Evolutionary design has gained significant attention as a useful tool to accelerate the design process by automatically modifying molecular structures to obtain molecules with the target properties. However, its methodology presents a practical challenge—devising a way in which to rapidly evolve molecules while maintaining their chemical validity. In this study, we address this limitation by developing an evolutionary design method. The method employs deep learning models to extract the inherent knowledge from a database of materials and is used to effectively guide the evolutionary design. In the proposed method, the Morgan fingerprint vectors of seed molecules are evolved using the techniques of mutation and crossover within the genetic algorithm. Then, a recurrent neural network is used to reconstruct the final fingerprints into actual molecular structures while maintaining their chemical validity. The use of deep neural network models to predict the properties of these molecules enabled more versatile and efficient molecular evaluations to be conducted by using the proposed method repeatedly. Four design tasks were performed to modify the light-absorbing wavelengths of organic molecules from the PubChem library. A typical example is high-throughput computational screening (HTCS), which involves the use of virtual chemical libraries for large-scale predictions of material properties using simulations or machine learning, allowing a rational sorting of potential candidates for subsequent chemical synthesis3. However, HTCS is a local optimization technique whose success relies on the quality of the chemical libraries, the development of which depends on researchers’ experience and intuition. Thus, HTCS has a low hit rate, and in most cases, several iterative enumerations are necessary to generate suitable target materials. In this regard, evolutionary algorithms, a type of exhaustive enumeration, can be a viable alternative for de novo design. These algorithms are generic population-based metaheuristic optimization techniques that use bio-inspired operators, such as reproduction, mutation, recombination, and selection5. As design tools for materials, they not only optimize the molecular structures but also provide hints for a promising chemical space by identifying genetic traits that favor the target properties while maintaining the unique genotypes of ancestors. Recent advances in machine-learning algorithms19 have led to the proposal of data-driven methodologies.The discovery of new functional molecules has led to many technological advances and is still one of the most crucial ways in which to overcome technical issues in various industries, such as those in the organic semiconductor, display, and battery industries. Although the trial-and-error approach has generally been considered as the most acceptable way to develop new materials, computer-aided techniques are increasingly being used to enhance the efficiency and hit rate of molecular design21, have widely adopted evolutionary design methodologies (EDM) in combination with property prediction techniques29 based on first-principles calculation and machine learning. Specifically regarding organic molecules, two major challenges of EDM are to (1) preserve the chemical validity of evolved molecules and (2) choose the best-fit individuals in each generation efficiently and accurately according to the fitness function. To address the first challenge, heuristic chemical knowledge is generally incorporated. Molecules expressed as graphs or ASCII strings evolve according to user-defined rules, such as adding, deleting, and replacing atoms, bonds, and substructures under chemical constraints. Notably, not only the fragment structures that serve as building blocks but also their attachment points are specified in advance based on previous experience. This method increases the likelihood of generating more valid and synthetically tractable molecules and sometimes accelerates overall stochastic searches by using in-depth domain knowledge. However, predefined chemical rules and fragment libraries can lead to bias, and therefore the entire optimization process is at risk of converging to local optima. Moreover, every time the application changes, new chemical rules would have to be specified. For some emerging areas, it is challenging to determine a well-established guide for structural changes. To address the second challenge, simple evaluation methods, such as structural similarity indices, quantitative structure–property relationship models, and semi-empirical quantum chemistries, are generally adopted as fitness functions to reduce the computational cost. However, more diverse and complex assessments are needed to evaluate the candidates precisely but promptly.The development processes of various materials, including organic molecules, metals, ceramics, composites, and carbon molecules30, which acts as a decoder. This approach enables us to prevent explicit chemical knowledge from intervening during a molecular evolution while ensuring the molecules are chemically valid. Moreover, deep neural network (DNN) models31, aided by quantum chemical calculations, are used to evaluate the evolved molecules with more complex criteria.To overcome these demanding limitations, we devised an evolutionary molecular design method based on deep learning. Instead of graphs or ASCII strings, a bit-string fingerprint vector is used as a molecular descriptor to evolve molecules. Then, the evolved fingerprint vectors are converted into actual molecular structures using a recurrent neural network (RNN) model32. The deep learning models learn implicit knowledge from this rich library of materials and successfully guide the automatic evolution of seed molecules without heuristic intervention.The effectiveness of the entirely data-driven evolutionary approach was validated by conducting various molecular design tasks on data in the PubChem library to change the wavelengths at which organic molecules absorb the maximum amount of lighte(∙), decoding function d(∙), and property prediction function f(∙). As illustrated in Fig. e(∙) transforms the molecular structure m, which exists in a canonical simplified molecular input line entry system (SMILES)33 string format, into the corresponding extended-connectivity fingerprint (ECFP)34 vector x. Then, the decoding function d(∙) converts the bit-string ECFP vector x into the SMILES string m to enable it to be recognized as a real chemical structure. The property prediction function f(∙) predicts the molecular property t using the ECFP vector as an input. The decoding and property prediction functions are derived by the RNN and DNN, respectively, and consequently, lead the overall workflow shown in Fig. The evolutionary design framework where a genetic algorithm (GA) finds the design route towards the target under the guidance of deep learning models is illustrated in Fig. m0 in SMILES format is transformed into the corresponding ECFP vector x0 using the encoding function e(∙). Then, the evolution procedure begins with the generation of a population of vectors P0 = {z1, z2,∙…∙, zL} through the mutation of x0. Following the conversion of each vector zi into a SMILES string mi by d(zi), the validity of the decoded SMILES strings is inspected in terms of grammatical correctness with the RDKit library, e.g., additional open/close parentheses, unclosed rings, and Kekulization feasibility. This serves to evaluate the fitness of the molecules by predicting the molecular properties with ti = f(e(mi)). Subsequently, the top three ECFP vectors based on fitness are selected as parents for further evolution into the new population of vectors Pn = {zn1, zn2,∙…∙, znL} via crossover and mutation. Notably, the assessment of the chemical validity and fitness evaluation are performed once again, and the best-fit molecule in the nth generation is chosen. Generated molecules that overlap with molecules that already exist in the database are removed from the candidate list. Successive iterations of this procedure gradually refine the properties and automatically optimize the molecular structures to meet the target.First, the molecular structure of a seed molecule During the evolution, additional constraints, such as the presence or absence of specific substructures, can be imposed on the molecular structure depending on the design purpose. However, in this study, three simple structural constraints were specified in the form of a blacklist. Newly generated molecular structures that included these blacklisted substructures were then excluded from the list of candidate materials. The first constraint specifies that the size of fused rings in molecules generated with such rings be limited to between four and seven. Second, the maximum number of fused rings in the molecules is restricted to six, and finally the maximum length of an alkyl chain is limited to six carbon atoms. Third, to maintain the form of the seed molecule, we remove the following newly generated molecules: those for which the maximum number of rings exceeds those of the seed molecule by two and those for which the minimum number of rings is two fewer than those in the seed molecule. These structural restrictions made it possible to obtain novel molecules with appropriate properties via natural evolution without significantly deviating from the shape of the initial seed.e(∙) uses a hash function to map the structural features of a molecule into a fixed-length ECFP vector representation. In this study, we employ an ECFP with a neighborhood size of 6 and a length of 5000. Therefore, the encoding function encodes each atom and its circular neighborhoods with a diameter of six chemical bonds for a molecule m and transforms the SMILES into a 5000-dimensional vector x. Regarding the decoding function d(∙), an RNN composed of three hidden layers with 500 long short-term memory units35 is modeled to obtain the SMILES string from the ECFP vector. SMILES represents a molecular structure as a compact variable-length sequence of characters using simple vocabulary and grammar rules. As proven in recent studies, the RNN can generate SMILES strings because it effectively captures the long-term dependence of sequences. This occurs via the recurrent connections of units across the sequence steps. We form an RNN as a language model that generates a single-step moving window sequence of three-character substrings for each SMILES string. Here, the next substring in the sequence is predicted by conditioning the current substring and the given ECFP vector. This conditional generation of three-character substrings usually reduces the ratio of invalid SMILES by imposing additional restrictions on the subsequent character. The grammatically incorrect SMILES strings are deleted in the inspection step.ECFP is a circular topological fingerprint that has been successfully applied to represent organic molecules in vector forms. The encoding function f(∙), a five-layer DNN was built with 250 hidden units in each layer to identify the nonlinear relationship between molecular structures and their properties36.To obtain the property prediction function 37 with a mini-batch size of 100 and 500 training epochs. Each input and hidden layer in the neural networks is followed by a dropout layer with a dropout rate of 0.5 to prevent overfitting. Suppose that a dataset of k molecules and their annotated properties D = {}1–ki= is given. Subsequently, the RNN is trained on DRNN = {(e(mi), mi)}1–ki= by minimizing the cross entropy between the softmax output and mi, aiming at fulfilling the functional relationship d(e(mi)) = mi. In addition, the DNN is trained on DDNN = {(e(mi), ti)}1–ki= by minimizing the mean squared error between f(e(mi)) and ti. All neural networks were implemented using the Keras library based on the GPU-accelerated Theano library.In the RNN, the output layer is a softmax activation function to indicate the probability distribution of substrings. As for a DNN, all hidden layers use a logistic sigmoid activation function, and the output layer employs a linear function. The inputs of the RNN and DNN are 5000-dimensional ECFP vectors. All neural networks are trained by backpropagation using the Adam optimizerP0 in Fig. N to elements chosen with a ratio of 0.01 in an individual ECFP vector. The overall evolution was terminated when (1) the number of generations reached 500 and (2) the fitness was not enhanced during 30 consecutive generations. The default values in the DEAP library were used for the additional settings.The GA procedure was implemented using the Distributed Evolutionary Algorithms (DEAP) library in Python. The size of the population, crossover rate, and mutation rate are set to 50, 0.7, and 0.3, respectively. Following an initial mutation in each generation  using the hybrid B3LYP functional and all-electron 6-31G basis sets. A single-point time-dependent DFT calculation was performed with this geometry to calculate the vertical excitation energies to the lowest singlet state (S1). Symmetry constraints were not imposed in any of the calculations.All quantum chemical calculations were performed with the Gaussian 09 program suite1 energy. Accordingly, the RNN and DNN models were trained with a chemical library that comprises 10,000 to 100,000 molecules (with molecular weights between 200 and 600 g/mol) randomly sampled from the PubChem database32. Each molecule was labeled with the excitation energy (S1), molecular orbital energies , and lowest unoccupied molecular orbital (LUMO)) by the DFT calculation. As the amount of training data increases, the performance of the RNN and DNN models improves accordingly, as summarized in Table 1, HOMO, and LUMO were 0.977, 0.948, and 0.96, respectively, in a tenfold cross-validation  and f(∙), enable successful molecular evolution by acquiring the knowledge latent in the molecular data. In most cases, multiple evolutions of the same seed molecule occur along different design paths owing to the randomness of GA. Therefore, more diverse offspring can be obtained using an iterative approach.As a result, the deep learning-based functions, 1 values smaller than 1.77 eV using data with an S1 distribution above 1.77 eV, we selected the 30 molecules with the smallest S1 values in the training data as seed molecules. Based on the sampled 30 molecular seeds, the process of generating new molecules was repeated 300 times to derive new molecules with S1 lower than 1.77 eV. We calculated the new molecules by DFT and then re-trained the RNN and DNN models, similar to the initial training process. We defined this iterative process as a “phase” and repeated it three times. When proceeding with the next phase, we selected the 30 molecules with the lowest S1 values as the seed, including the new molecules created in the previous phase. This yielded an average S1 value for the training data of 4.91 eV, and the variance is 2.11. However, the aforementioned process produces new molecules for which the S1 distribution is relatively lower than that of the training data, as shown in Fig. 1 of the molecules produced in the first phase is 2.20, and the variance is 1.40. The averages in the second and third phases are 2.22 and 2.31, with variances of 1.38 and 1.36, respectively. Although the average value rises slightly as the number of phases increases, the variance gradually decreases. This intensifies the creation of molecular structures with the desired physical properties.The data-driven approach is heavily influenced by the training data. The challenge would therefore be to obtain a group of molecules outside the scope of the specified target property. As a final experiment, to generate a molecular structure with properties in the extrapolation area, we added a process that repeatedly calculates newly generated molecules and re-trains the RNN and DNN models. To create a group of molecules with S1 values that are relatively lower than those in the previous phase increases. After the first phase, the number of newly generated molecules with S1 values lower than 1.77 eV is 12. In the second and third phases, this number becomes 37 and 58, respectively. This process makes it possible to secure a group of potential new materials with properties that are not in the training data without significant changes in the shape of the molecular structure , quantitative estimate of drug-likeness (QED), and the central nervous system multi-parameter optimization (CNS MPO) tasks. In addition, we also selected the 100 top-scoring molecules from the ChEMBL25 test dataset as conditional seed to compare with the baselines. The EDM model generated a single batch of 500 SMILES strings. The performance of the model was similar to that of the cRNN in that they generated SMILES strings by extracting the ECFP that satisfied the initially constrained properties. Overall, the validation results confirm that the EDM method delivers performance comparable to that of the cRNN and other algorithms by achieving the maximum score for all eight of the given tasks. These results are summarized in Table An entirely data-driven evolutionary molecular design methodology based on deep learning models was developed in this study. In the proposed method, a GA along with RNN and DNN models were used to evolve the fingerprint vectors of seed molecules. The RNN decoder reconstructed chemically valid molecular structures in the SMILES format from the evolved fingerprint vectors without resorting to predefined chemical rules. In addition, the DNN efficiently evaluated the suitability of the evolved molecules even within a more complex range of properties.The closed-loop evolutionary workflow guided by deep learning automatically and effectively derived target molecules and discovered rational design paths by elucidating the relationship between the structural features and their effect on the molecular properties. Furthermore, owing to the inherent nature of data-driven methodologies, the molecular design performance can be influenced by the characteristics of the training data. Therefore, the training data should be prepared carefully according to the design purpose and situation. Unlike the test cases used illustratively in this study, the data to train the RNN and DNN need not be the same and could perhaps be configured differently depending on the design target. Moreover, traditional generative models based on data-driven approaches have limited ability to design new molecules with properties that are not included in the training datasets. In contrast, the proposed method can be designed to produce a new group of candidates by repeating the generation and calculation in that direction even if the molecules with the desired range of chemical characteristics are not included in the training data.Lately, various computer-aided techniques for designing materials, such as inverse designs, exhaustive enumerations, and molecular structure optimization models, have been proposed. Because each method has its advantages and disadvantages, the methods may act synergistically when used together rather than alone. In this respect, our evolutionary design method is also expected to be a promising tool with which to explore the enormous chemical space and facilitate the discovery of novel materials.Supplementary Information.


Gynecological Teaching Associates (GTAs) and Male Urogenital Teaching Associates (MUTAs) are individuals trained to instruct health professional learners with their own body to conduct accurate, patient-centered breast, pelvic, urogenital, rectal, and/or prostate examinations. Evidence indicates that this results in improvements in technical competence and communication skills, but there is wide variability to how such programs are implemented and engaged within the curriculum. In this scoping review, we mapped evidence regarding (1) how GTA/MUTA programs are utilized with health professional learners, (2) how GTA/MUTA programs are implemented using the Association of Standardized Patient Educators (ASPE) Standards of Best Practice (SOBP) as a framework, and (3) what broad outcomes are addressed in publications.PubMed, ERIC, PsychINFO, CINAHL, and Sociological Abstracts were searched for all publications addressing instruction of physical examinations with a GTA/MUTA and/or administration of GTA/MUTA programs. Studies were charted in tandem until consensus was identified and then charted individually, using an iterative process. The scoping review protocol was registered prospectively.One hundred and one articles were identified, and nearly all highlighted positive results regarding GTA/MUTA programs. Most studies addressed medical students within the USA and Europe. During instructional sessions, three (SD=1.4) learners worked with each GTA/MUTA and an average of 32 min (SD=17) was allocated per learner. GTAs/MUTA instructed both independently (n=33) and in pairs (n=51). Thirty-eight articles provided detailed information consistent with one or more of the Domains of the ASPE SOBP, with six providing specific information regarding safe work environments.While studies demonstrate consistently positive outcomes for learners, there is wide variability in implementation patterns. This variability may impact learning outcomes and impact both physical and psychological safety for GTAs/MUTAs and learners. Terminology used to refer to GTAs/MUTAs is inconsistent and may obscure relevant publications. Additional research is indicated to explore the pedagogical variables that result in positive learning outcomes and examine methods to ensure physical and psychological safety of GTAs/MUTAs and learners.https://osf.io/x9w2u/.The online version contains supplementary material available at 10.1186/s41077-021-00172-2. Gynecological Teaching Associates (GTAs) “teach, assess, and provide feedback to learners about accurate pelvic, rectal and/or breast examination techniques. They also address the communication skills needed to provide a comfortable exam in a standardized manner, while using their bodies [to instruct] in a supportive, non-threatening environment” . Male UrThe seminal work on GTA methodology grew from the founder’s experience with standardized patient (SP) methodology , and GTATwo systematic reviews have been conducted in relation to GTAs. Jha, Setna, Quinton, and Roberts  evaluateHow are GTA and MUTA programs utilized within the education of health professional learners?How are GTA and MUTA programs implemented?What broad outcomes do GTA and MUTA publications address?The objective of this scoping review is to map the available evidence regarding implementation and utilization of GTA and MUTA programs in the education of health professional students, in alignment with Preferred Reporting Items for Systematic Review and Meta-Analyses extension for Scoping Review (PRISMA-ScR) guidelines . The folThe protocol was drafted using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols  and reArticles were included if they discussed (1) instruction of breast, pelvic, rectal, and/or urogenital examination with a GTA/MUTA or (2) administration of a GTA/MUTA program. No limitations were placed on study methodology or timeframe, but the full text had to be available in the English language. Articles where a healthcare professional was providing all instruction on a live human model who provided no feedback were excluded, as were Letters to the Editor that did not substantially contribute to the field.PubMed, ERIC, PsychINFO, CINAHL, and Sociological Abstracts were searched on January 23, 2019. PubMed and CINAHL, as the search engines with the most relevant articles, were searched again on May 22, 2020, to identify any recent publications. The search strategy used in PubMed is available in Fig. Each title and/or abstract was reviewed by an independent reviewer to determine whether inclusion criteria were met. The first thirty titles and/or abstracts were reviewed by two randomized reviewers to evaluate consistency among reviewers. Uncertainties and disagreements were resolved through discussion and group consensus. Once consensus was identified, the remaining articles were divided among the three reviewers who each screened independently.All articles that met inclusion criteria for step 1 were reviewed in full. The first twenty full-text articles were reviewed by two randomized reviewers to evaluate consistency among reviewers. Uncertainties and disagreements were resolved through discussion and group consensus. Once consensus was identified, the remaining articles were divided among the three reviewers who each screened independently.Reference lists of included articles were hand-searched for additional relevant articles.Data charting was completed within Google Forms/Sheets . The firWe charted data on each study’s characteristics , structure , implementation  reported positive outcomes, although such outcomes were variously defined. While many studies addressed a combination of outcomes, learner outcomes n=72) were the most common, followed by GTA/MUTA program outcomes (n=34), and GTA/MUTA individual-level outcomes (n=14). The studies on GTA/MUTA program outcomes included reports on unique programs, literature reviews, and surveys of several programs. This breakdown of outcomes is in alignment with the variety of study aims  was addressed in 48 studies whereas the number of learners per session  and session length  were more frequently quantified. Time allocated per learner varied by the exam being taught . GTAs most commonly taught pelvic examination techniques (n=75) followed by breast examination (n=35) and female rectal examination (n=13). MUTAs most commonly taught the male rectal/prostate examination techniques (n=21) and urogenital examination (n=17). Many studies incorporated additional details such as patient-centered communication techniques and eliciting a patient’s history, but details were insufficient to measure and quantify across studies.Thirty-three studies presented GTA/MUTA programs in which the GTA/MUTA instructed independently; 51 studies presented methods of paired instruction with either a peer GTA/MUTA or healthcare professional (most commonly a physician). The details of the paired instruction methods were variable: some programs had the GTA/MUTA experiencing the exam portray the “patient” role while the other GTA/MUTA portrays the “provider” role, demonstrating an exam and guiding learners; other programs had a second person  in the room to function only as a resource or chaperone.Of the 18 studies addressing training, the mean training duration for novice GTAs/MUTAs was 15 h (SD=10). Studies presenting their training in a matter of days and/or weeks were removed from this analysis due to ambiguity. Pay was addressed infrequently. Ten studies reported pay by the hour, but ranges were generally non-specific and/or widely variable across time and currency; these limitations resulted in omission from Online Supplemental Material: Additional file When compared to the five Domains within the ASPE SOBP , thirty-Despite publications presenting MUTA programs , 18 beinProgram descriptions also indicate that engagement patterns are similar in spite of wide variation in terminology. These variations in terminology challenge research efforts and obscure potentially beneficial publications. Kretzschmar’s  landmarkAlthough much work has been done to assess the learner’s response to the program (which is consistently positive), the characteristics that make GTA/MUTA programs most valuable have yet to be sufficiently identified and articulated. For example, GTA/MUTA preparation, learner objectives, learner preparation, and time allocated to the session will impact learner outcomes, but the importance of these individual variables is not yet known or fully understood. In most studies, these variables were either not explicit or insufficiently specific to evaluate. Many studies report that the GTAs/MUTAs provide “feedback” or “instruction,” yet both of those processes can be undertaken in a variety of methods. Real-time feedback during a session  is distinct from completion of a checklist on conclusion of the session. Adhering to the publication recommendations to enhance the quality of Standardized Patient research  will enhThere is some evidence exploring use of task trainers before and after GTA sessions , but theThe ASPE SOBP  was the This instructional methodology grew out of significant ethical concerns regarding the common practice of incorporating sex workers and/or unconsented anesthetized clients into instructional settings –29. GTAsThese practices highlight the critical importance of robust administrative processes including recruitment, hiring, and provision of rigorous training to support GTAs/MUTAs becoming effective instructors. Poor practices may increase the risk of harm to GTAs/MUTAs such as having poorly defined expectations; having poorly prepared or unprofessional learners; and placing the GTA/MUTA in a position where another individual in the instructional session holds greater power than they do, or a position where they are/perceived to be unable to decline work (in part or in whole).Sexual harassment is common within medical schools –35 and hOne survey asked GTAs if they “felt ‘used’”; fortunately they did not, but this is a legitimate concern  that reqBy addressing these concerns, we can emphasize the safe practice of these skills as they relate to the learner’s future patients. While GTAs/MUTAs are not typically portraying a patient role during their instructional session, they are human and so experience the same sensations, and potentially harms, as a patient may when receiving care. Making the transition from seeing the GTA/MUTA as an educational tool and focusing more on their experience as humans who instruct with their body may help facilitate a shift in the ethical or moral perspectives used to guide this work , 39. SafSeveral studies address, at least in part, the learner experience within a GTA/MUTA session, yet few address the characteristics of GTAs/MUTAs and how they are individually impacted by their work e.g., –44). Add. Add44])It is possible that publications have been omitted due to variations in terminology and/or indexing. GTA/MUTA methodology has been reported in several textbooks , 45 and This scoping review is not intended to provide recommendations on GTA/MUTA program structure as there are many contextual variables that inform decisions at the local level. This study is rather intended to map the current state of the literature, which has been accomplished. Studies about GTA/MUTA methodology have demonstrated overwhelmingly positive learner outcomes for over 60 years, yet there is wide variability in how the studies are presented in the literature. Use of standardized terminology  in studiAdditional file 1. Title of Data: Studies Meeting Inclusion Criteria.Additional file 2. Title of Data: Broad Outcomes of GTA/MUTA Studies.Additional file 3. Title of Data: Utilization of GTA/MUTA Programs.Additional file 4. Title of Data: Implementation of GTA/MUTA Programs.


Direct effects on [3H]2-deoxyglucose uptake, insulin receptor phosphorylation, and regulation of metabolic function were tested ex vivo in the liver, soleus muscle, and white and brown adipose tissues. Bromocriptine and the antagonists domperidone, D2R antagonist, and haloperidol, antagonist of both dopamine receptor 1 (D1R) and D2R, were used to disclose dopamine receptors’ involvement.Dopamine is a key regulator of glucose metabolism in the central nervous system. However, dopamine is also present in the periphery and may have direct effects on insulin-sensitive tissues. Dopamine receptor 2 (D2R) agonist bromocriptine is a FDA-approved drug for type 2 diabetes. Herein, we explored the role of peripheral dopamine and its receptors in regulating glucose uptake and metabolism on insulin-sensitive tissues. Peripheral dopamine effect in [3H]2-deoxyglucose uptake in insulin-sensitive tissues was tested in vivo. Ex vivo, only dopamine increased glucose uptake in the soleus, while bromocriptine increased it in the liver; the effects were reverted by haloperidol and domperidone, respectively. In adipose tissue, domperidone reverted dopamine- and bromocriptine-mediated potentiation of insulin-induced glucose uptake, but in turn increased the insulin receptor, Akt, AMPK, HSL, ACC, and ACL, phosphorylation. In the soleus muscle, AMPK-phosphorylation increased with bromocriptine and dopamine whose effects were suppressed by domperidone and haloperidol.Peripheral dopamine increases glucose uptake In conclusion, peripheral dopamine stimulates glucose uptake with its receptors being differentially involved in glucose uptake in insulin-sensitive tissues. Dopamine also has a role in lipid metabolism in white adipose tissue. Altogether, these results suggest that peripheral modulation of the dopaminergic system should be further evaluated as a putative therapeutic approach for metabolic disorders. Dopamine is the predominant catecholamine neurotransmitter in the mammalian central nervous system (CNS) regulating neuronal functions such as locomotor activity and movement coordination, cognition, emotion, positive reinforcement, food intake, and endocrine homeostasis . DopaminOutside the CNS, dopamine derived from the synaptic terminals of the peripheral nervous system regulates several processes such as hormone secretion, vascular tone, renal function, and gastrointestinal motility , also plIn the present study, we evaluated the role of peripheral exogenous dopamine in regulating glucose uptake, as well as its direct effect on glucose uptake, insulin signaling, and lipid metabolic function of insulin-sensitive tissues. Additionally, we characterize the dopamine receptors involved in these actions. We found that exogenous dopamine administration promotes glucose uptake. Moreover, dopamine directly acts on insulin-sensitive tissues through different receptors to regulate glucose uptake and insulin receptor phosphorylation and to modulate the metabolic function, namely, adenosine monophosphate kinase (AMPK) phosphorylation in the liver, adipose tissue, and the skeletal muscle and to regulate lipid metabolism–related proteins in the adipose tissue. Therefore, we postulated that the modulation of peripheral dopaminergic signaling in insulin-sensitive tissues must be further evaluated as a potential therapeutic approach to treat metabolic disorders.All experimental and care procedures conducted on the animals were approved by the Ethics Committee of Faculdade de Ciencias Médicas|Nova Medical School and by Direccao-Geral de Alimentacao e Veterinária (DGAV), the Portuguese National Authority for Animal Health Animal Care. Principles of laboratory care were followed in accordance with the European Union Directive for Protection of Vertebrates Used for Experimental and Other Scientific Ends (2010/63/EU).Experiments were performed on 8- to 10-week-old male Wistar rats (200–300 g) obtained from the vivarium of the Faculdade de Ciências Médicas|Nova Medical School, Portugal. Animals were kept under controlled temperature and humidity  with a 12-h light/dark cycle and ad libitum access to food and water.Animals were starved overnight and randomly divided into the control group (0.9% NaCl) versus dopamine -injected group (100 nmol). Dopamine or saline were administered in the tail vein in a bolus of 0.1 ml. Immediately after dopamine or saline administration, an oral glucose tolerance test (OGTT) with a bolus of 2-deoxy-D--glucose  mixed with glucose  was performed by gavage administration, to better mimic the food ingestion and all the molecular, anatomical, histological, and immunological characteristics of food ingestion. Glycemia was measured at the baseline and 1 h after glucose bolus administration with a glucometer  and test strips . In order to determine glucose-specific activity, 20 µL plasma (collected at the baseline and 1 h after glucose administration) was deproteinized with 200 µL ice-cold perchloric acid (0.4 N) and centrifuged, and its radioactivity was measured in a scintillation counter . Animals were euthanized 1 h post glucose bolus administration with sodium pentobarbital  and tissues, such as the liver, soleus muscle, and white  and brown adipose tissues (BAT), were collected (50–200 mg) and homogenized in 1 ml ice-cold perchloric acid (0.4 N). Tissue samples were centrifuged and 2-deoxy-D-[3H]glucose incorporation was measured in the supernatants by scintillation counting. Tissue glucose uptake  was calculated as described by 2/5%CO2-equilibrated tyrode solution . Three to four pieces of the tissue per Eppendorf tube (2 ml) were then incubated at 37°C for 15 min for stabilization and afterward moved to other tubes for incubation for 15 min per piece in different experimental conditions. Different tissues samples were incubated in the presence and absence of insulin (10 mU ml−1) together with dopamine (10 µM); dopamine plus domperidone (50 nM); dopamine plus haloperidol (500 nM), bromocriptine ); bromocriptine plus domperidone; and bromocriptine plus haloperidol. The samples were then incubated with 2-deoxy-D--glucose for 30 min and homogenized. Uptake of 2-deoxy-D--glucose was performed as described in the previous section. Results were expressed in cpm.mg tissue−1  and the liver, soleus, and WAT  (50–150 mg) samples were rapidly collected in an ice-cold 21%Otissue−1 .in vivo by our laboratory for the modulation of blood pressure and ventilation Drug concentrations were chosen with an aim to achieve the complete activation of receptors, and therefore were 10–100x higher than the respective EC50 for agonists and IC50 as described for antagonists. The dopamine concentration tested was based on previous data obtained , #4058S), adenosine monophosphate kinase , hormone sensitive lipase , acetyl CoA carboxylase , and ATP citrate lyase  were obtained from Abcam  or Cell Signalling Technology . Calnexin (AB0041-200) from SICGEN  was used as the loading control. Secondary antibodies of anti-rabbit and anti-goat samples were obtained from Bio-Rad (Spain) and Invitrogen or Thermo Fisher Scientific (United States), respectively. Membranes were revealed using an ECL substrate  in a VersaDoc system  and analyzed with ImageQuant® .For Western blot analyses, tissue explants were collected and immediately stored at−80ºC after a 15-min incubation under different experimental conditions. Tissue explants  were homogenized and processed for Western blot as previously described by Matafome . Primaryp < 0.05.In glucose uptake experiments, a piece of each evaluated tissue  was used and incubated with dopamine or its agonists and antagonists. For statistical analyses, all data were evaluated using GraphPad Prism Software, version 8  and presented as scatter plots or mean values with standard deviation (SD). Significance between the mean values was calculated by the one-way ANOVA with Dunnets’s and Bonferroni multiple comparison tests. Differences were considered significant at in vivo tissue glucose uptake. Dopamine (100 nmol) was administered intravenously immediately before oral glucose administration. Peripheral dopamine mediates vivo tissue uptake of glucose studies was performed; thus, insulin-sensitive tissues were incubated with selective dopamine receptor’s agonists and antagonists.Aiming to clarify the contribution of the specific dopamine receptors role in each insulin-sensitive tissue, ex −1) Dopamine effect on glucose uptake in the liver: Insulin significantly increased glucose uptake by 37% in the liver  in comparison with the control group  were determined by Western blot. As depicted in Dopamine induces InsR phosphorylation in the skeletal muscle: In the skeletal muscle, insulin and dopamine applied separately were able to induce InsR phosphorylation, by 8 and 18%, but when applied together the effect was not modified, suggesting that they act on the same signaling pathway . Haloper: Insulin significantly increased InsR phosphorylation by 24% in the mWAT and by 46% in eWAT, respectively  compared to control conditions , and ACL-Ser455. Insulin increased by 35% and HSL-Ser563 levels by 73% in mWAT and eWAT depots, respectively . InteresInterestingly, both dopamine and bromocriptine directly decreased by 50 and 66%, respectively, ACC-Ser79 phosphorylation levels in eWAT, meaning that these substances decreased ACC activity . This efAltogether, our data suggest that dopamine D2R is involved in glucose uptake–independent activation of InsR and in the regulation of pathways associated with lipid metabolism in WAT, by impacting on the expression of phosphorylated forms of AMPK, HSL, ACC, and ACL in both WAT depots.Herein, we described for the first time the direct effects of dopamine (mediated by different dopamine receptor activation) in glucose uptake and metabolism in insulin sensitivity tissues. We showed that peripheral dopamine stimulates glucose uptake in several tissues, in part through direct actions in the liver, the skeletal muscle, and WAT. Moreover, dopamine receptors, especially dopamine D2R, play a role in these tissues in the regulation of InsR, Akt, and AMPK phosphorylation. Also, dopamine D2R is involved in the regulation of lipid metabolism pathways in WAT, which could be relevant for unraveling new therapeutic approaches for obesity-related diseases. Herein, we also show that dopamine effects on glucose uptake occur preferentially through dopamine D1R in the skeletal muscle and through dopamine D2R in the liver and WAT .Dopamine effects on glucose metabolism were attributed for many years to the regulation of the central nervous system , supportin vivo that peripheral dopamine administration during an OGTT produces an overall enhancement of tissue glucose uptake by the insulin-sensitive tissues in the liver, WAT , the soleus muscle, and BAT suggesting that dopamine is involved in the peripheral glucose homeostasis. We also found that dopamine directly promotes peripheral glucose uptake in insulin sensitive tissues ex vivo, via its action on different dopamine receptor subtypes. Interestingly, increased in vivo glucose uptake in each tissue after dopamine administration does not resemble the ex vivo glucose uptake in the same tissues upon direct dopamine or bromocriptine stimulation, showing that dopamine may have direct and indirect effects on each of these tissues. For example, we can postulate that when administered systemically in vivo, dopamine may act to decrease sympathetic activation to efferent organs changing the metabolic turnover and the release of signaling molecules that will act distant from the releasing cells, as seen previously with other mediators  and the muscle and adipose tissue (insulin-dependent GLUT4)  , it seemInterestingly, domperidone induced InsR-Tyr972 phosphorylation in the liver and InsR-Tyr972 and Akt-Ser473 phosphorylation in WAT without any evident increase in glucose uptake, suggesting that the dopamine D2R impact on insulin signaling on these tissues exerts other metabolic effects in cells apart from glucose uptake. Altogether, the results of dopamine receptors modulation herein described point toward a major role of dopamine D2R in the peripheral control of glucose metabolism in the insulin-sensitive tissues with the exception of the soleus muscle where dopamine D1R appears to be a modulator of glucose uptake.Additionally, from our results, it is clear that dopamine D2R potentiated insulin–glucose uptake at mWAT, suggesting an interaction between insulin and dopamine signaling pathways to control glucose uptake in this tissue, while in the soleus muscle and the liver the effect seems to be additive. This is corroborated by the fact that dopamine- and bromocriptine-mediated glucose uptake in WAT is independent of InsR and Akt activation  since phosphorylation of both proteins is only stimulated by insulin. Dopamine is known to act on dopamine D1R and D2R to regulate cAMP levels inside the cell, regulating neuronal excitability through membrane availability of ion channels . Apart fvia dopamine D2R, while enhanced AMPK phosphorylation in the soleus muscle upon stimulation with dopamine and bromocriptine. Confirming the dopamine D2R modulation, the effects of dopamine D2R agonism on the soleus muscle AMPK phosphorylation were reversed by D2R antagonism.In the present study, we also describe for the first time that dopamine receptors regulate AMPK phosphorylation in eWAT and in the skeletal muscle. Interestingly, these effects were opposite since dopamine decreased AMPK phosphorylation in eWAT We also show that dopamine D1 receptors modulate glucose uptake in the skeletal muscle, suggesting different pathways mediating metabolism in the soleus muscle. Nevertheless, we cannot forget that AMPK also facilitates GLUT4 translocation to the plasma membrane  and therOn the other hand, dopamine D2R inhibition by domperidone in the presence of dopamine increased HSL, ACC, and ACL phosphorylation, and therefore activates HSL and ACL pathways and inactivates the ACC pathway, in both mWAT and eWAT showing dopamine involvement in lipid metabolism pathway modulation in the adipose tissue. HSL and ACL are involved in lipid catabolism pathways, so we can speculate that dopamine D2R inhibition promotes lipid oxidation in the adipose tissue. Regarding ACC, its main function is to catalyze lipogenesis, and therefore domperidone, by inactivating this enzyme, will promote a decrease in fatty acid synthesis and a shift of lipid metabolism in lipolytic pathways. This, together with HSL and ACL activation, will improve lipid metabolism in the adipose tissue and therefore will benefit metabolic disease treatment. Moreover, we recently described that bromocriptine treatment in GK rats, a type 2 diabetes rat model, upregulates catabolic pathways, improving the metabolic profile . AltogetOf note, we cannot forget the possibility of dopamine receptor heterodimerization on the interpretation of the results obtained in the present study, particularly between D1R and D2R. The formation of heterodimers between D2R and D1R are well established in the brain as described, for instance, by via D1R regulates glucose uptake in the soleus muscle, while via D2R regulates glucose uptake in the liver and WAT. Importantly, we found that dopamine also impacts lipid metabolism pathways in WAT, showing its importance for obesity. Nevertheless, the direct effects of dopamine only partially explain the tissue glucose uptake observed after its in vivo peripheral administration. Given that dopamine is not able to cross the blood–brain barrier, other peripheral indirect mechanisms of glucose homeostasis regulation by dopamine may be involved and should be addressed in the future. The present study adds knowledge about the peripheral dopaminergic signaling role over glucose uptake to the already described mechanisms. These results can support the effects of bromocriptine treatment in type 2 diabetes and contribute to the identification of new therapeutic strategies, based on the modulation of dopaminergic signaling, for the treatment of metabolic disorders such as type 2 diabetes, obesity, and metabolic syndrome.Overall, we found that peripheral dopamine modulates glucose uptake through direct and indirect actions in insulin-sensitive tissues. Dopamine D1R and D2R are differentially involved in glucose uptake and regulation of the insulin signaling pathway and AMPK phosphorylation in insulin-sensitive tissues. Particularly, dopamine


Dopamine has long been implicated as a critical neural substrate mediating anorexia nervosa (AN). Despite nearly 50 years of research, the putative direction of change in dopamine function remains unclear and no consensus on the mechanistic role of dopamine in AN has been achieved. We hypothesize two stages in AN– corresponding to initial development and entrenchment– characterized by opposite changes in dopamine. First, caloric restriction, particularly when combined with exercise, triggers an escalating spiral of increasing dopamine that facilitates the behavioral plasticity necessary to establish and reinforce weight-loss behaviors. Second, chronic self-starvation reverses this escalation to reduce or impair dopamine which, in turn, confers behavioral inflexibility and entrenchment of now established AN behaviors. This pattern of enhanced, followed by impaired dopamine might be a common path to many behavioral disorders characterized by reinforcement learning and subsequent behavioral inflexibility. If correct, our hypothesis has significant clinical and research implications for AN and other disorders, such as addiction and obesity. Worldwide, millions of people diet and struggle to lose weight, with high rates of relapse and weight rebound. This is not surprising considering that we evolved over millions of years to eat, a behavior that is highly reinforcing. Yet, evolution and a lifetime of reinforcement are seemingly overridden in a subset of dieters who develop anorexia nervosa (AN), an eating disorder characterized by unrelenting self-starvation.stop losing weight emerges, even when it is life threatening. Those with AN develop an obsessive preoccupation with being thin, often engaging in ritualistic, restrictive eating behaviors and vigorous, extended exercise. The nature of these behavioral changes have been compared to compulsions in obesity and addictive disorders . These Our hypothesis would suggest any gene variants that regulate how the dopamine system responds to weight loss, exercise or chronic stress may in turn modify AN risk. This might include dopamine-related genes or genes of other systems– such as leptin, ghrelin, HPA axis, insulin– that modulate dopamine. Risk modification may be stage specific such that some variants may render an individual more likely to develop AN (stage 1) or more likely to progress to severe, persisting AN (stage 2).Our hypothesis can be tested in humans by measuring dopamine  prior to and at timepoints following initiation of dieting in a prospective study. We predict that weight loss resulting from dieting, particularly in combination with exercise, will increase dopamine function in most participants, but those who develop AN will show a more pronounced dopamine increase. We predict that those with AN who develop behavioral rigidity and treatment resistance will subsequently exhibit impaired dopamine, while those who are successfully treated will not. Moreover, we predict that behavioral change in successful, non-AN dieters might be associated with a more modest, time-limited increase in dopamine, while unsuccessful dieters will exhibit a minimal change in dopamine during diet adherence. Impaired dopamine found in obese individuals  may rendOur hypothesis is specific to AN but reflects a broader pattern common to disorders marked by compulsive behaviorThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by NIDA, DA046058 (JB), NIMH, R21MH114182 (NB), US National Institute on Minority Health and Health Disparities of the NIH, G12MD007599 (NB), PSC-CUNY Awards jointly funded by the Professional Staff Congress and The City University of New York (JB and NB) and a Klarman Family Foundation Eating Disorders Grant (JB and NB).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vibrio vulnificus is known to cause necrotizing soft tissue infections (NSTIs). However, the pathogenic mechanism causing cellulitis, necrotizing fasciitis, muscle necrosis, and rapidly developing septicemia in humans have not been fully elucidated. Here, we report a multilayer analysis of tissue damage after subcutaneous bacterial inoculation as a murine model of V. vulnificus NSTIs. Our histopathological examination showed the progression of cellulitis, necrotizing fasciitis, and muscle necrosis worsening as the infection penetrated deeper into the muscle tissue layers. The increase in vascular permeability was the primary cause of the swelling and congestion, which are acute signs of inflammation in soft tissue and characteristic of human NSTIs. Most importantly, our sequential analysis revealed for the first time that V. vulnificus not only spreads along the skin and subcutaneous tissues or fascia but also invades deeper muscle tissues beyond the fascia as the crucial process of its lethality. Also, increased vascular permeability enabled V. vulnificus to proliferate in muscle tissue and enter the systemic circulation, escalating the bacterium’s lethality. Our finding may yield important clinical benefits to patients by helping physicians understand the impact of surgical debridement on the patient’s quality of life. Furthermore, this study provides a promising system to accelerate studies of virulence factors and eventually help establish new therapies. Vibrio vulnificus is a Gram-negative and highly motile bacterium that lives in the sea and brackish waters and is known as a major causative bacterium of necrotizing soft-tissue infections  and encapsulation, which helps resist phagocytic cells  inoculated healthy mice with the pathogen and quantified the degree of the skin and soft-tissue infection. In our thorough investigation of the murine wound infection model, we successfully observed dose- and time-dependent clinical signs, such as inflammation, edema, cellulitis, and necrotizing fasciitis by a multi-faceted approach. Here, we report for the first time that characteristic clinical signs primarily increased vascular permeability, signaling the spread in soft tissues and invasion in deep tissue by V. vulnificus. Our detailed analysis of the infection site may lead to potentially life-saving therapeutics against V. vulnificus NSTIs.To understand the pathogenic mechanism of culation . However in NSTI . HoweverAll animal studies were carried out in strict accordance with the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science JALAS; . The aniVibrio vulnificus CMCP6 and E4 was cultured aerobically in Luria-Bertani (LB) broth or on LB agar at 37°C. When required, the medium was supplemented with rifampicin (50 μg/ml) or ampicillin (100 μg/ml) for V. vulnificus.rtxA gene were amplified with primers: RTX up Fw (5'-GGA TCC TTG CCT TTT AAT TGA GCT TGC-3'), RTX up Rev (5'-TCT AGA TCT CAT TTA CCC TGT GAA GAA GTA TTC AAC-3'), RTX down Fw (5'-TCT AGA TGC TAA CGC ATG TTG GTG ATG-3'), and RTX down Rev (5'-GCA TGC ATG AGT AAT GAT GTT GGC TTT-3'). The underline shows the recognition sites of the restriction enzymes, BamHI, XbaI, and sphI, respectively. The amplicons were confirmed by Sanger sequencing, then digested with BamHI-XbaI and XbaI-SphI, and cloned into the suicide vector pYAK1, retaining sacB. E. coli BWλpir were used as a conjugal donor to V. vulnificus CMCP6 with spontaneous rifampicin resistance. pYAK1-rtxAKO was introduced into V. vulnificus CMCP6. Vibrio vulnificus retaining pYAK1-rtxAKO was cultured in LB broth containing 20% sucrose following the standard sacB-assisted allelic exchange method. Mutants were confirmed by PCR to detect expected changes in size at the rtxA locus. The plasmid pXen-13 containing a bacterial luminescent gene cluster (luxCDABE) was transformed into V. vulnificus via electroporation. Vibrio vulnificus was grown in LB medium supplemented with 100 μg/ml ampicillin with agitation (163 rpm) at 37°C.The gene deletion mutant was constructed as described previously . BrieflyFive-week-old female C57BL/6 and BALB/c mice were purchased from Charles River Laboratories Japan . C57BL/6 mice and BALB/c mice were bred and maintained under specific pathogen–free conditions at Kitasato University. The mice were housed in plastic cages in a group and were maintained on a standard laboratory diet  and tap water under a 12 h light and dark cycle. The ambient temperature during the study was maintained at about 21°C.600) with a microplate reader  with 0.1 ml of 5 mg/ml Evans blue dye . Whole thighs of the sacrificed mice were transected at the ankle and at the hip joint, peeled, divided into SC tissue with skin and muscle tissue, and incubated at 60°C for 24 h in three volumes of formamide. Quantitative analysis of extracts containing Evans blue dye was determined by measuring optical density of blue at 600 nm . For immunofluorescence staining, the paraffin-embedded sections were used. After deparaffinization, heat-based antigen retrieval and endogenous peroxidase blockade using 3% hydrogen peroxide were performed. Rabbit anti-neutrophil elastase  was added and incubated overnight at 4°C. Following a rinse in PBS, peroxidase (PO)-conjugated goat anti-rabbit IgG  was added and incubated for 1 h at room temperature. The site of the immunoreaction was visualized by the diaminobenzidine (DAB) reaction. Sections were counterstained with Mayer’s haematoxylin and mounted under coverslips. Image acquisition of the muscle tissue was performed using an inverted microscope  equipped with 5×/0.40, 10×/0.40, 20×/0.40, and 40×/0.40 objective lenses.Vibrio vulnificus was grown in LB medium supplemented with 100 μg/ml of rifampicin with agitation (163 rpm) at 37°C. Overnight cultures (100 μl) were inoculated into 2 ml of fresh LB medium and incubated for 2 h. Bacteria were harvested, washed with PBS (pH 7.2) containing 0.1% gelatine, and resuspended in fresh LB medium. Then, 106 colony-forming units (CFU)/mouse were SC inoculated in mice. Infected mice were sacrificed at 9 h post-infection. Whole-blood samples were collected in a syringe by cardiac puncture at multiple time points after infection and centrifuged at 1,200 g for 30 min, and sera were collected. Serum creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) concentrations were evaluated using Dimension EXL with the LM Integrated Chemistry System .Vibrio vulnificus pXen-13 was grown in LB medium supplemented with 100 μg/ml ampicillin with agitation (163 rpm) at 37°C. Overnight cultures (100 μl) were inoculated into 2 ml of fresh LB medium supplemented with 100 μg/ml ampicillin and incubated for 2 h. Bacteria were harvested, washed with PBS (pH 7.2) containing 0.1% gelatine, and resuspended in fresh LB medium. Then, 106 CFU/mouse were SC inoculated in BALB/c mice. Luminescence signals emanating from V. vulnificus were imaged at defined time points using an in vivo imaging system (IVIS) 200 imaging system  with a 1 min exposure time. The total photons emitted were acquired using the Living Image software package. Mice were anesthetized in chambers containing 2.0% isoflurane inhalant .Vibrio vulnificus was grown in LB medium supplemented with 100 μg/ml of rifampicin with agitation (163 rpm) at 37°C. Overnight cultures (100 μl) were inoculated into 2 ml of fresh LB medium and incubated for 2 h. Bacteria were harvested, washed with PBS (pH 7.2) containing 0.1% gelatine, and resuspended in fresh LB medium. Then, the bacteria were SC, intramuscularly (IM), or intravenously (IV) inoculated in mice. Infected mice were carefully monitored and sacrificed at endpoints to measure survival time or at defined time points to collect tissues. The collected muscles or spleen were suspended in cold PBS containing 0.1% gelatine, homogenized for 5 s with a lab mixer IKA EUROSTAR digital , and centrifuged at 800 rpm for 5 min. The supernatants were plated at 10-fold serial dilutions in duplicate on LB agar containing 50 μg/ml rifampicin and incubated for 12 h at 37°C. Vibrio vulnificus colonies were counted, and bacterial burden was determined by calculating the number of CFU/g.U test or Fisher’s exact test. Survival curves were analyzed using the log-rank test. A p value less than 0.05 was considered significant, and significance values are indicated as follow: *p < 0.05; **p < 0.01; and ***p < 0.001.Statistical analysis was performed using GraphPad Prism . Statistical differences between the two groups were analyzed using the Mann–Whitney Vibrio vulnificus NSTI is a fulminant infection that causes death within a few days in humans. The consistent assessment of the process of host death due to the pathogenicity of V. vulnificus is a prerequisite for an infection model. First, we investigated whether the wound infection model could assess the lethality of a clinical isolate strain. Clinical strain CMCP6 wild type (WT) and environmental strain E4 were SC inoculated into mice and observed for 72 h. Mice infected with CMCP6 WT caused dose-dependent death  at 12 h post-infection when the mice began to die  and significantly lower than that of CMCP6 WT (rtxA was frequently not detected from the spleen in the infected mice (n = 3 of 8), and the number of bacteria collected from the spleen was 4.59 × 102 CFU/g  seemed to have been the cause of death in mice via the SC routes of infection , and IV injections. IM-inoculated mice survived for the shortest amount of time, followed by SC- and IV-inoculated mice . Similarted mice . The hignfection , althoug with WT ,B. Thus,V. vulnificus wound infection; there are reports of cases of infection in healthy hosts (V. vulnificus has a mechanism to colonize, proliferate, and spread by evading innate immunity in the intradermal and SC tissues of healthy hosts. This study elucidated the relationship between bacterial proliferation and pathophysiological progression in the murine wound infection model at each infection step.Adequate infection models that mimic human disease are the key to analyzing the mechanisms of bacterial pathogenesis. In the hy hosts . The firhy hosts . The shoV. vulnificus (rtxA was reduced in resistance to neutrophils in a model of skin air sac infection (rtxA were a low ability to proliferate in the subcutaneous tissues compared with WT (V. vulnificus in the soft tissues. Thus, our infection model enables the investigating of the mechanisms for evading the host immunity and proliferation during the wound infection.Our wound infection model showed that a clinical isolate strain caused the lethal infection, whereas a strain lacking MARTX and an environmental strain lacking capsule were avirulent in the same dose . The MARlnificus . Lo et anfection . The MARnfection . The capnfection . The env with WT . These rV. vulnificus depends on flagellar-based motility and chemotaxis in the soft tissues (V. vulnificus WT in soft tissues shown by IVIS was the most extensive at 6 h post-infection after maximizing increased vascular permeability in the subcutaneous tissue at 4 h post-infection (V. vulnificus proliferates at the site where the vascular permeability is increased. This proliferation may be provided based on the sensing of nutrient sources and spread through the space induced by the increased vascular permeability. The increased vascular permeability caused by V. vulnificus infection plays a vital role in exacerbating the pathological condition of NSTI.The perivascular inflammation and excessive activation of neutrophils result in the release of neutrophil elastase, which also leads to increased vascular permeability . Our wou tissues . The sprnfection , 5A. Seqnfection , 5A. In nfection ; Table 1V. vulnificus NSTI but has never been reproduced in mouse models. In this study, we successfully reproduced the progression of necrosis from superficial layers to deep muscle tissue (V. vulnificus to evade host immunity, proliferate, and cause the pathologies.Necrosis is a characteristic pathological condition in e tissue . This pre tissue . Our wouV. vulnificus in soft tissue by IVIS and measurement of bacterial burden (rtxA was detected from the primary infection site by measurement of bacterial burden but could not be detected 9 h after infection by IVIS (rtxA survived (8 CFU/g and around 104 CFU/g in the systemic circulation (spleen; rtxA was not enough to increase the number of bacteria in the spleen. However, ΔrtxA has the potential to invade and proliferate in the muscle tissue depending on other mechanisms associated with the lethality. This point out was supported by the death of mice by the high dose of inoculation or by IM-inoculation of ΔrtxA (In the treatment for NSTI, extensive surgical debridement of the local infection site exhibiting multiple lesions and necrosis is generally performed to reduce the number of bacteria and prolong a patient’s life . Howeverl burden , 6. In al burden . In cont by IVIS , 6. Conssurvived . In our  spleen; . These iof ΔrtxA . Our datThe original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by Kyoko Shimabukuro The president of Kitasato University.KYa and TKad conducted experiments. KYa, TKas, and KYo designed the study. KYa analyzed data. KYa and TKas wrote the manuscript. SU supervised the study. All authors contributed to the article and approved the submitted version.This work was supported by the Japan Society for the Promotion of Science KAKENHI under Grant number 18H02350. The foundation had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. The authors have no additional financial interests.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study examined (1) the influence of ethical leadership on knowledge sharing, (2) the mediating role of psychological ownership, and (3) the moderating effect of professional commitment between ethical leadership (EL) and knowledge sharing (KS). Data were collected from 307 public listed Pakistani companies’ employees. Statistical analyses were performed by using SPSS Version 25 and AMOS version 22. The findings indicate a positive relationship between EL and KS behavior. Additionally, the impact of EL on KS was partially mediated by psychological ownership. Furthermore, professional commitment buffers the link between EL and KS. This study contributes to the body of knowledge in the field of leadership by confirming the role of ethics. The results show that ethical leaders develop employee attitudes  that contribute to employee KS behavior. Ethical leaders create and encourage a learning culture to enhance organizational performance. This study adds to the little data on the positive impact of EL on listed company’s employees and addresses the gaps in previous studies on the role of EL in changing environments. In addition, professional commitment as a moderator has not been previously investigated with ethical leadership antecedents. Leaders are credited with helping their organizations in getting an edge in the marketplace by managing their finances and teaching ethical values to the employees . Over thEL is defined as “the demonstration of normatively appropriate conduct through personal actions and interpersonal relationships, and the promotion of such conduct to followers through two-way communication, reinforcement, and decision-making”Social learning theory is one of the most frequently referenced theories to study the link between EL and followers conduct in the social learning workplace . We advo“Professional commitment is defined as loyalty, the desire to stay in a profession, and a sense of responsibility toward the profession’s particular problems and challenges”  extensively. This is the first study to examine how EL influences the development of workers’ ability to share knowledge during the changing environment. Second, we used social learning theory to show how strong employee professional commitment when combined with EL, leads to a willingness to share knowledge among colleagues. Third, by recognizing KS as a psychologically and interpersonally significant phenomenon, this study investigated the significance of EL as a predictor of information exchange in organizations. For the knowledge management literature, this is a fresh approach to understanding employees’ attitudes. From a practical perspective, our findings demonstrate that leaders play a critical role in promoting an information-sharing culture. Organizations that want to maximize the value of their intangible assets by increasing the use and retention of workers’ expertise and information e leader . This stvia observational learning, in which employees learn indirectly by seeing the activities and repercussions of ethical leaders’ actions  was found to be appropriate for this instrument. All items were measured in 5-likert scale.We used a 10-item scale created by a = 0.90) for the psychological ownership measure was satisfactory.α for professional commitment was (a = 0.95).Four scale items with 5-likert scale from a prior study on healthcare professional commitment were included in the member questionnaire to gage members’ level of professional commitment . Sample The employees’ KS behavior was assessed using In survey-based investigations, common method variance (CMV) should be investigated, particularly when data for independent and dependent variables are obtained simultaneously using the same technique at the same time. As advised by α values more than 0.7, and the composite reliability values in In the assessment criteria presented by χ2/df = 2.23, RMR = 0.03, GFI = 0.88, CFI = 0.90, and RMSEA = 0.02. See A confirmatory factor analysis (CFA) was conducted to assess the factor structure of the EL, KS, psychological ownership, and professional commitment scales. Different confirmatory factor analyses were performed through AMOS 22. The resulting 4 factor model demonstrated excellent fit, r = 0.668, p < 0.01). Furthermore, EL was positively related to both PO and PC . The Variance Inflation Factor was used to assess for multicollinearity in this study (VIF). The VIF values in Several variables were kept under control. Individual demographics  have been found to impact employees’ knowledge behaviors in the past . As a reR2 value of 0.35 revealed that EL explained 35% of the variation in KS behavior with F =165.70, p < 0.001. The findings revealed that EL was positively related to KS behavior . In via psychological ownership , showing that Hypothesis 2 was also accepted.β = −0.1046*), indicating that PC moderated the positive relationship between EL and employees’ KS behavior. In β = 0.1771, p = 0.0007) for employees with high PO, Thus, Hypothesis 3 is verified.The moderating effect of PC in the relationship between EL and KS was investigated using First, we found that EL was linked to employees’ willingness to share their knowledge, which is consistent with social learning theory . We alsoFirst, this study examines the effect of EL on employees’ knowledge-sharing behavior using a psychological perspective. In spite of previous research indicating that EL has a significant impact on employees’ risk-taking and pro-social behavior, such as employee voice, creativity and organizational citizenship, there is surprisingly little research on the impact of EL on employees’ KS . SharingOur findings also demonstrate that professional commitment is associated with an explanatory power that is comparable to psychological ownership in the EL- employee KS relationship. As Our research provides insight into how organizations might encourage their employees to share their knowledge in ways that are beneficial to the company as a whole, rather than just to themselves. First, according to our findings, ethical leaders are actively involved in stimulating information sharing among their staff. Employees who desire to boost KS at work may want to be mentally robust and to participate in professional development programs that encourage ethical conduct. Second, organizations could encourage EL by offering training programs for leaders, stressing the relevance of psychological concepts, and presenting examples of ethical behavior that leaders should demonstrate in their everyday behavior and management practices. Third, when it comes to encouraging information sharing within an organization, it is critical that employees understand the significance of both internal and external regulations. Consequently, organizations must regularly implement mental health programs to prevent interruptions in the flow of information among their employees.There are several possible drawbacks to this research as well. First, all factors, such as EL, psychological ownership, professional commitment, and KS, were derived from the same source. Because the data were acquired from the same source, a common technique bias may have emerged . Experimvia two-way communication, may contribute to followers’ good evaluations of their leader’s personality. This research examines and determined that EL is an effective approach to encourage KS among employees by using social learning theory. Our findings offer preliminary empirical evidence in favor of the theoretical model of EL as a foundation for learning organizations. To create an effective learning environment and encourage resource sharing among employees, these results emphasis the need for ethical leaders who not only support their followers psychologically but also act as a mentor to help them in developing their professional commitment.Most organizations find it challenging to motivate employees to share their knowledge and abilities with colleagues in a productive and effective manner. In the disciplines of organizational behavior and knowledge management, the results of this research may be used to better understand the links between EL, organizational performance, trust in leaders, and certain aspects of knowledge behavior in the workplace. The findings of this study support the hypothesis that EL is crucial in improving employees’ psychological wellbeing and developing employees’ loyalties to their jobs and organizations, allowing them to share knowledge with their colleagues. KS behavior can be strongly influenced by EL. The fact that a leader treats employee with dignity, honesty, fairness, and integrity, enables them to participate in decision-making; and encourages them to do so and the fact that the leader encourages normative and ethical behavior among his or her workers The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.JK, IS, and MZ contributed to the conception and design of the study. SZ organized the database. JK performed the statistical analysis. JK, IS, MZ, and SZ wrote the first draft of the manuscript. AV-M and NC-B wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.Funding for the publication fees of this article have been provided by Universidad Autónoma de Chile and Universidad Andrés Bello (NC-B and AV-M).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Kaposi sarcoma (KS) incidence has declined substantially since the advent of effective ART but it remains a frequent cancer among people living with HIV, including those on ART with a sustained undetectable HIV viral load and in late presenters. ART is responsible for KS improvement and resolution, but new onset (unmasking KS-IRIS) or sudden progression of preexisting KS  can occur, even in patients with a low degree of immunocompromise. Both paradoxical and unmasking KS-IRIS have been associated with significant morbidity and mortality. We carried out a literature review regarding the incidence, pathogenic mechanisms, risk factors, clinical presentation, and management strategies of KS-IRIS in the ART era.10 copies/mL, detectable pre-treatment plasma-KSHV, and initiation of ART alone without concurrent chemotherapy. Both paradoxical and unmasking KS-IRIS have been associated with significant morbidity and mortality, and thrombocytopenia  has been associated with death. KS-IRIS is not to be considered as ART failure, and an ART regimen must be pursued. Systemic chemotherapy for KS in conjunction with ART is recommended and, in contrast with management of IRIS for other opportunistic infections, glucocorticoids are contra-indicated. Despite our preliminary results, the place of targeted therapies in the prevention or treatment of KS-IRIS needs further assessment.People living with HIV (PLWH) with advanced immunosuppression who initiate antiretroviral therapy (ART) are susceptible to the occurrence of an immune reconstitution inflammatory syndrome (IRIS). Although ART is responsible for AIDS- associated Kaposi sarcoma (KS) improvement and resolution, new onset (unmasking KS-IRIS) or sudden progression of preexisting KS  can occur after a time delay of between a few days and 6 months after the initiation or resumption of ART, even in patients with a low degree of immunocompromise. KS-IRIS incidence varies from 2.4% to 39%, depending on study design, populations, and geographic regions. Risk factors for developing KS-IRIS include advanced KS tumor stage (T1), pre-treatment HIV viral load >5 log People living with HIV (PLWH) and with advanced immunosuppression who initiate antiretroviral therapy (ART) are susceptible to the occurrence of an immune reconstitution inflammatory syndrome (IRIS) . This syA wide spectrum of clinical manifestations can be observed during IRIS with two distinct temporal patterns recognized as (1) paradoxical IRIS, when there is a worsening or recurrence of previously treated opportunistic infections (OI) symptoms or other inflammatory auto-immune or neoplastic processes that occurs despite an earlier favorable response to therapy prior to ART, and (2) unmasking IRIS, when one or more of these events occurred following ART initiation ,7,8,9.KS has long remained the most common malignant condition in people living with HIV (PLWH) and is one of the most severe complications of full-blown AIDS.Herpesviridae family found in the lesions of all epidemiological forms of Kaposi’s sarcoma [KS is now recognized as a cytokine-mediated angioproliferative disease caused by a human herpesvirus  (HHV8),  sarcoma . KSHV en sarcoma ,15.The w sarcoma ,17.In resource-rich countries, KS incidence has declined substantially since the advent of effective ART ,19, but We carried out a literature review regarding the incidence, pathogenic mechanisms, risk factors, clinical presentation, and management strategies of KS-IRIS in the ART era.General IRIS case definitions, according to French et al. and Shelburne et al., are reported in International consensus case definitions have been developed for tuberculosis-IRIS (TB-IRIS)  and cryp10 in the HIV-1 RNA level, or with two of the following three minor criteria: (a) a 2-fold increase in the CD4+ T-cell count after ART, (b) an increase in the immune response (KSHV-antibodies), and (c) a spontaneous resolution of disease without specific chemotherapy with continuation of ART [Therefore, definitions of KS-IRIS have varied according to different studies. French et al. defines KS-IRIS as either an abrupt clinical worsening of a previously existing KS or a new presentation of a previously unknown KS in temporal association with initiation or reinitiation of ART or change to a more active regimen. For each of these clinical situations, KS-IRIS occurrence is associated with a concomitant reduction of at least 1 logn of ART .3 or ≥two fold rise in baseline CD4+ cell count. Furthermore, KS exacerbation was defined as having at least two of the following criteria: abrupt increase in the size or number of KS lesions, the appearance or exacerbation of lymphedema, and the appearance or increase of otherwise unexplained, gallium-negative, lung opacities on chest X-rays after starting ART [In the study conducted by Volkow et al., KS-IRIS diagnosis was considered if a clinical abrupt worsening of previously existing KS  or the development of KS (“unmasked”) occurred within the first 6 months after the initiation of ART, associated with a reduction of at least 1 log10 of HIV-1 RNA and/or an increase of ≥50 cells/mmting ART .Furthermore, in the absence of sufficient viral load reduction, Novak et al. proposed as an adequate response to ART a 50% or greater increase in CD4 cell count within 6 months after initiating a new or different ART regimen .According to Bower et al., the major factors used to diagnose IRIS-associated KS are the temporal relationship between rapid clinical progression of KS and the initiation of ART . Thus, There is also a need to distinguish between KS-IRIS and a recurrence/occurrence of KS despite virological control under ART. We and others ,17,32 haThe pathogenesis of KS involves KSHV, cytokines, and the host-immune response.3 regardless of CD4 T-cell count have been found to be associated with an increased risk of KS [In HIV-infected individuals, infection by KSHV is a necessary but not sufficient condition for KS development. Furthermore, not all HIV-KSHV coinfected individuals will develop the disease. Immunosuppression and HIV viral load have been found to be correlated with KS occurrence in PLWH ,38. Addisk of KS ,39.HIV-1 infection might promote KSHV pathogenesis through the effects of HIV-1 proteins (tat protein) on KSHV replication and indirectly through CD4 lymphocyte depletion and the production of inflammatory cytokines  ,12,40.KSHV induces viral and host cytokine production that promotes KS cell proliferation, differentiation and angiogenesis  ,41,42. PHence, successful control of KS obtained after ART initiation may be attributable to immune restoration and inhibition of HIV replication, thus decreasing HIV-1 angiogenic tat protein concentrations that promote KSHV survival and tumorigenesis within infected cells ,49, and IRIS results from the rapid expansion of antigen-specific CD4+ and CD8+ lymphocytes following initiation of ART, with an imbalance between pro- and anti-inflammatory immune responses . KS-IRISA meta-analysis  of cohorIRIS) [The incidence of KS-IRIS varies widely, from 2.4% to 39%, depending on study design, populations, geographic regions, and the type of IRIS investigated  ,55,56,57IRIS) ,53,54, aIRIS) . In two 3, and detectable baseline KSHV DNA in the plasma [KS-IRIS incidence and associated mortality were reported to be higher in low-resource settings compared to resource-rich countries . In a poe plasma . The pree plasma .However, in two urban ART clinics in South Africa  with IRIThe contribution to mortality of IRIS was also investigated among 2610 PLWH from the HOPS cohort in Washington. In this study, an increased risk of death was found in patients having experienced an IRIS event, with 23.2% of deaths versus 8.5% among those who had not, with a median delay of 40.6 months .Most studies have examined risk factors for paradoxical and unmasking IRIS. and few for KS-IRIS ,59.In the study by Achenbach et al., a greater median increase in CD4 T cell count was identified in patients with mucocutaneous KS-IRIS . A higheAlthough low CD4 counts at the start of ART drive the incidence of IRIS independently of the pathology involved ,36, KS-I5 Log/mL) [In a prospective cohort of 69 HIV infected adults conducted in Mozambique, Letang et al. identified four independent predictors of KS-IRIS including clinical pretreatment KS, detectable plasma KSHV DNA, hematocrit <30%, and high plasma HIV viral load (≥10  Log/mL) . Additio Log/mL) .The association between T1 KS stage and KS-IRIS risk found in this study confirms the hypothesis that higher KS disease burden increases the risk of KS-IRIS, as suggested for IRIS associated with other OI ,61,62,63However, in a study by Bower et al., neither tumor stage nor site of KS were associated with KS-IRIS risk, while KS-associated edema at baseline was more likely to be observed in patients with KS-IRIS . LikewisOf note, concurrent or recent use of glucocorticoids may lead to increased risk of KS-IRIS .Regarding KSHV DNA, detectable baseline plasma KSHV DNA was found to predict KS-IRIS. However, plasma KSHV DNA can be undetectable during KS-IRIS, as previously reported .The time of onset of KS-IRIS is variable, from a few days to 6 months after ART initiation, with the greatest risk within the first 2 months ,27,28,35A short onset delay after ART initiation does not seem to have any predictive value, and KS-IRIS can remain a time-limited phenomenon, controlled with a limited course of early systemic chemotherapy  or contiDepending on the clinical presentation and the location of the lesions, KS-IRIS can be life-threatening but also lead to functional or aesthetic consequences.Thus, paradoxical KS IRIS frequently presents as inflammation and enlargement of existing KS lesion with marked lesional swelling, increased tenderness, and peripheral edema or worsening edema . It is oKS may also extend or appear rapidly at new anatomical sites with variable symptomatology . In its Pulmonary manifestations include cough, dyspnea, hemoptysis, parenchymal nodular lesions, adenopathy, and pleural effusions that can necessitate thoracentesis and talc pleurodesis . The preMore atypical presentations of KS-IRIS have been reported, such as oral localization , chylousDiagnosis of KS can be confirmed with immunohistochemical staining using endothelial markers such as CD31, CD34, D2-40, and ERG, through skin biopsy or alternatively endoscopic, pleural ortransbronchial biopsy. No current guidelines recommend the use of virological diagnosis tools to detect the HHV-8 genome or antibodies for KS diagnosis, but HHV-8 can be identified by using LNA-1 antibody or by PCR. Imaging using modalities such as CT, MRI, and FDG-PET/CT scans can aid in diagnosing and staging cutaneous KS and lymph node involvement, as well as responses to treatment of KS lesions ,78.3 at 12 weeks) was found to be significantly associated with death [Both paradoxical and unmasking KS-IRIS have been associated with significant morbidity and mortality in patients with visceral disease, including persistence of mucocutaneous lesions, lymphedema and death ,79,80,81th death .More severe clinical presentations have been reported, with abrupt clinical exacerbation, fever, thrombocytopenia, anemia, hyponatremia, and hypoalbuminemia . HoweverIn contrast with the prevention of TB-IRIS which relies on a delay of ART initiation of at least four weeks after TB treatment initiation , ART shoThe best therapeutic approach for the prevention of KS-IRIS is still not currently defined. Management of AIDS-related Kaposi sarcoma depends on the location and extent of the disease, according to most guidelines ,91. IndiThus, decisions about whether to initiate chemotherapy together with ART to prevent KS-IRIS must consider the extent of the diseaseand the potential benefits and added toxicities of chemotherapy plus ART over ART alone, in particular for individuals with severe immunosuppression. Furthermore, potential drug–drug interactions between ART and chemotherapy need to be assessed . WhateveA meta-analysis of six randomized trials and three observational studies involving 792 HIV-infected patients with severe KS has shown that ART plus chemotherapy with paclitaxel, liposomal doxorubicin, or daunorubicin facilitated a significant reduction of disease progression, with no differences between the three drugs , but no However, Letang et al. reported, in a pooled analysis of three prospective cohorts of ART-naïve PLWH with KS from SAA and one from the UK, a positive impact of adjunctive chemotherapy on KS-IRIS risk . This reRecently, three randomized clinical trials conducted in SSA and Brazil evaluated different chemotherapy drug regimens for the prevention of KS-IRIS. However, liposomal anthracyclines, the gold standard of care, are not available in these countries, and only non-liposomal doxorubicin, bleomycin, vincristine, and etoposide may be currently administered, even though they are less effective and have worse side-effect profiles in cases of KS .A randomized trial conducted by Mosam et al. in South Africa found that the addition of chemotherapy with intravenous bleomycin/vincristine to ART did not affect rates of KS-IRIS . In a thIn a randomized trial evaluating the impact of initiating ART with either immediate or as-needed oral etoposide in PLWH with mild to moderate KS, immediate etoposide with ART provided early but non-durable clinical benefits , and redValganciclovir has been evaluated in the prevention of KS-IRIS due to the in vitro activity of this antiviral agent against KSHV and epidemiological data showing a reduction of KS incidence when given prophylactically for CMV infection ,104,105.KS-IRIS does not indicate the failure of ART or a need for changes in ART regimen ,58, and The staging for AIDS-related KS was developed in adults by the AIDS Clinical Trial Group (ACTG) of the National Institutes of Health in the pre-ART era  and was Currently, liposomal anthracyclines and paclitaxel are the two reference treatment for patients suffering from extensive and/or rapidly progressive KS, and liposomal anthracyclines are the gold standard of care. However, to our knowledge, the best therapeutic approach for KS-IRIS has not yet yet evaluated in controlled trials.In contrast with management of IRIS for some opportunistic infections, glucocorticoids may be harmful for KS-IRIS and are contra-indicated, because of the potential for life-threatening KS exacerbation ,109,110.In recent years, multiple targeted therapies for KS have been evaluated in clinical trials but, to our knowledge, none in patients with KS-IRIS ,118,119,Although no cases of irAEs related to anti-PD-1 therapy have been observed in a group of  HIV posiMoreover, a case of KSHV-associated B-cell lymphoproliferation, possibly attributable to pembrolizumab, has been observed in a heavily pretreated patient with KS and a history of elevated peripheral circulating cell-associated KSHV and KICS; this emphasizes the need for careful monitoring of these patients during treatment with targeted therapies .Although KS-IRIS is less frequent than other IRIS-related opportunistic infections, close clinical supervision is warranted for patients with KS who are initiating, changing, or resuming ART, as the onset of IRIS-associated KS flare can be observed in as little as three weeks. Particular vigilance is recommended in the case of KS with visceral involvement and peripheral edema, including in those with minimally altered immune status. Clinicians should remain cautious as IRIS-KS is often not readily recognized and may occur in patients resuming ART after a long interruption period. No preventive treatment for KS-IRIS has yet been validated, and glucocorticoids may be harmful for KS-IRIS treatment, therefore they should be limited to life-threatening cases only.