. The joint therapy sessions contributed to strengthening the couple’s relationship, created a shared experience, and a break from the caregiver-care recipient roles that had been created as a result of the dementia.When Sophie and Mark sang the aria Habanera from Bizet’s opera Carmen together, it was easily possible to forget that Sophie had an advanced case of vascular dementia. In the therapy sessions the spouses sang songs together, Sophie as a soprano and Mark as a tenor-baritone. The restlessness and depression Sophie was coping with on a daily basis lessened and for the first time she was fit to participate in a joint activity with Mark. Mark had a huge smile on his face when Sophie joined him in song. Although it was without vocal warmup and sheet music, these were precious moments where he could put aside the heavy role of PC. They simply went back to being a couple, partners in a meaningful activity that gave them a sense of connection and enjoyment.During the therapy sessions it was suggested that the couple practice using music together to help them on a daily basis. The joint practice made it possible to involve the PWD in therapy, fine-tune the intervention, and increase the PWD’s degree of cooperation.Jonathan’s walking was very slow and stiff since he was living with advanced Alzheimer’s and Parkinson’s, making it difficult for him to move from one place to another. However, when his wife Yael learned to use rhythm in the therapy sessions to encourage walking, it became easier, both technically and emotionally. Together we explored what was right for Jonathan: Whether to walk to the sounds of a marching tune, to the percussive sound of a regular and steady beat, or to a rhythmic count: left-right, or one-two. The practice was accompanied by laughter and enjoyment. Yael hummed a military march and humorously used military terms like: “Yes, sir” and saluted when Jonathan asked to go home for the weekend, and said “we are returning to base” when she guided him back to the couch, and “soldier at ease” when she helped him sit down. The practice became an experience, and Jonathan, whose speech was usually repetitive and meaningless, suddenly declared: “want a hug.” Jonathan’s involvement in the practice made him feel loved and equal and made the use of rhythm an inseparable part of the couple’s daily life.Concurrent to the joint therapy sessions, the PC spouse was individually addressed in a separate session, where topics that were inappropriate or impossible to discuss together could be raised and processed, giving this spouse an empathetic ear, understanding, and support. The support sessions made it possible for the spouse to share and ask for advice on the daily challenges common with dementia, to vent emotions, and to regain strength.Observing the spouse losing cognitive function and changing was intolerable for Tamar. Robbie was frustrated and depressed. Although music always played in their home, Tamar preferred to watch the musical activity rather than partake. The disconnection protected her. “I’ve lost a friend. I used to be able to consult and speak with him. Now I cannot,” Tamar shared for the first time, expressing the loneliness and pain she felt in living with her husband Robbie’s Alzheimer’s disease. We were already in the middle of the therapeutic process and Tamar felt safe to express these feelings out loud. She did not want to burden her children or close friends with these hard feelings. Outwardly, Tamar presented a strong and optimistic facade, kept busy, and was socially active. “But inside I’m a very sad person,” she expressed with honesty.The validation, understanding, and empathy she received for her feelings during the phone sessions helped her to process and come to terms with the dramatic turning point in their lives. The therapy sessions created an opportunity for Tamar to return to the joint sessions and to experience positive and pleasant quality time with her spouse in a non-threatening manner. Also, the sense of distress Tamar described at the onset of the intervention lessened after she was given an opportunity to grieve the loss of their couplehood.The phone support sessions were held with the PC spouse separately. However, topics that emerged during the calls made it possible to fine-tune the intervention in the therapy sessions and to suggest different ways of using music. In this way, the phone sessions went from theory to applied practice.Singing and listening to classic Israeli songs turned out to be extremely meaningful for Eliraz. “There’s hope!” Motti, her husband, declared whenever she responded verbally and coherently after a musical activity. Motti was elated because Eliraz was living with dementia and spent most of the day sitting in her wheelchair, barely able to speak and understand instructions. However, when Motti tried to recreate the success on his own, he encountered great difficulty. He was frustrated that Eliraz did not respond to music on a daily basis in the same way she did during the joint therapy sessions. A joint examination revealed that without musical support, Motti had a hard time producing song and tended to recite the words without a melody. Also, he tended to ask Eliraz questions about the background of the songs, which elicited frustration and distress in Eliraz. Joint practice in the therapy sessions helped Motti learn singing techniques and how to address Eliraz in order to elicit past memories. Toward the end of the intervention, Motti succeeded in using music effectively. His singing was good, and he managed to create moments of closeness and connection, which the couple sorely lacked.When the therapy sessions ended, the participants continued to receive support for an additional 3 months. During this period, therapy sessions and phone support sessions were conducted with each spouse once every other fortnight, so that the same type of session occurred at a frequency of once a month. This frequency of sessions and calls made it possible for the couples to gradually wean off the support while still providing a small amount of assistance. In this way, the couples were encouraged to continue using music independently without being left to cope on their own.Elizabeth did not believe that a day would come when she and Victor would once again stand face to face, hold hands, and sway to the sounds of Tango. Victor, who was living with advanced dementia and required long-term care, was uncommunicative most of the time. The musical activities during the therapy sessions were a shared couple activity and elicited in Victor rare coherent verbal responses, which surprised and pleased Elizabeth. The phone support that Elizabeth received helped her to use musical strategies also during the week. Together we examined the couple’s challenges and how music can assist. Thus, Elizabeth learned to sing and play music in order to stimulate Victor, who spent most of the day sitting passively in the armchair. However, the musical work required effort from Elizabeth. She stated that the therapy sessions and the phone counseling boosted her motivation and encouraged her to use music more often. Without support, it was harder. In the follow-up phone session, about a month after the intervention ended, Elizabeth said: “After I speak to you, I also use singing, now I use it less.”The current study examined whether a work model that included extensive support for a total period of 26 weeks enabled the independent implementation of music, used in daily life and over time for PWD and their PCs. The work model made it possible to address the needs of both spouses, separately and together, while maintaining the required balance. Moreover, the MT’s presence enabled better implementation of the musical strategies independently and this was maintained during the intervention and the support. The research literature shows that addressing the roles of the MT for PWD and their spouses presents two widespread approaches. In the first approach, the MT focuses on treating the PWD but does not address the PC. The dependence on the therapist is high and the use of music is preserved only during the therapy sessions . In the As can be seen in The MT’s primary role is to provide therapy The therapy is given jointly while addressing the couple as one unit, contrary to focusing on the PWD and providing separate support for the PC . StrengtThe MT’s second role is to provide separate counseling and support for the PC. The phone counseling provided a platform for discussing topics that could not have been raised in the joint sessions, for giving tools to cope with the daily challenges, and for fine-tuning the therapeutic intervention. This model is consistent with other studies that see the importance and significance of addressing the PC spouse, in order to enable them to continue to provide optimal care , 16.The MT’s third role is to enable the spouses to continue using music beyond the therapy session. In the current work model, musical strategies were given internally, as part of the joint therapy, which addressed the needs of both spouses. Musical strategies were also provided separately to the PC in a way that made it possible to fine-tune the therapeutic intervention and to practice during the session together with the MT. Unlike studies that include musical strategies conducted by the PC with limited guidance and support from the MT , in the The study findings indicated that the MT’s extensive support helped to implement the use of music in the daily lives of PWD and their spouses. This extensive support requires an inclusive solution of couple’s therapy, counseling for the PC, and providing musical strategies. This inclusive solution requires extensive training of MTs. Alongside the resources required to enable the ongoing presence of a MT, it is a model that: (1) Provides a therapeutic space for the couples. (2) Enables good implementation of musical strategies. (3) Provides a stable foundation that makes it possible to reduce the frequency of the sessions, which saves resources, yet still provides support in using music independently. This study examined a limited number of couples and additional studies are required to further validate and develop the work model. In addition, a longer follow-up period is needed to examine the sustainability of the couple’s use of musical strategies and to explore whether music has been integrated into their daily lives.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving humans were approved by Department of Music at Bar-Ilan University (E.MUS.2018-7). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.MR, AD, and AG conceived of the study, participated in its design, and took part in drafting the manuscript. MR, the music therapist, was in charge of coordination, data collection and coding as part of her Ph.D dissertation. AD and AG supervised the work. All authors contributed to the article and approved the submitted version.


Eu ions’ chemi- and physisorption to both graphenes may lead to structural deformations, drop of symmetry of low-dimensional lattices, interlayer fusion, and mutual slides of TBG graphene fragments. The frontier bands in the valence region at the vicinity of the Fermi level of both spin-polarized 2D Eu/NPBG(AA) and Eu/TBG lattices clearly demonstrate flat dispersion laws caused by localized electronic states formed by TBG Moiré patterns, which could lead to strong electron correlations and the formation of exotic quantum phases.Advanced two-dimensional spin-polarized heterostructures based on twisted (TBG) and nanoporous (NPBG) bilayer graphenes doped with Eu ions were theoretically proposed and studied using Periodic Boundary Conditions Density Functional theory electronic structure calculations. The significant polarization of the electronic states at the Fermi level was discovered for both Eu/NPBG(AA) and Eu/TBG lattices.  Quantum phases ,2,3 are The connection between localized electronic states and electronic correlations is a fundamental concept in condensed matter physics and materials science ,9,10,11.In crystalline lattices which are characterized by flat electronic bands, the electronic states are typically highly localized in real space . This meThe unique physical properties of 2D bigraphenes (BGs)  make theAt particular magic angles with the largest one of around 1.05° , the Moic is a strongly correlated Mott insulator [c~0 K [c around 1.7 K with the formation of 2D superconducting quantum phase.Twisted bilayer graphene at magic angles and at higher temperatures Tnsulator , which ir [c~0 K , the inssp-metals, such as Li, Ca, and Al [Recently, it was shown that superlattices of potential bumps driven by folded holes in nanoporous bigraphene may induce visible spin polarization by breaking the balance between two graphene sublattices, thereby creating unpaired spins . Dai et , and Al . It coulThe spin polarization of graphene-based low-dimensional materials can be achieved by the creation of either complex heterostructures or defect lattices based on transition metal dichalcogenides/graphene  heterostHigh magnetoresistance in graphene was discovered in graphene coordinated to EuO and EuS surfaces . It was 2/Si substrate. There are two main options for its synthesis: the intercalation of Eu under the graphene sheet or the adsorption of Eu on top of the material [6 stoichiometry for single-layer graphene.Experimental data demonstrated that both EuS/graphene  and EuO/material ,47,48,49material . It is pmaterial ,52. At amaterial . As a reThe main goal of this investigation is theoretical design and the study of complex heterostructures based on nanoporous and twisted bigraphenes with either adsorbed or intercalated Europium ions using Density Functional Theory Periodic Boundary Conditions electronic structure calculations. It was shown that Eu-doping causes spin polarization with a different spatial localization of α and β spin states and fundamental alteration of their band structures with the formation of flat bands in the vicinity of the Fermi level, which may promote strong electronic correlations with the formation of quantum phases, which are promising for advanced spin- and quantum-related applications.k-point sampling was implemented, and the k-point mesh contained 3 × 3 × 1 k-points along with a, b, and c directions, respectively, being used for carbon-based media optimization. For all elements involved in the electronic structure calculations, PAW potentials were used. For carbon, 4 outer electrons, and for Eu, 8 outer electrons, were treated as valence electrons. For Eu, semi-core f-electrons were treated as core states despite being higher in energy than other valence states. For the density of states calculations, the 6 × 6 × 1 k-point mesh was used. A vacuum interval of 20 Å was set normal to the plane to avoid artificial interactions between adjacent unit cell images. In all calculations, the cut-off energy was equal to 600 eV. During the optimization procedure, the maximum force acting on atoms less than 0.001 eV/Å was used as a stopping criterion for structural minimization.The electronic structure calculations of low-dimensional crystalline lattices were performed using the Vienna Ab-initio Simulation Package (VASP) ,55,56 wia = b and γ = 120°, but during structural optimization, a drop of symmetry had been found for some of them. The unit cells of all heterostructures are presented in the Initial atomistic models of the heterostructures were developed using (i) AA and AB bigraphenes  with eitFor the sake of comparison, the optimization of the 2D atomic lattices of parent TBG-21 and both AA/NPBG and AB/NPBG was performed using the same PBC GGA-PBE + U PAW D3 approach. Structural optimization revealed small effects of DFT + U corrections on structure and electronic properties of all 2D lattices. The Parent TBG-21 lattice suffered small structural distortions constituting graphene fragments, which drastically increased with the Ei adsorption.xy direction. In both BG (AA) and BG (AB) cases the Eu interlayer led to the bending of the graphene fragments induced by repulsion with Eu ions.The PBE PBC atomic structure optimization of Eu/BG-AA and Eu/BG-AB experienced drastic cell distortion. In both cases, the lattice symmetry dropped from hexagonal to monoclinic with some slides of graphene fragments  along thh-BN, g-C3N4, g-C4N3, MoS2, and MoSe2 lattices [Topological stability of complex low-dimensial crystalline lattices with multiple non-equivalent sublattices is a key issue for the design and study of 2D periodic atomic lattices from both from theoretical  and expelattices ,68,69 malattices  the lattThe lattices of the graphene fragments of Eu/TBG demonstrated significant reconstruction during structural optimization, with the formation of corrugated carbon lattices formed by triangles, pentagons, hexagons, heptagons, octagons, and even nonagons . In partThe analysis of Total (TDOS) and Partial (PDOS) Density of Electronic States for Eu/BG a unequivf = 7.5 eV and Jf = 0.6 eV parameters [f-electrons. The results of DOS and band structure calculations  is similar to the shape of the second valence band for the β spin state (2b). This fact can indicate the correspondence of the −2a band to the 2b band, induced by the Eu coordination atop TBG since the DOS at the Fermi level is fully determined by the TBG fragment (The band structure of spin-polarized Eu/NPBG (AA) and Eu/TBG heterostructures are presented in fragment d. Tuninga) looks fairly similar to the first flat conduction band of the β spin state (1b), so the −1a band-to-1b band correspondence can occur. The flat nature of the band dispersion law for boundary CB and VB demonstrates their localized nature and the absence of chemical interactions between the states. In case of flat bands, the electronic transitions between VB and CB would not experience phonon relaxation, which could enhance the transition performance.The band shift could be identified in the case of the Eu/NPBG (AA) interface as well. The first flat valence band of the α spin state (−1The spin density distribution for Eu/NPBG (AA) and Eu/TBG are presented in In particular, for Eu/TBG, the spin density is fully localized at the TBG fragment c,d. NeitIn this study, the key Eu-doped low-dimensional 21.8° twisted bilayer graphene and both AA and AB nanoporous bigraphenes were theoretically proposed and studied using the PW PBC DFT approach. Based on electronic structure calculations, it was shown that Eu coordination on top of both the bigraphene and twisted bilayer graphene induces the glide of graphene fragments and drastic structural deformation for Eu/TBG. In accordance with the Topology Conservation Theorem, corrugated TBG graphene fragments form structural waves coupled with interlayer graphene fusion, which would stabilize the local structure and the long-order topology of 2D graphene lattice. The electronic structure calculations revealed that both Eu/NPBG (AA) and Eu/TBG are spin-polarized direct flat band semiconductors with a spin density mostly localized at graphene fragments. The localization of uncompensated spins highlights the leading role of carbon fragments in the spin polarization of the heterostructures. Heterostructure DOSes clearly demonstrate the similar distribution of electronic density of both valence and conductivity bands in the vicinity of the Fermi levels. The total density of states of the valence bands is mostly formed by carbon contributions, while conduction bands have some contributions to the Eu partial electronic states. The distinctive spin polarization and flat bands of the proposed graphene-based low-dimensional quantum materials may promote advanced spin- and quantum-related applications.


Report. A 49-year-old male presented with recurrent dyspnea on exertion, easy fatigue, and chest pain after several visits to different health facilities over 5 months. A diagnosis of pulmonary sarcoidosis was made after a series of laboratory and imaging investigations were done revealing bilateral reticonodular opacifications, noncaseating granulomata, elevated serum ACE and calcium levels consistent with sarcoidosis.  Sarcoidosis is a complex disease with nonspecific etiology and clinical presentation. Its diagnosis is often delayed due to the absence of a single specific investigation modality. A multidisciplinary approach is necessary for its diagnosis.  Sarcoidosis, although a rare presentation in our setting, may easily be overlooked or misdiagnosed if a holistic or multidisciplinary approach is not employed in its diagnosis. Nonspecificity of symptoms contributes to the delayed diagnosis. Sarcoidosis is a granulomatous disease that commonly affects the lungs, and its worldwide prevalence is estimated to be between 4 and 6 in 100,000  on accouData on the disease burden of sarcoidosis in Ghana is not readily available. However, a number of cases have been reported such as that by Bajantri et al. who reported on a 53-year-old woman with neurosarcoidosis  and two The elusiveness of the etiology makes the condition a subject of ongoing research . Severalα leading to massive macrophage activation. Other cells involved include monocytes and lymphocytes. Cytokines and chemokines also play a role in the pathogenesis of pulmonary sarcoidosis . In cases of steroid resistance, a weekly low-dose methotrexate therapy, azathioprine, infliximab, and leflunomide may be used , 20.The recommended period for follow-up is 6-12 months. Pulmonary function tests, especially vital capacity and chest X-ray, can be used to assess for resolution of symptoms or for progression of symptoms . It is aThe classification of sarcoidosis will depend on the outcome of the treatment. It is termed acute if the symptoms resolve between 2 and 5 years after the start of treatment. If symptoms persist after 5 years of treatment, it is termed chronic, and the classification of refractory sarcoidosis is used if symptoms worsen after the start of treatment .Delayed diagnosis is likely and seen in about 50% of cases on account of nonspecificity of the clinical signs and symptoms; however, a high index of suspicion should be entertained and a holistic multidisciplinary approach employed for its diagnosis in a low-resource setting.


However, it is unknown if the activation of TrkB in PV+ interneurons is important for other plasticity-related behaviors, especially for learning and memory. Here, using mice with heterozygous conditional TrkB deletion in PV+ interneurons (PV-TrkB hCKO) in IntelliCage and fear erasure paradigms, we show that chronic treatment with fluoxetine, a widely prescribed antidepressant drug that is known to promote the activation of TrkB, enhances behavioral flexibility in spatial and fear memory, largely depending on the expression of the TrkB receptor in PV+ interneurons. In addition, hippocampal long-term potentiation was enhanced by chronic treatment with fluoxetine in wild-type mice, but not in PV-TrkB hCKO mice. Transcriptomic analysis of PV+ interneurons after fluoxetine treatment indicated intrinsic changes in synaptic formation and downregulation of enzymes involved in perineuronal net formation. Consistently, immunohistochemistry has shown that the fluoxetine treatment alters PV expression and reduces PNNs in PV+ interneurons, and here we show that TrkB expression in PV+ interneurons is required for these effects. Together, our results provide molecular and network mechanisms for the induction of critical period-like plasticity in adulthood.Critical period-like plasticity (iPlasticity) can be reinstated in the adult brain by several interventions, including drugs and optogenetic modifications. We have demonstrated that a combination of iPlasticity with optimal training improves behaviors related to neuropsychiatric disorders. In this context, the activation of TrkB, a receptor for BDNF, in Parvalbumin-positive (PV Learning and memory dysfunction is a common neuropsychological symptom of neuropsychiatric and neurological diseases. It has been proposed that chronic treatment with antidepressants (ADs) improves impaired learning and memory in animal models –3 via in+) fast-spiking interneurons primarily targeting the perisomatic area of pyramidal neurons [+ fast-spiking basket cells exhibit plasticity by dynamically changing their states in response to recent experience: a state characterized by low PV expression in the PV+ interneurons is involved in plastic networks while a state with high PV-expression in PV cells promotes memory consolidation in the hippocampal CA3 region. This leads to a lower and a higher number of excitatory synaptic inputs onto PV interneurons, respectively, regulating experience-dependent network plasticity [The activation of the brain-derived neurotrophic factor (BDNF) and its receptor TrkB is a key factor in neuronal plasticity. The binding of BDNF to TrkB causes the autophosphorylation of TrkB and leads to the activation of intracellular signaling pathways involved in neuronal differentiation, survival, and growth, as well as synaptic plasticity in neurons , 10. Thi neurons –13. Donaasticity . Furtherasticity , an extrasticity , 17.+ interneurons is necessary and sufficient for iPlasticity in the visual cortex [+ interneurons combined with learning processes can improve learning and memory more generally.We have demonstrated that ADs induce a critical period-like plasticity in the adult brain (iPlasticity), which allows brain networks to better adapt to environmental stimuli, such as training or rehabilitation, and consequently ameliorate neuropsychiatric symptoms , 10, 18.l cortex . Therefo+ interneurons on reversal learning, we treated PV+ interneuron-specific heterozygous TrkB knockout (PV-TrkB hCKO) mice with fluoxetine in the fear extinction paradigm and in IntelliCage apparatus. We also examined the dependency of LTP on the expression of TrkB in PV+ interneurons by studying local field potential activity in the hippocampal CA1 of PV-TrkB hCKO mice. We then performed a transcriptomic analysis specifically for PV+ interneurons using translating ribosome affinity purification (TRAP) after chronic treatment with fluoxetine, and found intrinsic changes in PV+ interneurons, especially in genes related to the formation of PNNs. Finally, we immunohistologically confirmed the plastic changes in PV+ interneurons after chronic treatment with fluoxetine.In order to assess the effects of TrkB in PVDetails of material and methods are provided in the + interneurons , and littermate control mice that normally express TrkB  were produced by mating females from a heterozygous PV-specific Cre line [cre/wt; Pvalb-IRES-Cre, JAX: 008069, Jackson Laboratory) with males from a homozygous floxed TrkB mouse line (TrkBflx/flx) [cre/wt) to obtain the mice (2 months old) expressing GFP-L4 specifically in PV interneurons. The room temperature was kept at 23 ± 2°C, and all mice were kept in a room with a 12-h light/dark cycle (lights on at 6:00 a.m.) with access to food and water ad libitum. All experiments were carried out in accordance with the European Communities Council Directive 86/6609/EEC and the guidelines of the Society for Neuroscience and were approved by the County Administrative Board of Southern Finland (License number: ESAVI/38503/2019).Heterozygous mice with reduced expression of TrkB specifically in PVCre line  (PVcre/wflx/flx)    were croThe fear-conditioning paradigm was conducted following a protocol described previously .IntelliCage  is an automated device that allows housing, performance and measurements of specific tasks in a fully automated manner, removing the need for a human operator and operator-derived bias , 25. We Population spike-free field excitatory postsynaptic currents (fEPSPs) were recorded in an interface chamber using ACSF-filled electrodes (2–4 MΩ) positioned within the CA1 stratum radiatum . Notably3 until cutting on a vibratome . Free-floating sections (40 μm) were processed for fluorescence immunohistochemistry following a protocol described previously [Animals treated with control or fluoxetine were perfused transcardially with PBS followed by 4% PFA in PBS and the brains were isolated. The brains were post-fixed overnight and stored in PBS with 0.02% NaNeviously . The stahttps://fiji.sc/) [+ interneurons subgroups expressing low, intermediate low, intermediate high, and high PV according to the distribution pattern . PV/PNN was imaged in different sections containing CA3b region in the dorsal hippocampus (between −1.94 and −2.18 mm in the anterior-posterior axis relative to Bregma). The multilayer confocal images were stacked , and the fluorescence intensity of PV, PNN and TdTomato were analyzed with Fiji software  (iji.sc/) . We arbi+ interneuron (see above), after 2 weeks of treatment with 0.1% (w/v) saccharine in drinking water (control) or 0.1% (w/v) saccharine and 0.080% (w/v) fluoxetine in drinking water . The isolated hippocampi were stored at −80 °C until the TRAP experiments. After lysating the hippocampus, the tagged ribosomes were precipitated using magnetic beads covered with anti-GFP antibodies. Actively translated mRNA from tagged ribosomes co-precipitated and were sequenced using HiSeq2500  and SMART-Seq v4 Ultra Low Input RNA Kit  for making cDNA library. Primary statistical analysis was carried out with Negative Binomial Distribution with a significance limit of P < 0.05 and the pathway analysis was done from these significant genes using Fisher’s exact test in DAVID with a significance limit of P < 0.1.TRAP analysis was performed according to a previously published protocol . Brieflyt-test are shown in Supplementary Table P value < 0.05 was considered statistically significant.Biochemical and behavioral data were analyzed by two-way ANOVA, taking sessions, genotype, and treatment with fluoxetine, followed by post hoc test. All results of two-way ANOVA and + interneurons thereby reducing the proportion of PV+ interneurons expressing PNN [+ interneurons and would therefore be blunted in PV-TrkB hCKO mice  = 21.94, P < 0.0001; Genotype, F  = 4.049, P < 0.0452; post hoc, wild-type vs PV-TrkB hCKO in Trial 3, P = 0.0037)  = 1.988, P = 0.0288; Ext2, F  = 9.624, P < 0.0001), indicating that the freezing response decreased during the extinction training  = 34.34, P < 0.0001; Ext2, F  = 39.68, P < 0.0001)  = 0.8726, P < 0.0001), but not after day 1  = 0.8743, P = 0.5660). In PV-TrkB hCKO mice, fluoxetine treatment failed to significantly enhance extinction when compared to water-treated mice  = 3.866, P = 0.0502; Ext2, F  = 3.776, P = 0.0530)   = 14.05, P = 0.0003; Sidak’s post hoc test “water-treated vs Fluoxetine-treated” Trial 1, P = 0.0387)  = 4.585, P = 0.0370; Sidak’s post hoc test “water-treated vs Fluoxetine-treated” P = 0.0229)  = 1.876, P = 0.1739)  = 4.585, P = 0.0370, Sidak’s post hoc test “water-treated vs Fluoxetine-treated” for PV-TrkB hCKO P = 0.8618)  = 14.56, P = 0.0002; Sidak’s post hoc test “water-treated vs Fluoxetine-treated” Trial 1, P = 0.0159)  = 7.465, P = 0.0086; Sidak’s post hoc test “water-treated vs Fluoxetine-treated” P = 0.0122)  = 16.61, P < 0.0001, Sidak’s post hoc test “water-treated vs Fluoxetine-treated” Trial 1, P = 0.7996, Trial 2, P = 0.0195, Trial 3, P = 0.0243)  = 7.465, P = 0.0086; Sidak’s post hoc test “water-treated vs Fluoxetine-treated” P = 0.4948)  Fig. . The wiling Fig. . Moreove01) Fig. . Both co30) Fig. . Moreove08) Fig. , suggest87) Fig. , as well29) Fig. . The fre39) Fig. , and in 18) Fig. . In addi59) Fig.  (Treatme22) Fig. . Interes43) Fig. , but the48) Fig. . The absF  = 8,520, P < 0.0001; Treatment, F  = 1.021, P = 0.3133)  = 9.462, P < 0.0001; Treatment, F  = 16.37, P < 0.0001)  = 6.015, P < 0.0001; Genotype, F  = 30.09, P < 0.0001)  Fig. . The PV 33) Fig. , but int01) Fig. . The watF  = 4.212, P = 0.0002)  = 6.794, P = 0.0098)  = 3.550, P = 0.0013) , 02) Fig. , but the98) Fig. . These r13) Fig. . These rF  = 50.20, P < 0.0001). There was, however, no effect of fluoxetine treatment on LTP in hPV-TrkB CKO mice  = 0.2726, P = 0.6019)  Fig. . These r+ interneurons   (Supplementary Table P = 0.0268) and heparan biosynthesis  pathways  were significantly downregulated. These are associated with chondroitin sulfate proteoglycans, which are an integral part of PNNs [P = 0.0384) and glycerophospholipid metabolism  were downregulated. These pathways are involved in the regulation of lipid composition of the cellular membrane, which is highly related to AD effects [P = 0.0863), including G Protein Alpha Inhibiting Activity Polypeptide 3 (Gnai3), G protein subunit gamma 4, 8, and 13 , which are coupled with GABA type B receptor, and mediate slow and prolonged inhibitory action [A) receptors and influences the recycling of the receptor by inhibiting its degradation [A receptors are thought to be a plastic event resulting in the maintenance of the excitatory/inhibitory balance [In order to investigate gene expression in PV interneurons after chronic treatment with fluoxetine, we conducted a TRAP analysis, a system that allows the precipitation mRNA bound to ribosomes, to investigate ongoing protein translation specifically in PVons Fig.   Fig. , and the of PNNs . Also, g effects . Furthery action . Huntingradation . Such mo balance .Fig. 4TR+ interneurons, such as synaptic formation and turnover of PNNs through the regulation of gene expression after fluoxetine treatment.Overall, our TRAP analysis provides insight into the observed phenomena of increased neural plasticity in PVF  = 1.757, P = 0.1934; Genotype, F  = 1.203, P = 0.2801; Fisher’s LSD post hoc test, control vs Flx: wild-type, P = 0.0138; CKO, P = 0.7571)  = 4.785, P = 0.0298; PV intensity, F  = 16.92, P < 0.0001; Treatment, F  = 3.103, P = 0.0796; Fisher’s LSD post hoc test, control vs Flx: Low, P = 0.6365; High, P = 0.0281)  = 1.326, P = 0.2520; PV intensity, F  = 28.88, P < 0.0001; Treatment, F  = 2.564, P = 0.1122; Fisher’s LSD post hoc test, control vs Flx: Low, P = 0.6722; High, P = 0.1075)  Fig. . Interes81) Fig. . However75) Fig. . These r+ interneurons promotes reversal learning in fear and spatial memory. We also showed that both the potentiation of LTP in Schaffer collaterals to CA1 synapses, and the shift in the configuration of the PV and PNN network to a more plastic state require TrkB expression in hippocampal PV+ interneurons. These alterations involve changes in the gene expression patterns related to GABAergic synapses and PNN formation.Here, we demonstrate that iPlasticity induced by pharmacological activation of TrkB in PVIt has previously been shown that heterozygous PV-hTrkB CKO male mice exhibit slightly impaired extinction consolidation but not fear acquisition/conditioning in the auditory fear paradigm . In thisWe show here that fluoxetine treatment decreases cued and contextual fear responses in wild-type mice as previously shown , but not+ interneurons plays a role in the basal level of spatial learning as well as in the reversal learning improvement promoted by fluoxetine. However, we cannot exclude the possibility of the TrkB-independent effects of fluoxetine, acting on spatial memory. For instance, serotonin receptor inhibitors might shed light on possible SSRI effects. More research is needed to elucidate the detailed mechanism of the effect of chronic fluoxetine treatment on spatial memory.To test whether fluoxetine treatment can improve behavioral flexibility or spatial reversal learning through TrkB receptors in PV interneurons, we used the IntelliCage system. IntelliCage is an automated setup that allows behavioral experiments without direct handling of mice except when changing cages and water bottles, which results in higher reproducibility and reliability . Previou+ interneurons, which was also observed in visual cortex plasticity [We found that fluoxetine treatment mainly affects the high PV-expressing PV interneurons, which have been demonstrated to be born earlier during embryonic development and to be responsible for memory formation of recent experiences , 36. Forasticity . These eHippocampal LTP is widely regarded as the cellular substrate underlying learning and memory, enabling plasticity processes to take place . PreviouIn addition to the genes related to the formation of PNNs, we found regulation of genes related to the composition of the cellular membrane, regulatory proteins of GABArgic receptors and genes related to axonal growth guidance.In a previous study from our lab, we observed that the optogenetic activation of TrkB in PV neurons modulates the expression of genes related to GABAergic signaling . This reAmong our data, we also found upregulation of Hap1, which is known to co-localize with TrkB and takes part in the reuptake of BDNF . LiteratMoreover, several studies show how the Trk family is involved in the development of thyroid cancer  and TrkBOverall, the observation of the upregulation of genes involved in TrkB signaling or in processes regulated by TrkB supports the notion that the BDNF/TrkB pathway has indeed been activated by the fluoxetine treatment.+ interneurons responding to environmental stimuli.All significant DE genes related to axon guidance were upregulated. For instance, Sem3D is a receptor of Sema3A, which is known to be localized in PNNs , and Srg+ interneurons [+ neurons in iPlasticity. Importantly, while the activation of TrkB in pyramidal neurons promotes their excitability, in PV+ neurons TrkB activation reduces excitability through the downregulation of Kv3-family potassium channels [+ interneurons does not counteract the concomitant TrkB activation in pyramidal neurons but synergizes with it by disinhibiting pyramidal neurons, thereby orchestrating an enhanced state of cortical plasticity that underlies iPlasticity. Our present data suggest that a similar kind of state of enhanced plasticity, involving reformation of GABAergic signaling and reduction in PNNs, is underlying the effects of fluoxetine on behavioral flexibility and reversal learning.We have recently shown that fluoxetine directly binds to the transmembrane domain of TrkB dimers and increases TrkB retention in the plasma membrane, thereby allosterically promoting BDNF signaling . Furtherrneurons . Taken tchannels . TherefoSupplementary noteSupplementary Table 2Supplementary Table 3Supplementary Table 4


The capacity to stop impending or ongoing actions contributes to executive control over behavior. Action-stopping, however, is difficult to directly quantify. It is therefore assayed via computational modeling of behavior in the stop signal task to estimate the latency of stopping  and, more recently, the reliability of stopping in terms of the distribution of SSRTs  and the frequency with which one outright fails to react to a stop signal . Critically, the validity of computational estimates remains unknown because we currently have no direct readouts of behavior against which to compare them. Here, we developed a method for providing single-trial behavioral readouts of SSRT and trigger failures. The method relies on an adaptation of the stop signal task in which participants respond by moving a computer mouse. In two online experiments, we used movement kinematics to quantify stopping performance , and then applied the standard Race Model and recent BEESTS model in order to examine the convergent validity of the methods. Overall, we demonstrate good correspondence between kinematics- and model-based estimates of stopping performance at the group and individual level. We conclude that the new method provides valid estimates of stopping performance that, unlike model-based estimates, can be read out at the level of single trials. Our approach might therefore be useful for interrogating single-trial neurophysiological correlates of stopping and for large-scale, online studies of behavioral stopping. Action-stopping is an important aspect of executive control, helping to ensure we behave appropriately in a given situation. For example, resisting the urge to swear when angry or reach for a doughnut when dieting. Stopping is typically studied with the stop signal task, in which participants prepare motor responses but are sometimes then cued to stop the impending response. Since successful stopping results in the omission of a response, behavioral stopping cannot be directly observed or quantified. Instead, stopping research relies on a computational model, the Race Model  shorter than Race Model estimates  using movement kinematics, the Race Model and the BEESTS model in order to examine the convergent validity of the methods.https://Prolific.co) and completed the experiments online. The experiments were approved by the UCSD Institutional Review Board.All participants were healthy, adult humans who provided informed consent and were compensated $6.50/hour. They were recruited via an online participant database (r = 0.5 as the minimum meaningful relationship of interest between the estimates of SSRT. We estimated that a sample size of ≥ 23 (difference score) and ≥ 26 (correlation) would be required to detect such effects with an alpha of 0.05 and power of 0.8. We therefore decided to recruit 40 participants, assuming an attrition rate of 25% after filtering data based on performance criteria (see below), to leave us with a final sample size of ~30. Note that in some cases we were predicting a null result , therefore we also computed Bayes factors to enable interpretation of null results, i.e., the strength of evidence for the null hypothesis .No previous study has directly contrasted kinematic- and model-based estimates of stopping performance. Therefore, for the first experiment, we chose a 25-ms difference between estimates of SSRT as the minimum meaningful difference of interest  and TF to enable comparisons at the group level with the same estimates produced by the Race Model and kinematic methods.We report the mean and 95% confidence interval of the population level mean parameters, focusing on the SSRT [μ + τ], SD-SSRT [√ from each performance metric . Under the assumption that our summary average for each individual reflected the ‘true’ value, we asked how many trials would be required to obtain values within an arbitrary ± 5 % of this true value. We ran the simulation 1000 times at each increment of x trials.In order to investigate how many trials are required to obtain reliable measures of stopping performance with our kinematic approach, we ran post hoc simulations on the data. We randomly sampled https://osf.io/d6a92/ ). The code used to run the tasks is also available.We provide the current data set and Matlab code used to perform the main behavioral analyses on the Open Science Framework . However, for output from the BEESTS model we report the posterior mean of the group-level data and the associated 95% credible intervals. Instead of using t tests when contrasting estimates from BEESTS with those produced by the other methods, we evaluated whether or not our Race Model and kinematic estimates fell within the credible intervals associated with the posterior distributions for each variable. This was to account for the fact that there is uncertainty in point estimates from the model  were computed for t tests and correlations, and were interpreted as follows: 1/10-1/3, substantial evidence for the null hypothesis (H0); 1/3-1, anecdotal evidence for H0; 1-3, anecdotal evidence for the alternative hypothesis (H1); 3-10, substantial evidence for H1; 10-30, strong evidence for H1; 30-100, very strong evidence for H1; and >100, extreme evidence for H1. Response times and single-trial SSRTK estimates were considered outliers and removed if they exceeded 1.5 times the inter-quartile range of the first and third quartiles, or if they were <100 ms.Analyses were performed in Matlab 2021a (The Mathworks Ltd.). Most data are reported as mean ± standard deviation and were analyzed using paired Overall, the results from experiments Performance on the primary go task was good, with overall very few omissions, missed targets, false alarms, and choice errors Table . As expeAlthough our primary aim here was to develop direct, single-trial measurements of stopping latency and trigger failures using movement kinematics, we begin by presenting the results of the Race Model analyses. The Race Model forms the backbone of most stopping research but is rarely used to study reaching movements or the stopping of ongoing, as opposed to planned, movements [though see . pStop was close to 50% for both staircases . Stopping latency estimates derived via the Race Model . This implies that the stopping of planned and ongoing actions relies on a common process, an idea that is strongly supported by the very high correlations between home staircase Race Model and kinematic estimates that we come to shortly.The staircasing procedure for the stopping aspect of the task worked well. The different stop performance criteria for the two staircases led to an expected greater SSD for the target versus home staircase , where amplitudes were measured as the maximum resultant displacement of the cursor relative to the point at which the cursor left the home pad.Here we assayed the stopping of ongoing movements shortly after their initiation, and therefore close in time to the planning stage evaluated when applying the Race Model to the same staircase. Despite the trials of interest being classified as failed stops according to the home staircase criterion, individuals still stopped before reaching the target in nearly every trial  of movement and during the early stages of movement execution (SSRTK-H) relies on heavily overlapping processes. Our kinematics-based measurement therefore appears to provide sensible readouts of the latency of stopping and does so on a single-trial basis. This is useful, because it therefore allows a direct estimate of the intra-individual variability in the latency of stopping across trials, which was ~60 ms in both experiments . Movement amplitudes tended to increase as a function of SSD too, consistent with predictions of the Race Model  but differed slightly in experiment t = 15.9, p < 0.001, BF10 > 100, mean difference 32 ±42 ms; Table The pattern of results here was very similar to that of the home staircase. As expected, movement amplitudes were smaller for successful stops of the target staircase compared to go trials . BEESTS produced similar SSRTs to those of the other methods  for each parameter in each data set , and ~240 trials for SD-SSRTK.The total number of stop trials required to obtain a precise estimate of SSRTWe developed a version of the stop signal task that required participants to respond by making reaching movements with a computer mouse and used movement kinematics to provide single-trial readouts of key performance metrics. Despite using a different mode of responding (reaching movements) to standard tasks and assaying the stopping of ongoing (as well as planned) movements, we found that behavior in the task was well explained by the standard Race Model and BEESTS model. Across the two experiments, we found that kinematically derived estimates of SSRT, SD-SSRT, and TF corresponded well with model estimates at the group and individual level, particularly SSRT which showed very strong correlations across the different methods. Overall, we conclude that our approach has good face and convergent validity and offers a range of practical benefits over computational methods of assaying action-stopping.The data were well described by both the Race Model and BEESTS, suggesting that stopping in our task and standard versions of the task share common underlying processes and principles. This is important because both models have typically been employed to study the stopping of planned saccades or finger movements [e.g.  is highly compatible with SSRT measured during the execution phase  [and see  movements can be interrupted during the planning and execution phases [see Fig. Our Race Model-based estimates of SSRT (~330 ms) were broadly consistent with that of previous work utilizing continuous measurement of movement kinetics/kinematics to directly quantify SSRT [~300 ms , and the opposite is true for the target staircase. Additionally, the Race Model only provides a good approximation of ‘true’ SSRT when model assumptions hold, SSRT is constant, go RTs are not heavily skewed, and the trigger failure rate is zero . In fact, most people did not have any trigger failures at all, meaning that most trigger failures at the group level were attributable to a sub-sample of individuals. We speculate that this heterogeneity may be of relevance to individual differences in real-world behavioral control, though this idea remains to be tested.Although BEESTS estimates of trigger failures did not overlap with our kinematic estimate, the absolute values were in fact very close (~0 and ~1%). The reason for the lack of overlap can be explained by two factors. First, the task design afforded relatively few trigger failures overall, particularly compared to the 4-18% trigger failures indicated by BEESTS in the standard button pressing version of the task  and different stop signals in experiment We note that our approach can only be used to estimate trigger failures at short stop signal delays . We assume that the proportion of trigger failures remains constant across all delays, but this may not be true. BEESTS also makes this assumption but, with sufficient data quality could be used to test this assumption.The present method permits precise quantification of stopping performance indices with seemingly few trials: SSRT and TF can be estimated with ~100 trials in total. Admittedly, our approach to estimating measurement precision was somewhat crude, using an arbitrary 5% criterion, and does not speak to the accuracy of the estimates (though this is supported by the similarity with BEESTS estimates). As a point of reference, standard applications of the Race Model require at least 200 trials to produce reliable estimates (Verbruggen et al., A further benefit is that the current method does not require any special equipment to collect data, such as a strain gauge [e.g., (Morein-Zamir et al., A potential downside of studying the stopping of ongoing movements is that biomechanical factors might play a bigger role in the absolute SSRT and SD-SSRT values than for the stopping of planned movements. For example, inertial movements might take some time to ‘brake’ and decelerate, and this could prolong the detection of the stop process in the movement kinematics. Approaches based on changes in acceleration might help to minimize any delay between the onset of the stop process at the level of the muscle and the kinematics. However, even these will be susceptible to factors such as the force-length and force-velocity profiles of the involved agonist-antagonist muscle pairs that undergo coordinated suppression/recruitment (Atsma et al., Biomechanical confounds are also present in simple button pressing tasks (Jana et al., We introduced a novel version of the stop signal task that provides direct readouts of stopping behavior. In addition to the standard performance metric, SSRT, the task also provides estimates of the variability of SSRT and the proportion of trigger failures – measures that are not possible using the standard Race Model approach. These additional descriptors of stopping performance, along with the inter-individual heterogeneity they exhibit, makes this task a good vehicle for future individual differences studies into the psychology of behavioral control. It is possible that although SSRT correlates only poorly with self-reported behavioral control in the literature, the standard deviation of SSRT and trigger failures might fare better. Finally, our approach offers some advantages over the use of model-based estimations of stopping performance since it requires fewer trials to obtain a reliable estimate and offers single-trial readouts of performance.


Under a strain of 40%, the sensor still showed a sensitivity of 42.7 ± 3.1 mV/log [NH4+]. The proposed highly stretchable and robust electrochemical sweat sensor provides a new choice for wearable-device-based personal daily healthcare management beyond hospital-centric healthcare monitoring.Wearable electrochemical sensors have attracted tremendous attention and have been experiencing rapid growth in recent years. Sweat, one of the most suitable biological fluids for non-invasive monitoring, contains various chemical elements relating abundant information about human health conditions. In this work, a new type of non-invasive and highly stretchable potentiometric sweat sensor was developed based on all-solid-state ion-selective electrode (ISE) coupled with poly and polyurethane (PU). This highly stretchable composite of PDMS-PU allows the sensor to be robust, with the PDMS providing a flexible backbone and the PU enhancing the adhesion between the electrodes and the substrate. In addition, graphene–carbon nanotube (CNT) network 3D nanomaterials were introduced to modify the ion selective membrane (ISM) in order to increase the charge transfer activity of the ISEs, which also could minimize the formation of water layers on the electrode surface, as such nanomaterials are highly hydrophobic. As a result, the sensor demonstrated a wide detection range of NH Under a strain of 40%, the sensor still turned out to have a sensitivity of 42.7 ± 3.1 mV/log [NH4+]. We anticipated that this highly flexible and robust sensor could be applied as a personal daily healthcare management solution based on a wearable sensor.In this paper, graphene–carbon nanotube (CNT) network 3D nanomaterials have been introduced to modify the ion-selective membrane (ISM) to increase the charge transfer activity of ISEs. The introduction of the 3D Graphene–CNT network also minimized the formation of a water layer on the electrode surface, as such nanomaterials are highly hydrophobic, which also enhances the accuracy of ammonium detection and long-term stability. In addition, a highly stretchable composite material -polyurethane (PU)) was used as the substrate, in which PDMS provided a flexible backbone while PU enhanced the adhesion between the electrodes and the substrate. As a result, the proposed sensor turned out to have a high sensitivity of 59.6 ± 1.5 mV/log 3−/4− containing 0.1 M NH4Cl solution as redox probes. p) of the bare carbon electrode and the 2D Graphene and 3D Graphene–CNT deposited electrodes, repectively, at a sweep rate of 200 mV/s. The Graphene–CNT modified electrode responded to a higher current value than that of both the bare carbon electrode lectrode a and thelectrode b. Based 4Cl solution. The results of the EIS are illustrated as a Nyquist plot in ctR).The properties of electrodes with different types of modified surfaces were examined by EIS in 0.1 M NH4+, in which the ions move into the ISM phase and electrons travel through the Graphene–CNT layer to reach the electrode. The reduced diameter of the semicircle on the EIS of the 3D Graphene–CNT modified electrode with ISM also indicates a lower ctR compared with that of the bare carbon electrode with the ISM. Thus, a more effective ion-to-electron transduction has been demonstrated using high-surface-area carbon materials, e.g., 3D Graphene–CNT. The EIS results were also fitted and verified by the equivalent circuit model (See ctR—with regards to the 3D Graphene–CNT modified electrode with the ISM (~5.8 × 106 Ω)—was much reduced compared to that of the bare carbon electrode with the ISM (~1.47 × 107 Ω).In odel See , in whic4+ can “size-fit” the complex and combines with the chosen ionophore perfectly. On the contrary, the wall-like ionophores prevent the formation of both wrapping complexes of crown ether with small ions and complexes with large ions.An ISE exhibits selectivity because the chosen ionophore has a much larger free energy of complexation with the specific target ion than with other ions , as show4Cl solution with different concentrations from 10−6 M to 10−1 M, the sensor exhibited an almost ideal Nernstian response, with a sensitivity of 59.6 ± 1.5 mV/log [NH4+]  and an LOD lower than 10−6 M—as depicted in The potentiometric technique was employed to characterize the performance of the sweat sensor in ammonium ion detection. In this detection, both the working electrode and reference electrode were screen printed. The potential difference between the working and reference electrode was recorded using the Open Circuit Potential–Time (OCPT) technique using a CHI600D analyzer. In the tests using the NH4+ using the Graphene–CNT modified electrode, the sensors with the two types of electrodes (with and without the Graphene–CNT coating) were dipped into the same set of NH4Cl solutions for 50 s, at concentrations varying from 10−4 M to 0.1 M. The tests were conducted for two cycles of concentration variation. The potential signal was also recorded by the OCPT technique using the CHI600D analyzer. 4+ concentration. In addition, the potential signal was also not consistent between the two cycles of measurement. As shown in 4+ concentration, with reduced potential variation between cycles. This comparative experiment demonstrated that the sensor with the modified Graphene–CNT electrode achieved higher reversibility, which is one of the critical performance metrics of wearable sensors. Such enhanced reversibility was probably attributed to the hydrophobic properties of the 3D Graphene–CNT layer , in which the output signal looks very stable. 4+. The curves are generally linear, even under the highest strain level of 40% (Min R2 = 0.9108). Under a strain of 40%, the sensor still turned out to have a sensitivity of 42.7 ± 3.1 mV/log [NH4+]. The stretchability test provides evidence that the Graphene–CNT ion-selective membrane is structurally stronger than the bare carbon ion-selective membrane, which could still work stably under a 40% strain—indicating that leakage issues are not significant here.−6 M. Under a strain of 40%, the sensor still turned out to have a sensitivity of 42.7 ± 3.1 mV/log [NH4+]. Additional selectivity studies are critical for the proposed sensor for application in the detection of ammonium in real sweat samples. Thus, our future work is to conduct selectivity tests using samples made of a mixture of NH4+ and other ions in sweat, such as Na+, K+, etc. At the final stage, we shall conduct a test by recruiting volunteers to wear our sensor and detect the NH4+ in their sweat. If further integrated with a wireless communication system, this next-generation sensor could be applied as a key component of a wearable monitoring device used for daily healthcare management.A non-invasive and stretchable potentiometric sweat sensor was proposed to measure the ammonium concentration in human sweat. This wearable sensor was based on highly stretchable composite material substrate of PDMS and PU. With modifications of the 3D nanomaterials of the Graphene–CNT network on the ISEs, the charge transfer activity of the ISEs was enhanced and the water-layer issue was minimized, respectively. This highly stretchable and reliable sensor turned out to have a high sensitivity of 59.6 ± 1.5 mV/log and an LOD lower than 10


Teleogryllus oceanicus to either low or high‐humidity environments while holding temperature constant. We measured water loss and the expression of both pre‐ and postmating reproductive traits. Males exposed to a low‐humidity environment lost more water than males exposed to a high‐humidity environment. A male's cuticular hydrocarbon profile (CHC) did not affect the amount of water lost, and males did not adjust the composition of their CHC profiles in response to hydric conditions. Males exposed to a low‐humidity environment were less likely to produce courtship song or produced songs of low quality. Their spermatophores failed to evacuate and their ejaculates contained sperm of reduced viability. The detrimental effects of low‐humidity on male reproductive traits will compromise male fertility and population persistence. We argue that limits to insect fertility based on temperature alone are likely to underestimate the true effects of climate change on insect persistence and that the explicit incorporation of water regulation into our modeling will yield more accurate predictions of the effects of climate change on insect declines.Global declines in insect abundance are of significant concern. While there is evidence that climate change is contributing to insect declines, we know little of the direct mechanisms responsible for these declines. Male fertility is compromised by increasing temperatures, and the thermal limit to fertility has been implicated as an important factor in the response of insects to climate change. However, climate change is affecting both temperature and hydric conditions, and the effects of water availability on male fertility have rarely been considered. Here we exposed male crickets  The thermal limit to fertility has been implicated as a factor in the response of insects to climate change. However, climate change is also affecting hydric conditions. We demonstrate strong effects of hydration stress on mating behavior and male fertility that will adversely affect insect reproduction under climate change. The stock is reseeded annually with ~50 field‐collected individuals and is housed in a constant temperature room set at 26°C with a relative humidity ~50% and a 12 h:12 h light: dark photoperiod. Male nymphs at the eighth instar were collected from stock culture and held individually in plastic boxes (7 × 7 × 5 cm), provided with cat chow and water ad libitum. Crickets were monitored daily and used in experiments 7–10 days after the molt to adulthood.2.2n = 26). The mean annual 15.00 h relative humidity in Carnarvon, where these crickets were sourced, is 55% (range 52%–59%) . The experimental humidities thereby lie 28% below and 24% above the current normal humidities experienced by these insects in their natural population. Crickets were assigned haphazardly to one of the humidity treatments, and were maintained in their humidity treatments for 48 h before being assayed.The lids of individual cricket containers were removed and replaced with fly screen lids to facilitate free airflow between container and the external environment. Water bottles were removed, and crickets in their containers were placed into one of two humidity‐controlled environments. Crickets within their individual containers were placed inside 149‐L storage boxes, on a wire rack that rested above a tray containing a humidifying agent. Once crickets had been stacked inside these boxes, the boxes were sealed with plastic food wrap before securing the lid. High‐humidity was maintained using a saturated solution of potassium chloride. Low‐humidity was maintained using silicon dioxide (silica gel), which was dried in an oven at 60°C and changed every 2 days. Humidity within the storage boxes was recorded periodically throughout the course of the experiment, which lasted for 68 days: the high‐humidity treatment had a mean (±SE) relative humidity of 79.0 ± 0.2% (range 77%–83%) while the low‐humidity treatment had a mean humidity of 26.7 ± 0.3% (range 25%–31%) , once before and once after exposure to his respective humidity treatment. Thus, before being placed into their treatments, crickets were weighed and then sedated by placing them into a refrigerator at 4°C for 30 min. Crickets were then rubbed the full length of their bodies with a clean SPME fiber (Agilent) for a total of 2 min at a stroke rate of approximately 1 stroke per second; 60 strokes on the ventral surface and 60 strokes on the dorsal surface. The fiber was then emersed and agitated in 1 mL of Hexane (Merck) to dissolve the CHC sample. Samples of CHC were sealed in Chromacol vials (Agilent) and stored at −20°C until analysis. Fibers were washed twice before reuse, by emersion and agitation for 1 min in each of two beakers containing 50 mL of clean Hexane. Forty‐eight hours after being placed into their respective humidity treatments, crickets were reweighed and their CHCs resampled following the procedures outlined above. Crickets were then frozen, dried in an oven at 60°C for 12 h, and their dry weight and pronotum widths determined.Hexane extracts were transferred to new Chromacol vials containing 250 μL glass inserts with polymer feet , the samples dried and then reconstituted to 100 μL. We injected 1 μL of this sample via an Agilent 7693 Autosampler into a gas chromatograph and mass spectrometer (Agilent GCMS 7890B/5977E) operating in the split mode (ratio 2:1) and fitted with an Agilent VF‐WAXms column of 30 m × 0.25 mm internal diameter with 0.5 μm coating . Helium was used as the carrier gas at a flow rate of 1 mL/min. We optimized separation of the extract using a column temperature profile in which the analysis began at a temperature of 150°C for 1 min and rose to 250°C by 25°C/min for 16 min. Final temperature was set at 255°C for 8 min. The MS transfer line, the ion source, and the quad‐pole temperatures were 280, 230, and 150°C, respectively. The certified saturated alkanes standard  was used for concentration calibration and retention index calculation.Agilent Mass Hunter Software was used to acquire and analyze extracts. These were randomized and analyzed in two independent iterations. The mass spectra of unknown compounds were deconvoluted and identified by NIST MS Search 2.4 and AMDIS_32 with the NIST MS database 2020. The retention index was also used to help identify the compounds. Quantitation of metabolite features, peak detection, deconvolution, filtering, scaling, and integration were all processed by Mass Hunter Quantitative Analysis for GCMS (Ver. 7.045.7).x) was used because not all individuals contained every compound. This transformation has been used previously for hydrocarbon data in T. oceanicus  was placed into each male's container such that the container was divided into two equal‐sized sections. A sexually mature virgin female, 7–14 days post adult eclosion, was placed on the opposite side of the partition to the male who was able to contact the female through an array (5 × 10) of holes in the partition, 5 mm in diameter and spaced 5 mm apart. The pair was left for 2 h or until the male began to court the female, at which time his song was recorded. One minute after the onset of courtship singing, males were recorded for 2 min using a Marantz PMD660 Professional digital recorder via a Røde NTG2 condenser microphone. Sampling frequency was 48 Hz and sampling depth 16 bit. All recordings were made in an anechoic room at 26°C and were saved as WAV files.Songs were analyzed using Raven 1.6 (Cornell Laboratory of Ornithology). Recordings were filtered to remove noise at <3.5 and >6 kHz. The courtship song has two elements, a chirp followed by a trill . The evacuating tube was severed with scissors and the ejaculate was allowed to evacuate completely. If the ejaculate did not begin to evacuate within 20 min, the ampulla of the spermatophore was ruptured with forceps to release the ejaculate into the saline. The ejaculate was mixed gently with the saline on the slide. A 5 μL aliquot was then mixed with 5 μL of SYBR 14 (1 mM), diluted 1:50 with Beadle saline and the sample incubated in the dark for 10 min. Then 2 μL of propidium iodide (2.4 mM) was added and incubated in the dark for a further 10 min. The sample was then viewed using a fluorescence microscope, and the number of live (stained green with SYBR 14) and dead (stained red with propidium iodide) sperm in the first 500 sperm counted. The proportion of live sperm in the sample was used as a measure of ejaculate quality.Immediately after they had been recorded, or after 2 h of exposure to their allocated female if they had not produced courtship song, males were checked for the presence of a spermatophore in the genital pouch by gentle squeezing of the abdomen. Males produce a fresh spermatophore at the start of each scotophase Loher,  so that After checking for spermatophores, males were sedated for their second CHC sampling as described above before being frozen. All statistical analyses were conducted in JMP 15.33.129–C35 and represented a measure of the relative abundance of all CHC compounds (Table 31–C33) with both shorter (≤C31) and longer (C35) compounds . CHC profiles both before and after a male was exposed to its humidity treatment were available for 108 males. We putatively identified 29 hydrocarbon compounds in these CHC profiles that ranged in relative abundance from <0.01% to 26.7%  and a marginal within‐subject effect of sampling time . There was no significant interaction effect between treatment and sampling time . Males tended to have higher, though not significantly higher, scores on PC1 48 h after the first sampling  and a statistically significant within‐subject effect of sampling time . There was no significant interaction effect between treatment and sampling time . Males scored higher on PC2 48 h after the first CHC sample was taken  as might be expected. Humidity treatment had a significant effect on water loss  with males in the low‐humidity environment losing more water than those in the high‐humidity environment  and no interaction between PC1 and treatment . Quantitatively similar patterns were found when using PC2 in the model . Identical results were obtained when PC scores from the post‐treatment CHC sample were used.We examined water loss during a male's time spent in the different humidity treatments and the effect of a male's CHC profile on water loss, by entering the change in a male's wet weight over the 48 h period he spent in the humidity treatment, with treatment as the main effect and PC1 (from the pretreatment CHC sample) and dry weight as dependent variables. Heavier males (dry weight) lost more water . Of those surviving, 23 (48.9%) males from the low‐humidity treatment produced courtship song compared with 53 (96.4%) males from the high‐humidity treatment .Only 49 (89%) males survived to be assayed for courtship song in the low‐humidity treatment, while all 55 (100%) males survived in the high‐humidity treatment . Males from the low‐humidity treatment produced songs with positive scores on PC1 and negative scores on PC2, while the reverse was true for males from the high‐humidity treatment . Although there was no significant difference in spermatophore production, spermatophore function was affected by humidity treatment. Only 33 (70%) of the spermatophores produced by males from the low‐humidity treatment evacuated their ejaculate compared with 51 (93%) of the spermatophores produced by males from the high‐humidity treatment . Moreover, the viability of sperm in the ejaculates of males from the low‐humidity treatment was lower  also produced a spermatophore compared with all 55 (100%) males that survived the high‐humidity treatment  with one of two experimentally controlled humidities, either low (RH30%) or high (RH80%). Males from the low‐humidity treatment lost around 30% of their body weight due to water loss, some three times more than males from the high‐humidity treatment. Our manipulation of humidity was, therefore, successful in affecting differences in hydration stress between treatments. The composition of male CHC profiles did not change in response to hydration stress. Males from the low‐humidity treatment were significantly less likely to produce courtship song, and those that did, produced song of lower quality, having a lower duty cycle (sound per unit time) and shorter trill elements. Moreover, although males from the low‐humidity environment were equally likely to produce a spermatophore, their spermatophores were more frequently dysfunctional, failing to ejaculate their contents, and the ejaculate contained a lower proportion of live sperm. The effects of hydration stress on pre‐ and postmating reproductive traits that we have observed are expected to impact directly a male's ability to reproduce.We asked how male reproductive traits, including both premating courtship traits and postmating ejaculate traits, might be affected by hydration stress. Male T. oceanicus show strong mating preferences for males able to deliver energetically costly courtship songs, rejecting the courtship attempts of males delivering songs with a low duty cycle; songs that contain few sound pulses particularly in the trill element of the song, and sound pulses that are both short and delivered slowly are unattractive to females ; data curation ; formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); resources (lead); writing – original draft (lead); writing – review and editing . Maxine Lovegrove: Data curation ; investigation (supporting); writing – review and editing . Melissa Thomas: Data curation (supporting); investigation (supporting); methodology (supporting); writing – review and editing . Xin (Bob) Du: Data curation (supporting); investigation (supporting); methodology (supporting); writing – review and editing . Yonglin Ren: Investigation (supporting); resources (supporting); writing – review and editing .The authors declare no competing interests.


ChatGPT has shown impressive performance in national medical licensing examinations, such as the United States Medical Licensing Examination (USMLE), even passing it with expert-level performance. However, there is a lack of research on its performance in low-income countries’ national licensing medical examinations. In Peru, where almost one out of three examinees fails the national licensing medical examination, ChatGPT has the potential to enhance medical education.We aimed to assess the accuracy of ChatGPT using GPT-3.5 and GPT-4 on the Peruvian National Licensing Medical Examination . Additionally, we sought to identify factors associated with incorrect answers provided by ChatGPT.We used the ENAM 2022 data set, which consisted of 180 multiple-choice questions, to evaluate the performance of ChatGPT. Various prompts were used, and accuracy was evaluated. The performance of ChatGPT was compared to that of a sample of 1025 examinees. Factors such as question type, Peruvian-specific knowledge, discrimination, difficulty, quality of questions, and subject were analyzed to determine their influence on incorrect answers. Questions that received incorrect answers underwent a three-step process involving different prompts to explore the potential impact of adding roles and context on ChatGPT’s accuracy.GPT-4 achieved an accuracy of 86% on the ENAM, followed by GPT-3.5 with 77%. The accuracy obtained by the 1025 examinees was 55%. There was a fair agreement (κ=0.38) between GPT-3.5 and GPT-4. Moderate-to-high-difficulty questions were associated with incorrect answers in the crude and adjusted model for GPT-3.5  and GPT-4 . After reinputting questions that received incorrect answers, GPT-3.5 went from 41 (100%) to 12 (29%) incorrect answers, and GPT-4 from 25 (100%) to 4 (16%).Our study found that ChatGPT (GPT-3.5 and GPT-4) can achieve expert-level performance on the ENAM, outperforming most of our examinees. We found fair agreement between both GPT-3.5 and GPT-4. Incorrect answers were associated with the difficulty of questions, which may resemble human performance. Furthermore, by reinputting questions that initially received incorrect answers with different prompts containing additional roles and context, ChatGPT achieved improved accuracy. ChatGPT (OpenAI), a large language model (LLM) trained with over 175 billion parameters, has gained growing attention owing to its performance in different tasks, including mathematics, economics, and medicine  to passiIn the Peruvian context, low-quality medical education is evidenced by high failure rates 42.8%) in the Peruvian NLME  % in the , leadersENAM is a professional requirement for Peruvian medical doctors and international physicians who aspire to practice medicine within Peruvian borders. Since its introduction in 2003 by the Peruvian Society of Medical Schools, this examination has served as a key evaluation of doctors’ readiness to practice medicine in the country . ENAM isThe passing score on the ENAM is 10.5 on a vigesimal scale (95/180). Over the years, the examination has gained even more significance owing to the regulatory measures that have made it a critical element in the selection process for Rural Service positions . AdditioBearing this in mind, we hypothesized that if ChatGPT can pass the ENAM, it may be used as a medical tutor to enhance medical students’ experience. Thus, in this study, we aimed to assess the accuracy of ChatGPT (GPT-3.5 and GPT-4) on the ENAM and identify factors associated with incorrect answers provided by ChatGPT.Our primary data source was the 2022 ENAM question set obtained directly from the official website of the Peruvian Society of Medical Schools (ASPEFAM) . The datThe 2022 data set was chosen for two main reasons: first, the ENAM blueprint ensures that each examination evaluates the same construct, thereby allowing a single year’s data to be representative; second, since ChatGPT’s training information only covers knowledge up to September 2021, the 2022 data set assures that the selected questions were not part of the model’s training data. Therefore, we assert that our data set selection strategy offers a degree of generalizability to the ENAM. The ENAM 2022 data set is available in We carefully collected the exam questions and divided them into four parts: (1) stem, the main problem or story ; (2) lead-in, the question asked ; (3) response options, the different answers provided for each question; and (4) the correct answer, as given by the exam creators .Two ChatGPT versions were used, namely, GPT-3.5 and GPT-4. Our approach involved the development of three distinct prompts to guide the artificial intelligence (AI) response. To create these prompts, two authors (JAF-C and JG-A) engaged in discussions to ensure they accurately represented the cognitive processes an examinee would typically use when answering a multiple-choice question. After reaching a consensus, we designed a three-step prompt that, to the best of our understanding, mimics this thought process effectively.The prompt was, “Analyze the following question, determine what is being assessed, and provide the correct answer/explanation.” With this prompt, we followed the same process as Kung et al , inputtiOpen-ended prompt: We removed response options, thus providing only the stem and lead-in with the prompt.Multiple-choice question with no justification: We provided the whole question with a stem, lead-in, and response options. In the prompt, we asked only to provide the correct answer with no further explanation.Multiple-choice question with justification: We provided the whole question with stem, lead-in, and response options. In the prompt, we asked for a lengthy explanation.Five of us  entered the questions into ChatGPT. Students received training on how to use ChatGPT through a prerecorded video, and their proficiency was assessed to ensure consistency in the application of prompts. A new chat session was initiated for each question to eliminate any potential memory retention bias. In situations where ChatGPT initially failed to deliver a clear response, we reattempted the question up to three times. The responses were then transferred to a structured Google Spreadsheet for further examination. The first (GPT-3.5) data extraction process was conducted between March 15 and 20, 2023, and the second (GPT-4) was conducted on May 5, 2023.On May 20, 2023, we conducted a second run, which incorporated three prompts following incorrect answers in GPT-3.5 and GPT-4. After providing the question and lead-in without instructions, if an incorrect answer was provided, we asked, “Are you sure? Pretend to be a junior doctor with expertise in clinical practice and exam solving and retry.” If an incorrect answer was provided, the following final prompt was provided: “Are you sure? Re-assess the question and pretend to be a Peruvian junior doctor with expertise in clinical practice and exam solving and retry.”Additionally, we obtained the results of 1025 examinees who took the ENAM as a progress test in a national preparation course. The examinees comprised final-year medical students and medical doctors preparing to undertake the ENAM in 2023. Using this data set, we analyzed questions using classical test theory to calculate the difficulty and discrimination index using the psychometrics package in RStudio . The difficulty index was calculated as a quantitative assessment of the proportion of examinees answering each question correctly, estimating the individual question’s difficulty level. The discrimination index refers to the question’s capacity to differentiate between high and low performers on the overall test . These tThe outcome was the performance of ChatGPT (GPT-3.5 and GPT-4) on the ENAM measured as correct or incorrect answers. We classified answers as correct if the answer provided by both versions matched the official answers provided by ASPEFAM.Independent variables were as follows: (1) type of objective, which was categorized as recall, whenever a question only required factual knowledge, or application, whenever a question required application of knowledge through clinical, therapeutic, communication, or professional decision-making; (2) Peruvian-specific knowledge ; (3) discrimination index; (4) difficulty index; (5) quality of questions; and (6) subject, which was categorized into basic sciences, internal medicine, surgery, obstetrics and gynecology, pediatrics, emergency medicine and critical care, and public health by two physicians with experience in assessing and preparing candidates for the ENAM. Both the discrimination and difficulty indices were calculated using classic test theory for the sample of 1025 examinees. For the discrimination index, we considered the question to provide good discrimination if the index was ≥0.25. For difficulty, questions were classified as hard (<0.30), moderate (0.30-0.70), or easy (>0.70). The quality of questions was measured by JAF-C and JG-A using a 5-point Likert scale with the question, “What is the quality of this question?”. Using this approach, we estimated the overall quality of the questions including the stem, lead-in, and response options using a tool based on the National Board of Medical Examinees’ item writing flaws .We downloaded the data as Microsoft Excel files and exported the data to RStudio for analysis.For descriptive analyses, we used absolute and relative frequencies for categorical variables and measures of central tendency and dispersion for numerical variables.To compare the agreement between GPT-3.5 and GPT-4, we used Cohen κ. To evaluate factors associated with incorrect answers from GPT-3.5 and GPT-4, we used a logistic regression model to calculate the odds ratio (OR) and 95% CI.We used the variance inflation factor (VIF) and Hosmer-Lemeshow test for goodness of fit to assess multicollinearity among predictors. All variables of interest were entered into the multivariable model, and this process was conducted for GPT-3.5 and GPT-4. The predictive accuracy of each version of ChatGPT was assessed using the receiver operating characteristic (ROC), from which we calculated the area under the curve (AUC). The data set and the RStudio script are available in This study adhered to the Helsinki Declaration. No humans were involved during the study. Therefore, evaluation by the ethics committee was not considered necessary.The performance of GPT-4 was 86% (155/180). GPT-3.5 scored 77% (139/180), 73% (133/180), and 60.5% (109/180) for multiple-choice questions with justification, multiple-choice questions with no justification, and open-ended prompts, respectively. The historical performance of the examinees was 54% (97.5/180), and the examinees’ performance in our data set was 55% (99/180). Additionally, we calculated that 7.7% (79/1025) and 22.53% (231/1025) of examinees scored better than GPT-3.5 and GPT-4, respectively, as shown in As shown in We used Cohen κ to assess the agreement between GPT-3.5 and GPT-4; the overall agreement was κ=0.38 . The agrWhen analyzing the odds for incorrect answers on GPT-3.5 and GPT-4, we found that high- and moderate-difficulty questions presented higher odds for incorrect answers in the adjusted model both for GPT-3.5  and GPT-4 , and low-quality questions were associated with correct answers in the GPT-3.5 adjusted model , as shown in Finally, we reinput prompts for incorrect answers following a three-step process, as shown in Here we showed that ChatGPT (GPT-3.5 and GPT-4) can pass the ENAM with expert-level performance. Furthermore, GPT-4 surpassed almost 90% of examinees in our data set with an accuracy of 86.1%, and GPT-3.5 surpassed 80% of examinees with an accuracy of 77.2%. These results are in concordance with the findings of Nori et al . HoweverWhen analyzing differences between the two versions, GPT-4 outperformed GPT-3.5 in almost all areas; however, we observed fair agreement between versions. The agreement was higher for high-difficulty questions, for which both versions failed all questions, and low-difficulty questions, for which both versions answered all questions correctly. These results suggest that the improvement in performance from GPT-3.5 to GPT-4 is due to enhanced reasoning rather than randomness .Although previous studies reported the likelihood of lower accuracy in GPT-3.5 for higher-order problem-solving , we founTo our knowledge, this is the first study to assess the agreement between GPT-3.5 and GPT-4 in the context of medical education and to examine factors linked to incorrect answers. We demonstrated that reformulating incorrect answers by varying prompts and changing roles and contexts improved the accuracy of ChatGPT.However, certain limitations of this study should be considered when interpreting our results. First, our study was confined to the Peruvian medical education system and involved a relatively limited number of questions. Therefore, the results may not be generalizable to other educational settings or a wider range of questions. We recommend future research with larger sample sizes, more diverse examinations, broader question sets, and different factors to identify reasons for wrong answers, such as the date of the questions.Second, while GPT-4 exhibited expert-level performance on the ENAM, this finding must be cautiously interpreted. The competencies required by a medical professional, as defined by frameworks such as CanMEDS or the Accreditation Council for Graduate Medical Education core competencies, extend beyond the confines of a licensing examination. These examinations assess knowledge and its application under controlled conditions, which may differ substantially from real-world clinical scenarios. Furthermore, more valid assessment tools, such as entrustable professional activities, represent the gold standard in medical education. Consequently, despite GPT-4’s promising performance, it is premature to suggest that it could replace human doctors. We encourage additional research to assess the potential use of ChatGPT in different roles or as a supportive tool for medical practitioners.Finally, our study did not evaluate the use of “mega-prompts”—large, intricate prompts detailing specific roles, contexts, and tasks, which might elicit more sophisticated and targeted responses—or other novel methods, such as chain-of-thought prompts  or threeThis study has several implications for both medical education and research on ChatGPT and AI. First, we demonstrated that ChatGPT can pass the ENAM with expert-level performance, surpassing 9 out of 10 examinees. Although our sample does not represent the real score in the ENAM, a previous study [Second, we found that incorrect answers provided by ChatGPT using GPT-3.5 and GPT-4 were associated with question difficulty, which opens further research directions to identify reasons for why ChatGPT fails some questions and inform new directions to understand the behavior of LLMs. Also, to our knowledge, this study is the first to apply psychometrics to ChatGPT, and further studies could explore different theories, such as cognitive diagnostic modeling or other diagnostic classification models with larger data sets, searching for a more in-depth understanding of the reasoning process of ChatGPT.Third, by reinputting incorrectly answered questions and adjusting prompts with more complexity , we found that ChatGPT may perform better. This requires further research on prompt engineering in medical education with tailored prompts for specific tasks, such as the development of assessment tools, curriculum development, communication with patients, or tutoring students. Additionally, tailored LLMs trained with specific and curated medical knowledge are needed for these different applications.Finally, despite the outstanding performance of ChatGPT in the ENAM, as previously stated by Thirunavukarasu , practicOur study found that ChatGPT (GPT-3.5 and GPT-4) can achieve expert-level performance on the ENAM, outperforming most of our examinees. We found fair agreement between both versions. There was an association between high-to-moderate-difficulty questions and wrong answers in both versions of ChatGPT. Furthermore, we observed enhanced performance by reinputting new prompts for incorrectly answered questions and adding roles and context for ChatGPT. Despite the outstanding performance of ChatGPT, we note that being a doctor goes beyond passing a licensing examination.