Antibody-mediated rejection (AMR) remains one of the major barriers for graft survival after kidney transplantation. Our previous study suggested a gut microbiota dysbiosis in kidney transplantation recipients with AMR. However, alternations in gut microbial function and structure at species level have not been identified. In the present study, we investigated the metagenomic and metabolic patterns of gut microbiota in AMR patients to provide a comprehensive and in-depth understanding of gut microbiota dysbiosis in AMR.We enrolled 60 kidney transplantation recipients, 28 showed AMR and 32 were non-AMR controls with stable post-transplant renal functions. Shotgun sequencing and untargeted LC/MS metabolomic profiling of fecal samples were performed in kidney transplantation recipients with AMR and controls.l-pipecolic acid, taurocholate, and 6k-PGF1alpha-d4 directly correlated with changes in gut microbial species and functions. Specific differential fecal species and metabolites were strongly associated with clinical indexes , and could distinguish the recipients with AMR from controls as potential biomarkers.Totally, we identified 311 down-regulated and 27 up-regulated gut microbial species associated with AMR after kidney transplantation, resulting in the altered expression levels of 437 genes enriched in 22 pathways, of which 13 were related to metabolism. Moreover, 32 differential fecal metabolites were found in recipients with AMR. Among them, alterations in 3b-hydroxy-5-cholenoic acid, Altogether, our findings provided a comprehensive and in-depth understanding of the correlation between AMR and gut microbiota, which is important for the etiological and diagnostic study of AMR after kidney transplantation.The online version contains supplementary material available at 10.1186/s12967-022-03825-6. Kidney transplantation is considered to be the preferred therapeutic option for patients with end-stage renal disease, as it can significantly prolong the lifespan and improve the quality of life in patients . HoweverClostridiales could be potentially used as biomarkers to distinguish the recipients with AMR from the controls  rectale and Bifidobacterium pseudocatenulatum could help to maintain intestinal barrier and suppress inflammation activation through inhibiting CD83 and TLR4/NF-κB, respectively [Lactobacillus counts were observed in patients with chronic kidney disease and recipients with AMR [Lactobacillus fermentum, Lactobacillus johnsonii and Lactobacillus acidophilus in recipients with AMR after kidney transplantation using shotgun metagenomic sequencing, all of which were demonstrated to have the effect of enhancing immune response, especially antibody response [The top five differential species based on relative abundance were l cancer . Generalng IL-10 , 29. Simectively , 31. Incwith AMR , 32. Herresponse –35. Possl-glutamine, alpha-ketoglutarate, N1-methyl-2-pyridone-5-carboxamide, etc.) up-regulated, and 21 metabolites  down-regulated in fecal samples from recipients with AMR. A serum metabolomics study of the acute graft rejection in human renal transplantation based on liquid chromatography-mass spectrometry have revealed comprehensive metabolic abnormalities in acute graft rejection [Changes of gut microbiota usually resulted in functional alteration. In the present work, we totally identified 437 differential KOs between recipients with AMR and the controls, which were enriched in 22 pathways. The differences in metabolic pathways  caused by alteration of gut microbiota in AMR were the most obvious, which was similar to the prediction from our previous study by PICRUSt analysis . Thus, wejection . Metabolejection .l-pipecolic acid, taurocholate, and 6k-PGF1alpha-d4 were directly correlated with altered gut microbial species and the related functional genes of enzymes. Both 3b-hydroxy-5-cholenoic acid and taurocholate were metabolites involved in bile acid metabolism [l-pipecolic acid, and 6k-PGF1alpha-d4 have been published, therefore further research is necessary to demonstrate their role in AMR. Based on the above results, we hypothesize that changes of gut microbiota structure and function could result in the alteration of the fecal metabolites, and in turn may impact the pathogenesis and progression of AMR. It is still noteworthy that causal conclusions cannot be drawn from our data, and further Mendelian randomization studies are needed to confirm this hypothesis. This will have important implications for understanding the precise role of gut microbiota in AMR.Among the differential fecal metabolites we identified, 3b-hydroxy-5-cholenoic acid, tabolism , 38. Contabolism . Moreovetabolism . Taken t+ catabolism. Previously, Rutkowski et al. have suggested that high serum concentrations of N1-methyl-2-pyridone-5-carboxamide in chronic renal failure resulted from kidney function injury, since the serum concentrations of N1-methyl-2-pyridone-5-carboxamide were approximately 20-fold higher in patients with advanced renal failure than in healthy controls, which could decline after dialysis or kidney transplantation [Besides the metabolites mentioned above, N1-methyl-2-pyridone-5-carboxamide and aminopterin should also be noted, since they exhibited high correlation with multiple clinical indicators of kidney function. N1-methyl-2-pyridone-5-carboxamide is an end product of NADantation . Strong antation . Accordiantation , 44. Howantation . InteresBanff criteria, a combination of serologic (circulating DSA), histologic (primarily microvascular inflammation and transplant glomerulopathy), and immunohistologic (C4d staining in peritubular capillaries) criteria, is the gold standard for the diagnosis of AMR after kidney transplantation . HistoloThere are some limitations in the present study that must be recognized. Firstly, our findings warrant further confirmation with an external cohort. Secondly, the data extracted from non-transplant fecal samples was absent. Comparing the findings of this study to the data extracted from non-transplant fecal samples will provide a metagenomic and metabolic background for the allograft recipients, and further studies will be required to address this important issue. Thirdly, we didn’t take account of the compositional nature of microbiome datasets in the selection of the analysis methods. The counts of sequencing reads assigned to organisms were normalized to a constant area. Thus, our results could reflect only changes in the relative abundance of the microbiota but not the absolute abundance.In conclusion, our current study highlighted the gut microbiota dysbiosis at the species level in AMR after kidney transplantation, which was associated with an alteration of the gut microbiota functions and a change in fecal metabolites. Moreover, specific species and metabolites were well associated with kidney function indicators, and could be used as biomarkers to distinguish kidney recipients with AMR from the ones with stable kidney functions. Altogether, these findings provided a comprehensive and in-depth understanding of the correlation between AMR and gut microbiota, which is important for the etiological and diagnostic study of AMR after kidney transplantation.Additional file 1: Table S1. Histopathological characteristics according to the Banff 2019 criteria of AMR cases. Table S2. Comparison of relative abundance of gut microbiota between AMR and control groups at the species level. Table S3. Comparison of relative abundance of gut microbiota between AMR and control groups at the phylum level. Table S4. Comparison of relative abundance of gut microbiota between AMR and control groups at the genus level. Table S5. Comparison of relative abundance of KOs between AMR and control groups. Table S6. Interrelationship between AMR-associated gut microbial species, functions and metabolites. Table S7. Spearman correlation between species and clinical indicators. Table S8. Spearman correlation between metabolites and clinical indicators. Table S9. Area under the ROC curves of microbial biomarkers. Table S10. Antibiotic resistance and virulence genes of key species associated with AMR after kidney transplantation. Table S11. Area under the ROC curves of metabolic biomarkers


LE) and charge-transfer (CT) excitations within a restricted orbital space including two occupied and two unoccupied orbitals for each molecular monomer. We study energy profiles and disentangle inter-state couplings to disclose the (CT) character of singlet and triplet exciton states and assess the influence of inter-molecular orientation by displacing one molecule with respect to the other along the longitudinal translation coordinate. The analysis shows that (CT) contributions are relevant, although comparably less effective for triplet excitons, and induce a non-negligible mixed character to the low-lying exciton states for eclipsed monomers and for small translational displacements. Such (CT) contributions govern the a/LbL state inversion occurring for the low-lying singlet exciton states of naphthalene dimer and contribute to the switch from H- to J-aggregate type of the strongly allowed bB transition of both oligoacene aggregates.Intermolecular interactions modulate the electro-optical properties of molecular materials and the nature of low-lying exciton states. Molecular materials composed by oligoacenes are extensively investigated for their semiconducting and optoelectronic properties. Here, we analyze the exciton states derived from time-dependent density functional theory (TDDFT) calculations for two oligoacene model aggregates: naphthalene and anthracene dimers. To unravel the role of inter-molecular interactions, a set of diabatic states is selected, chosen to coincide with local ( Electronic and electro-optical materials based on organic molecules have been the subject of numerous investigations ranging from basic materials science to possible technological applications ,2,3. OliAlthough the assessment of the nature of singlet exciton states in crystals and aggregates of organic chromophores has been the subject of several studies ,32,33,34LEs) and charge transfer (CT)  excitations. Such intra- and inter-molecular excitations can be considered as a suitable basis of diabatic states describing electron promotions between occupied molecular orbitals to unoccupied molecular orbitals of the same  or neighboring (ionic) molecules (or sites), respectively [The exciton states of molecular materials are superpositions of local (intra-molecular) excitations  calculations, a diabatization procedure can be used to determine the superposition of on state ,36,55,57on state ,38.LE and CT contributions. The approach is however extended to include a larger orbital space, required to correctly describe the orbital nature of the low-lying oligoacene excited states. Oligoacene dimers in their triplet and singlet exciton states are perfect model systems for the investigation and understanding of triplet-triplet and singlet-singlet interactions between π-conjugated molecules, and how these interactions are modulated by different intermolecular organizations. Specifically, here we focus on the longitudinal translation coordinate  of naphthalene and anthracene are collected in notation . Tables  the TDA ,77. Notably, in between the four ordinate . The numThe protocol employed to analyze the intermolecular interactions leading ultimately to the character of exciton states, transforms the amplitudes of TDDFT calculations carried out on the aggregate, from the basis of single excitations between aggregate’s orbitals  basis) to the basis of single excitations between molecular  [De/h(n) , but als Ve . In addiFor aggregates characterized by a symmetric arrangement of chromophores, as is the case for those investigated here, when the molecules approach each other, intermolecular interactions mix the ) states ,79,80 ofThe SA diabatic states are then obtained as linear combinations of In the following sections we analyze the TDA computed singlet and triplet exciton states of oligoacene dimers to determine their character  the strongest absorption peak has moved back almost toward the isolated molecule value, although not completely owing to some still non-negligible exciton interactions. Indeed the distance , in contThe ground state monomer structure of naphthalene and anthracene was optimized at the ωB97X-D/6-31G* level of theory. The distance between the planes of different monomers was set to 3.4 Å, as used in previous investigations on dimers of other polycyclic aromatic hydrocarbons ,30. ExciI dimers ,38, and I dimers . To analyze the exciton character, we followed the approach described in previous works ,38 and wained as ,30,85: whose oIntermolecular interactions determine the nature of exciton states which ultimately govern the optoelectronic properties and the outcome of photoinduced processes in molecular materials. In this work we have analyzed the character and modulation of singlet and triplet exciton states of two oligoacene homo-dimers formed by naphthalene and anthracene, along the intermolecular longitudinal translation coordinate.The character of exciton states computed with TDDFT and the relevant inter-molecular and inter-state interactions were determined with a diabatization procedure successfully employed in previous investigations of PDI and here extended to include a larger orbital space, mandatory to correctly describe the low-lying excited states of oligoacenes. The analysis in terms of localized excitations shows that Concerning the singlet spin manifold, the study shows that such We believe that this study provides useful insights on the magnitude of inter-molecular interactions occurring in molecular materials and determining the nature of exciton states of both singlet and triplet spin multiplicity, paving the way to future investigations on more complex aggregates and inter-molecular organizations.


The concept of personalized medicine refers to the tailoring of medical treatment to each patient’s unique characteristics. Scientific advancements have led to a better understanding of how a person’s unique molecular and genetic profile makes them susceptible to certain diseases. It provides individualized medical treatments that will be safe and effective for each patient. Molecular imaging modalities play an essential role in this aspect. They are used widely in screening, detection and diagnosis, treatment, assessing disease heterogeneity and progression planning, molecular characteristics, and long-term follow-up. In contrast to conventional imaging approaches, molecular imaging techniques approach images as the knowledge that can be processed, allowing for the collection of relevant knowledge in addition to the evaluation of enormous patient groups. This review presents the fundamental role of molecular imaging modalities in personalized medicine. Hippocrates of Kos (c. 460—ca. 370 BC) stated that it is more important to understand the type of person with a disease than to identify the type of disease the patient has. This statement by the father of modern medicine is considered the platform of personalized medicine (PM). The term “personalized medicine” refers to a relatively new field of medicine that aims to enhance diagnostic precision and reduce therapeutic failures. There is wide use of molecular imaging modalities in screening, diagnosis, treatment, assessment of disease heterogeneity, progression planning, molecular characteristics, and long-term follow-up for various diseases. As opposed to conventional imaging techniques, molecular imaging approaches images as data that can be mined and used to extract additional information as well as assess large populations of patients ,2.Molecular imaging has become widely used in many diseases, with a particular focus on cancer care. It refers to the in vivo characterization and measurement of key biomolecules and molecular events underlying malignant conditions. As applied to oncology, this article discusses both established and emerging methods of molecular imaging. Current molecular imaging techniques offer advantages for improving clinical cancer care as well as drug development ,2,3.Cancer is one of the challenging issues in public health care that affects millions of people around the world and leads to high mortality rates worldwide. According to the World Health Organization, since cancer is positively correlated with patients’ ages, even if it affects younger people, cancer incidence prevalence and mortality are expected to significantly raise due to population growth and aging. This increases the importance of promoting and advancing health care systems and strategies and developing conventional therapy methods, including chemotherapy, radiation therapy, and surgery, to eliminate cancer cells, increase the survival rates of patients, and ensure sustainability, providing satisfactory global healthcare based on prevention, accurate diagnosis, and effective treatment that has less multi-drug resistance, high selectivity, and less cytotoxicity ,5,6.Normal healthy cells do not evolve to be cancerous but are established from an aggregation of DNA damage to cells. Cancer cells have a complex pathophysiology and determining the main cause for each case is difficult to achieve; therefore, there are three main categories that are used to determine cancer causes. First, genetic inheritance and epigenetic factors; second, physical agents, such as exposure to genotoxic chemicals, UV light, and ionizing radiation; third, physiology, namely metabolic changes and telomerase enzyme activity changes .Annually, there are 24 million new patients who are diagnosed with cancer worldwide; usually, there are multiple causes, manifesting differently over time, varying from one patient to another, which makes treating cancer very challenging. Conventional cancer therapy is non-targeting and damages healthy cells due to the accumulation of therapy on them, leading to the reduced efficiency of treatment. Therefore, it is desirable to develop targeted drug delivery systems that can deliver the therapeutic agent to the target tumor to reduce the adverse effects and improve efficacy ,6.Significant rapid advances in molecular biology, cancer biomarkers, and radio-genomics help to have a better understanding of cancer, resulting in developing personalized medicine and molecular imaging since both are strongly dependent on the collaboration of different clinical disciplines. Personalized medicine is a comparatively new emerging practice of medicine that focuses on providing the tumor genetic profile to proffer individual prevention, diagnosis, and treatment, which reflects on cancer treatment by improving the anti-cancer therapeutic efficiency and reducing the adverse effects. Molecular imaging is used widely in screening, detection and diagnosis, treatment, assessing disease heterogeneity and progression planning, molecular characteristics, and long-term follow-up. Moreover, it is able to detect very tiny tumors and assess their activity numerically, which makes molecular imaging one of the most scientific reasons that contributes greatly to expanding and developing the personalized medicine, research, clinical trials, and medical practice of cancer fields, evolving a new generation of platforms with greater accuracy and sensitivity for in vivo quantification and characterization of various biological processes ,10,11,12This paper will review a wide range of published research on personalized medicine and molecular imaging to define the role of molecular imaging  in personalized medicine. In addition, the article discusses the importance of molecular imaging to the emerging field of theranostics and how molecular imaging may one day be integrated with other diagnostic techniques to improve cancer treatment efficiency and effectiveness ,2,3.Nowadays, personalized medicine involves non-invasive imaging methods to detect pathologies and treat patients. These imaging methods can be divided into two groups: morphological/anatomical imaging and molecular imaging based on nanobodies. Multidisciplinary collaboration across several domains, including radiology, nuclear medicine, pharmacology, chemistry, molecular and cell biology, physics, mathematics, and engineering, has led to the creation and translation of molecular imaging ,15,16,17Ultrasound is a high-resolution structural imaging technique that is one of the most widely used diagnostic clinical imaging modalities. Ultrasound is an imaging technique and biological system approach that can be used as two highly efficient methods for thermal cancer therapy therapies (thermoablation and high-intensity frequency ultrasound treatment (HIFU) that produce hyperthermia or hypothermia). It can also be used for diagnosis in clinical trials due to its beneficial properties such as its safety due to no ionizing irradiation, wide availability, portability, real-time imaging/acquisition time (min), high spatial resolution, external or internal application (endoscopy), inexpensiveness, high sensitivity, and ability to be combined with contrast agents to separate contrast and morphological imaging (with use of harmonics). These characteristics improve interest in wide ultrasound application and its role in personalized medicine ,19,20,21An ultrasound method called speckle tracking echocardiography (STE) is employed to evaluate myocardial function. This technique examines how distinctive speckle patterns, natural myocardial acoustic markers, move during the cardiac cycle. Myocardial velocities and intrinsic cardiac deformation can be calculated offline (strain and strain rate). This strategy should be used in patient follow-up even though it necessitates specialized software and depends on better image quality .Another method called ultrasound elastography (USE) is a new tool for measuring tissue stiffness and has demonstrated the importance of tissue elasticity for the diagnosis of tumors. It measures the spatial variation of the mechanical response and monitors local changes in tissue pressure during anti-stromal therapy and hyaluronic acid depletion ,24. In tThis imaging technique has played a role in therapeutics and theragnostic by determining the presence and degree of molecular targets for a certain disease and confirming the effective administration of these drugs; it is used for the targeted delivery of drugs, including genetic material, which is proliferating. It is also used for focal disruption of the blood–brain barrier to enable access to the brain for hydrophilic diagnostic and therapeutic agents, in blood pool enhancement, perfusion imaging, characterization of lesions, phase and metabolism, echocardiography, monitoring and quantifying arthritis, identifying the phase of this heterogeneous disease, and detecting sites of atherosclerosis pathogenesis before lesions occur by accumulating the microbubbles in ECAM-rich sites ,45,46,47Otherwise, with all the incredible use of ultrasound contrast agents but still limited to the vasculature, they produce a high background signal and “tether” to a surface, which limits their ability to oscillate, and thus their echogenicity is slightly hampered. The FDA reported microbubble-based injectable suspensions  and Optison (Perflutren Protein-Type A Microspheres for Injection) in 2007 due to the risk of serious cardiopulmonary reactions during or within the first 30 min after administration. However, these warnings were edited in 2008, but these microbubble contraindications limited their clinical use. Moreover, the US has poor penetration specificity and limited sensitivity, meaning it detects tumors without being able to distinguish between malignant and non-malignant tumors. It also has poor image quality since the blood is a poor scatterer at clinical diagnostic transmission frequencies. Currently, it is not possible to use US to scan the full human body, and it is considered operator dependent. All the listed limitations can weaken US applications in personalized medicine ,48,49,50Contrarily, a lot of work is being put towards expanding US’s function in molecular imaging and personalized medicine. Some potential uses are the incorporation of sound pulses into MRI pulse sequences to broaden US’s use of the molecular field, drug delivery by increasing the rate of lipid exchange and the tendency for fusion or improved contact between the nanoparticles and the targeted cell membrane while using the safe level of US waves, drug-loaded albumin-based carriers, and controlling drug release. Additionally, researchers have improved contrast-enhanced ultrasounds to detect image molecular markers and to boost ultrasound image quality, making them appropriate for ultrasound therapy ,53,54,55Molecular imaging is a turning point in the era of personalized imaging. The term was introduced in the last decade, referring to a variety of scientific disciplines, from biomedicine to clinical medicine, and it explains numerous clinical issues. The development of multimodality imaging, such as multiparametric magnetic resonance imaging (MRI) and PET/MRI, which are used more frequently for cancer diagnosis, staging, and surveillance, is one of the main components of molecular imaging. This type of imaging is becoming more important since it supports how physicians formulate diagnoses and arrange treatments in cases where ineffective surgical operations and harmful treatments might be avoided ,48,56,57Magnetic resonance imaging (MRI) is based on the interaction of specific nuclei, usually protons, with molecules neighboring each other in intercellular tissue. The relaxation times of various tissues vary, which causes an endogenous contrast. External contrast agents can further improve this by specifically reducing the length of either the longitudinal T1 relaxation or the transverse T2 relaxation ,18,52.All types of testing for analyzing tumor behavior have been demonstrated to be affected by molecular imaging techniques. Signaling mechanisms that support cancer cell reprogramming are essential because the clonal heterogeneity of the tumor determines the sort of treatment required for each individual cell, thus “personalizing” the course of treatment. Fluorodeoxyglucose, the glucose analogue on a combined PET-MRI modality, is a significant molecular marker used in the in vivo visualization, characterization, and quantification of biological processes in a tumor at the molecular and cellular level. In cancer, when information on the morphology and function of a diseased lesion is co-registered, the innovation involved in merging these modalities enhances its accuracy ,47,58,59Functional molecular imaging offers a distinctive perspective on various conditions. We are able to examine both the underlying biochemistry and the geographical and temporal changes in biomarkers using techniques such as magnetic resonance imaging. When imaging and molecular diagnostics are used together, it becomes possible to measure abnormal cellular signalling pathways in unprecedented depth. This succinct essay demonstrates how radiotracers and nuclear imaging techniques are being developed to track drug effectiveness and, at the same time, promote the objective of individualized healthcare ,59,60,61SPECT is one of the molecular imaging techniques that has really helped to visualize, characterize, and measure abnormal biologic processes of cancer at the macro and micro levels by emitting gamma rays at various energies to detect the radiopharmaceuticals  that proBy boosting sensitivity with molecular targeting, anatomic specificity, and resolution, hybrid technologies such as SPECT/CT, PET/SPECT, and SPECT/MRI enable further advancement in molecular imaging. PET/SPECT is an infusion technology that uses ligands with short half-lives to provide long-term cell vision for up to 14 days. It has the potential to identify molecular targets that are significant in the progression of the disease and theragnostic strategies. Nanomolecular diagnosis of the severity and distribution of the disorders is provided by SPECT/MRI. SPECT/MRI has received less focus in pre-clinical research than PET/MRI ,67,69,71The benefits of SPECT/CT include a quick acquisition time of 1–2 min, low radiation exposure to the patient, and a good signal-to-noise ratio that allows for the use of the scout image for reasons other than attenuation correction. These factors provide the necessary pretherapy information on biodistribution, dosimetry, the limiting of critical organs or tissues, and the maximum tolerated dose, making the tailored therapy and imaging of personalized medicine safe and appropriate. The main strength of SPECT/CT is the features for individual modalities that increase the molecular and anatomical integration systems. SPECT/CT have a significant impact on clinical management by better guiding subsequent operations, avoiding unnecessary procedures, and providing predictive information that aids in guiding change in both intra- and inter-modality therapy ,64,67,70PET is the gold standard in clinical molecular imaging because it possesses the high sensitivity required for deep tissue penetration and visualization of most interactions between physiological targets and ligands. Due to this, non-invasive detection up to the picomolar level is achievable. By producing quantitative images and 3D morphological images at quick scan times, which enables dynamic imaging (time-resolved images to be generated), it has become the fastest-growing clinical imaging technology and is now a current tool in cancer diagnoses and cancer treatment planning. The basis of the PET technique is the phenomenon of positron–electron annihilation, resulting in the formation of two high-energy photons (511 keV) emitted in opposite directions (180°). PET using biomarkers are labelled with positron (a positively charged electron)-emitting radioisotopes, primarily nitrogen, oxygen, carbon, and fluorine, which are short-lived elements 2–110 min) used to image the molecular interaction of biological processes such as cell proliferation, glucose metabolism, amino acid uptake, and membrane biosynthesis. They also deliver information about biomarker expression and tissue biochemical characteristics, provide the exact location of a lesion frequently before symptoms arise, determine molecular phenotypes, provide valuable molecular, functional, and metabolic information, and aid in determining the tumor biology of neoplasms by creating quantitative imaging that is capable of transforming collected gamma rays into quantitative terms. These quantitative images support safer surgical resections that minimize morbidity and mortality as well as increase the cost-effectiveness of healthcare with a measurable return on investment. They also aid in the diagnosis, optimization, and personalization of treatment for a variety of diseases. Moreover, the use of several tracers in PET technology is one of the technique’s distinctive advantages . Over th–110 min Due to the special characteristics of PET and the quick development and growth of hybrid PET in recent decades, the scope of PET clinical applications has increased. By advancing the clinical use of PM, PET clinical applications will continue to support the role of molecular imaging in the era of personalized medicine. PET has been used in oncology using antimetabolic image information for diagnosis and identifying undetected distant metastases, stages, and volume in cancers and the presence of inflammatory infiltrate. By providing personalized medicine, such as personalized chemotherapy, immunotherapy, targeted therapy, and dosage, as well as personalized evaluation of response early in treatment due to changes in glucose metabolism and evaluation of the antiangiogenic therapeutic result, PET scanning can improve cancer management. PET scans have many advantages in toxicology studies because they are an important tool in personalized drug discovery and development, screening, identifying new drug candidates, and evaluating individual patient susceptibility to treatment by nanocarrier systems. PET imaging is ideal for radiopharmaceutical micro-dosing research and drug therapy development. Additionally, it contributes to minimizing expenditure on medication development and animal use in preclinical toxicological research. In order to determine whether the drug concentration delivered to the target is sufficient to elicit a pharmacologic response, the PET imaging protocol can be used to measure both AR levels (in the sense of a predictive biomarker for estimating response to therapy and monitoring drug-target engagement) and AR activity using a PD biomarker ,90,91,92Hybrid imaging adds value to imaging data and provides efficient diagnosis, radiogenomics, and therapy planning. PET combined with various modalities such as US, MRI, optical imaging systems, immune probes, and CT, are most commonly used in clinical settings today. PET-US uses radiolabelled microbubble shells such as 18F-labeled, albumin-shelled, and VEGFR2-targeted, which have a short half-life and are several micrometres in size. This modality can be used for investigating the biodistribution of microbubbles after i.v. injection and offers better quantification, which is particularly true in biodistribution analyses and can be used for targeted drug delivery, such as delivering VEGFR2 in breast cancer ,54,80.PET-MR units are currently in development and being used in pre-clinical environments. This dual modality allows high spatial resolution, temporal resolution and accuracy, superior soft tissue contrast and multi-planar capabilities, and less ionizing radiation exposure. These features allow it to perform translational research from a cell culture setting to pre-clinical animal models to clinical applications, which is advantageous for the drug discovery and evaluation process that could help optimize the development of new drugs non-invasively and develop radiotracers. Additionally, it has been used to measure processes as diverse as blood flow and volume, tissue oxygenation, tissue pH, protein synthesis, cellular proliferation, enzyme kinetics, endogenous metabolite concentration, water diffusion, tissue anisotropy, vascular permeability, and better treatment response, providing information on downstream effects from multiple pathways, even though it is more limited with respect to the number of molecular processes that can be imaged, and provides additional opportunities for facilitating targeted biopsy and the determination of its efficacy. Hybrid PET/MR systems provide complementary multi-modal information about perfusion, metabolism, receptor status, and function, together with excellent high-contrast soft tissue visualization without the need to expose the patient to additional radiation, which makes them very useful for precision medicine cancer care in cardiac sarcoidosis, degenerative diseases such as Alzheimer’s disease, and cancers such as pharyngeal and ovarian cancer ,80,93,94PET and optical imaging have been combined and demonstrated in vitro, ex vivo, or in vivo in recent years. The principal benefits are related to the combination of increased tissue penetration of radiation from positron emitter radionuclides that enables non-invasive quantitative imaging and tumor detection and light generated by the fluorescent probe for optical imaging during surgery, in particular, robotic surgery. This allows for effective, targeted drug delivery in vivo without causing systemic toxicity, and both the administered dose and therapeutic efficacy can be precisely monitored non-invasively over time. In order to image and evaluate the concentration and function of the target without having an impact on it, the probe is utilized in extremely low mass amounts during PET imaging (tissue concentrations of around femtolitres per gram of tissue). Similarly to PET/MRI, this dual imaging is used in the drug development process to identify, accurately measure, and assess medications’ performance in vivo in mice models and human patients. This will make it possible to discover drugs more successfully using a systems-based approach that is driven by molecular imaging and diagnostic techniques ,59,94.Despite not being fully realized, the combination of radiation therapy and immunotherapy has the potential to change the field of oncology. Immuno-PET imaging could play a critical role in providing the crucial information required to help understand this sophisticated connection. Nowadays, immuno-PET is a safe multimodality treatment strategy that helps to move toward precision medicine using radio-labelled antibodies and targets that combine with the high sensitivity and quantitative potential of PET non-invasively to provide quantitative, high quality, high spatial, and temporal resolution images that help to estimate the antigenic expression level of immuno-PET such as immune checkpoints and effector molecules, or the detection and tracking of immune cell populations such as T-cell subsets and chimeric antigen receptor T-cells, in identifying diseases and stages, responses to therapy, and whole-body bio-distribution in real-time, which leads to improvement in cancer patient management. In contrast, the long half-life of intact antibodies hampers their use as imaging agents due to the several days required for blood and background clearance in order to achieve a good signal-to-noise ratio. These emerging methods in PET may improve patient selection and target delineation and, ultimately, may become a useful tool for adaptive radiation planning as we collectively strive toward personalized medicine in radiation oncology ,92.The most widely available and widest molecular imaging modality used in oncology is PET-CT due to its non-invasive nature and high accuracy in its application and management in oncology. PET-CT is a quantitative technique that provides information about morphologically relevant, physiologic, and pathologic processes at the molecular level, as well as biodistribution, dosimetry, the limiting or critical organ or tissue, and the maximum tolerated dose (MTD). It could detect and quantify abnormal molecular activity throughout the body and have high accuracy in differentiating malignant tumors from benign ones. It can also be used to evaluate the response rates of chemotherapy to allow easy management and early detection of tumor recurrences. This is useful in order to identify non-responders as soon as possible and to modify treatment. Furthermore, radiation planning with a PET-CT scan can be more beneficial by modifying the radiation dose for patients with situs dose deposition in the tumor. It has the ability to determine the more active and metabolic areas within the tumor to direct more aggressive radiation to reduce the chance of converting to more aggression, which fulfils the potential of personalized medicine. Moreover, there are also dynamic PET/CT scans, which are a new technology of PET/CT scan that allows new opportunities for personalized nuclear medicine by providing better image quality in a short scan time that can be used to optimize administered radioactivity and for pediatric patients and sick patients who cannot remain still for long periods ,59,75,95PET-CT scans have high sensitivity and specificity, allowing them to use radiopharmaceutical tracers such as F-18 fluorocholine, Ga-68, and C-11 methionine to measure cellular characterization and biological processes in a tumor at the molecular and cellular level. The ability to quantify the disease at a molecular level, tumor hypoxia, and bone metastases may help assess the global inhibitory effect of such multi-targeted therapeutic approaches. Notwithstanding, there is a lack of personalized radiotracers in PET-CT radiotracers, which presents a major limitation to the molecular imaging role in personalized medicine ,95,96,97In conclusion, personalized medicine aims to enhance diagnostic precision and reduce therapeutic failures. Molecular imaging, which has emerged as a dynamic and exciting field of study, plays a great role in personalized medicine


The human placenta represents a unique non-neuronal site of monoamine transporter expression, with pathophysiological relevance during the prenatal period. Monoamines  are crucial neuromodulators for proper placenta functions and fetal development, including cell proliferation, differentiation, and neuronal migration. Accumulating evidence suggests that even a transient disruption of monoamine balance during gestation may lead to permanent changes in the fetal brain structures and functions, projecting into adulthood. Nonetheless, little is known about the transfer of dopamine and norepinephrine across the placental syncytiotrophoblast. Employing the method of isolated membranes from the human term placenta, here we delineate the transport mechanisms involved in dopamine and norepinephrine passage across the apical microvillous (MVM) and basal membranes. We show that the placental uptake of dopamine and norepinephrine across the mother-facing MVM is mediated via the high-affinity and low-capacity serotonin (SERT/SLC6A4) and norepinephrine (NET/SLC6A2) transporters. In the fetus-facing basal membrane, however, the placental uptake of both monoamines is controlled by the organic cation transporter 3 (OCT3/SLC22A3). Our findings thus provide insights into physiological aspects of dopamine and norepinephrine transport across both the maternal and fetal sides of the placenta. As monoamine transporters represent targets for several neuroactive drugs such as antidepressants, our findings are pharmacologically relevant to ensure the safety of drug use during pregnancy. In the early fetal brain, monoamine receptors develop before the inception of synaptogenesis, highlighting their relevance for behavioral development2. In addition, they participate in cell proliferation and differentiation processes, neurite outgrowth, modulation of the cell cycle, and neuronal migration7. Moreover, norepinephrine is a pivotal monoamine for the fetal cardiovascular system, lung function, and glucose mobilization9 and plays a detrimental role in postnatal adaptation8. On the other hand, dopamine affects placental endocrine functions by regulating the production of human placental lactogen and chorionic gonadotropin11.The human placenta represents a unique non-neuronal site for the metabolism and transport of dopamine, norepinephrine, and serotonin. As messengers on the placenta-brain axis1. Altered dopamine homeostasis is linked to higher risks of neurological disorders, such as schizophrenia, autism spectrum disorder, bipolar affective, or attention deficit hyperactivity disorder after birth13. In addition, monoamines are vasoactive agents, and inappropriate levels have been related to changes in uterine blood flow, resulting in hypoxia and preterm delivery15. Thus, the placental transport of monoamines has crucial pathophysiological importance during the prenatal period.Accumulating evidence suggests that even a transient disruption of monoamine balance during gestation may lead to permanent changes in the fetal brain structures and functions, projecting into adulthood+/Cl− gradient and an outward K+/H+ gradient18. However, their substrate specificity is not unambiguous, and a significant overlap exists in substrate affinity20. In addition, a low-affinity but high-capacity organic cation transporter 3 (OCT3/SLC22A3), also known as the extraneuronal monoamine transporter, mediates monoamine uptake in the brain22. To some extent, monoamines can also pass biological membranes by passive diffusion24.Specific transport proteins of the solute carrier family (SLC) serve as the primary mechanism for the active uptake of monoamines into cells. The three neuronal monoamine transporters—serotonin transporter (SERT/SLC6A4), norepinephrine transporter (NET/SLC6A2), and dopamine transporter (DAT/SLC6A3)—are high-affinity, low-capacity membrane transporters, driven by an inward Na25. Current knowledge of monoamine transport in the placenta is restricted to the MVM, where functional expression of SERT and NET was reported27. Several studies have failed to detect DAT in placental cells30, speculating that dopamine must use different transport system(s) to enter the placenta from the maternal circulation. Surprisingly, despite increasing fetal monoamine production towards the term15, limited evidence exists on the transport of monoamines across the BM. Our team has recently characterized a serotonin clearance mechanism in the human and rat placenta, mediated by SERT in the MVM and OCT3 in the BM; this synchronized transporter activity in conjunction with the intracellular metabolism by monoamine oxidase A (MAO-A) regulates serotonin levels in the fetoplacental unit31. Nonetheless, little is known about the placental handling of dopamine and norepinephrine. The only other report of this kind dates back to 1984; it indicates a non-specific extraction of norepinephrine from the fetal circulation, followed by enzymatic degradation via MAO-A and catechol-o-methyltransferase (COMT)32. As OCT3 in neuronal tissues exhibits affinity for dopamine and norepinephrine33, we hypothesized that OCT3 might also be responsible for placental uptake of these monoamines from the fetal circulation.The functional unit of the human placenta is the syncytiotrophoblast—an epithelial layer polarized into the apical, maternal-facing microvillous membrane (MVM) and basal, fetal-facing membrane (BM)Thus, in the present study, we aimed to characterize dopamine and norepinephrine transport in the human placental syncytiotrophoblast. Experiments were conducted in purified MVM and BM vesicles simultaneously isolated from the human term placenta, providing an ideal system to investigate transport characteristics of both membranes in parallel. Collectively, our findings provide insights into physiological aspects of dopamine and norepinephrine transport across the placenta. As monoamine transporters represent targets for several neuroactive drugs, typically antidepressants, our findings are pharmacologically relevant to ensure the safety of drug use during pregnancy.3H-norepinephrine, 20 Ci/mmol, 3H-serotonin, 80 Ci/mmol, and 3H-dopamine, 60 Ci/mmol were purchased from American Radiolabeled Chemicals, Inc. . Dopamine hydrochloride, norepinephrine hydrochloride, serotonin hydrochloride, sodium L-ascorbate, paroxetine hydrochloride, venlafaxine hydrochloride, nisoxetine hydrochloride, GBR 12935 dihydrochloride, decynium-22 and hydrocortisone were purchased from Sigma-Aldrich . Tri Reagent solution was obtained from the Molecular Research Centre . Bicinchoninic acid assay (BCA assay) reagents were purchased from Thermo Fisher Scientific . All other chemicals were of analytical grade.Term placentas from uncomplicated pregnancies  were collected at the University Hospital in Hradec Kralove, Czech Republic. All the experiments conducted in the study conformed to the ethical standards established by the Declaration of Helsinki, and all the women provided signed informed consent. The study protocol was approved by the University Hospital Research Ethics Committee (201006 S15P).According to the manufacturer's instructions, RNA was isolated from weighed placenta tissue samples using the Tri Reagent solution . Total RNA concentration and purity were calculated using NanoDrop™ 1000 Spectrophotometer . 1 µg of total RNA was reversely transcribed to cDNA in a total volume of 20 µl using the iScript Advanced cDNA Synthesis Kit  and Bio-Rad T100™ Thermal Cycler , according to the manufacturer's protocol.31. Briefly, the duplex reaction mixture consisted of 10 µL ddPCR™ Supermix for probes , 1 µL of the predesigned probe assay , and 1 µL of cDNA (25 ng/µL), in a total volume 20 µL. Expression levels of the following target genes were quantified: NET/SLC6A2 (Hs00426573_m1), DAT/SLC6A3 (Hs00997374_m1), SERT/SLC6A4 (Hs00984349_m1), and OCT3/SLC22A3 (Hs01009571_m1)—all TaqMan assays were obtained from Thermo Fisher Scientific . On the other hand, β-2 microglobulin (B2M) was used as the reference gene, obtained from Bio-Rad . Droplets were created using QX200 Droplet Generator and amplified to the endpoint using the T100™ Thermal Cycler, following the thermal conditions recommended by the manufacturer. Droplets were counted in QX200™ Droplet Reader, and the concentration of the target gene was calculated using the QuantaSoft™ Software. Only samples with more than 13 000 generated droplets were used for final data analysis. Expression levels are reported in the number of transcripts/ng of transcribed RNA. The whole system for ddPCR reaction and analysis was obtained from Bio-Rad .The quantification of monoamine transporter expression in human term placentas was investigated using duplex droplet digital PCR (ddPCR) analysis, as described previously31. Briefly, maternal decidua and chorionic plate were removed. Next, placental villous tissue (80–100 g) was chopped into small pieces and washed with 0.9% NaCl to remove blood. The purification method involved different steps: homogenization, differential centrifugation, precipitation of non-microvillous membranes with Mg2+, and a sucrose gradient step. Microvillous membranes (MVM) and basal membranes (BM) were resuspended in intravesicular buffer  and vesiculated by 15 passages through a 25-gauge needle. Protein concentration in the placental homogenate, MVM, and BM vesicles, was analyzed using the BCA assay.Syncytiotrophoblast plasma membranes were simultaneously prepared from fresh healthy term placentas as described previously34. β-adrenergic receptor expressed in the basal membrane was assayed as a basal membrane marker by measuring [3H]dihydroalprenolol, levo- hydrochloride binding35. Purity and enrichment results are listed in Supplementary Table The purity and enrichment of MVM were determined enzymatically by assaying the activity of alkaline phosphatase, according to a well-established protocol3H-dopamine, 3H-norepinephrine, and 3H-serotonin uptake by syncytiotrophoblast vesicles (MVM or BM) was performed using the MultiScreen™ Vacuum Manifold 96-well system. Briefly, 3 µL of extravesicular buffer  were added into 3 µL of MVM or BM vesicles (10 mg/ml) at room temperature. Uptake was initiated by adding 3 µL of 3H-dopamine, 20 µCi/ml (112 nM) or 3H-norepinephrine, 20 µCi/ml (332 nM) or 3H-serotonin, 24 µCi/ml (100 nM). Uptake was stopped after 1 min or pre-defined time points (time-dependency studies), by adding 200 µL of ice-cold stop solution , consequently, transferred into 1 800 µL stop solution and filtered through a 0.45 µm mixed cellulose ester filter . Finally, filters were washed ten times with 300 µL of stop solution and cut using the MultiScreen Multiple Punch system . Protein-free controls were analyzed in parallel to determine tracer binding to the filter, and this background value was subtracted from the total vesicle count.3H-dopamine, 3H-norepinephrine, and 3H-serotonin were used as a tracer. The total substrate concentration was supplemented with increasing levels of unlabelled dopamine hydrochloride, norepinephrine hydrochloride, or serotonin hydrochloride. To assess 3H-dopamine or 3H-norepinephrine binding and/or diffusion in MVM and BM vesicles, uptake experiments were performed at 4 °C at 0 and 1 min incubation times, respectively. On the other hand, to evaluate the effect of intracellular K+ on 3H-dopamine or 3H-norepinephrine uptake, vesicles were preloaded with 140 mM KCl overnight at 4 °C before the uptake experiments.For concentration-dependent uptake and interaction studies, 3H-dopamine and 3H-norepinephrine uptake was assessed by preincubating MVM and BM vesicles with the selected inhibitors for 10 min at room temperature. Subsequently, 3H-dopamine and 3H-norepinephrine uptake was assayed in the presence of the inhibitor. The following inhibitors were used: paroxetine 100 µM 36, venlafaxine 100 µM 37, nisoxetine 20 nM (NET inhibitor)37, GBR 12935 50 nM (DAT inhibitor)38, hydrocortisone 100 µM (OCT3 inhibitor)33, and decynium-22 10 µM (OCT3 inhibitor)39. Inhibitors' concentrations were chosen based on previously published data41.The effect of specific transport inhibitors on 3H-norepinephrine, 20 Ci/mmol, 3H-serotonin, 80 Ci/mmol, and 3H-dopamine, 60 Ci/mmol were measured by liquid scintillation counting using a Tri-Carb 2910 TR instrument. Non-specific tracer binding to the filter was determined by measuring protein-free controls.Filter-associated concentrations of + on 3H-dopamine and 3H-norepinephrine uptake by MVM and BM was assessed by the Mann–Whitney test. Kruskal–Wallis test was used to evaluate the effect of inhibitors on 3H-dopamine and 3H-norepinephrine uptake by MVM and BM. IC50  values for monoamine interactions were obtained by fitting concentration-inhibition curves through nonlinear regression analysis. All statistical analyses were implemented in GraphPad prism 9.3.1 software . Asterisks in the figures indicate significance levels: * (p ≤ 0.05), ** (p ≤ 0.01), and *** (p ≤ 0.001).The effect of incubation temperature and intracellular KNET expression was predominant among the four monoamine transporters tested, albeit revealing a high inter-individual variability. SERT and OCT3 showed comparable expression levels, whereas DAT expression in whole placental tissue was negligible, with transcript levels below 0.2 transcripts/ng RNA.ddPCR analysis was employed to determine and compare the transcript levels of monoamine transporters in the human term placenta Table . NET exp3H-dopamine and 3H-norepinephrine uptake was determined at pH 7.4 over a period of 20 s to 60 min. As shown in Fig. 3H-dopamine and 3H-norepinephrine accumulation. Contrary to 3H-norepinephrine, whose uptake increases with time , the intravesicular 3H-dopamine concentration was 1.97 times higher than at 60 min. On the other hand, uptake of 3H-dopamine and 3H-norepinephrine by BM vesicles was fast and increased with time, reaching equilibrium in 60 min . The time course of ime Fig. c, 3H-dopon” Fig. a, previovershoot 5 min, th3H-dopamine and 3H-norepinephrine, indicating a transporter-mediated uptake mechanism. However, at a concentration range of 1 to 1000 µM, we observed different saturation kinetics between both membranes  and extraneuronal OCT3) monoamine transporters in MVM and BM, we investigated the effect of intracellular K monoamin3H-dopamine  in the BM, only inhibitors related to the extraneuronal transport system (OCT3) were tested in this membrane. We found that paroxetine, venlafaxine, and nisoxetine significantly inhibit ine Fig. a and 3H-ine Fig. c uptake ine Fig. b and 3H-ine Fig. d uptake 3H-dopamine, 3H-norepinephrine, and 3H-serotonin uptake was investigated  values are shown in Table To investigate the interactions of monoamines on placental monoamine transporters, the effect of unlabelled monoamines on ted Fig. a–f. Thesine Fig. a and 3H-ine Fig. c uptake nin Fig. e uptake ine Fig. b and 3H-ake Fig. f. The ha43. Our findings expand on the pioneering work by Ganapathy et al., who were the first to identify the functional expression of monoamine transporters in the placenta44. The importance of SERT and NET for maternal-placental handling of dopamine and norepinephrine has been clearly established. However, despite increasing fetal monoamine production towards term15, limited attempts have been made to elucidate dopamine and norepinephrine transport across the fetal-facing basal membrane. Using an established ex vivo model of human placenta membrane vesicles, we demonstrate a novel physiological mechanism of dopamine and norepinephrine transport across the fetal-facing membrane into the placenta by OCT3.This study characterizes the transport mechanisms involved in dopamine and norepinephrine transfer across the apical and basal membranes of the human placenta Fig. . Uptake 1. Although polar molecules, dopamine and norepinephrine were reported to cross the placental barrier from the maternal to the fetal circulation46. Moreover, increased fetal monoamine secretion during pregnancy results in elevated cord blood levels15. This intrauterine neuroendocrine milieu requires a finely tuned clearance mechanism to control monoamine levels in the fetoplacental unit. The current state of knowledge has primarily focused on the MVM, where functional expression of neuronal monoamine transporters (SERT and NET) is recognized as critical to intrauterine monoamine homeostasis27. Our findings show that these transporters share a similar transcriptional profile in the human placenta. In agreement with previous studies29, we demonstrate that NET mediates norepinephrine uptake by MVM in a time-, concentration- and Na+-dependent manner. Nonetheless, inhibition of norepinephrine transport in the presence of paroxetine (a selective SERT inhibitor) indicates the potential involvement of SERT in total norepinephrine uptake by MVM, although with significantly lower affinity.Increasing evidence supports the placenta as a dopaminergic and adrenergic organ30. Interestingly, we observed that dopamine uptake by MVM vesicles reveals an overshoot phenomenon over time, which largely mirrors that of serotonin, previously reported by our team31. On the contrary, norepinephrine uptake does not exhibit such a phenomenon, thus, suggesting SERT as the key transporter responsible for dopamine uptake by MVM. However, the sensitivity of the uptake to selective SERT (paroxetine) and NET (nisoxetine) inhibitors suggests an overlapping substrate specificity whereby both SERT and NET contribute to placental uptake of dopamine across the maternal-facing membrane. Indeed, accumulating evidence reveals that the SLC6A members share structural and mechanistic properties48. Consequently, dopamine has already been described as an alternative substrate of SERT and NET in neuronal tissue, albeit with a mechanistically distinct mode of substrate translocation50.In contrast, DAT expression in the human placenta is negligible and, based on the functional studies, does not contribute to monoamine uptake. This was previously suggested by Ramamoorthy et al., who attributed dopamine transport across placental MVM to NET31. Since OCT3 recognizes dopamine and norepinephrine as substrates33, we speculated that this transporter might also mediate the uptake of these two monoamines. Using several OCT3 inhibitors39, we confirm that this transporter is involved in the placental uptake of fetal dopamine and norepinephrine. Nonetheless, we identified a non-saturable fraction in the transport kinetics across BM. Although not widely acknowledged in the literature, several studies indicate that dopamine and norepinephrine may cross biological barriers also by passive diffusion24. Such a mechanism was also previously speculated in the placenta32, particularly in the BM, where water permeability through a lipid diffusive mechanism is significantly higher than in MVM51.Importantly, our findings reveal a concentration-dependent placental uptake of dopamine and norepinephrine across the fetal-facing BM. Isotope displacement kinetics revealed different profiles between MVM and BM, indicating the involvement of a high-capacity transporter in BM. Moreover, intracellular potassium concentrations did not affect dopamine and norepinephrine uptake by BM vesicles, thus excluding the involvement of neuronal monoamine transporters in the fetus-facing membrane. We have recently shown that the low-affinity but high-capacity OCT3 mediates placental serotonin uptake from the fetal circulation32. Later, this was also reported in the ovine model, highlighting the role of the placenta in fetal norepinephrine clearance52. Similarly, we have shown that via a synchronized activity of transporters (SERT and OCT3) and metabolizing enzyme (MAO-A), the placenta contributes significantly to serotonin clearance from the fetal circulation31. Thus, we propose that the human placenta plays a neutralizing role in preventing elevated monoamine levels in the fetoplacental unit under physiological conditions.Fetal-to-maternal transfer of norepinephrine was first described by Sodha et al. using a human placenta perfusion system. The authors demonstrated that up to 50% of extracted fetal norepinephrine is metabolized within the trophoblast via COMT and MAO-A and released on the maternal side in the form of inactive metabolites44; here, we describe a similar behavior also in the BM. It is thus logical to assume potential competition of monoamines for their transporters on both the maternal and fetal sides of the placenta. However, we report that the calculated IC50 values are notably higher than the physiological plasma concentrations of these monoamines in healthy pregnant women55, suggesting that mutual interactions and competition for uptake systems do not occur under physiological conditions. Nonetheless, this phenomenon may be detrimental in placental pathologies such as preeclampsia, associated with altered monoamine transporter expression27 and elevated plasma monoamine concentrations56. Moreover, inhibition of monoamine transporters by illicit drugs  and pharmaceuticals  used during pregnancy, or endogenous molecules , could compromise monoamine levels in the fetoplacental unit36, contributing to the adverse effect on fetal development and programming.Monoamine transporter promiscuity in the placenta was previously suggested in the MVM57. Thus, an intact cell system is required to identify the contribution of specific transporter(s) on monoamine transplacental flux in vivo. Moreover, cellular metabolism should be considered when estimating the total clearance capacity.The strength of this study is using simultaneously isolated MVM and BM from the same placenta, thus minimizing the influence of clinical and genetic heterogeneity when comparing the transporter characteristics in each membrane. Nonetheless, while isolated human placental membranes provide an ideal model to investigate syncytial transport aspects , this is done in the absence of intracellular constituents and regulatory factorsIn conclusion, the placenta provides an efficient clearance system for dopamine and norepinephrine from both maternal and fetal circulations. Although not investigated in this study, we speculate that this transport function plays a multifaceted role in placental physiology in conjunction with intracellular metabolism. Ultimately, placental monoamine transport and metabolism may constitute essential mechanisms implicated in fetal programming of adulthood disorders.Supplementary Table S1.


Haloperidol (HPL) is a typical antipsychotic drug used to treat acute psychotic conditions, delirium, and schizophrenia. Solid charge transfer (CT) products of HPL with 7,7,8,8-tetracyanoquinodimethane (TCNQ) and picric acid (PA) have not been reported till date. Therefore, we conducted this study to investigate the donor–acceptor CT interactions between HPL (donor) and TCNQ and PA (π-acceptors) in liquid and solid states. The complete spectroscopic and analytical analyses deduced that the stoichiometry of these synthesized complexes was 1:1 molar ratio. Molecular docking calculations were performed for HPL as a donor and the resulting CT complexes with TCNQ and PA as acceptors with two protein receptors, serotonin and dopamine, to study the comparative interactions among them, as they are important neurotransmitters that play a large role in mental health. A molecular dynamics simulation was ran for 100 ns with the output from AutoDock Vina to refine docking results and better examine the molecular processes of receptor–ligand interactions. When compared to the reactant donor, the CT complex [(HPL)(TCNQ)] interacted with serotonin and dopamine more efficiently than HPL only. CT complex [(HPL)(TCNQ)] with dopamine (CTtD) showed the greatest binding energy value among all. Additionally, CTtD complex established more a stable interaction with dopamine than HPL–dopamine. Atypical antipsychotics or serotonin–dopaminergic antagonists, the fourth class of antipsychotic drugs, can improve the so-called positive symptoms of schizophrenia, such as hallucinations, delusions, and agitation, as well as negative symptoms, such as catatonia and flattening of the ability to feel emotion. Each agent in this group has a unique profile of receptor interactions. Almost all antipsychotics block dopamine receptors and reduce dopamine transmission in the forebrain. Moreover, atypical antipsychotics have an affinity for serotonin receptors. Atypical antipsychotics are related to chlorpromazine and haloperidol (HPL), and HPL is used to treat acute psychotic conditions, delirium, and schizophrenia . The formation of highly colored charge-transfer (CT) complexes that absorb light in the visible region is often related with molecular interactions between electron donors and acceptors ,3. CT coDonor–acceptor complexation plays an important role especially in the field of biochemical energy transfer process . The forSolid CT products of HPL with 7,7,8,8-tetracyanoquinodimethane (TCNQ) and picric acid (PA) have not been reported till date. Therefore, we conducted this study to investigate such reactions. The molecular docking software AutoDock Vina was used to investigate the interactions between ligands (HPL and synthesized CT complexes) and receptors (serotonin and dopamine). Hydrophobic, ionizability, aromatic, and hydrogen bond surfaces were studied as well as binding energy. The best molecular docking data were submitted to molecular dynamic simulation at 300 K for 100 ns to give a more effective mechanism for illustrating receptor–ligand interactions. In terms of residue flexibility, structural stability, solvent accessible surface area, structure compactness, and hydrogen bond interactions, the dynamic properties of the complexes were compared.−1 cm−1 mol−1, respectively. The low conductance values of the synthesized CT complexes deduced the formation of D+ and A− datives anions based on the association of donor–acceptor chelation. The electronic spectra of synthesized CT complexes of HPL-PA and HPL-TCNQ refer to the association of new electronic absorption bands , which did not exist in the spectra of free reactants. The infrared spectrum of HPL-PA complex was assigned upon intermolecular hydrogen bonding between the –OH group of the PA acceptor and basic oxygen atom center of HPL donor  was 1:1. The conductivities of HPL-PA and HPL-TCNQ CT complexes were 45 and 53 ΩPL donor . In the PL donor , the –OHThe bonding of the –OH group of HPL and the –OH group of PA to the –CN of TCNQ acceptors via intermolecular hydrogen bonding was confirmed by proton NMR spectra of the free HPL donor and its HPL-PA and HPL-TCNQ complexes. The activation energy (E) was used to calculate the thermal stability of both HPL-PA and HPL-TCNQ complexes using Coats–Redfern and Horowitz–Metzger techniques ,9,10.−1 and 98 kJ mol−1, respectively, and the variant data might be influenced by the acceptor type. The activated complexes had a more ordered structure than the reactants, and the activation of entropy (ΔS ∗) had negative values, indicating that the reaction rates were slower than normal.The average activation energies for the [(HPL)(PA)] complex and the [(HPL)(TCNQ)] complex were 132 kJ molg), which refers to the minimum transition energy, was determined based on the electronic absorption spectra. Optical absorption near the edge of the absorption band can be used to estimate Eg and confirm the formation of CT complexes. The absorption coefficient (α) can be estimated from the transmittance (T) of the complex according to the following equation:g based on the following equation  and [(HPL)(TCNQ)] against serotonin (PDB ID: 6A94) and dopamine (PDB ID: 6CM4) were determined. For comparison, HPL was employed as the control. CT complexes have a larger potential binding energy than HPL in both receptors . Among tThe illustration of molecular docking for ligand–receptor interactions depicted in Molecular docking of HPL drug with serotonin and dopamine revealed the potential binding energies as −10.0 and −10.9 kcal/mol, respectively. The higher binding energy value of HPL–dopamine (HPLD) signifies stronger interaction with dopamine compared to that with serotonin. The best docking position with dopamine (HPLD) is shown in The best docked location of HPLD and CTtD with the highest docking score at 100 ns molecular dynamics (MD) generated by AutoDock Vina was used. Only the best docking output was used to build up this method in a high-throughput manner for analyzing the binding mechanism of the ligand at the active site of protein under clearly defined aqueous conditions. The root mean square deviation was computed to determine structural stability from MD data (RMSD). HPLD and CTtD formed stable conformation after ~80 ns and ~65 ns, respectively, with an appropriate RMSD value of 3.04 and 2.41 Å, respectively, as seen in the RMSD plot .The RMSD value range of <3.0 Å is the most acceptable . CTtD prRR Distance Maps creates a distance map through Chimera 1.15 software by a structural comparison tool. The map can display Cα-Cα distances within a single protein chain, as well as averages and standard deviations for many chains. The white diagonal on the map represents no distance between the two residues, but the red and blue on the map reflect residue pairings with the biggest distance differences between the two conformations .The average radius of gyration (Rg) values for HPLD and CTtD were 22.647 and 22.586 Å, respectively. Along the simulation time, Rg decreased, indicating that the structures became more compact .The number of hydrogen bond interactions between HPLD and CTtD was displayed against time using a grid-search on a 27 × 18 × 21 grid with rcut = 0.35 . There wOverall, the receptor–protein interaction enhanced the number of hydrogen bonds substantially, and it was more in CTtD.The values of solvent accessible surface area (SASA) altered as the ligand bound to the receptor . When th3OH was allowed to react with 3 mmol of each acceptor (PA and TCNQ) in 10 mL CHCl3 solvent, and both mixtures were stirred at room temperature for 45 min. Following this, the yellow and green solid complexes were isolated, washed three times with a minimum amount of CHCl3 solvent, and dried under vacuum over anhydrous CaCl2.Previously, synthesis and characterizations  of the two solid HPL CT complexes were performed . For preA Perkin–Elmer Precisely Lambda 25 UV/Vis spectrometer using a 1 cm quartz cell was used to measure the electronic absorption spectra of charge transfer complexes produced in the presence of methanol at the range of 200–800 nm. The Jenway 4010 conductivity device was also used to measure the molar conductivity in the presence of newly prepared dimethyl sulfoxide solutions. http://openbabel.org/wiki/Main Page; accessed 1 March 2022) [The structures of [(HPL)(PA)] and [(HPL)(TCNQ)] were obtained in PDBQT format using the OpenBabelIGUI tool ch 2022) . The RCS . The RCch 2022) . The Geich 2022) . The resSimulations were conducted using processor Intel(R) Xeon(R) CPU E5-2680 v4 @ 2.40GHz, 64 bit. MD simulation was performed using the optimal receptor–ligand complex position and an evaluation of the conformational space and inhibitory potential. MD simulation analysis with the GROMOS96 43a1 force field was performed using the Gronningen machine for chemical simulations .+ and 46 Cl− ions  were administered to neutralize the systems and reproduce physiological salt concentrations  and constant pressure (1.0 bar) over 100 ns were used for simulations using the leap-frog MD complement in the NPT/NVT equilibration run . The ste0 K and cIn the present research, we looked into how haloperidol (HPL) interacts with two key neurotransmitters (serotonin and dopamine) that are vital in mental health. These findings were compared with the synthesized charge transfer complexes of TCNQ and PA with HPL. The [(HPL)(TCNQ)] coupled with serotonin and dopamine more efficiently than HPL alone. Also, [(HPL)(TCNQ)]–dopamine has a higher binding energy value than HPL–dopamine. The molecular dynamic simulation at 100 ns demonstrated that the [(HPL)(TCNQ)]–dopamine complex had a more stable interaction with the dopamine receptor than the HPL–dopamine complex.


TM) is a novel non-invasive system to quantify the lung fluids, which may correlate to intracardiac filling pressures. We aimed to understand the variation in ReDS measurements by chronotype in patients with chronic heart failure. Methods: The patients who were hospitalized for heart failure exacerbations between November 2021 and March 2022 were prospectively included. ReDS values were measured at clinically stable conditions at the following three time points during the day: 5:00 (morning), 12:00 (noon), and 21:00 (night) . Results: Twelve patients were included. The median age was 84  years and four patients (33%) were men. The median plasma B-type natriuretic peptide was 235  pg/mL. The median ReDS value was 38%  in the morning. The ReDS value decreased significantly at the noon measurement, down to 28%  (p = 0.005) and again increased significantly at the night measurement, up to 31%  (p = 0.002). The patients were clinically stabilized during the observational period. Conclusions: the lung fluid level varied considerably in patients with chronic heart failure following clinical stabilization.Background: The variation in lung fluid levels dependent on chronotype in patients with chronic heart failure is unclear. Remote dielectric sensing (ReDS Variations in human physiology, as exhibited in changes in blood pressure, heart rate, and metabolism, occur over the course of sleep-wake cycles with specific regulation by circadian sleep-wake cycles . PatientPulmonary congestion may worsen later in the day, as the effect of neurohormonal agents wane for those who take medical therapy in the morning, whereas pulmonary congestion may be at a nadir mid-day, following the true effect of medical therapy and drops in morning cortisol levels. Of note, changes in lung fluid levels, which often correlate with intracardiac filling pressures and degree of pulmonary congestion , by chroTM, Sensible Medical Innovations Ltd., Netanya, Israel) system is a novel electromagnetic-based method to quantify lung fluid levels with robust inter-rater and intra-rater reliability , 12:00 (noon), and 21:00 (night). In the morning, the ReDS value was measured under fasting conditions before taking any medications, including diuretics.ReDS values were measured at sitting position, according to the manufacture-recommended methodology . ReDS emp values less than 0.05 were considered significant.All continuous data are presented as median with interquartile range. Categorical data are presented as numbers and percentages. The trends of the ReDS values were analyzed using the Friedman test and post-hoc Wilcoxon signed-rank test. Statistics were performed using SPSS Statistics 23.0 software . Two-sided A total of 12 patients who were hospitalized for decompensated heart failure were included . The medp = 0.001; p = 0.005), and then increased significantly at the night measurement up to 31%  (p = 0.002). The systolic blood pressure and heart rate showed a similar trend. The systolic blood pressure was 129  mmHg, 121  mmHg, and 125  mmHg (p = 0.035). The heart rate was 79  bpm, 76  bpm, and 77  bpm (p = 0.022).The trend of the ReDS values varied considerably during the day, following initial clinical stabilization .Seven patients had a ReDS value of >35% in the early morning, which had no significant association with the baseline characteristics  and 38% (morning) during the observed measurement period. These unique time-varying differences may inform the clinician as to when therapies may be dosed to ensure longer periods of clinical stability.


For many years, the Molecular Mechanical (MM) force field (FF) methods have been classical tools for modeling molecular crystal structures, nanomaterials, and surfaces. Being computationally inexpensive, the FF methods are also extensively used in the modeling of the biological systems. Yet, having a set of approximations and based on many parameters derived from the experimental data, the conventional FF methodss are tending to suffer from deficiencies and contain some restrictions. For example, when the energy difference between crystal polymorphs does not exceed several kJ/mol the FFs fail to sufficiently describe well the hydrogen bonding, strong inter/intramolecular interactions, anisotropic electrostatics, or large dispersion contributions. With the rapid development of polarizable FFs, the improvement in treatment of molecular interactions can be achieved but computational cost increases dramatically. Therefore, the dilemma between computational workload and accuracy has to be resolved.Nowadays, the majority of electronic structure studies are performed with the aid of Quantum Mechanical (QM) methods, which are based on periodic density functional theory (DFT). It should be noted that calculations involving periodic DFT with either plane-wave or atom-centered Gaussian-type basis sets are quite accurate and do not demand high computational cost, once the dispersion corrections are taken into account. However, the tasks involving crystal structure prediction are susceptible to the high extend to the choice of the dispersion correction and type of applied DFT functional.An alternative approach of electronic structure methods offers a large group array of fragment-based techniques . The fraHowever, there are some drawbacks that are inevitable upon fragment partitioning. Fragmentation destroys the hydrogen bond cooperativity and impacts the dispersion and induction of many-body interactions. To circumvent this deficiency, the DFT embedding scheme needs to be introduced to mimic interaction between the fragments. The embedding formalism is non-trivial, since it implies an embedding potential common to all subsystems, and therefore it reflects the influence of the environment of the system’s nearest neighbors.This Research Topic of articles will accustom readers with the leading-edge advances in the performance and development of fragment-based methods. Topics covered in this issue encompass a variety of QM-based methods, such as time-dependent density functionals (TD-DFT), embedded-cluster models, and many-body molecular interactions in molecular solids.Hartman et al., a molecular correction (MC) approach to the gauge-including plane augmented wave (GIPAW) method is used to calculate electric field gradients (EGF) in molecular solids. It combines the benefits of periodic calculations and single-molecule techniques in the context of electric field gradients (EFG) tensors in molecular solids, which are used to derive nuclear quadrupolar coupling constants and anisotropy parameters. Applying molecular corrections to periodic DFT calculations of NMR parameters of molecular crystals, the authors benchmark the performance of several basis sets and DFT functionals on a relatively large set of crystals, which is important for a sufficient statistical significance of the results. The results of an extensive set of calculations show a noticeable improvement in the calculated NMR observables by including the molecular correction, with minor changes relative to basis set.In the first paper by Shen et al. tested the performance of another fragment-based method - the electrostatically embedded generalized molecular fractionation with conjugate caps (EE-GMFCC). The efficiency of the EE-GMFCC scheme has been proven for the excited state description of the fluorescent RNA aptamer systems. Their results evidenced that EE-GMFCC calculations, which are one order of magnitude faster, are in excellent agreement with the properties of excited state obtained by traditional full-system time-dependent ab initio calculations for so complicated systems. That allowed to describe properties of different fluorescent RNA-aptamer systems.In the second paper, Larsson and Veryazov present the convergence of the electron density with regard to the size of the embedded clusters of MgO and Ni-doped MgO ionic crystals. The main focus of the paper is on the quality of an embedding protocol for ionic oxides and comparison of periodic and cluster calculations.In the third study, The branch of fragment-based electronic structure methods invoked to treat solid-state materials is very large and diverse, and this special research selection elucidates only a small part number of manifold applications and recent developments in this Research Topic. Nevertheless, we hope that the selected papers will contribute to further development of fragment-based methods and lead to relevant insights into the balance between accuracy and computational cost, serving as a valuable benchmark for the embedded approaches.


Along wOver the past decades, elaborate molecular mechanisms for regulating the phenylpropanoid pathway at multiple levels have been extensively studied. Phenylpropanoid metabolism has been revealed to be modulated by multiple regulatory mechanisms, including transcriptional, post‐transcriptional, post‐translational, and epigenetic regulation, and through a variety of signaling pathways, such as phytohormone, biotic stress, and abiotic stress signaling pathways . This ReWang et al. (in this Research Topic) identified and quantified 133 flavonoids within the seed coat of four different testa-colored peanut cultivars and proposed several MYB-like transcription factors (TFs), an anthocyanidin reductase (ANR), and a UDP-glycosyltransferase (AhUGT236) were implicated in the testa pigmentation based on RNA-seq and gene co-expression network analysis.Phenylpropanoids exhibit extraordinary complexity and high‐level plasticity in different species, developmental stages and in response to environmental stimuli. Significantly, many of phenylpropanoids can be specific to only one or a few plant species; it’s, therefore, necessary to have comprehensive analyses of phenylpropanoids among various species to expand our knowledge gained mainly from model plant species. Cheng et al. (in this Research Topic) have characterized a novel bHLH TF, VvibHLH93, as a negative regulator in the proanthocyanidins (PAs) biosynthesis route of grapevine, and found that VvibHLH93 could target a wide range of structural genes and TFs genes in flavonoid pathway. The findings of this study essentially complement existing knowledge on the regulation of PAs. Lu et al. (in this Research Topic) have found a cluster of R2R3-MYB TF, XsMYB113s, and showed they likely control the progressive color changes during yellowhorn flower developments through positively regulating the anthocyanin biosynthesis genes.A group of TFs including MYBs, NACs, MBW ternary complex composed of three classes of regulators, namely R2R3‐MYB TFs, basic helix‐loop‐helix (bHLH) TFs, and WD40 Repeat (WDR) proteins, and other TFs were believed to be critical in regulating the structural genes of lignin or flavonoid biosynthesis. Lu et al. (in this Research Topic) showed the methylation status of CHH on the transposon element near the XsMYB113-1 influenced its expression and dynamic epigenetic regulation of the XsMYB113-1 affected anthocyanins buildup along with color changes during yellowhorn flower development.In addition to transcriptional regulation, there is growing interest in the role of epigenetic regulation in controlling phenylpropanoid metabolism. Epigenetic regulations, such asDNA methylation and demethylation, have been implicated in anthocyanin biosynthesis and associated with pigmentation. Chen et al. (in this Research Topic) studied the relationship between lignin biosynthesis and 19 environmental factors in natural birch. They discovered that the lignin content in birch wood was negatively correlated with climate temperature. They also showed that DNA methylation levels in the promoter regions of two key NAC TFs, BpNST1/2 and BpSND1, were variable in birch trees grown in different environments, and suggest that DNA hypermethylation may repress the expression of these genes and thus negatively regulate lignin biosynthesis. This study provides evidence that environmental signals can lead to epigenetic variations that cause changes in lignin biosynthesis.Environment stimuli frequently trigger phenylpropanoids biosynthesis. Therefore, lignin biosynthesis can be affected by environmental variations due to climate change. What these four research articles have in common is that their research objects are all non-model plants of significant economic and ecological value. As more plant species for which full-genome sequences are becoming available, it will benefit the studies to explore the functions of phenylpropanoid metabolites in more plants and enrich the diversification of phenylpropanoid research on a multi-species level.via the exploration of metabolic diversity, transcriptional regulation, epigenetic modification, and environmental interaction. In the future, the ability to bridge the fields of molecular biology, epigenetics, chemistry, evolutionary biology, and molecular ecology to resolve the complexity of phenylpropanoid metabolism cooperatively will provide high-resolution information on its metabolic regulatory networks.Phenylpropanoid metabolism is often connected to a complex interaction between cell signaling and environmental influences. The studies in the current Research Topic have greatly advanced our understanding of phenylpropanoid metabolic regulation in plants All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


The ventral tegmental area (VTA) is a principal source of NAc dopamine. To investigate how high frequency stimulation (HFS) of the rodent VTA or nucleus accumbens core (NAcc) modulates the acute effects of cocaine administration on NAcc tonic dopamine levels multiple-cyclic square wave voltammetry (M-CSWV) was used. VTA HFS alone decreased NAcc tonic dopamine levels by 42%. NAcc HFS alone resulted in an initial decrease in tonic dopamine levels followed by a return to baseline. VTA or NAcc HFS following cocaine administration prevented the cocaine-induced increase in NAcc tonic dopamine. The present results suggest a possible underlying mechanism of NAc deep brain stimulation (DBS) in the treatment of substance use disorders (SUDs) and the possibility of treating SUD by abolishing dopamine release elicited by cocaine and other drugs of abuse by DBS in VTA, although further studies with chronic addiction models are required to confirm that. Furthermore, we demonstrated the use of M-CSWV can reliably measure tonic dopamine levels  Despite the increasing interest and resources devoted to addiction research, there has been little improvement in the clinical care and prevalence of substance use disorder (SUD) . In the via neuromodulation, as has been done for neurological diseases such as Parkinson’s disease, is potentially an effective strategy for the treatment of these pathologies. Indeed, previous attempts have been made to stimulate targets within the mesolimbic dopaminergic pathway as a means to rectify dysfunctional dopamine dynamics  and substantia nigra pars compacta are major producers of dopamine in the mesolimbic dopaminergic pathway . A majorin vivo measuring methods such as the use of microdialysis have limited the continuous measurement of dopamine as a useful biomarker for interventive therapy, until recently . This method is able to measure tonic extracellular dopamine levels with unprecedented temporal resolution (10 s) and minimal trauma to the neural tissue when used in combination with carbon fiber microelectrodes (CFM) . This teNAc DBS has shown promising results for the treatment of SUD . Here wead libitum food and water. The present studies were approved by the Institutional Animal Care and Use Committee (IACUC), Mayo Clinic, Rochester. The NIH Guide for the care and use of laboratory animals guidelines  were followed for all aspects of animal care.Male Sprague-Dawley rats  were used for this study. Rats were kept in social housing in an association for assessment and accreditation of laboratory animal care international (AAALAC) accredited vivarium following a standard 12-h light/dark cycle at constant temperature (21°C) and humidity (45%) with in vivo.Carbon fiber microelectrodes were fabricated using an established standardized CFM design at Mayo Clinic . Each miEach rat was anesthetized with urethane  and administered buprenorphine  for analgesia. Following anesthesia, they were placed in a stereotaxic frame . Respiratory rate  and hind-paw and tail pinch were used to monitor the physiological state and depth of anesthesia, respectively. Using a standard rat brain atlas , three tThe depths of the stimulating electrode in the VTA and CFM in the NAcc were first adjusted to obtain a robust stimulation-evoked dopamine signal as measured by FSCV . After another 30 min of observation, the stimulation was turned off. Post-stimulation, the animal was observed for another 30 min before being sacrificed using Fatal-Plus injection .Once the optimal electrode depths were identified, the system was switched to the M-CSWV sensing technique see . After 6N = 5), alpha-methyl-p-tyrosine , a tyrosine hydroxylase inhibitor, was given to further confirm the recording of dopamine by M-CSWV. Tyrosine hydroxylase is the rate limiting enzyme of catecholamine biosynthesis, converting tyrosine into L-DOPA, the precursor to dopamine. Thus, AMPT administration, acting as a negative control, was expected to decrease the voltammetric signal if the signal indeed arose from dopamine.In a separate group of animals . For comparison, the levels were all measured by averaging over 10 data points, i.e., 10 s. In cases where i.v. drug was administered, the 10 data points centered at peak within 10 min of injection.Statistical analysis was performed using repeated measures one-way ANOVA and two-tailed paired t-tests were used to demonstrate sequential changes in the post hoc analysis. In the control experiments, pre-injection baseline tonic dopamine concentrations were compared to the post-saline levels, and the post-saline levels were compared with the post-cocaine peak levels. In the NAcc stimulation experiments . In the post hoc analysis, as expected, saline administration did not evoke a statistically significant change in peak tonic dopamine concentration compared to pre-saline levels ; time to peak = 6.8 ± 0.8 min; In the positive control experiments, after implanting the CFM at the optimal position within NAcc , we elicited cocaine-induced dopamine changes by first administering i.v. saline and then i.v. cocaine, while tonic dopamine levels were recorded using M-CSWV. One-way ANOVA test among the three levels  showed significant differences . In the post hoc analysis, i.p. AMPT administration (250 mg/kg) did acutely reduce tonic NAcc dopamine concentrations over 30 min [N = 5; paired t-test, p = 0.004; change = −34.5 ± 5.7 nM (−27%); time to stable baseline = 25.3 ± 2.2 min; N = 5; paired t-test, p = 0.513; In the negative control experiments, AMPT, a tyrosine hydroxylase inhibitor, was applied intraperitoneally (i.p.) to reduce dopamine production. This was compared against i.p. saline. One-way ANOVA test among the three levels showed significant differences . With further post hoc analysis, NAcc HFS elicited an initial decrease in tonic dopamine concentration [N = 5; paired t-test, p = 0.011; change = −28.3 ± 6.3 nM (−20%); Here, saline and cocaine administration was repeated on the background of NAcc stimulation . With one-way ANOVA test, there were significant differences among the six different levels   with continuous HFS, and, as before, did not significantly affect NAcc tonic dopamine concentrations over 30 min . With further post hoc analysis, VTA HFS elicited a decrease in tonic dopamine concentration which persisted over the 30 min [N = 5; paired t-test, p = 0.002; change = −47.3 ± 7.0 nM (−42%); Here, saline and cocaine administration was repeated on the background of VTA stimulation . With one-way ANOVA test, there were significant differences among the five different levels   with continuous HFS, and, as before, did not significantly affect NAcc tonic dopamine concentrations over 30 min  are also possible, but it is difficult to ascertain how this may interact with the dopaminergic neurons in this case. A recent voltammetry study has shown that electrical stimulation leads to multi-synaptic modulation of dopamine release, as a gamma-aminobutyric acid (GABA) antagonist increased electrical stimulation-evoked release of dopamine, compared to optogenetic stimulation, which only targeted dopaminergic terminals . In contBoth the NAcc  and shelOther possibilities may include down regulation of active dopaminergic transporters. The diminished response is consistent with a preclinical study where DBS of the NAc (shell) increased cocaine self-administration . This maIn contrast to NAc HFS, VTA HFS led to a decrease in NAcc dopamine levels that did not recover over the course of the experiment . There aMicrodialysis studies have shown that extracellular dopamine release in the NAc is regulated by GABA (inhibitory), dopamine (inhibitory), glutamate (excitatory), and acetylcholine (facilitatory) receptors in the VTA . It is pFunctional magnetic resonance imaging (fMRI) in rodent and in swine models have shown that electrical stimulation of the VTA not only induced dopamine release in the NAc (phasic release as detected by FSCV) but also led to increased blood-oxygen-level-dependent (BOLD) responses . HoweverIn addition to SUD, NAc DBS has been of great interest for application to a number of neuropsychiatric diseases, such as depression , TourettAlthough the VTA is vital in the expression of a number of drug-related behaviors, such as behavioral sensitization , VTA DBSOptogenetic studies have provided insight into the possible behavioral effects of VTA stimulation. One rodent study demonstrated continuous (“tonic”) optogenetic stimulation of VTA dopaminergic neurons can reduce ethanol self-administration . In addiGiven the reduction in tonic dopamine levels and attenuation of the cocaine-induced response, VTA DBS may be helpful in not only treating SUD but also pathologies associated with hyperdopaminergic states such as mania, schizophrenia, and dopamine dysregulation syndrome, where excessive dopamine in the system may lead to excessive risk-taking behavior . In addiIt should be noted the current study utilized anesthetized naïve rodent models with acute administration of cocaine. Larger animals and chronic addiction models will be necessary to verify the dopamine-attenuating effect of HFS. It would be useful to know the effects of HFS on models of SUD of other substances, especially those that are not psychostimulants, such as opioids, and alcohol. Also, the effect on behaviors associated with SUD, such as craving and withdrawal, needs to be explored. Further mechanistic studies such as manipulation of dopamine transporter availability and other biochemical essays are also warranted.Previous literature also suggested there are persistent exposure of drugs does not necessarily lead to addictive behavior and dopamine is likely to be only one contributing factor to the behavioral changes observed. Therefore, one must consider the impact of other biological processes, such as changes in synaptic plasticity and other neurochemicals  .In summary, this study elucidated the tonic dopaminergic dynamics with NAc and VTA HFS with high spatiotemporal resolution. HFS appeared to have an alleviating effect on the elevations in tonic dopamine levels associated with cocaine administration. This may explain how NAc DBS was found to be therapeutic in both preclinical models and patients suffering from SUD. Dopamine, measured by M-CSWV, may provide a useful closed-loop biomarker for DBS, given the pivotal role of dopamine in many neuropsychiatric pathologies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This animal study was reviewed and approved by the Mayo Clinic IACUC.KL, YO, and JY conceptualized the study. JY conducted experiments, collected the data, and drafted the first manuscript. AR manufactured the 3D-printed electrode holder. JY, YO, and HS designed the analyses. JY and HS conducted the analyses. KL, HS, and YO supervised all aspects of the work. JY and YO drafted the figures. All authors critically reviewed and revised the manuscript and accepted the final version of the manuscript.


Klinefelter’s syndrome (KS) is considered as a genetic risk factor for intellectual disability and specifically for impairment on verbal skills. Prenatal and perinatal morbidity concurrent with this diagnosis determine the entity and typology of neurodevelopmental deficits.To describe the intellectual and adaptive functioning of a patient with KS and prenatal and perinatal difficulties, assessed in the Service of Psychiatry, Clinical Psychology and Mental Health at La Paz University Hospital (Madrid).A descriptive study was conducted of a 6 year and 7-month-old boy with diagnosis of KS (karyotype XXY), extremely low weight at birth (752 gr.), Arnold Chiari type I malformation with post-hemorrhagic hydrocephalus and periventricular leukomalacia, bronchopulmonary dysplasia, intra-atrial septal defect, left kidney agenesis with iatrogenic adrenal insufficiency, and acute ventriculitis due to E. Coli. Neuropsychological evaluation was carried out in October 2021. Leiter-3 scale was selected due to the absence of expressive language; and his parents completed the questionnaire Adaptive Behavior Assessment System-II.The nonverbal intelligence quotient was observed in the very low range . The result of general adaptive behavior (z = -2.83), showed very high functional disability both in the conceptual, social, and practical domains.The high number of causes of disability in this patient is consistent with a high degree of functional disability. Efficient evaluation sessions of intellectual performance, adaptive functioning, and necessary supports, due to the absence of expressive language and limited receptive language, are required. A specific neuropsychological evaluation profile should be established for KS.No significant relationships.