The neuromodulation effect of low-intensity focused ultrasound (LIFU) is highly target-specific. Unintended off-target neuronal excitation can be elicited when the beam focusing accuracy and resolution are limited, whereas the resulted side effect has not been evaluated quantitatively. There is also a lack of methods addressing the minimization of such side effects. Therefore, this work introduces a computational model of unintended neuronal excitation during LIFU neuromodulation, which evaluates the off-target activation area (OTAA) by integrating an ultrasound field model with the neuronal spiking model. In addition, a phased array beam focusing scheme called constrained optimal resolution beamforming (CORB) is proposed to minimize the off-target neuronal excitation area while ensuring effective stimulation in the target brain region. A lower bound of the OTAA is analytically approximated in a simplified homogeneous medium, which could guide the selection of transducer parameters such as aperture size and operating frequency. Simulations in a human head model using three transducer setups show that CORB markedly reduces the OTAA compared with two benchmark beam focusing methods. The high neuromodulation resolution demonstrates the capability of LIFU to effectively limit the side effects during neuromodulation, allowing future clinical applications such as treatment of neuropsychiatric disorders. Compared with traditional stimulation techniques such as transcranial magnetic stimulation, LIFU has the potential to reach deep targets inside the brain and achieve reversible neuromodulation effects with millimeter-level focusing resolution while being markedly less invasive than deep brain stimulation4.Low-intensity focused ultrasound (LIFU) neuromodulation, an emerging therapeutic tool for altering neuronal activity, has received increased attention for treating various neuropsychiatric disorders6, even with a separation of merely 1 mm. In addition, neuromodulatory effects are not limited to the target location only. Off-target focal pressure peaks can occur during stimulation due to standing waves, which potentially resulted in the reported neuromodulatory effects in many previous experiments7. Opposite neuromodulation effects can also occur at the same target using focused and unfocused ultrasound stimulation, due to differences in the number of neurons affected8.Recent studies have shown that the LIFU neuromodulation effect is sensitive to the stimulated region. Different target locations of ultrasound power focusing can result in different sensations and motor responses9. Hence, to quantitatively evaluate the unintended neuronal excitation, this work proposes to computationally model the off-target activation area (OTAA) of LIFU neuromodulation, which indicates the region with unintended neuronal spiking responses to LIFU while the target is successfully excited simultaneously10. The computational modeling of OTAA is mainly based on the interaction between the ultrasound propagation model and the multi-scale optimized neuronal intramembrane cavitation (SONIC) model that allows quantitative interpretation of LIFU neuronal excitation effects11. To effectively minimize the OTAA, this work considers using phased array transducers for LIFU stimulation, which allows more flexible control of the ultrasound radiation pattern compared to the single-element transducers used in most literature. A new beam focusing algorithm, named constrained optimal resolution beamforming (CORB), is proposed to minimize unintended neuronal excitation effects on off-target brain regions while ensuring effective stimulation of the target point. The OTAA with CORB used is computationally evaluated in a 2-D human head model12 given three potential transducer deployments . Compared to traditional beam focusing algorithms, CORB method elicits much smaller OTAA while ensuring successful stimulation to the target neurons.Although such unintended neuromodulation effects can be potentially observed in many scenarios, there is still a lack of quantitative evaluations on the corresponding influence, i.e., the specific brain regions affected. Accordingly, the methods for minimizing the unintended neuronal excitation have also not been well investigated yet, even though the dependence of the ultrasound focal spot size on transducer design parameters has been understood13. The SONIC model can then be applied to calculate the spiking pattern of neurons in each brain region, as in Fig. 11. To demonstrate the influence of unintended neuronal excitation, this work evaluates OTAA, i.e., the off-target area of neurons excited. The OTAA measures the highlighted area in Fig. This work computationally models the unintended neuronal excitation during LIFU neuromodulation relying on a unified simulation framework of ultrasound propagation and neuronal responses. The modeling methods are illustrated in Fig. 18. In addition, although the neuronal spike count generally increases with intensity in both the continuous-wave and pulsed-wave scenarios, such an increase with pulsed waves of low duty cycles is slower and non-monotonic, which could influence the neuronal excitation resolution as discussed in further numerical evaluations.The SONIC model can simulate excitatory neuronal spiking responses to ultrasound stimulation, which rely on several factors, including the type of neuron, stimulation signal parameters , etc. In the current study, the spiking pattern of regular spiking neurons is modeled using SONIC, the analysis of which can be extended to various types of neurons in future research. Other factors, including brain region and anesthesia state, are beyond the scope of the current study. Simulated neuronal spiking patterns under continuous and pulsed LIFU stimulation using different ultrasound intensities are shown in Fig. 19. The off-target suppression benchmark maximizes the power focused to the target point while simultaneously limiting the power delivered to all off-target brain regions20. The details of these benchmark methods are introduced in the This work evaluates the unintended neuronal excitation elicited with three beam focusing methods, including two benchmark methods, i.e., the conjugate beamforming benchmark and the off-target suppression benchmark, and the proposed CORB method. The conjugate benchmark maximizes ultrasound intensity at the target point, which has similar mechanism to most focusing methods used in literature, e.g., time-reversal methodM-element transducer array operating at frequency f is used for neuromodulation. An input sine signal with constant amplitude m, generating the acoustic field during a pulse with duration T or a continuous wave stimulation17. The acoustic pressure generated by the waveform of each single element m is denoted by G in the brain tissue region. If the wave equation in k-Wave13 is considered linear, m to the point G. The cumulative acoustic signal at a given point can be modeled as a linear combination of signals from all active transducer elements, i.e., m, where G. Therefore, the average ultrasound intensity at any point G, which is used in CORB to calculate neuronal spiking responses to LIFU stimulation, is22G are represented by c(G) , respectively.The CORB method optimizes transducer array beam focusing relying on the ultrasound propagation and intensity model in tissues, which is described as follows. It is assumed that an G in the off-target brain region, the intensity needs to be smaller than a relatively “conservative” threshold 18. The values of The CORB method is designed to estimate neuronal spiking responses based on the ultrasound intensity model in the previous section, and minimize the unintended neuronal excitation area, which is subject to the excitation thresholds as illustrated in Fig. I(G) higher than the “conservative” threshold Given the neuronal excitation thresholds, the unintended neuronal excitation can be modeled by OTAA, which can further be approximated by P is defined as a scaling of the sum power of the transducer array, and 23. To solve this problem, the step function is first approximated using a logistic function, which yields the approximated objective function24. Because G according to –(i) in Figs. In Fig. The current study introduces novel methodology involving the use of the OTAA to evaluate unintended neuronal excitation during LIFU neuromodulation and proposes the CORB method for minimizing unintended neuronal excitatory effects while ensuring the excitation of target neurons. The lower bound of OTAA is theoretically derived and validated computationally. Simulations within a 2-D human head model are conducted to numerically evaluate the OTAA of three beam focusing methods with three different transducer setups. Numerical results show that the OTAA increases with stimulation intensity, duration, and duty cycle. Also, the proposed CORB method can significantly reduce areas with unintended neuronal spiking responses compared with benchmark methods using all three array setups.27 typically include modulation areas from greater than 20 mm29. The resolution of transcranial magnetic stimulation in each dimension is also usually on a cm scale30. Traditional ultrasound neuromodulation methods, either with single-element or phased array transducers, have focusing mechanisms similar to the conjugate benchmark, which is significantly outperformed by the proposed CORB method. The achievable FWHM resolution of traditional LIFU neuromodulation in human applications is usually several millimeters laterally and can achieve cm scale along the transducer axis, given the low frequency range (250–500 kHz)16. The estimated equivalent OTAA, given the higher stimulation intensity used3 and the simulated neuronal response patterns in Fig. Simulation results show that LIFU is a promising technique for high-resolution neuromodulation with reduced invasiveness in clinical applications. Although the resolution metric is difficult to unify, a simple comparison between this work and previous results reported in the literature implies that LIFU with CORB may achieve higher neuromodulation resolution than many other neuromodulation techniques. Methods based on transcranial electrical currents31 has created a cranial window so that the transducer can be placed directly on dura. Another work32 investigates ultrasound transducer miniaturization for implantation, and has proposed to consider implanting multiple transducers into a rat. Literature33 has already shown the high resolution of multi-array transducer setups as well. Therefore, despite few current research using intracranial multi-array transducer setups, this direction could be explored in the future, and the proposed CORB method should be able to support this setup.This study has also sought to establish stimulation parameters and protocols for clinical applications involving neuromodulation resolution requirements. By simulating the neuronal spike pattern with the SONIC model Fig. , the stu35. Given the similar excitatory effect of LIFU, high-resolution focus of ultrasound energy to the internal globus pallidus using CORB may serve as a non-invasive treatment for Parkinson’s disease after experimental validations.The high-resolution neuronal excitation effect demonstrated in this study could potentially be used to treat neuropsychiatric disorders. For example, a previously reported therapy for Parkinson’s disease stimulates the internal globus pallidus with electrical currents, with the probable mechanism being suppression of the thalamus activity via excitation of the internal globus pallidus neuronsThe fundamental mechanism of ultrasonic neuromodulation is still not completely clear. Although the definition and evaluation of OTAA in this work is based on SONIC model, the results, e.g., comparison of OTAA between different methods, will not be qualitatively different when other ultrasonic neuromodulation mechanisms are involved. The reason is that CORB method only relies on two intensity thresholds, 36. The SONIC model supports the spike pattern simulation of multiple types of neurons including subthalamic nucleus neurons, which allows the OTAA model to account for brain region specificity11. In addition, neuronal activation elicited by ultrasound stimulation can be subsequently transmitted to other functionally connected brain regions37. Thus, a comprehensive understanding of the spike-level response of brain circuits to ultrasound stimulation could guide the design of beam focusing techniques.The proposed computational model of unintended neuronal excitation does not address other important considerations in evaluating neuromodulatory outcomes, including other types of neuromodulation effects, differential effects across brain regions, and the impact of brain anesthesia state on proper neuromodulation resolution metrics. Recent reports of the complexity of neural responses to ultrasound stimulation can benefit the generalization of the model through integration of corresponding excitation/inhibition mechanisms, such as the neuron-type-selectivity of responses to ultrasound PRFin vivo setup and further validate the effectiveness of the proposed method, future work could extend the current framework to 3-D scenarios and phantom experimental setups. The skull is also modeled as homogeneous medium based on one-layer assumption in this work, leaving heterogeneity to be considered in the next step38. In addition, the acoustic parameter selection and computational accuracy can influence the effectiveness of the designed beam pattern39. Thus, a sensitivity analysis of neuromodulation resolution based on the acoustic parameters of brain tissue will be the focus of future studies.To better approximate the Supplementary Information.


Hospice and Palliative Care (HPC) is in high demand in China; however, the country is facing the shortage of qualified HPC nurses. A well-suited competence framework is needed to promote HPC human resource development. Nevertheless, existing unstandardized single-structured frameworks may not be sufficient to meet this need. This study aimed at constructing a comprehensive multi-structured HPC competence framework for nurses.This study employed a mixed-method approach, including a systematic review and qualitative interview for HPC competence profile extraction, a two-round Delphi survey to determine the competences for the framework, and a cross-sectional study for framework structure exploration. The competence profiles were extracted from publications from academic databases and interviews recruiting nurses working in the HPC field. The research team synthesized profiles and transferred them to competences utilizing existing competence dictionaries. These synthesized competences were then subjected to Delphi expert panels to determine the framework elements. The study analyzed theoretical structure of the framework through exploratory factor analysis (EFA) based on a cross-sectional study receiving 491 valid questionnaires.The systematic review involved 30 publications from 10 countries between 1995 and 2021, while 13 nurses from three hospitals were interviewed. In total, 87 and 48 competence profiles were respectively extracted from systematic review and interview and later synthesized into 32 competences. After the Delphi survey, 25 competences were incorporated into the HPC competence framework for nurses. The EFA found a two-factor structure, with factor 1 comprising 18 competences namely Basic Competences; factor 2 concluding 7 competences namely Developmental Competences.The two-factor HPC competence framework provided valuable insights into the need and directions of Chinese HPC nurses’ development.The online version contains supplementary material available at 10.1186/s12904-023-01318-x. In mainland China, there is a high demand for hospice & palliative care (HPC) due to the ageing population, rising burden of chronic diseases, and poor quality of death. China’s seventh national census of 2020 reported the number of individuals aged ≥ 65 years was 190.64 million, accounting for 13.50% of the total population , hospice nurses . Th. Th64]. From a design perspective, our study extracted competencies from international publications and synthesized the attitudes of two key stakeholders: academic and clinical experts in the HPC field, and nurses working on the HPC frontline. Therefore, we are confident in stating that this 25-competencies framework has broad applicability, and nurses trained with these competencies as a target should be competent in clinical palliative care work in most countries. Furthermore, we have grouped the 25 competencies into Basic Competences and Developmental Competences based on clinical nurses' ratings. This grouping suggests that a continuous HPC education program may consider a two-stage design. We propose including Basic Competences in HPC training for general nurses or in the initial training for novice HPC nurses. Developmental Competences may be reserved for the continuous education program targeting experienced HPC nurses seeking career development.The strength of this study included 1) extracting competence from multi-resources including published literatures, clinical nurses’ interviews, and experts’ opinions, maximized the comprehensiveness and clinical usefulness of the extracted content; 2) exploring the underline structure of the constructed HPC competence framework that deepened the understanding of the Chinese nurses’ attitude on HPC competences, and increase the implementation potential of the framework. This study has a limitation due to the nature of Delphi survey. Although the Delphi panel members had diverse background and rich experience in HPC, they may not be adequate to represent all opinions of the HPC experts in China. Therefore, the online survey recruiting a large sample was conducted for more opinion collection. Another limitation was that clinical nurses may not have a holistic view or a clear understanding of the future HPC development in China, so the structure of the competency framework derived from the clinical nurse perspective alone may need to be modified in consultation with relevant government department officers and experts in the HPC field. Considering that clinical nurses reflected current realities of clinical palliative and hospice care, it was recommended using the known framework structure until further results are available.By extracting and synthesizing HPC related competence profiles for nurses from publications and interview transcripts and pursuing consensus from experts, we constructed a 25 HPC competence framework for nurses. This framework may have two underline factors that one about basic competence (18 competences) contributing to direct care and self-regulation, another one about developmental competence (7 competences) contributing to advanced practice and career development. This framework has promising implementation potential that researchers may consider using it as foundation to develop competence assessment or self-evaluation tools and education programs.Additional file 1. Systematic review searching keywords and strategy.Additional file 2. Qualitative Interview Outline.Additional file 3. Table of evidence of the systematic review.


The ongoing pandemic due to coronavirus disease (COVID-19) is not only causing casualties amongst patients but is also putting an enormous strain on healthcare workers worldwide, especially those in frontline of the COVID-19.This study examined the influence of pandemic fatigue on physicians’ mental health, with resilience as a mediator.This was a descriptive, cross-sectional study involving frontline young doctors at two university hospitals of Sfax, Tunisia. The Pandemic Fatigue Questionnaire, Brief resilience scale, Maslach Burnout Inventory and Satisfaction on call duty scale were used to collect data through an online survey. The survey was carried out through an anonymous questionnaire using Google Forms. Collected data was treated on SPSS program to make all the statistical analysis. The level of statistical significance was set at p<0.05.A total of 261 young doctors responded to the online survey. The mean pandemic fatigue score was 25.09 (out of 50). Terminal years of residency experience , being vaccinated  and staff inadequacy  were associated with elevated score of pandemic fatigue. Resilience partially mediated the relationships between (a) pandemic fatigue and different dimensions of burnout , depersonalization  and personal accomplishment  and (b) pandemic fatigue and satisfaction on call duty .Resilience reduces the effects of pandemic fatigue on young doctors’ mental health. Implementing resilience-promoting measures is essential to support physicians’ mental health and foster their well-being therefore improves the quality of care provided.None Declared


Artificial intelligence (AI) and radiomics algorithms in radiology and nuclear medicine have demonstrated a good performance as diagnostic, predictive or prognostic markers for several diseases with a high potential to be used as clinical tools. However, these algorithms should be further validated in clinical practice to spread their routinary use worldwide.https://www.frontiersin.org/research-topics/15427/artificial-intelligence-in-positron-emission-tomography) a wider and more comprehensive Research Topic on the role of AI and radiomics in radiology and nuclear medicine was launched in 2022. This Research Topic comprises 12 articles.After the successful publication of a previous Research Topic about Radiomics in Positron Emission Tomography (PET) on Frontiers in Medicine in 2021 . However, the authors demonstrated that there is currently no clear consensus on how XAI should be used in order to close the gap between medical professionals and deep learning algorithms for clinical implementation. Furthermore, De Vries et al. also suggested a systematic technical and clinical quality assessment of XAI methods.For successful employment of AI and deep learning algorithms in the clinical practice, explainable artificial intelligence (XAI) could be introduced for several imaging modalities as clearly discussed in the comprehensive review of De Vries et al. including 75 articles .Radiomic features could be very useful for a better prognostic stratification in patients with glioblastoma. The study of Chiesa et al. including 90 patients with glioblastoma applied a radiomic analysis focusing on healthy tissue ring around the surgical cavity on post-operative magnetic resonance imaging. This study provided a preliminary model for a decision support tool for a customization of the radiation target volume in glioblastoma patients to achieve a margin reduction strategy  images in the preoperative discrimination between lung invasive adenocarcinomas and non-invasive adenocarcinomas among 1,185 pulmonary nodules. The authors found that radiomics based on CT images showed good predictive performance in discriminating between these tumoral entities, especially in part solid nodule group. Furthermore, radiomics based on contrast enhanced CT images provided no additional value compared to non-contrast enhanced CT images .18F]FDG) PET/CT radiomic features to predict overall survival in 50 patients with locally advanced uterine cervical carcinoma. The authors found that standardized uptake value peak (SUVpeak) and the textural feature gray-level run-length matrix (GLRLM) presented the best performance to predict overall survival in patients with cervical cancer undergoing chemo-radiotherapy and brachytherapy .A brief research report evaluated the performance of fluorine-18 fluorodeoxyglucose .A retrospective study assessed the predictive ability of [18F]FDG PET/CT radiomics in the identification and correct classification of spine multiple myeloma lesions and bone metastases. The radiomics model constructed based on [18F]FDG PET/CT images achieved satisfactory diagnostic performance for the classification of multiple myeloma and bone metastases. In addition, the radiomics model showed significant improvement in diagnostic performance compared to human experts and PET conventional parameters .Another retrospective study on 131 patients explored the application of FDG PET/CT to predict event free survival in 163 patients with Hodgkin lymphoma. The Uptake Formula showed a similar performance to total metabolic tumor volume in predicting event free survival in Hodgkin lymphoma .A retrospective study from Ghezzo et al. tested on a cohort of 85 prostate cancer patients a recently proposed convolutional neural network for the automatic segmentation of intraprostatic cancer lesions on prostate specific membrane antigen PET images. The authors demonstrated that the AI model could be used to automatically segment intraprostatic cancer lesions to define the volume of interest for radiomics or deep learning analysis. However, more robust performance is needed for the generation of AI-based decision support technologies to be proposed in clinical practice .Yan et al. have summarized the application of radiomics for predicting recurrent pancreatitis, evaluating the clinical severity of pancreatitis, differentiating pancreatitis from pancreatic adenocarcinoma, and functional abdominal pain from pancreatitis, identifying pancreatis, its risk factors and complications .Beyond oncological indications of imaging methods, AI may be also applied for other indications. For instance, the review article of Flaus et al. developed a deep learning-based [18F]FDG PET image enhancement method using simulated brain PET to improve visualization of epileptogenic lesions. However, the authors recommended further evaluation to generalize their method and to assess its clinical performance in a larger cohort .Gu et al. proposed a weakly supervised model, Chest L-Transformer, to take these characteristics into account. The authors demonstrated a significant segmentation performance improvement over the current state-of-the-art while achieving competitive classification performance .Weakly supervised deep learning models have gained increasing popularity in medical image segmentation. However, these models are not suitable for the critical characteristics presented in chest radiographs: the global symmetry of chest radiographs and dependencies between lesions and their positions. In their study, Quak et al. including 67 patients demonstrated that the degradation of image quality on PET due to a reduction in injected activity at the end of the 68Ge/68Ga generator lifespan can be effectively counterbalanced by using AI-based PET denoising .Lastly, an original article by Finally, we would like to underline that AI and radiomics tools are widely used for research purpose in the fields of radiology and nuclear medicine. Nevertheless, large validation protocols and real-life experience are needed to allow an increasing use of these tools in clinical practice, with possible benefit for patients' treatments and outcomes.SA and GT drafted the manuscript and revised the final version. All authors contributed to the article and approved the submitted version.


Balance recovery often relies on successful stepping responses, which presumably require precise and rapid interactions between the cerebral cortex and the leg muscles. Yet, little is known about how cortico-muscular coupling (CMC) supports the execution of reactive stepping. We conducted an exploratory analysis investigating time-dependent CMC with specific leg muscles in a reactive stepping task. We analyzed high density EEG, EMG, and kinematics of 18 healthy young participants while exposing them to balance perturbations at different intensities, in the forward and backward directions. Participants were instructed to maintain their feet in place, unless stepping was unavoidable. Muscle-specific Granger causality analysis was conducted on single step- and stance-leg muscles over 13 EEG electrodes with a midfrontal scalp distribution. Time-frequency Granger causality analysis was used to identify CMC from cortex to muscles around perturbation onset, foot-off and foot strike events. We hypothesized that CMC would increase compared to baseline. In addition, we expected to observe different CMC between step and stance leg because of their functional role during the step response. In particular, we expected that CMC would be most evident for the agonist muscles while stepping, and that CMC would precede upregulation in EMG activity in these muscles. We observed distinct Granger gain dynamics over theta, alpha, beta, and low/high-gamma frequencies during the reactive balance response for all leg muscles in each step direction. Interestingly, between-leg differences in Granger gain were almost exclusively observed following the divergence of EMG activity. Our results demonstrate cortical involvement in the reactive balance response and provide insights into its temporal and spectral characteristics. Overall, our findings suggest that higher levels of CMC do not facilitate leg-specific EMG activity. Our work is relevant for clinical populations with impaired balance control, where CMC analysis may elucidate the underlying pathophysiological mechanisms. Performing daily activities  constantly challenges postural balance. With different postures and activities, continuous adaptation through contraction and relaxation of specific muscles allows the control of balance and the execution of corrective steps whenever these are needed. In the past decade, many studies using mobile EEG have provided evidence that the cortex plays an important role in postural control –3. This Postural perturbations are known to elicit several event-related potentials, suggesting cortical involvement in the ensuing balance recovery responses. The initial P1 response (30–90 ms) is thought to represent proprioceptive sensory afferents , followeCortico-muscular coupling (CMC) is a powerful analysis tool to investigate functional connectivity between the cerebral cortex and muscles across the body. CMC is known to take place with the Tibialis Anterior (TA) and Soleus (SO) muscles at beta 15–25 Hz) frequencies during isometric contraction and gamma (40–80 Hz) frequencies during isotonic contraction , 14. In –25 Hz frThe aim of this study was to investigate CMC from cortex to muscle through spectral Granger causality coupled to key events of the stepping response in balance. We hypothesized that CMC would occur over multiple frequency bands during stepping responses. In addition, we expected CMC to differ between the step and stance leg according to their differential muscle recruitment patterns inherent in executing the step response. In particular, we expected that stronger CMC would be most evident for the muscles involved in generating the greatest biomechanical contribution for the stepping movement (depending on the step and stance leg), and that an increase in CMC would precede changes in EMG activity in these muscles relative to foot off and foot strike event. Therefore, we conducted separate CMC analysis time-locked to perturbation onset, foot-off and foot strike event.Twenty young healthy adults participated in this study. We analyzed a total of 18 datasets  due to technical issues in two other datasets. All participants received ample information about the experiment and signed an informed consent document prior to the start of the experiment. Participants were financially compensated after completion of the study. None of the participants had a history of neuromuscular disease or any other impairment that could affect their performance in the experiment. The experimental procedure was approved by the Research Ethics Committee of the Radboud University Medical Center . The experiments were conducted in line with the Declaration of Helsinki.Data used in this study were derived from a protocol to investigate theta power modulations related to balance monitoring by imposing leaning angles prior to perturbation  Figure . Partici2 with a higher resolution at lower accelerations in both forward and backward sway direction . The initial experiment contained 15 sequences with 29 balance perturbations each . The first perturbation of a sequence always consisted of a low-intensity dummy trial and was not included in the analyses. For the analysis of the present study, we only considered the neutral stance conditions with forward and backward perturbation directions .Balance perturbation profiles consisted of 300 ms platform acceleration, 500 ms constant velocity and 300 ms deceleration . PlatforParticipants took a small break of 5 min after three consecutive sequences and were seated on a chair to prevent fatigue. After nine perturbation sequences, participants were given a 20-min resting break. The active experiment time was 2.5 h and the preparation time was 2.5 h, the complete lab visit lasted a maximum of 6 h (including resting breaks).We recorded high-density EEG using a cap with 126 Ag-AgCl electrodes . The electrodes were fixed in the cap and distributed across the scalp according to the five percent electrode system . The EEGWe recorded electrical activity bilaterally from five leg muscles [see Body movement was recorded using an 8-camera 3D motion analysis system  at a sample rate of 100 Hz. For this purpose, a total of 23 reflective markers  were attached to anatomical landmarks on the participants' body.post-hoc alignment of EMG, EEG and motion data.Ground reaction forces were recorded from two force plates  embedded in the moveable platform. Trials were recorded from−2 to +5 s relative to the platform perturbation. Synchronization triggers were generated by the platform controller and recorded for The EMG signal was preprocessed in MATLAB using low-pass filtering with the ‘filtfilt.m' function , and downsampling to 250 Hz. EMG was separately preprocessed for EMG envelope visualization and Granger causality.2 for feet-in-place responses. This normalization allowed us to evaluate the difference in activation between step and stance legs during stepping trials since both legs contributed equally in the feet-in-place trials. The acceleration of 0.7 m/s2 was the maximum platform acceleration where all participants were able to respond with feet-in-place in either direction.For EMG envelope visualization, the data were band-pass filtered , full-wave rectified, low-pass filtered , and normalized per muscle per subject to the maximum muscle activation at 0.7 m/sPrior to Granger causality computation, we downsampled and normalized (z-score) the EMG activity. Then we subtracted the ensemble average EMG from the single-trial EMG data per participant and muscle. This procedure is necessary for Granger causality analyses involving event related potential to facilitate model-fitting and reduce non-stationarities [; see FigFor the preprocessing of EEG and EOG data, MATLAB functions of the EEGLAB toolbox were used . ContinuOur and other studies indicated that midfrontal cortical activity plays a major role during the initial phase of the balance response regarding the monitoring and cortical control of the balance response , 17, 22.We collected a total of 2,491 trials and rejected 149 trials, based on flat lines and artifacts in EEG and EMG data. Of the remaining 2,342 trials , 1,177 were forward and 1,165 were backward perturbations. In the forward direction there were 675 feet-in-place responses vs. 502 step responses. In the backward direction, we recorded 565 feet-in-place responses and 600 step responses.t0 predicts EMG activity up to sample t100 (up to 100 ms ahead).We applied Granger causality analysis to compute the directional coupling from the cerebral cortex to the muscles between individual EEG channels and EMG data over 1 to 100 Hz with a resolution of 0.05 Hz. For the analysis we used the spectral Granger causality Matlab MVGC toolbox , 29. We Granger Causality analysis benefits from large amount of data. To optimize the Granger causality outcome we conducted the analysis over a time series of 4 s with a consistent amount of data for all frequency bands and individual muscles. Second, we included all step trials per participant, which resulted in a substantial amount of 500 (forward) and 600 (backward) trials (see 2.5 Data inclusion). In addition, we removed the ERP from both the EMG and EEG data to improve the signal stationarity (see 2.4 EEG processing). The sliding window approach of the Granger causality analysis provided by the MVGC toolbox , 29 alsoWe conducted spectral Granger causality relative to three time-locking events to get a good alignment with the EMG activity. Due to the temporal sliding window of the Granger causality analysis, between-signal comparisons can only be made at specific time locked events. Therefore, we time-locked to three specific events at which we can temporally accurately compare EMG with Granger causality at time 0 s. First, perturbation onset time-locked Granger causality analysis was done to investigate coupling with muscles immediately after perturbation. Secondly, Granger causality time-locked to foot-off event was computed to investigate CMC prior to step initiation and during stepping response. Finally, Granger causality time-locked to foot strike was done to investigate CMC prior to and after foot landing. For each time-locking event we averaged the Granger causality data over all 13 EEG electrodes, meaning that we analyzed one average cortical Granger causality time-frequency map for each muscle.p = 0.07; medianforward = 451 ms, IQRforward = 210 ms, medianbackward = 400 ms, IQRbackward =202 ms). In addition, foot strikes latencies did not differ for step direction . For convenience, we rounded the 75th percentile values up to foot off = 600 ms, foot strike = 800 ms. This resulted in the following analysis time windows; perturbation onset to 600 ms, −200 to 250 ms relative to foot off and−250 to 200 ms relative to foot strike. Please note that there is temporal overlap between these time windows. To determine whether significant EMG activity and CMC occurred relative to baseline activity in response to balance perturbations, multiple sample-wise t-tests were conducted time-locked to perturbation onset for each muscle per step and stance leg separately. Given the multiple tests computed in the time domain, the corresponding p-values were corrected for false discovery rate [FDR; . Temporal windows for significance testing were determined by foot-off and foot strike latencies.To determine differences between step and stance leg, significant differences between muscle specific step and stance leg EMG activity were computed using a sample-wise lo: 25–40 Hz) and high gamma (γhi: 50–85 Hz) ranges, and are indicated with dashed boxes.Time-frequency analysis averaged over all 13 midfrontal EEG electrodes and muscles in the forward and backward direction revealed increases in Granger causality over multiple frequency bands in response to perturbation onset . The freOverall, EMG activity significantly increased compared to baseline in both perturbation directions , TA and RF (larger in stepping leg), consistent with their differential roles in the stance and stepping leg. Observed CMC dynamics did not follow similar patterns to EMG activity within time locked events. Theta dynamics were mainly increased for stance leg muscles and the lower leg muscles of the step leg. Alpha band CMC only increased bilaterally in the SO and ST muscles. In the beta frequency, increased CMC compared to baseline was observed in the step leg SO and ST. Increased CMC in the low-gamma frequency band is observed around the foot off event for all but the TA muscles. The high-gamma band dynamics are overall symmetrical for all muscles and mostly not significantly increased relative to baseline.Time-locked to the foot strike event, all muscles showed elevated muscle activity relative to baseline. We observed greater EMG activity in step leg TA compared to the stance-leg TA muscle and the other stance leg muscles show increased EMG activity relative to the step-leg.Although significant CMC dynamics are observed, these did not follow similar activity patterns from the EMG activity. Significant increase in theta CMC was observed prior to foot strike in the step-leg SO and following foot strike event in the stance-leg ST and BF. Alpha band CMC dynamics were limited to both SO muscles. In the beta frequency, step-leg SO and both ST leg muscles showed increased CMC prior to foot strike. In addition, all but the BF muscles showed asymmetrical beta CMC increase following the foot strike event. Low-gamma CMC dynamics were mostly increased symmetrically prior to foot strike in the posterior leg muscles. In addition, both low and high gamma showed asymmetric activation patterns for all except the TA muscles following foot strike.In the backward perturbation direction, EMG activity of all muscles was increased relative to baseline, yet only modestly so in SO. This increased EMG activity is sustained throughout all time-locked events .Time-locked to perturbation onset, EMG activity transiently and symmetrically increased for all muscles over the initial 200 ms. Similar transient and symmetrical CMC dynamics are observed in the theta range for all muscles. Only stance leg ST and RF showed significant Alpha CMC increase following perturbation onset. Beta CMC dynamics followed a corresponding transient and symmetrical pattern in the lower leg muscles which only resulted in a significant increase for the stance-leg SO. Gradual and modest increase of low-gamma dynamics are observed in posterior leg muscles, albeit not always reaching significance. High-gamma band dynamics overall show similar symmetrical transient cortical interaction patterns as the theta band in all muscles.Time-locked to the foot off event, all muscles showed increased EMG activity relative to baseline. Prior to foot off, a symmetrical increase in muscle activity was observed in all lower leg muscles. However, upper leg muscles show greater EMG activity in the step leg hamstring and stance leg RF muscles, in line with their agonist roles in backward step initiation. Overall, CMC theta dynamics follow similar symmetrical patterns for the lower leg muscles in comparison to the EMG activity. Yet, dissimilar theta band CMC dynamics compared to EMG were observed in the upper leg muscles. Only increased alpha CMC was observed in the stance leg TA. Please note that the alpha CMC seems elevated for the RF throughout the foot off event while a significant effect remains absent. However, this is caused by strongly increased CMC in one participant specific for this muscle and frequency band. Removal of this participant's data did not influence the observation of additional significant difference effects. Little significant CMC dynamics were observed in the beta band. In the low-gamma band, almost all leg muscles, except for the RF, showed a gradual increase in CMC activity. In the high-gamma frequency band, all lower leg muscles show similar CMC patterns compared to the EMG activity. However, the upper leg muscles overall show little dynamics in comparison to the EMG activity during foot-off event with only slight high-gamma band CMC increases over stance-leg RF and BF muscles.Time-locked to the foot strike event, all muscles show increased EMG activity and step leg muscles show greater muscle activation. In addition, all muscles showed CMC over multiple frequency bands, yet the observed asymmetrical EMG activation patterns are not mirrored in the CMC patterns in the various frequency bands. All stance leg muscles except the ST muscle show increased CMC in the theta frequency. In addition, the step leg ST and BF showed increased theta CMC following foot strike. Alpha band CMC dynamics were limited to the stance leg SO and the upper leg muscles that showed increased EMG activity prior to the foot strike event. CMC dynamics in the beta band were primarily observed following foot strike for the upper leg muscles that showed increased EMG activity prior to foot strike. Overall, relatively similar CMC dynamics were observed over both the low and high gamma bands.Overall, asymmetrical activation patterns in EMG and CMC between step and stance leg were observed time locked to foot off and foot strike events in both stepping directions. Following a brief symmetrical recruitment of the various muscles shortly after perturbation onset , the activation patterns of stance- and stepping leg muscles started to differ depending on their differential functional roles. Time locked to foot strike, all muscles show difference in CMC activity primarily over beta and gamma frequency bands. Only the RF muscles show similar asymmetrical activity over beta and gamma frequencies compared to the EMG activity following foot strike.In the backward perturbation direction , the lowOur aim was to identify cortical interactions with various leg muscles through spectral Granger causality during a reactive balance task. We hypothesized that CMC would occur during reactive stepping responses, indicating the contributions of the cerebral cortex to the execution of these responses. In line with our hypothesis, our results illustrated significant CMC increase relative to baseline over multiple muscles and frequency bands following balance perturbations in both directions. In addition, we expected CMC dynamics to differ between step and stance leg. In particular, we expected that stronger CMC would be most evident for the agonist muscles involved in generating the stepping movements, and that an increase in CMC would precede an upregulation of EMG activity in these muscles. Contrary to our expectations, our data illustrated that step and stance leg CMC generally did not show similarity with EMG data . Our findings shed new light on cortical involvement of dynamic postural responses with implications for future studies involving clinical populations with deficient CMC (such as stroke).Granger causality analysis during reactive step responses revealed cortical interaction over multiple frequency bands for all muscles in both stepping directions, showing that on average the cortex becomes actively involved in the execution of reactive stepping response at ~180 ms post perturbation. Overall, we observed significant CMC increases in the theta, beta and both gamma frequency bands relative to baseline for individual muscles in either perturbation direction and throughout the three time locked events. In the backward direction, notable broadband interaction was present in upper leg muscles following the foot strike event. Although such broadband CMC following foot strike may suggest that observed dynamics were caused by an artifact, increased broadband CMC in the stance leg muscles during forward stepping and the absence of similar broadband CMC in other step leg muscles (the leg that receives most movement impact that may cause artifacts) support the interpretation of a causal cortical interaction that spanned multiple canonical frequency bands. We propose that the broadband interaction with specific muscles may emphasize the importance of these respective muscles in maintaining a stable posture following the stepping response and we will elaborate on our arguments below. As multiple studies investigated distinct functional roles of cortical rhythms, we will separately discuss their potential roles during the reactive stepping response.Strong transient cortico-muscular interactions in the theta frequency band were observed following perturbation onset in both perturbation directions for almost all leg muscles, indicating that theta dynamics may play a general role in the initial reactive stepping response. Interestingly, after the foot strike event in the backward stepping direction CMC in the theta frequency band mostly involved the muscles that showed increased muscle activity around foot off , suggesting that the leg muscles that primarily showed increased EMG activation also require relatively more cortical interaction in the later phase of the response. Our findings are in agreement with an increase in CMC in the theta frequency band observed in lateral pull perturbations while standing, which was speculated to facilitate muscle recruitment of the feet-in-place balance response . Thus, tIncreased dynamics in the alpha band has been coupled to sensorimotor processes and motor readiness, and it may facilitate a similar role during the reactive balance response. Several studies reported increased alpha coupling during standing compared to walking , 31, 32.increase in beta band CMC is linked to increase in muscle activity, while a simultaneous power decrease of the cortical beta rhythm  is observed during sustained muscle contraction  is known to be mediated by subcortical circuits . While subtracting the ERP from the single trial data, we may have induced minor non-stationarities. Therefore, we recommend future studies to include perturbation intensities of a single intensity that is strong enough to elicit step responses.On the other hand, our study shows novelty and strengths in several aspects. First of all, CMC at key events throughout the stepping response elicited temporal evolution of distinct spectral dynamics with respect to EMG. Future studies may further investigate the specific role of the spectral dynamics with respective muscles throughout the balance response. In specific, it is of interest to investigate the role of each frequency band with respect to the functional muscular role during the reactive step response. In addition, the current study is the first to conduct separate analysis of step- and stance-leg CMC in comparison to the temporal evolution of EMG activity. The surprising dissimilarity that we observed in CMC and EMG patterns between the legs indicates that the cortex does not appear to facilitate EMG recruitment for executing the balance recovery step in a refined leg- and muscle specific manner.We conclude that reactive balance responses require direct interactions from the cortex with the individual muscles, yet without substantial leg-specific differences in CMC patterns. Our work is relevant for clinical populations with impaired balance control, where CMC analysis may elucidate the underlying pathophysiological mechanisms.https://data.donders.ru.nl/) via di.dccn.DSC_4220000.06_843, ‘EEG of human balance control'.The original contributions presented in the study are publicly available. This data can be found here: The Donders Repository . The patients/participants provided their written informed consent to participate in this study.MS: designed research, analyzed data, and wrote paper. TS-E, MC, and VW: supervised MS throughout the study and provided feedback. All authors contributed to the article and approved the submitted version.


Cutaneous melanoma is a highly aggressive form of skin cancer. The development of immune checkpoint inhibitors (ICIs) has revolutionized the management of advanced melanoma, led to durable responses, and improved overall survival. However, the success of ICIs in melanoma treatment is influenced by the tumor microenvironment (TME) which plays a critical role in regulating the immune response to the tumor. Understanding the mechanisms underlying this interaction is crucial to optimizing the efficiency of ICIs. Electrochemotherapy (ECT) has been shown to enhance the efficacy of ICIs in melanoma treatment by inducing tumor cell death and facilitating the release of tumor antigens which can subsequently be recognized and targeted by the immune system. Moreover, ECT has been reported to modulate the TME, leading to increased infiltration of immune cells and a more favorable immunological profile. In this review, we summarize the available knowledge of changes in TME after ECT of melanoma cutaneous metastasis and highlight the differences in tumor-infiltrating immune cells between immunocompetent and immunosuppressed organisms. In addition, we showed that ECT can be an effective and safe procedure for organ transplant recipients. Furthermore, repeated ECT may enhance immune activation and probably induce a bystander effect by trained immunity. Malignant melanoma is one of the immunogenic tumors with a high potential to elicit a specific adaptive antitumor immune response . Since tElectrochemotherapy (ECT) is a very safe and efficient local anticancer treatment frequently used in treating in-transit melanoma metastases and displays immunostimulatory properties through immunogenic cell death . A comprTumor-infiltrating immune cells participating in the control of malignant cells with the extracellular matrix and tumor vasculature, which together represent the tumor microenvironment (TME), continue to be areas of intense research. Innate and adaptive immune responses are both responsible for the immune surveillance of melanoma . High raThe most efficient combination of local therapeutic options and systemic therapies is still unknown. Although tremendous effort has been made to explore the TME, it still presents challenges of high heterogenicity, complexity, and plasticity. To address this challenge, we integrate the available knowledge of changes in TME  after ECT of melanoma cutaneous metastasis, with special emphasis on differences in tumor-infiltrating immune cells between immunocompetent and immunosuppressed organisms. Considering that less attention has been devoted to changes in TME and clinical results after ECT in organ transplant recipients on immunosuppressant medication, we present a case of advanced melanoma in which repeated treatment with ECT with bleomycin-induced a complete clinical response of in-transit metastases.The host immune system is considered to be an essential part of the response to ECT. In 1997, Sersa et al., observed both a twofold longer tumor growth delay and higher curability rates in immunocompetent C57B1/6 mice than in immunodeficient nude mice when mice were treated with ECT with cisplatin . SimilarIn a study on murine melanoma, markers of immunogenic cell death were studied after ECT with cisplatin and oxaliplatin. Calreticulin (CRT) exposure was observed after ECT with both drugs used in vitro and in vivo. The release of CRT shifts nonimmunogenic cell death to immunogenic cell death attracts antigen-presenting cells (APCs) and consequently induces an adaptive immune response. As a result, after ECT, a 4-fold increase in tumor infiltration by granzyme B-positive (GrB) immune cells, representing mainly NK cells and CTLs, were observed. This finding demonstrated that exposure of melanoma cells to ECT with cisplatin or oxaliplatin induces immunogenic cell death which correlates with an increase in tumor-infiltrating lymphocytes (TILs) after ECT . In addiCRT surface expression was also detected in human melanoma metastases after ECT with bleomycin. Moreover, immediate and late changes in T-cell infiltration in ECT-treated lesions using fluorescent immunohistochemistry were also investigated. Ten patients (median age 80.5 years) who had not been eligible for systemic therapies have had histologically confirmed cutaneous or subcutaneous melanoma metastases located in the limb and have been undergoing ECT with bleomycin were included. Skin biopsies were performed on different lesions prior to ECT and one and fourteen days after ECT. Before ECT, Th cells were the most represented T cells in the perilesional border, while Tregs were present in both the perilesional dermis and within the tumor. After ECT, Tregs significantly decreased in number, while CTLs significantly increased in the perilesional dermis 14 days after ECT. In contrast, the frequency of Th remained constant before and after ECT .Another immunohistochemical analysis of the inflammatory infiltrate in sequential punch biopsies  of human melanoma cutaneous metastases after ETC showed similar results. In this study, only two patients were enrolled. To minimize differences, two sets of biopsies were obtained in one patient, one set from a single large nodule and another set from smaller lesions. Unfortunately, the authors did not report if any differences in these two sets of biopsies were observed. However, they described differences in histologic findings as well as in immune infiltration in all time lapses after ECT. Early presence and a progressive increase in inflammatory infiltrate were observed with T lymphocyte prevalence in all samples taken at different time points after ECT. From the earliest phases after ECT, CTLs increased in number and surrounded the tumor cells, and residual CTL infiltration was observed two months after ECT. Research has demonstrated that CTLs are activated early after ECT and are key elements in the late inflammatory response . In contTo observe DC activation and migration, sequential biopsies before ECT with bleomycin and seven and fourteen days after ECT in nine patients with histologically confirmed skin melanoma metastasis were performed. Prior to ECT, the most represented subgroup of DCs was Langerhans cells (LCs), located mainly in the epidermis and expressing a typical immature immunophenotype. Epidermal DCs significantly decreased seven days after ECT, but LCs repopulated fourteen days later, suggesting that ECT may stimulate LCs to drain lymph nodes . These f®) underwent excision of pT3b melanoma from the right lower limb in March 2019. Due to a positive sentinel node and after FDG PET CT excluded visceral involvement, groin dissection was performed. In total, 3 out of 23 nodes were positive. After one week of receiving targeted therapy, it was discontinued due to side effects. Immunotherapy was contraindicated due to organ transplantation. After approximately one year, in-transit melanoma metastases were cytologically confirmed. During a six-month period, three ECT sessions with i.v. bleomycin application wasperformed, and one ECT session was performed after nine months on only a small number of nodules.A 68-year Caucasian woman, 18 years after liver transplantation, and taking the calcineurin inhibitor tacrolimus , pulse duration 100 μs and frequency 5 kHz were applied and for hexagonal electrodes 12 pulses with voltage 730 V, pulse duration 100 μs, and frequency 5 kHz were applied. The amplitudes were automatically checked after each application. If needed, several applications were performed to cover the entire nodule volume. Electric pulses were generated by a Cliniporator  device and applied using either plate or hexagonal electrodes, depending on the size of the tumor lesion. Bleomycin , at a standard dose of 15,000 IU/mRegression of treated lesions was observed while new metastases also appeared C. FDG PEAll specimens were fixed in 10% buffered formalin overnight. Histological analysis of morphology and estimation of tumor-infiltrating lymphocytes (TILs) were performed on 2–3 μm thick, formalin-fixed paraffin-embedded (FFPE) sections stained with hematoxylin-eosin (HE).Immunohistochemical (IHC) characterization of the tumor inflammatory infiltrate was performed on 2–4 µm FFPE tissue sections dried at 56 °C for 2 h using the fully automated IHC staining platform Benchmark Ultra . Epitopes were retrieved onboard employing heat-mediated epitope retrieval using high pH Cell Conditioning Solution one  for 88 min at 100 °C. Retrieved epitopes were detected using commercially available monoclonal primary antibodies, as presented in The amount of TIL was estimated semi-quantitatively according to the MIA scoring  independHistologic examination of vital in-transit CM nodules excised five months after the fourth ECT showed metastasis of CM in the dermis with expansion into the subcutaneous fat A. The peTwo biopsies of sites of prior cutaneous in-transit lesions treated by ECT showed only fibrosis and dermal melanosis —completeIn this study, we showed that ECT can be an effective and safe procedure in organ transplant recipients. Repeated ECT may enhance immune activation and probably induce a bystander effect by trained immunity. Furthermore, in viable excised metastases, the number of CD163-positive macrophages was prevalent, an observation that might contribute to ECT resistance.To our knowledge, no studies describing the TME in immunocompromised melanoma patients after ECT are available. However, histological and immunohistochemical immune characteristics of the TME of mainly primary cutaneous melanoma and some melanoma metastasis biopsy specimens in 20 renal transplant recipients who developed post-transplantation melanoma were reported. Testing adaptive immunity, little or no expression of CD8+, FoxP3, PD1, and PD-L1 was observed, confirming a deserted TME and the absence of T-cell activation . MechaniIn contrast to a prior study describing the immune characteristics of the TME in renal transplant recipients, Quaglino et al., investigated the distribution and phenotype of TILs of in-transit metastases before ECT in 15 stage III melanoma patients. A high number of Th cells and CTLs within the TILs was observed in nine patients (60.0%), while more than 10% of Tregs were found in five patients (33.3%). Furthermore, brisk distribution TILs with a high number of CTLs were associated with higher response rates to ECT and a lower incidence of visceral progression .Comparison of the results of previous studies with immunohistochemical characteristics of excised vital in-transit lesions after the fourth ECT in our patient should be conducted with caution. Based on the spatial distribution of CTLs, the excised vital nodule could be described as immune inflamed. In addition, TIL phenotyping with IHC staining for CD3, CD4, CD8, and FoxP3 showed a favorable CD8:FoxP3 ratio. As previously described, these features correlate with improved CTL function and better clinical response rates ,30,31. DIn our case, the patient was taking the immunosuppressant tacrolimus which is a calcineurin inhibitor that blocks the activation of the nuclear factor of activated T cells (NFAT) and leads to a decreased synthesis of IL-2. IL-2 is a key cytokine required for T-cell activation of the primary immune response and throughout memory cells as well as T-cell proliferation ,33. CalcThe innate immune system is less vulnerable to the immunosuppressive effect of tacrolimus, so it is not surprising that we found 10–15% positivity of CD 163 (macrophage- and monocyte-specific transmembrane protein) in excised vital in-transit metastasis . This maInterestingly, histological examination of sites previously treated for in-transit CM metastasis by ECT showed only fibrosis and dermal melanosis, confirming the clinically observed complete response after ECT. However, after the first two ECT procedures, out-of-field progression of a high number of in-transit metastases was observed, while after the third ECT, only six in-transit metastases appeared, and after the fourth ECT, only one in-transit metastasis escaped of immune control. These findings suggest that repeating ECT potentiates antitumor immune effects as previously reported ,39. Our Tumor response to ECT is very complex and depends on many factors. Response to ECT is dependent mainly on tumor size, type, and previous oncological treatment. Therefore, the comparison of different studies with their inherent risk of selection bias is challenging. Due to intermetastatic heterogeneity, significant variations among different metastatic lesions in the same patient as well as the primary tumor, including the composition of the TME, can be observed ,42. We aOur case report with a review of the literature contributes to the understanding of changes in the melanoma tumor microenvironment, especially immune cell infiltration, even in the case of immunosuppression. This shows that ECT is a safe and effective procedure for organ transplant recipients. Repeated ECT may enhance immune activation and probably induce a bystander effect by trained immunity.ECT could be utilized to enhance the activation of melanoma-specific immunity and maintain the long-term antimelanoma immune response as part of a combined therapeutic approach in the era of ICI treatment.


Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) are responsible for many drug-resistant infections in the U.S. We undertook two parallel studies aimed to investigate the differences in the microbial communities of individuals colonized with MRSA (or VRE) as compared to their respective non-colonized counterparts matched for age, sex, race, ethnicity, unit of admission, and diagnostic-related group, when available.Hospital-acquired infections present a major concern for healthcare systems in the U.S. and worldwide. Drug-resistant infections result in increased costs and prolonged hospital stays. Methicillin-resistant Enterococcus genus communities in the VRE colonized samples. Our findings for both MRSA and VRE studies suggest a strong association between 16S rRNA gene alpha diversity, beta diversity, and colonization status. When we assessed the colonized microbial communities in isolation, the differences disappeared, suggesting that the colonized microbial communities drove the change. Isolating Staphylococcus, we saw significant differences expressed across colonization in specific sequence variants.The VRE study showed considerably more the Staphylococcus (or Enterococcus) genus, the removal of which results in the disappearance of any differences in the diversity observed across the match-pairs.The differences seen in the microbial communities from MRSA (or VRE) colonized samples as compared to non-colonized match-pairs are driven by the isolated communities of  Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) are routine in many hospitals in the United States due to the associated risks to patients. MRSA alone accounts for over 94,000 annual infections and almost 19,000 deaths, with 86% of the cases associated with healthcare [Programs of infection prevention for pathogens such as methicillin-resistant althcare . It is aUnderstanding the factors contributing to the colonization and persistence of MRSA and VRE in human populations is crucial for the development of effective prevention and control strategies. Prior to 2020, the Medical University of South Carolina (MUSC) utilized an active surveillance testing program designed to identify patients colonized with MRSA and VRE and exercise the use of contact precautions for those found to be colonized. Under this program, a majority of patients admitted for over 24 hours are tested for MRSA using nasal swabs, producing approximately 15,000 specimen tests for the pathogen annually. High-risk hospitalized patients undergo active surveillance testing for MRSA and VRE weekly until the patient becomes positive for MRSA, VRE, or is discharged.Investigating the microbiome’s role in MRSA and VRE colonization may shed light on the complex interactions between bacteria and their human hosts. There are important scientific and clinical implications for our research. Firstly, by characterizing the nasal and perianal microbiome in MRSA and VRE colonization, we aim to identify microbial signatures that could serve as potential signatures for colonization risk. These biomarkers may help identify those who are more likely to become colonized and may facilitate the implementation of focused preventative therapies to limit the spread of antibiotic-resistant pathogens in healthcare settings. Secondly, understanding the variations in microbial diversity and community structure among individuals colonized and uncolonized may provide insight into the mechanisms underlying antibiotic resistance. Studies such as ours could help elucidate how specific microbial species or groups interact with MRSA/VRE and affect the likelihood of colonization. This may lead to the development of novel interventions aimed at specific microbial communities to reduce colonization and subsequent infection rates.We utilized infrastructure from the Living μBiomeBank to enroll, process, sequence, and perform downstream analyses . The MUSTwo parallel studies were undertaken to assess the differences in the microbial communities of those colonized with MRSA and VRE as compared to their uncolonized matched counterparts. The methods describe the parallel and similar processes for enrolling specimens from these populations and downstream handling and processing. Daily reports using the electronic medical health record platform Epic identified nasal specimens, reported MRSA/VRE status, demographic variables, admission unit, admission diagnosis, and other identifiers. MRSA/VRE positive specimens were captured on an ongoing basis, de-identified, and stored at −80°C for batch processing. Once a MRSA/VRE positive specimen was identified, a one-to-one matched MRSA/VRE negative specimen was sought based on age (± 5 years), sex, race, ethnicity, unit of admission, and diagnostic-related group, when available. To minimize potential biases due to seasonal microbiome variability, matching MRSA/VRE negative samples were at most sought within four weeks of the original MRSA/VRE positive specimens’ collection dates. If a match was not had within the prespecified window, the positive specimen was discarded, and another positive specimen was recruited.Samples were processed for bacterial DNA extraction using QIAamp DNA Mini Kit from Qiagen  according to the manufacturer’s instructions. The quality and purity of extracts were determined using the QIAxpert system (Qiagen). Bacterial 16S rRNA variable region V2 was amplified with Illumina  Nextera XT Index Kit v2 index adapters (CTGTCTCTTATACACATCT) , 11. BarAll statistical analysis have been performed in R (v. 4.0.3) . InitialStaphylococcus or Enterococcus have been removed; and (c) in which all ASVs except those classified as genus Staphylococcus or Enterococcus have been removed. Within each of these three scenarios, figures were generated and analytical tests performed including univariate and multivariate testing. Non-bacterial ASVs were removed as an initial step. Compositional bar charts, alpha diversity figures, and statistical assessments of alpha diversity (paired t-tests) were performed prior to any filtering. Filtering threshold was set at to retain ASVs with at least a 1% relative abundance and observed in more than one sample. Following filtering, univariate and multivariate analysis were performed. Relative abundance data were transformed and normalized using centered log-ratio (CLR) calculations to use the geometric mean of the filtered sample vector as the reference. Multiple clustering methods were utilized: Jensen-Shannon divergence and Bray distance matrices utilized relative abundance data, whereas the Euclidean distance was used with the CLR-transformed abundances. Match-stratified multivariate data were analyzed using A flowchart of the downstream analysis is presented as Supplementary Figure S1. Three analysis scenarios were considered: (a) in which all microbial ASVs are present; (b) in which all ASVs identified as genus ERMANOVA . ICD9 anERMANOVA . Gap staERMANOVA  to assest- and chi-squared tests) were observed in age, race, sex, and Elixhauser Comorbidity Index between the cases and controls for the MRSA and VRE studies.Samples (N = 108) were successfully sequenced and generated all downstream data. Staphylococcus genus. This same trend was not observed in the VRE study; although in the cases we observe the Enterococcus genus with the majority abundance, this is not the case in the matched negative controls (in 1b top facet).For the MRSA study, 55 samples generated a total of 1,704,344 non-chimeric reads . These comprised 740 ASVs, five (5) of which were eukaryotic in origin and were only observed across four samples. For the VRE study, 53 samples generated a total of 1,648,085 non-chimeric reads . These reads comprised 962 ASVs, only one (1) of which was eukaryotic in origin and was observed across four samples. The eukaryotic ASVs were removed prior to analysis. Sequencing depth between MRSA/VRE colonized and uncolonized samples were assessed to ensure no significant read quality differences were present in any of the covariates. Case-control matched visualization of the top-10 most abundant genus-level communities revealed an interesting trend  and b; ut-test of observed, Shannon, and Simpson diversity measures, all three of which were found to be significantly associated with subjects’ colonization status . ASVs identified as Staphylococcus were removed and processed for the same analysis. This time only the observed alpha diversity count was seen to be significant (p = .02). Subsequently, Staphylococcus ASVs were isolated and processed using the same assessment of alpha diversity and no significance was observed on a 0.05-level. Similar trends were observed in the VRE study in which observed, Shannon, and Simpson diversity measures were found to be significantly associated with subjects’ colonization status  when all ASVs were included. ASVs identified as Enterococcus were removed and processed for the same analysis leading to significantly different alpha diversity measures again . Subsequently, Enterococcus ASVs were isolated and processed using the same assessment of alpha diversity and no significance was observed on a 0.05-level. We have included visualized alpha diversity measures as Supplementary Figs. 3a and b.Alpha diversity of samples was assessed  prior toStaphylococcus genus) as statistically significant . These two ASVs are also the most abundant overall. When corrected for multiple comparisons using FDR approach, q-values were observed to be non-significant (on a .05 level). A table containing all 104 ASVs is included as Supplementary Table 1a. A similar comparison was performed on genus agglomerated CLR data , resulting in 104 ASVs that were used for univariate and multivariate assessments. CLR-transformed data was assessed in ASV-wise manner across colonization status . This asCLR data . We do nEnterococcus genus and ASV20 belongs to the Klebsiella genus. A table containing all 176 ASVs is included as Supplementary Table 1b. A similar comparison was performed on genus agglomerated CLR data , resulting in 176 ASVs that were used for our downstream analyses. CLR-transformed data was assessed in ASV-wise manner across VRE colonization status . AlthougCLR data  which shStaphylococcus ASVs 3 and 4 to be widely present in both cases and controls, whereas ASVs 1 and 2  are almost exclusively observed in the cases (MRSA colonized). On the other hand, Staphylococcus ASVs 9 and 11 are observed largely in the controls (non-colonized for MRSA). We have included a larger heatmap including all 104 AVSs within the MRSA study as Supplementary Fig. 4. Assessing these differences across colonization using Pearson’s correlation test shows significance . In 2b (VRE study) we see Enterococcus ASVs 1 and 3 to be primarily present in the cases (VRE colonized), whereas ASV 2 (Escherichia-Shigella), ASV 13 (Staphylococcus), and ASV 18 (Peptoniphilus) are primarily observed in the controls (non-colonized for VRE). We have included a larger heatmap including all 176 AVSs within the VRE study as Supplementary Fig. 5. Assessing these differences across colonization using Pearson’s correlation test shows no significance .The composition of the top 30 most abundant ASVs across samples is displayed using a heatmap in Staphylococcus genus  of the Bray-transformed relative abundance data  is influenced by these two genera. In other words, the inclusion of Staphylococcus or Enterococcus genera indicate important sources of observed variation in our alpha diversity analysis. Our ASV-level univariate analysis results showed that Staphylococcus genus ASV 1 and 2 are significantly different in MRSA colonized vs uncolonized match pairs (paired t-test corrected using FDR). A similar trend was not observed in our VRE study once corrected for multiple testing. Genus-level agglomerated univariate testing also showed no significant differences even in the case of Staphylococcus that was previously observed on the ASV-level. This may be indicative of differences in behaviors of species within the same genus such as Staphylococcus.The findings in both the MRSA and VRE studies indicated alpha diversity measure differences, notably within the ASVs of genus Staphylococcus or Enterococcus); and is markedly present in colonized genus in isolation (only Staphylococcus or Enterococcus). The variance obtained from the PCoA as well as our own robust Enterococcus) genus.We performed PCoA of the beta-diversity revealing distinct clustering profiles of colonized cases and non-colonized matched controls. A similar pattern is observed in MRSA and VRE studies in which a significant difference is seen when all ASVs are present; this difference disappears entirely when the colonized genus is removed (all ASVs except While the study provides important insights into the compositional microbiome differences of the groups, there are several limitations including the relatively small sample size, which could have made it difficult to detect some of the differences between the two groups. Additionally, the study was conducted in a single hospital, which may limit the generalizability of the findings. The study may also suffer from selection bias given we relied on surplus clinical specimens and there may be inherent biases associated with subjects who are hospitalized or sampled.Staphylococcus and Enterococcus genera were found to be significant contributors to these differences which may have implications for understanding the colonization dynamics and developing potential interventions for MRSA and VRE infections.The findings from this study shed important light on the variations in the microbiome composition of subjects colonized with MRSA/VRE compared to uncolonized matches. The


Dear Editor,+ tumor-infiltrating lymphocyte (TIL)-dependant antitumor immune response. The release of tumor neoantigens and danger-associated molecular patterns (DAMPs) also participates in making the TME more immunogenic. RT can also play immunosuppressive roles. For instance, it can aggravate hypoxia and ROS synthesis in tumors and stimulate the secretion of immunosuppressive cytokines. Overall, this results in the inhibition of immune effector cells and the accumulation of immunosuppressive cells such as Treg, MDSC and TAM2, both monocyte-derived populations [Radiation therapy (RT) is now known to be a powerful immunomodulatory agent. Tumor irradiation leads to the release of double-stranded DNA, sensed via the cGAS/STING pathway . This trulations . Most ofulations ) which sulations .Fig. 1Ba+ TIL-mediated immune response. CRT was responsible for an accumulation of CD8+ TILs in the tumor whereas SRT mainly triggered an ingress of Treg. Moreover, infiltrated CD8 + T cells had enhanced effector functions after CRT but not after SRT.They first showed that CRT was more effective than SRT at delaying tumor growth and improved survival in a dose and fractionation independent manner due to a more effective acute CD8ACT) phenotype after CTR, characterized by lower levels of Ly6C and CD115 and higher levels of major histocompatibility complex II (MHC II), CD86 and PD-L1. Surprisingly, this monocyte and monoACT infiltration resulted in a monocyte-derived cGAS/STING-independent IFN-I secretion promoting monocyte accumulation and monocyte activation again following a positive feedback loop. These populations were therefore critical for activating polyfunctional CD8+ TILs after CRT. Finally, normal tissues received significant dose after SRT (but not CRT), resulting in an increased gut permeability, gastrointestinal damage and the relocation of monocytes into the colon whilst not causing lymphopenia which might have accounted for the differences between the two types of RT. This suggests that discrepancies between CRT and SRT probably lie in the relocation of monocytes to healthy tissues after they receive a significant dose.To explain these differences which occur even though the same dose was delivered through both RT modalities, the authors pinpointed that CRT elicited a greater early recruitment of monocytes in the TME than SRT. Moreover, whereas monocytes tended to differentiate into TAMs after SRT, they mostly acquired an “activated monocyte” (mono3) and the immune TME described might be different from that obtained after the irradiation of much larger tumors.These results demonstrate that the RT technique and ballistics not only affect the toxicity linked to unintentional healthy tissue irradiation, but also extensively impact the radio-induced immune response and eventually RT efficacy. This underlines the importance of more accurately describing the irradiation technique used in forthcoming preclinical studies: although RT dose, fractionation and the type of particles used are often found in the methods section, the precise ballistics and dose-volume histograms for organs at risk are often missing although they probably largely impact the outcomes as highlighted here. Tumor volume (TV) also appears to be key in studying the radio-induced immune response. Indeed, TVs in the present study were rather small when RT started (i.e. 50–100 mmThis work also paves the way to a new understanding of immune mechanisms underlying RT efficacy. ‘Classical’ pathways largely described to explain the radio-induced immune response, such as the cGAS/STING pathway, albeit important, may not account for the majority of radio-induced effects. Beyond the interest of the improvement of knowledge on the matter, this aspect is paramount, as promising results from the combination of STING agonists and RT found with SRT in preclinical studies might be disillusioning once in the clinic .+ TIL-related efficacy of conformal RT through the accumulation and activation of monocytes and their secretion of IFN-I. This was not found when healthy tissues were irradiated, demonstrating that the exposure of organs at risk has a negative therapeutic effect on the antitumor response.Altogether, Tadepalli et al. bring a brand new antigen-independent and dendritic cell-independent model to the polyfunctional CD8