Neurofeedback allows for the self-regulation of brain circuits implicated in specific maladaptive behaviors, leading to persistent changes in brain activity and connectivity. Positive-social emotion regulation neurofeedback enhances emotion regulation capabilities, which is critical for reducing the severity of various psychiatric disorders. Training dorsomedial prefrontal cortex (dmPFC) to exert a top-down influence on bilateral amygdala during positive-social emotion regulation progressively (linearly) modulates connectivity within the trained network and induces positive mood. However, the processes during rest that interleave the neurofeedback training remain poorly understood. We hypothesized that short resting periods at the end of training sessions of positive-social emotion regulation neurofeedback would show alterations within emotion regulation and neurofeedback learning networks. We used complementary model-based and data-driven approaches to assess how resting-state connectivity relates to neurofeedback changes at the end of training sessions. In the experimental group, we found lower progressive dmPFC self-inhibition and an increase of connectivity in networks engaged in emotion regulation, neurofeedback learning, visuospatial processing, and memory. Our findings highlight a large-scale synergy between neurofeedback and resting-state brain activity and connectivity changes within the target network and beyond. This work contributes to our understanding of concomitant learning mechanisms post training and facilitates development of efficient neurofeedback training. Self-regulation refers to the ability to manage one’s own emotional, cognitive and behavioral functions and can be learned through neurofeedback4. Neurofeedback is a form of training that enables individuals to learn voluntary self-regulation of brain circuits implicated in specific behavior or pathology4. Importantly, improvements in clinical symptoms are observed directly after neurofeedback training and persist for weeks5 or even months10 after the training. Neurofeedback is associated with post-training resting-state functional connectivity (rs-FC) modulations in target brain areas and beyond them, as detected immediately after neurofeedback training12, several days after training13 or weeks/months after training16. Alterations in rs-FC have been reported for different types of fMRI feedback types, specifically for activity18, functional connectivity19 and decoding20. In studies with emotion regulation neurofeedback, rs-FC modulations were also consistently observed after neurofeedback training between amygdala and the (pre)frontal–temporal-limbic areas21.Resting-state functional magnetic resonance imaging (rs-fMRI) has sparked widespread interest after observation that spontaneous blood oxygenation level-dependent (BOLD) signal oscillations are organized into large-scale networks27. This concept has also been demonstrated during the regulation of positive emotions30. A comprehensive understanding of positive emotion upregulation in social situations  is critical as impairment of this ability may constitute a key factor contributing to the anhedonic and emotion dysregulation symptoms associated with depression and posttraumatic stress disorder34. Recently, it has been shown that positive-social emotion upregulation is associated with the direct influence of the prefrontal cortices on bilateral amygdala35. Consistently, positive-social emotion regulation based on effective connectivity neurofeedback enhances top-down emotion regulation capabilities and results in concomitant behavioral changes29. Effective connectivity was estimated using dynamic causal modeling (DCM) as a degree of a top-down network model dominance  onto bilateral amygdala) over a bottom-up one . Neurofeedback-induced behavioral changes implied increases in valence ratings of emotional stimuli after as compared to before training.According to emotion regulation models, successful regulation is achieved through the modulation of bottom-up emotional responses in limbic areas by cognitive top-down processes originating in prefrontal cortices19. However, authors did not observe significant progressive rs-FC modulations between the trained areas, as well as their resting-state scans were not acquired right after the neurofeedback training. Similar resting-state scans were acquired in patients with borderline personality disorder, and progressive increases in amygdala-dlPFC rs-FC was observed during neurofeedback as a mechanism to induce amygdala downregulation36. Gradual recovery processes were observed during inbuilt rest of visual-spatial attention neurofeedback runs that consisted of interleaved neurofeedback and baseline conditions with 1 min rest epochs37, however progressive rs-FC modulations were not addressed. Thus, the role of rest in neurofeedback learning and concomitant resting-state neural modulations remains unexplored, where filling this knowledge gap could help understand learning mechanisms and improve design efficiency of neurofeedback training paradigms.Previously, training a bidirectional connectivity change between primary motor and lateral parietal cortices in healthy participants led to progressive rs-FC modulations of large networks across short resting-states at the beginning of the training sessions29 and networks engaged in emotion regulation and neurofeedback learning. To test this hypothesis, we employed a comprehensive and multidimensional approach to previously unpublished resting-state data from our positive-social emotion regulation neurofeedback study29. To exhaustively elucidate brain network resting-state activity and connectivity changes given the novelty of this study, we examined orthogonal, yet complimentary analyses. Five analyses were carried out, pertaining to (i) model-based effective connectivity of target brain network areas based on spectral DCM (spDCM), (ii) whole-brain data-driven spatiotemporal patterns of spontaneous brain activity based on tensorial Independent Component Analyses (tICA), (iii) whole-brain FC estimates based on the eigenvector centrality (EC), (iv) FC density (FCD), and (v) seed-based FC. Importantly, these measures provide unique information regarding various aspects of network dynamics. Specifically, by means of the spDCM40, we investigated whether learning positive-social emotion regulation using effective connectivity neurofeedback leads to progressive (linear) increase in resting-state effective connectivity within the trained network. By means of the data-driven tICA42, we investigated whether such neurofeedback training leads to a progressive increase of whole-brain spatiotemporal patterns of spontaneous resting-state activity. To examine whether neurofeedback training modulates emotion regulation and neurofeedback learning FC networks, conventional rs-FC analyses were performed based on voxel-wise Pearson correlations. We performed whole-brain EC analyses to reveal modulations of brain areas that are central within the small‐world network and strongly correlated with other nodes43. We performed whole-brain FCD analyses to identify modulations of brain areas with large long-range correlations. Finally, seed-based FC analyses unveiled modulations of resting-state dmPFC connectivity with the rest of the brain.Here, we hypothesized that short resting-state periods at the end of positive-social emotion regulation neurofeedback training sessions would reflect progressive neural modulations of the trained network model 29. We then continue with new results associated with intermediate rest periods in terms of the progressive (linear) increase of resting-state (i) network model-based effective connectivity, (ii) whole-brain data-driven spatiotemporal spontaneous activity and (iii) whole-brain functional connectomics.We first briefly describe neurofeedback task-related learning effects of the positive-social emotion regulation based on effective connectivity neurofeedback training, which were thoroughly reported and discussed in a previous publication29. The ANOVA on valence ratings revealed the main effect of group 1 = 6.93,The slope of the learning curve indicated learning success via Pearson rho values, which indexed the linear relationship between logarithmic Bayes factors and neurofeedback training runs. Participants in the experimental group learned to increase the dominance of the top-down effective connectivity model compared to the bottom-up model . Positive self-connectivity values, indicate increased self-inhibition and estimated negative self-connectivity values indicate decreased self-inhibition (or disinhibition)45. Positive and negative connectivity parameters between network nodes indicate positive and negative directional influence (or interaction), respectively. Positive parameters in group differences indicate higher connectivity strengths in the experimental as compared to the control group, and vice versa for negative parameters.We applied a three-level hierarchical model to evaluate the progressive increase of resting-state effective connectivity during neurofeedback training using spDCM and Parametric Empirical Bayes (PEB) approachesure Fig. A. BetweeL = − 0.51, Pp = 100%; for AMYR = − 0.32, Pp = 100%), as well as a negative top-down influence from dmPFC onto the bilateral amygdala . The average effect in the experimental as compared to the control group was associated with larger self-inhibition of dmPFC  and AMYL , as well as lower self-inhibition of AMYR . The progressive effect across four resting-state runs in both groups was associated with a significant dmPFC self-inhibition in both groups  in both groups, and significantly lower dmPFC self-inhibition in the experimental group as compared to the control group . The average , which indicates that larger learning slope is associated with larger dmPFC self-inhibition and AMYR disinhibition. In this interaction, for the progressive effect, we found a significant AMYR self-inhibition , which indicates that larger learning slope is associated with larger progressive self-inhibition of AMYR. In the interaction group × valence difference, for the average effect, we found a significant dmPFC disinhibition  and a negative AMYL to AMYR connectivity . Surprisingly, in this interaction, for  the progressive effect, we found a significant AMYR disinhibition , which indicates that larger valence difference due to neurofeedback training is associated with larger progressive disinhibition of AMYR.In the interaction group × neurofeedback slope, for the average effect, we found a significant dmPFC self-inhibition  and AMYFor both groups, we found that the average and progressive effects across four resting-state runs were best explained by the top-down connectivity model  with a specific spatial map and time-series is weighted for each run. To assess the progressive modulation of ICs, we then computed a linear regression of each IC weights across four resting-state runs. For each decomposition in the dimensionality range from #50 to #80 ICs, in the experimental group we found a highly consistent single IC that showed a significant increase of presence across resting-state runs over neurofeedback training . These ICs were highly spatially consistent across the performed decompositions . In addition, for post-hoc comparison, among these 29 ICs, 21 ICs had a significantly different increase of presence across resting-state runs in the experimental compared to the control group . To report key findings, we selected IC3 of the decomposition #58  with maximum 7.9% of total variance across 21 ICs , while negative subIC3 had an overlap with bilateral amygdala  anatomical ROI masks. Note that decomposition #58 was consistent with conventional template resting-state networks (RSNs)46, as shown by cross-correlation between positive and negative subIC3s and RSN templates (Table 47). Notably, IC3 showed significant spatial overlap with the visuospatial RSN (rho = 0.31).We observed the progressive increase of the IC3 presence across four resting-state runs over neurofeedback training in the experimental group  but not in the control group  .43. For whole-brain EC estimates, we found an average increase of the EC across resting-state runs in the experimental as compared to the control group in several brain areas, including dmPFC, IFG, vlPFC, SPL, ITG, and MTG  mapping to reveal resting-state modulations of brain areas that are central within the small‐world network and strongly correlated with other nodes35. We did not observe progressive FC modulations in the control group.We used conventional seed-based FC analysis to investigate modulations of resting-state dmPFC connectivity with the rest of the brain. We found larger average increase of FC across resting-state runs in the experimental as compared to the control group in dmPFC, hippocampus, vmPFC, IPS/IPL, MTG, PCC, MFG, and BF Table , Fig. 4CTo directly link learning effects and resting-state EC, FCD and seed-based FC estimates, we identified brain areas associated with an interaction between the neurofeedback slope and valence difference covariates and the corresponding main effects of group  effects associated with learning effective connectivity-based neurofeedback related to positive-social emotion regulation Fig. . Most imR self-inhibitions. We found that larger learning slopes were associated with larger average dmPFC self-inhibition and average AMYR disinhibition, which is characteristic of progressive dmPFC self-inhibition in the experimental group ; to the motor cortex (motor predictions); as well as to sensory cortices (sensory predictions), including somatosensory and primary visual cortices69. Identified brain regions were also consistent with those typically engaged in neurofeedback, e.g. vlPFC, dlPFC, ACC, premotor, temporo-parietal and occipital cortices, anterior insula, putamen, caudate and thalamus59.To complement the hypothesis-driven model-based effective connectivity analysis, we used the data-driven exploratory analysis to investigate progressive modulations of whole-brain spontaneous spatiotemporal activity patterns Fig. . Highly 70. In addition, vlPFC could be implicated in emotion regulation processes by selecting or inhibiting goal-appropriate responses and interpretations to emotional situations52 as well as in diminishing self-reports of negative affect via impacting amygdala reactivity50. SPL and MFG are considered a part of the dorsal attention network, and areas including IPL are a part of the ventral attention network, critically implicated in visuospatial function71. Increased prefrontal and limbic connectivity along with increased connectivity within visuospatial RSNs, as also indicated by tICA, may indicate increased integration of visuospatial information in the scene during positive-social imagery. Decrease of connectivity between dmPFC and IPL (posterior PG) could be associated with strong referencing to one’s self during the control of one’s own emotions30 and related to visuospatial processing by shifting focus towards distant space63. In addition, the posterior PG has been shown to be involved in shifting the attention system toward stimuli that have high salience72. Thus, a progressive decrease of dmPFC-IPL connectivity could indicate a counter-mechanism against this shift of visuospatial and attention systems during learning self-referential emotion regulation and top-down dmPFC control over bilateral amygdala.The findings of ROI network model-based effective connectivity and whole-brain data-driven spatiotemporal activity insights are consistent with broadly used functional connectivity analyses based on voxel-wise Pearson correlations. The average increase of rs-FC across runs was observed in multiple areas in the experimental group as compared to the control group, most notably in the dmPFC, hippocampus, vmPFC, IFG BA44), vlPFC (BA45), SPL, MTG and MFG, in terms of the EC and FCD estimates  increased valence ratings, (ii) increased top-down regulation mechanisms exerted from dmPFC onto bilateral amygdala during training, transfer runs and resting-states at the end of training sessions, (iii) increased dmPFC activity during training and transfer runs, (iv) decreased right amygdala activity during training, (v) lower dmPFC self-inhibition during resting-states at the end of training sessions in the experimental as compared to the control group, (vi) increased resting-state functional connectomics in emotion regulation, neurofeedback learning, visuospatial and memory networks, and (vii) linked behavioral, task-related and resting-state alterations. We suggest that lower progressive dmPFC self-inhibition in the experimental group could be associated with its increased connectivity with other regions, as evidenced by progressively increased patterns of spatiotemporal activity and functional connectomics. Our findings highlight a large-scale synergy between neurofeedback task-related and resting-state connectivity during intense neurofeedback training and moves towards better understanding of neurofeedback learning mechanisms.77, imagery learning78, as well as cognition and emotion regulation79. Since neurofeedback training typically requires multiple training runs spanned over daily training sessions with intermediate and between-day rest and sleep periods, it could be assumed that specific restoration and consolidation processes might take place between the neurofeedback training sessions. It was also suggested that larger increases in the neurofeedback signal across training sessions on different days than within daily training sessions could indicate a between-day consolidation effect80. Further research is needed to investigate whether resting-states at the end of neurofeedback training sessions could reflect the beginning of specific restoration and consolidation effects. Investigation of rest and sleep processes between neurofeedback training periods could be used to assess the efficacy of intermediate rests, and to help in optimizing the number of training sessions and rest days required for effective neurofeedback-based therapy.Between-day restoration and consolidation effects related to rest and sleep take place within non-neurofeedback studies, for example in studies focusing on memory processing29. The present study data sample is based on previously unpublished resting-state data from the published positive-social emotion regulation neurofeedback study29. The sample size of this preliminary study was selected based on the practical considerations, such as availability of scanner time and funding. Healthy participants were assigned sequentially to experimental  and control  groups, which allowed us to derive control (sham) feedback values based on values of the best performing participants in the experimental group. Experimental participants successfully acquired the positive-social emotion regulation through neurofeedback as compared to control participants29. Participants gave written informed consent to participate in the experiment, all methods used in this study were performed in accordance with the relevant guidelines and regulations of the University Hospital of Geneva, all experimental procedures used in this study were approved by the Ethics Committee of the University Hospital of Geneva. At the beginning of the experiment, participants were provided with written instructions describing experimental procedures, which stated that they would be asked to control the emotion regulation networks using induction of positive-social emotions. Behavioral testing included psychological questionnaires before the experiment and rating positive-social stimuli for valence and arousal before and after the experiment using the self-assessment manikin (SAM) scale .Fifteen healthy volunteers from the local student/staff community  with normal or corrected-to-normal vision and no prior history of neurological or psychiatric diseases performed a 3-day neurofeedback training experiment2*-weighted EPI sequence  .To test whether learning positive-social emotion regulation using DCM connectivity-based neurofeedback led to specific changes in resting-state spontaneous activity, conventional eyes-closed resting-state fMRI runs (6.1 min run duration) were acquired prior to neurofeedback training and after each training session . Isotropic 1.8 mm3 fMRI scans were downsampled to isotropic 3 mm3 images to facilitate the computationally intensive eigenvector centrality mapping while keeping data preprocessing identical for all applied analyses. Physiological noise of fMRI runs was reduced by regressing out nuisance signals voxel-wise and band-pass filtering in the 0.008–0.15 Hz range. We regressed out the average and first principal component of white matter (WM) and cerebrospinal fluid (CSF) signals, the first-order polynomial trend, as well as 6 head motion parameters, derivatives for head motion parameters and scans with excessive motion . Framewise displacement across scans was estimated as the combined difference of head motion displacements between subsequent scans85. The moderate low-pass frequency was set to 0.15 Hz, because of preceding neurofeedback training with relatively short regulation and baseline blocks (12 s). The preprocessing and analysis details associated with the neurofeedback training and transfer runs have been published before29.Resting-state fMRI data were preprocessed using SPM12  and in-house MATLAB scripts. The first 10 volumes were discarded to account for T1 saturation effects. The fMRI data preprocessing included realignment to the mean scan of each fMRI run, coregistration to the standard MNI structural template using DARTEL29. Such a comparison was expressed in terms of the logarithmic Bayes factor and allows to test which model architecture explains the acquired real-time time-series best, i.e., dominates. In our previous publication the experimental and sham groups were found to be significantly different in terms of the applied neurofeedback task-related effective connectivity estimates and concomitant learning effects.To provide the effective connectivity-based neurofeedback, two DCM model alternatives were compared in real-time, namely, top-down network model  versus a bottom-up one  using task-related bilinear DCM and Bayesian model comparison87 approach, as implemented in SPM1240. In contrast to task-related DCM, resting-state spDCM is developed to analyze effective connectivity during rest and based on fitting the cross-spectra of the observed time-series. To extract time-series for spDCM, we used individual static dmPFC and bilateral amygdala regions of interest (ROIs) defined as the disjunction of consistent dynamic ROIs in the last 3 neurofeedback training runs for each participant of the experimental group and first 3 neurofeedback runs for each participant of the control group. Specifically, for DCM-based neurofeedback training, most active voxels within preselected anatomical ROIs were identified dynamically using incrementally estimated general linear model (GLM) and updated across neurofeedback trials. The size of dynamic dmPFC ROIs did not differ across neurofeedback runs .Here, to investigate whether learning positive-social emotion regulation using task-related effective connectivity-based neurofeedback leads to progressive increase in resting-state effective connectivity within the trained network, we applied rest-related effective connectivity metrics based on three-level hierarchical model. We characterized within-run, between-run and between-subject effective connectivity using spectral DCM (spDCM) and Parametric Empirical Bayes (PEB)At the first-level, to infer the effective connectivity that best explains time-series for resting-state runs per participant, we estimated fully-connected DCM models consisting of three brain regions, namely: dmPFC and bilateral amygdala static ROIs Fig. A. Becaus87.Finally, to estimate which model architecture best explains the commonalities and differences between experimental and control groups, we defined a set of three candidate (reduced) models, which varied in terms of their A-matrix connections i.e., fully-connected, top-down and bottom-up models Fig. D. Connec42. It was carried out within the MELODIC toolbox . Pooled resting-state runs of all participants were preprocessed, including masking of non-brain voxels, voxel-wise demeaning and normalization of the voxel-wise joint data variance. FSL whitens and reduces the dimensionality of the data using principal component analysis as an internal processing step before the ICA criterion is optimized. tICA explains observations using a tensor structure (time × space × run domains), such that a component with a specific spatial map and time-series is weighted for each run. Estimated IC maps were z-scored by dividing by the standard deviation of the residual noise and thresholded (posterior probability p > 0.5) by fitting a mixture model to the histogram of intensity values42.DCM is a hypothesis-driven approach that requires prior knowledge about the target brain network dynamics and the modulatory influences. To investigate without prior assumptions whether learning positive-social emotion regulation via network model-based effective connectivity neurofeedback leads to a progressive increase of whole-brain spatiotemporal patterns of spontaneous resting-state activity, we performed data-driven tICA89. It is known that higher model orders lead to more fragmented IC maps that might capture more local changes, while lower model orders are capturing more widespread effects. Thus, we checked the dimensionality range from #50 to #80 decompositions that explained between 78 and 95% of the total variance. Note that spatial–temporal ICs can encompass spatially anticorrelated subcomponents within a single time-series (subICs)42. The polarity of these components was synchronized across decompositions using cross-correlations across IC time-series as a reference. For consistent interpretation, anticorrelated spatial subICs were justified as positive (z > 0) and negative (z < 0) across all comparisons based on the cross-correlations between IC time-series.The selection of the optimal number of ICs  remains a challenge for techniques such as ICA and PCAFor each IC and per decomposition, we computed a linear regression of IC weights (loadings) across four resting-state runs per participant using Pearson’s rho. To investigate within and between group effects, we used t-tests on Pearson rho values. Results were corrected for multiple comparisons using False Discovery Rate correction  across 2015 comparisons (sum of ICs for 31 tICA decompositions).46. Thus, positive and negative subIC spatial maps were compared to RSN templates using spatial cross-correlation with a threshold for Pearson rho > 0.25 (as implemented in FSL)47.For the decomposition that revealed IC with significant progressive changes across resting-state runs, we demonstrated the consistency of decomposed ICs with well-established resting-state networks (RSNs)43. EC mapping was applied to preprocessed whole-brain resting-state data using LIPSIA v3.1.043 based on positive correlations without thresholding, similarly to previous studies92. Voxel-wise FCD was used to identify resting-state modulations of brain areas with large long-range correlations. Positive FCD maps were estimated voxel-wise as the number of Pearson correlations with the rest of the brain, which were above 0.7. For long-range FCD maps, local FCD maps (10 voxels extent threshold) were subtracted from total FCD maps.To study the spatiotemporal structure of whole-brain functional components related to emotion regulation and neurofeedback learning networks, we performed conventional rs-FC analyses based on voxel-wise Pearson correlations. For each resting-state run, we estimated voxel-wise eigenvector centrality (EC), long-range FC density (FCD) and ROI seed-based FC maps. Whole-brain EC and FCD are methodologically different analyses with minimal parametrization, which were performed to study different aspects of functional connectomics. Voxel-wise EC analysis was performed to reveal modulations of brain areas that are central within the small‐world network and strongly correlated with other nodesSeed-based FC analysis was used to investigate modulations of resting-state dmPFC connectivity with the rest of the brain. As dmPFC FC seeds, we used individual static dmPFC ROIs as for resting-state effective connectivity analyses. To reduce calculations for the FC analysis, we limited them to grey-matter voxels. For dmPFC seeds , we estimated whole-brain FC maps based on Pearson correlations without thresholding between the average seed time-series and the rest of the brain. Pearson correlations were Fisher-transformed.48.For the whole-brain group-level analysis of FCD, FC and EC maps, we performed a three-way ANOVA with the fixed factors ‘run’ and ‘group’, and the random factor ‘subject’, as implemented in SPM12. Individual neurofeedback learning slope and valence difference, age and gender were modeled as covariates for all analyses, as well as size of dmPFC ROI covariate was additionally included for seed-based FS analysis. Statistical maps were corrected for multiple comparisons using whole-brain family-wise error correction . Anatomical labels were validated using Neurosynth.org and the SPM Anatomy ToolboxSupplementary Information.


Together, our understanding of the biological mechanisms of ZIKV exposure would aid in the development of anti-ZIKV therapies targeting host cellular and/or viral components to combat ZIKV infection and its neurological manifestations. In this present work, we review the current understanding of ZIKV entry mechanisms into the CNS and its implications on the brain. We also highlight the status of the drug repurposing approach for the development of potential antiviral drugs against ZIKV.Zika virus (ZIKV) is a mosquito-borne, single-stranded RNA virus belonging to the genus Flavivirus. Although ZIKV infection is usually known to exhibit mild clinical symptoms, intrauterine ZIKV infections have been associated with severe neurological manifestations, including microcephaly and Guillain Barre syndrome (GBS). Therefore, it is imperative to understand the mechanisms of ZIKV entry into the central nervous system (CNS) and its effect on brain cells. Several routes of neuro-invasion have been identified, among which blood–brain barrier (BBB) disruption is the commonest mode of access. The molecular receptors involved in viral entry remain unknown; with various proposed molecular ZIKV-host interactions including potential non-receptor mediated cellular entry. As ZIKV invade neuronal cells, they trigger neurotoxic mechanisms  Zika virus (ZIKV) is a mosquito-vectored flavivirus, consisting of three structural proteins , seven non-structural (NS) proteins  and a single-stranded RNA genome of positive polarity . ZIKV haThe entry mechanism of ZIKV into human cells, particularly neural cells, remains poorly understood. Previous studies have demonstrated several ZIKV entry factors, including dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), AXL receptor tyrosine kinase (AXL), TYRO3 protein tyrosine kinase (TYRO3) and though to a lesser extent, T-cell immunoglobulin and mucin domain 1 (TIM-1), mediate entry of ZIKV into human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells ; Table 1However, recent findings demonstrated that ZIKV still showed effective invasion, infection, and replication in AXL-depleted cerebral organoids , neural via tight junction proteins (TJP), which are associated with pericytes, astrocytes, and microglia (The blood–brain barrier (BBB) comprises endothelial cells strongly adhered icroglia . The tigicroglia  transcelicroglia  infectedicroglia  paracellicroglia  retrograicroglia  blood-toicroglia . Among ticroglia .in vitro  and iPSC-derived BBB models without significantly altering the endothelial barrier integrity and permeability in vitro . Despitein vitro . These fin vitro .in vitro studies have demonstrated ZIKV infection and release from both apical and basolateral surfaces of brain endothelial cells without compromising the barrier permeability and integrity  and receptor for advanced glycation end (RAGE) expressed on ZIKV-infected monocytes. In a state of inflammation, the natural ligand of CCR7, chemokine ligand 19 (CCL19), and the danger-associated molecular pattern (DAMP) molecule, nuclear high mobility group box 1 (HMGB1) are upregulated. This receptor-ligand interaction induces dysregulation of the endothelium layer associated with disorganization of cadherins and actin fibers, thereby increasing the membrane’s permissiveness . The proin vitro study using transfected HBMECs (THBMEC) infected with ZIKV demonstrated vascular leakage enhancement by ZIKV through disruption in the cytoskeleton and tight junctional proteins arrangement. This occurs via the upregulation of genes that are involved in the production of proinflammatory cytokines, such as interleukin-6 (IL-6), TNF-α, cell adhesion molecules (CAMs), and growth factors , interleukin-8 (IL-8), IL-6, and TNF-α], chemokines [C-C motif ligand-2 (CCL2/MCP-1) and CCL5], and CAMs [vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1)]. ZIKV infection of pericytes showed upregulation of chemokines (CCL5 and CXCL10), cytokines , as well as Toll-like receptor 3  inhibitor (CDKi), p21Cip1/Waf1 leading to downregulation of G2/M phase regulator, cyclin B1, and augmentation of the proapoptotic pathway with the increased B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax)/Bcl-2 ratio  pathway of ATP production . Mitocho by ZIKV . That sa by ZIKV . In the  by ZIKV .In vitro and in vivo studies have shown an increase in ER stress proteins as well as expression of key molecules of the UPR, such as glucose regulatory protein 78 (GRP78), calreticulin, calnexin, and protein disulfide isomerase (PDI) in infected neural cells (via eIF2α phosphorylation and SG proteins exploitation (Zika virus is capable of modulating the host endoplasmic reticulum (ER) structure for its replicative benefits . The viral cells , resultial cells . In paraoitation . Reticuloitation . Besidesoitation .Modulation of the host autophagy mechanism has been demonstrated by flaviviruses, such as DENV , HCV Si, and ZIKvia ZIKV’s upregulation of pre-mRNA-processing-splicing factor 8 (Pprf8), an anti-apoptotic factor demonstrated in Picornavirus-infected neurons  host-directed antivirals, which focus on modulating host cellular processes used for viral life cycle or (ii) direct-acting antivirals, which target viral components. In order to expedite the development of effective antivirals against ZIKV, much focus has been given to drug repurposing or drug re-profiling . RecommeMYD1, an engineered decoy AXL receptor, has a high-affinity binding to Gas6, preventing the interaction between ZIKV viral particles and the AXL receptor . AXL kinNanchangmycin, an antibacterial and insecticidal polyether, potently inhibits the early entry stage in the ZIKV replication process and infection in primary cells of uterine, placenta, umbilical vein, BBB, and neuron-glial mixed midbrain at 0.1–0.4 μM with low cytopathic effects .via cholesterol oxidization, demonstrated antiviral activity by preventing ZIKV entry through internalization and fusion of ZIKV envelope with host endosomal membrane, not via ZIKV attachment (25-hydroxycholesterol (25HC), an endogenous oxysterol formed tachment . It decrtachment . 25HC ditachment .in vitro BBB model), hNPCs, and mouse neurospheres from ZIKV infection -retinamide (4-HPR or Fenretinide), known for its anticancer properties, is found to also exhibit anti-ZIKV activity, presumably facilitated by a host factor, in significantly reducing ZIKV RNA production without effect on viral polymerase or membrane-related replication complexes (in vivo (omplexes . 4-HPR w(in vivo .50) at 24 and 27 nM, respectively , and PHA-690509, a CDKi, are able to suppress the caspase-3 pathway as well as improve hNPCs and astrocyte viability . Their eectively . Bithionectively . It is rectively .In various cell types, including hNPC, NGI-1 targets the ER-membrane multiprotein complex, and oligosaccharyltransferase (OST) complex, which is responsible for the catalysis of N-linked glycosylation of new proteins, by inhibiting viral RNA production and replication but independent of OST complex’s catalytic activity .de novo production pathway of purine, resulting in a smaller pool of nucleotides to be used for viral replication  was tested on epithelial and human neuronal cells, revealing efficient, dose-dependent inhibition of ZIKV infection, and lower cytotoxicity for neuronal cells . It robulication . 6MMPr ilication . Howeverlication .Ribavirin is a guanosine analog approved to treat influenza A and B, severe respiratory syncytial virus, Lassa fever virus, and hepatitis C . SimilarHeparin, an anticoagulant, exhibited anti-apoptotic activity in hNPCs, potentially through inactivation of caspase-3 . Its ultin vitro and in vivo studies here demonstrated neuroprotective effects of memantine, Dizocilpine/MK-801, agmatine sulfate, and ifenprodil in ameliorating ZIKV-induced apoptosis without impacting the viral replication process  receptors are expressed in microglial cells, IL-1 receptor antagonist (IRA) reduces fetal neuroinflammation by inhibiting fetal microglial activation . IRA indin vitro findings using glioblastoma SNB-19 cells , an FDA-approved anthelmintic therapy. Consistent with previous 19 cells , NIC is 19 cells . Using hn hiNSCs . Togethen hiNSCs . Both can hiNSCs . All excn hiNSCs .Sofosbuvir, which is a uridine nucleotide analog and anti-HCV drug, uses its 2′-F radical to form a covalent bond with amino acid residue Asn612 . The forvia attachment to polymerase  has been shown to promote neuronal cell growth and ameliorate apoptosis . It uprelymerase .A study using a mice model revealed an engineered AH peptide with D-enantiomer (AH-D) confers protection against ZIKV by stimulating viral liposome rupture . Therapein utero transmission and neuropathogenesis. In addition, systematic and long-term follow-ups are imperative to determine the unknown long-term neuropathological and behavioral consequences in both new-borns and adults.Following the sudden increase in the number of new-borns with microcephaly during the Brazillian outbreak of ZIKV, numerous studies have been conducted and they verify the causal relationship between ZIKV infection and neurological disorders. ZIKV possesses the ability to cross the placental barrier and BBB, targeting brain cells, particularly in the developing brain. The virus crosses the BBB through (i) transcellular pathway, (ii) monocyte transmigration through Trojan horse pathway, (iii) vascular leakage enhancement, and (iv) disruption of the choroid plexus epithelial layer. Upon crossing the BBB and entering the brain, ZIKV infects various cells in the developing brain, causing neuropathogenesis through (i) cell cycle perturbation, (ii) mitochondrial dysfunction, (iii) ER stress and UTP, (iv) modulation of host autophagy, and (v) neuronal apoptosis. Despite major advances in this field, many important questions remain answered: (i) Why only a small number of fetuses born to infected mothers develop microcephaly? What are the long-term outcomes of infected neonates without detectable abnormalities at birth? (ii) Is an intact BBB permissive to ZIKV infection? Can ZIKV target astrocytes in the adult brain? Will there be any long-term effects in adults infected with ZIKV? and (iii) Since the innate immune responses and mitochondria functions are connected, how does mitochondrial dysfunction upon ZIKV infection affect the cellular immune responses? Therefore, more studies are required to fully understand the mechanisms of cross-talk between ZIKV and the human brain, as well as the host factors involved in vitro. It is also critical to evaluate their efficacy in appropriate animal models to better predict their clinical outcome. In addition, the ability of these drugs to prevent ZIKV CNS invasion needs to be evaluated. Importantly, ZIKV drugs should be clinically safe for use in pregnant women and fetuses.Importantly, despite its severe implications and potential future outbreaks, there are still no vaccine or antiviral drugs available against ZIKV. The development of new therapeutic approaches should be one of the priorities for future research. The drug-repurposing strategy offers a promising avenue to identify potential antiviral drugs against ZIKV within a shorter development timeline and known safety. This approach is also particularly appealing for mosquito-borne viruses, which receive less attention from the affluent regions and pharmaceutical companies. A number of host-targeting and virus-targeting agents with potential inhibitory activity against ZIKV have been identified. However, the majority of these drugs have only been tested LT and VB: conceptualization. LT and TK: methodology and writing—original draft preparation. LT, TK, WJ, and VB: writing, review, and editing. VB and WJ: supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To the Editor,We report on a 74-year-old patient with severe pneumonia who had negative results on the first two reverse transcription-polymerase chain reaction (RT-PCR) examinations . His medical history and computed tomography findings strongly suggested coronavirus disease-2019 (COVID-19)  RNA, has been the primary diagnostic tool for COVID-19. However, several reports have criticized this method, particularly its sensitivity [It is thought that the primary target of SARS-CoV-2 is lung epithelial cells. Conversely, previous post-mortem analysis showed viral elements within endothelial cells and accumulation of inflammatory cells, with evidence of endothelial and inflammatory cell death [SARS-CoV-2 infection has been associated with endothelial glycocalyx injury . The endA previous study suggested that the endothelial glycocalyx was already injured in diabetic patients and that widespread inflammatory cell migration aggravated inflammation . Thus, tThe direct relationship between COVID-19 and endothelial glycocalyx injury requires further investigation. Future studies are needed to establish the diagnostic and therapeutic importance of the endothelial glycocalyx.


Inflammatory bowel disease (IBD) is an incurable disease of the gastrointestinal tract with a lack of effective therapeutic strategies. The proinflammatory microenvironment plays a significant role in both amplifying and sustaining inflammation during IBD progression. Herein, biocompatible drug-free ceria nanoparticles (CeNP-PEG) with regenerable scavenging activities against multiple reactive oxygen species (ROS) were developed. CeNP-PEG exerted therapeutic effect in dextran sulfate sodium (DSS)-induced colitis murine model, evidenced by corrected the disease activity index, restrained colon length shortening, improved intestinal permeability and restored the colonic epithelium disruption. CeNP-PEG ameliorated the proinflammatory microenvironment by persistently scavenging ROS, down-regulating the levels of multiple proinflammatory cytokines, restraining the proinflammatory profile of macrophages and Th1/Th17 response. The underlying mechanism may involve restraining the co-activation of NF-κB and JAK2/STAT3 pathways. In summary, this work demonstrates an effective strategy for IBD treatment by ameliorating the self-perpetuating proinflammatory microenvironment, which offers a new avenue in the treatment of inflammation-related diseases.The online version contains supplementary material available at 10.1186/s12951-022-01319-7. Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), are characterized by immune-mediated refractory chronic inflammation in the gastrointestinal tract . To dateAccumulating evidence strongly suggests that the proinflammatory microenvironment in the intestine plays a significant role in both amplifying and sustaining inflammation during IBD progression –7. The c3+ (reduced) and Ce4+ (oxidized) sites on the particle surface -DPA-714 dynamic PET/CT imaging were performed to allow for the evaluation of both active inflammation and active macrophage density as a function of CeNP-PEG treatment. The possible signaling pathways were also investigated to identify the potential mechanism of action associated with CeNP-PEG treatment for IBD. These findings collectively document that CeNP-PEG treatment disrupts the circuitry of the proinflammatory microenvironment in IBD, which offers a new therapeutic concept for any other chronic inflammatory conditions.Herein, we demonstrate the correlation between proinflammatory macrophages and disease severity in UC patients based on computed tomographic enterography (CTE) data, endoscopic images, biochemical measurements, and intestinal tissue biopsies. Polyethylene glycol (PEG)-capped ultrafine CeNP (CeNP-PEG) with excellent ROS scavenging activity were prepared. To identify the ability of CeNP-PEG in ameliorating the proinflammatory microenvironment of IBD, the ROS levels, barrier function of the intestinal epithelium, proinflammatory cytokine levels, intestinal macrophage function, and the Th1/Th17 response were determined by using dextran sulfate sodium (DSS)-induced colitis murine models. In vivo CTE and  DPA-714 is suitable for studying the localized inflammation associated with rodent IBD models [18F] DPA-714 concentration observed for the DSS group was significantly greater that the concentrations observed for the PBS and DSS + CeNP-PEG groups  are both involve in IBD pathogenesis , 28. FloIn line with previous reports showing that ceria nanoparticles were nontoxic in vivo at the therapeutic doses, we did not observe any toxicity of CeNP-PEG in current study . The bio2•− and H2O2 scavenging capacities, clearly reduce the levels of multiple proinflammatory cytokines and the proinflammatory features of macrophages. Furthermore, CeNP-PEG significantly attenuated the intestinal inflammatory microenvironment in a DSS-induced IBD model by decreasing the proinflammatory responses of macrophages and Th1/Th17 cells. The molecular mechanism by which CeNP-PEG protect against IBD may be attributed to suppression the co-activation of the NF-κB and JAK2/STAT3 pathways to ameliorate the proinflammatory microenvironment. Our study not only helps to explain the therapeutic mechanism of CeNP-PEG for IBD treatment but also provides a new strategy to address the unsatisfactory clinical requirement of treating inflammation-related diseases by ameliorating the proinflammatory microenvironment.In summary, we developed the ultrasmall drug-free nanozyme CeNP-PEG with multiple ROS scavenging activities for the efficient treatment of IBD. CeNP-PEG possess robust •OH, OAdditional file 1. Supplementary Material.


Brain-computer interface-assisted motor imagery (MI-BCI) or transcranial direct current stimulation (tDCS) has been proven effective in post-stroke motor function enhancement, yet whether the combination of MI-BCI and tDCS may further benefit the rehabilitation of motor functions remains unknown. This study investigated brain functional activity and connectivity changes after a 2 week MI-BCI and tDCS combined intervention in 19 chronic subcortical stroke patients. Patients were randomized into MI-BCI with tDCS group and MI-BCI only group who underwent 10 sessions of 20 min real or sham tDCS followed by 1 h MI-BCI training with robotic feedback. We derived amplitude of low-frequency fluctuation (ALFF), regional homogeneity (ReHo), and functional connectivity (FC) from resting-state functional magnetic resonance imaging (fMRI) data pre- and post-intervention. At baseline, stroke patients had lower ALFF in the ipsilesional somatomotor network (SMN), lower ReHo in the contralesional insula, and higher ALFF/Reho in the bilateral posterior default mode network (DMN) compared to age-matched healthy controls. After the intervention, the MI-BCI only group showed increased ALFF in contralesional SMN and decreased ALFF/Reho in the posterior DMN. In contrast, no post-intervention changes were detected in the MI-BCI + tDCS group. Furthermore, higher increases in ALFF/ReHo/FC measures were related to better motor function recovery (measured by the Fugl-Meyer Assessment scores) in the MI-BCI group while the opposite association was detected in the MI-BCI + tDCS group. Taken together, our findings suggest that brain functional re-normalization and network-specific compensation were found in the MI-BCI only group but not in the MI-BCI + tDCS group although both groups gained significant motor function improvement post-intervention with no group difference. MI-BCI and tDCS may exert differential or even opposing impact on brain functional reorganization during post-stroke motor rehabilitation; therefore, the integration of the two strategies requires further refinement to improve efficacy and effectiveness. Robot-assisted motor imagery (MI) and brain-computer interface (BCI) system is an advanced technique for improving motor function recovery in post-stroke rehabilitation. Motor imagery allows the imagination of movements without physical execution, and electroencephalography-based brain-computer interfaces (EEG-BCIs) enables interaction with the environment using brain signals . By combTranscranial direct current stimulation (tDCS) is another promising technique for stroke rehabilitation. tDCS is a non-invasive brain stimulation technique that uses constant, low direct current delivered via electrodes (anode and cathode) on the head to provide a reversible and focal way for neuroplasticity modulation , 2000. tResting-state functional magnetic resonance imaging (rs-fMRI) provides an indirect measure of neuronal activity and connectivity based on regional blood-oxygen-level-dependent (BOLD) signal fluctuations without any imposed task . VariousNevertheless, little work has been done to examine the brain functional changes in stroke after MI-BCI and tDCS-based intervention. Most of the previous work focused on cross-sectional brain ALFF/ReHo/FC changes in stroke patients in comparison with healthy controls. In terms of regional activity, some studies reported lower ALFF in the motor cortex and lower ALFF/ReHo in the default mode network (DMN) in stroke patients compared to healthy controls  while otIn this study, we examined the alterations in brain functional activity and connectivity in chronic subcortical stroke patients using ALFF, ReHo, and FC. The changes in the neural function of stroke patients pre- and post-intervention were investigated in the MI-BCI only group and MI-BCI combined with tDCS group separately. We hypothesized that 1) lowered ALFF/ReHo and disrupted FC in the motor cortex and DMN would be observed in stroke patients compared to healthy controls at baseline; 2) stroke patients would have higher ALFF/ReHo/FC in the motor cortex and DMN post-intervention, the degree of which would differ between the MI-BCI and the MI-BCI + tDCS groups. Lastly, we investigated whether these alterations in ALFF/ReHo/FC post-intervention would correlate with motor function recovery measured by Fugl-Meyer Assessment (FMA) scores.ClinicalTrials.gov . Written inform consent was obtained from all participants.Forty-two stroke patients aged from 21 to 70 years with moderate to severe motor impairments and 11 age-matched healthy controls were recruited in this study. The inclusion criteria for stroke patients were (1) first-ever subcortical stroke > 9 months before the study enrollment; and (2) unilateral moderate to severe upper extremity motor impairment measured by the upper extremity component of the FMA (score range 11–45). The exclusion criteria for stroke patients were (1) epilepsy, neglect, cognitive impairment, and other neurological or psychiatric diseases; (2) severe arm pain; (3) severe spasticity measured by the Modified Ashworth Scale in the shoulder or elbow (score > 2); (4) contraindications to tDCS; (5) weak grip strength measured by a dynamometer (weight < 10 kg); and (6) participation in other interventions or trials that target post-stroke motor recovery. Twenty-three stroke patients were excluded because they either declined to participate in the study or failed to meet the inclusion or BCI performance criteria. Detailed demographics of the remaining 19 patients and 11 healthy controls who completed the training and scans were provided in n = 10) and MI-BCI group (n = 9) with matching pre-training FMA scores. The MI-BCI + tDCS group received 20-min real tDCS, in which direct current was applied using a saline-soaked pair of surface sponge electrodes at an intensity of 1 mA with the anode placed over the M1 motor cortex of the ipsilesional hemisphere and the cathode placed over the contralesional M1 while the MI-BCI group received 20-min sham tDCS, in which current ramped up and down to give subjects the sensation of the stimulation. Both groups completed 10 sessions of MI-BCI training within 2 weeks. Each MI-BCI training was 40 min and consisted of 160 trials. In each trial, subjects were prepared with a visual cue and instructed to perform motor imagery by an instruction cue. The EEG segment of 0.5– 4.5 s from the instruction cue was then extracted to classify the EEG segment to perform online detection of MI or the idle condition using the Filter Bank Common Spatial Pattern (FBCSP) algorithm , and a resting state T2∗-weighted echo planar imaging . During the 7-min rs-fMRI, participants were instructed to remain relaxed and awake with their eyes closed and not engage in any specific activity.MRI data were collected using a 3T scanner  with a 32-channel head array coil. The scan protocol included a T1-weighted magnetization prepared rapid gradient-echo (MPRAGE) sequence  and analysis of functional neuroimaging (AFNI) following our standard protocol . The preQuality of co-registration and normalization were visually inspected. One post-intervention image with movement more than 4 mm was discarded. Thus after quality control, data of 19 stroke patients and 11 healthy control remained at baseline. Data of 10 subjects in the MI-BCI + tDCS group and 8 subjects in the MI-BCI group remained for pre- and post-intervention analysis.The ALFF was calculated using a toolbox for Data Processing & Analysis for Brain Imaging (DPABI) . For eacThe ReHo maps were also produced using DPABI toolbox. ReHo was calculated in a voxel-wise manner using Kendall’s concordance coefficient (KCC) across the time series of a centered cubic cluster of 27 voxels. A large ReHo value for a given voxel indicates a high local synchronization of rs-fMRI signal among neighboring voxels and vice versa.FC analysis was performed for two networks, default mode network and somatomotor network, which were previously reported to show changes in ALFF and ReHo analysis in our study. DMN and SMN were defined using Region of Interest (ROI) from a FC-based 114-node parcellation . 24 ROIst-tests. Voxelwise analyses  were thresholded using a height threshold of p < 0.01 (uncorrected) and extent threshold of p < 0.05 (familywise-error (FWE) corrected).Baseline differences in ALFF, ReHo, and FC between stroke patients and healthy controls were examined using two-sample Post hoc tests were subsequently performed to examine intervention effects in each group separately. Voxelwise analyses  were thresholded using a height threshold of p < 0.01 (uncorrected) and extent threshold of p < 0.05 (FWE corrected).To examine the effects of intervention as well as interaction effects between intervention and group on FMA scores, ALFF and ReHo maps, we conducted mixed-effect Analyses of Variance (ANOVA) with intervention as within-subjects factor and group as between-subject factor. Finally, associations between functional activity/connectivity and clinical outcomes (motor function improvement measured by FMA scores) were examined using Pearson’s correlation. Eight-mm spheres created in MARSBAR  with peap = 0.002) but no interaction effect between intervention and group (p = 0.450), suggesting that both groups gained significant motor function improvement post-intervention with no group difference observed either immediately or 4 weeks after training. Post hoc tests further showed that significant FMA gains compared to baseline were only found at 4 weeks after training  but not immediately post-training  .There was no difference between the MI-BCI group and MI-BCI + tDCS group at baseline in terms of age, post-stroke time, or FMA scores . A 3 (in = 0.11) . FurtherCross-sectional comparisons conducted at baseline indicated abnormal ALFF and ReHo patterns in stroke patients compared to healthy controls. Stroke patients showed lower ALFF in the ipsilesional precentral gyrus and postcentral gyrus and higher ALFF in the precuneus, posterior cingulate gyrus, angular gyrus, middle frontal gyrus, and contralesional insula . Thus, tStroke patients had higher ReHo in the precuneus and posterior cingulate gyrus and lower ReHo in the ipsilesional superior temporal gyrus and insula . CollectSignificant interaction effects between intervention  and group  were found in ALFF and ReHo based on mixed-effect ANOVA, suggesting differential brain functional activity and connectivity changes in the MI-BCI group and MI-BCI + tDCS group. Specifically, differential ALFF patterns post-intervention were found in the contralesional regions within the DMN and SMN, including supplementary motor area, contralesional precentral gyrus, middle frontal gyrus, precuneus, and angular gyrus . On the Post hoc analyses revealed that the MI-BCI group showed increased ALFF in the contralesional supplementary motor area and precentral gyrus post-intervention and decreased ALFF in the precuneus, inferior parietal lobule, and middle occipital gyrus  (post hoc effects) was found.Given the above ALFF/ReHo patterns mainly in the SMN and DMN, we then investigated if the FC in these two networks was disrupted in stroke patients and whether there was any intervention effect. As expected, stroke patients showed lower FC within the SMN at baseline compared to healthy controls ( = 0.93)  while noTo find the association between changes in brain functional activity/connectivity and motor function post-intervention, we investigated the correlation between the ALFF/ReHo/FC changes and FMA changes.r = 0.639, p = 0.044) between ALFF changes in the precentral gyrus and FMA changes was observed in the MI-BCI group  between ReHo changes at precuneus and FMA changes was found in the MI-BCI group  between inter SMN-DMN FC changes and FMA changes in the MI-BCI group  was reported in the MI-BCI + tDCS group (Differential associations of the FC changes between SMN and DMN and FMA changes were observed in the MI-BCI group and MI-BCI + tDCS group. We found a positive correlation (CI group  while a CS group . The conThis study investigated the brain functional activity and connectivity changes in chronic subcortical stroke patients pre- and post-intervention with MI-BCI and tDCS. We found that stroke patients had lower ALFF in the ipsilesional SMN at baseline and had increased ALFF in the contralesional SMN in the MI-BCI group, providing new evidence for the compensation effect. Furthermore, the higher ALFF/ReHo in the DMN at baseline were decreased after MI-BCI training, illustrating the re-normalization of brain activity and connectivity post-intervention. More importantly, the divergent associations between SMN-DMN FC changes and FMA changes in the MI-BCI group and MI-BCI + tDCS group suggested differential mechanisms in the two approaches.The ALFF findings suggest the possible beneficial effect of brain activity in the contralesional motor cortex after MI-BCI-based intervention. Stroke patients had lower ALFF in the ipsilesional SMN at baseline; and then the MI-BCI group showed increased ALFF in the contralesional SMN post-intervention. Importantly, increased regional brain activity in the contralesional motor cortex was positively associated with motor function improvement, suggesting a compensation effect of the contralesional SMN.A predominant hypothesis of interhemispheric interaction is that the motor cortex of one hemisphere inhibits the contralateral homolog . In lineIn parallel, recent BCI-related researches have shown contradictory results against the predominant hypothesis. Patients were reported to gain significant motor function improvement by utilizing the contralesional motor cortex in BCI therapy . AlthougStroke patients showed lower ALFF/FC in SMN and higher ALFF/ReHo in the DMN compared to healthy controls at baseline. Post-intervention, the MI-BCI group showed increased ALFF in the contralesional SMN and decreased ALFF/ReHo in the DMN. Our results match with previous findings on alterations of brain activity and connectivity in the DMN and the brain re-normalization hypothesis.Alterations of brain activity and connectivity in the DMN were widely reported in stroke patients with impaired cognitive function . Stroke More importantly, re-normalization of the brain activity and connectivity were observed post-intervention. The higher ALFF and ReHo in the precuneus and posterior cingulate gyrus at baseline reduced post-intervention in the MI-BCI group, which refers to re-normalization of the local activity and connectivity. Brain re-normalization in previous studies focused on alterations in FC. Increased FC after BCI rehabilitation therapy in the SMN and DMN where FC was lower at baseline was reported, which also hints at re-normalization of brain connectivity .The efficacy of MI-BCI on improving motor function in stroke rehabilitation has been widely reported . StudiesThis is a pilot study of stroke rehabilitation involving complex multimodal design of neuroimaging, MI-BCI and tDCS, which is limited by the small sample size. Due to the limited sample, we were not able to control for stroke severity and lesion type or location in our analysis. Also, the possible inter-variability in conventional occupational therapy was not taken into account. Future research with large sample size should incorporate these potential important factors to evaluate the long-term rehabilitation effect. Another limitation is the lack of a tDCS-only group which would help to understand the mechanism of tDCS to facilitate the development of stroke rehabilitation strategies. Although the tDCS stimulation parameters applied in this study follow the standard protocol, it would be to study how stimulation parameters such as sites and placement of electrodes could impact the efficiency. Furthermore, while the current study focused on subcortical chronic stroke patients, it is crucial to design effective rehabilitations for stroke patients in acute or subacute phase moving forward.In conclusion, patients with chronic subcortical stroke showed lower ALFF/FC in the SMN and higher ALFF/ReHo in the DMN compared to controls at baseline. Such brain functional vulnerability patterns in the two networks was partially re-normalized post-intervention in the MI-BCI group while the MI-BCI + tDCS group showed no significant changes. Divergent associations between SMN-DMN FC changes and FMA changes were found in the MI-BCI group and MI-BCI + tDCS group. Our results suggested the potential differential or even opposite network-specific brain functional mechanisms underlying MI-BCI with and without tDCS in stroke recovery.The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.The studies involving human participants were reviewed and approved by the National Healthcare Group Domain-Specific Review Board. The patients/participants provided their written informed consent to participate in this study.MH codesigned the study with JZ, EC, and CG performed all the data analysis, and prepared the manuscript. H-JC, FJ, and JC assisted with data preprocessing and manuscript revision. JZ, CG, and XJ supervised and revised the manuscript. EC provided the data. ZL, WH, KA, KP, and H-JC assisted in data collection and preparation. All authors reviewed the manuscript.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.


Beyond providing a precise non-invasive read-out of renal function, such radiotracers may also be used to assess renal inflammation. This manuscript will provide an overview of renal molecular PET imaging and will highlight the transformation of conventional scintigraphy of the kidneys toward novel, high-resolution PET imaging for assessing renal function. In addition, future applications will be introduced, e.g. by transferring the concept of molecular image-guided diagnostics and therapy (theranostics) to the field of nephrology.In recent years, a paradigm shift from single-photon-emitting radionuclide radiotracers toward positron-emission tomography (PET) radiotracers has occurred in nuclear oncology. Although PET-based molecular imaging of the kidneys is still in its infancy, such a trend has emerged in the field of functional renal radionuclide imaging. Potentially allowing for precise and thorough evaluation of renal radiotracer urodynamics, PET radionuclide imaging has numerous advantages including precise anatomical co-registration with CT images and dynamic three-dimensional imaging capability. In addition, relative to scintigraphic approaches, PET can allow for significantly reduced scan time enabling high-throughput in a busy PET practice and further reduces radiation exposure, which may have a clinical impact in pediatric populations. In recent years, multiple renal PET radiotracers labeled with  Figure 1). These are (1) radiotracers that are mainly filtered by the glomerulus to monitor glomerular filtration rate (GFR); (2) radiotracers that undergo tubular secretion for the measurement of effective renal plasma flow (ERPF); and (3), radiotracers that are taken up into the tubules so that morphological and/or tubular uptake function can be assessed  Renal functional imaging with single-photon-emitting radiotracers has been used as a noninvasive diagnostic technique since the mid-1960s e (GFR);  radiotraTable 2 provides an overview of advantages and disadvantages for different imaging modalities used for renal functional imaging.Over the last decades, other imaging modalities for assessing renal function beyond renal radionuclide imaging have emerged and include, but are not limited to, ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI). Ultrasound provides a fast and easily obtainable read-out of kidney morphology, while computed tomography (CT)-angiography provides in-depth renal vascular information. Renal MRI (rMRI), however, provides information with superior soft-tissue contrast to characterize renal masses or parameters on renal flow 68Ga-ethylenediaminetetraacetic acid (68Ga-EDTA), 11C-para-aminobenzoic acid (11C-PABA) and 2-deoxy-2-[18F]fluoro-D-sorbitol (18F-FDS) Recent years have also witnessed a shift from single-photon emission computed tomography (SPECT) to higher-resolution positron emission tomography (PET), which is supported by the overwhelming success and increased availability of PET imaging for oncological applications para-aminohippuate. Given the expensive and time-consuming nature of these diagnostics tests, they are nowadays seldomly performed. Normally, the kidneys receive 20% of cardiac output, with renal plasma flow averaging 600mL/min. The kidneys clear waste products and other substances from the plasma, and their clearances are determined by both glomerular filtration and tubular secretion. GFR is defined as the volume of filtrate by both kidneys per minute and is normally approximately 125 mL/min, i.e. 20 % of renal plasma flow delivered to the kidneys. A decline in GFR to <60 mL/min/1.73 m2 for ≥3 months is a common criterion for defining chronic kidney disease and such a loss in GFR has been well associated with a higher risk of all-cause mortality 51Cr-labeled ethylenediaminetetraacetic acid (EDTA) and 125I-labeled iothalamate are radiolabeled exogenous substances that offer more precise measurements of GFR, but are rarely used in currently clinical practice because of high costs and inability to provide split renal function para-aminohippuate (PAH) or ortho-iodohippurate (OIH), which are 20% filtered by the glomerulus and 80% secreted in the tubules. As a result, effective renal plasma flow (ERPF) is used as a biomarker in clinical practice The most important quantitative characteristics for renal function are GFR and ERPF, and the gold standard for measuring both biomarkers is the clearance of exogenous substances, such as inulin and 99mTc-diethylenetriaminepentaacetic acid  and 99mTc-mercaptoacetyltriglycine  is routinely performed to provide comprehensive information about the kidney. Due to its high accumulation and low background accumulation, 99mTc-MAG3 allows for high contrast renal images 99mTc-DTPA can provide an assessment of GFR because of its nature of radiotracer filtration at the glomerulus and minimal tubular uptake Instead of blood clearance of exogenous substances, dynamic renal scintigraphy with As a major drawback, renal scintigraphy has only limited spatial resolution of > 1cm when compared with other imaging modalities. Assessment is mainly based on planar dynamic images. Compton scatter and soft-tissue attenuation can further reduce the image resolution as well as quantification reliability In contrast to scintigraphy with single-photon-emitting radiotracers, PET provides multiple advantages including high temporal resolution and sensitivity allowing dynamic three-dimensional acquisition, superior spatiotemporal resolution, absolute quantification with radiotracer kinetics modeling and three-dimensional co-registration with CT images. PET imaging for assessment of the urinary tract using standard scanners with an axial field of view of 20 cm cannot cover the entire urinary system in a single field of view for a continuous three-dimensional dynamic san Table 3). 18F and 11C, recognized as common standard PET isotopes, are synthesized by a cyclotron, whereas 68Ga is typically produced by a generator. Longer half-life of 18F (110 min) is a major advantage as it allows radiotracer delivery from central cyclotron facilities and flexibility in study design in clinical practice (e.g. delayed imaging protocols). Because of its shorter half-life (20min), 11C-labeled radiotracers are difficult to use in clinical routine 68Ga has a relatively longer half-life (68 min) but it requires an in-house 68Ge/68Ga generator 68Ga-labeled radiotracers for somatostatin-receptor (SSTR) and prostate-specific membrane antigen (PSMA) imaging for neuroendocrine tumors and prostate cancer are frequently used in the clinic, the availability of such generators has improved 68Ga, however, has a relatively high positron energy with long positron range when compared to 18F which results in reduced spatial resolution 68Ga to 18F-labeled radiotracers and one may speculate that this trend will also further fuel the development and increased clinical use of 18F radiotracers for renal radionuclide imaging Various positron-emitting radionuclides have been used clinically and their characteristics are defined by different half-lives of the isotope, positron range, and production source . GFR quantified by 68Ga-EDTA clearance derived from three organ compartments including the kidneys, ureter and the bladder correlated well with GFR from plasma sampling (correlation co-efficient 0.9). In subjects with a 51Cr-EDTA GFR of more than 150 mL/min, 68Ga-GFR was significantly lower and the authors explained such findings by plasma binding, tubular secretion, or dissociation in vivo. Furthermore, in 11 patients, 99mTc-dimercaptosuccinic acid (99mTc-DMSA) was conducted and read-out included a renal static scintigraphy approach, planar imaging, and SPECT/CT. Good agreement between 99mTc-DMSA SPECT/CT and excretory phase (2-10 minutes) from 68Ga-EDTA dynamic PET was noted. The latter imaging modality, however, allows more in-depth information on the entire urinary tract system in a 3-D capability relative to SPECT Hicks et al. first revealed the excellent kinetics of  phase 2- minutes 68Ga-NOTA is a 68Ga-labeled metal chelate whose clearance reflects GFR, similar to 68Ga-EDTA. Lee et al. measured serum protein binding of 68Ga-NOTA and performed small animal PET studies for comparison with 51Cr-EDTA. 68Ga-NOTA showed the lowest serum protein binding compared to the other 68Ga-labeled chelating agents . Furthermore, both 68Ga-NOTA and 51Cr- EDTA showed very similar biodistribution patterns and almost the same GFR 68Ga-IRDye800-tilmanocept demonstrated receptor-mediated renal accumulation with evidence of glomerular uptake. Histologic examination demonstrated colocalization of CD206 which resides on the surface of mesangial cells, and IRDye800-tilmanocept within the glomerulus. Furthermore, molecular imaging of diabetic and nondiabetic mice was performed with fluorescence-labeled 99mTc-tilmanocept, and hyperfiltration of tilmanocept was observed in diabetic mice. Given the important role of mesangial cells during the progression of diabetic nephropathy, PET or SPECT renal imaging with radiolabeled tilmanocept may provide noninvasive quantitative assessment of glomerular function even in early disease stages Another GFR-reflecting agent, radiolabeled tilmanocept has been reported as a unique radiotracer for evaluating diabetic nephropathy. The number of diabetic patients continues to increase, with diabetic nephropathy being the leading cause of chronic renal failure and occurring in 40% 18F-FDS, which is a sorbitol derivative filtered through the glomerulus, has shown excellent pharmacokinetics and is readily available by a simple one-step synthesis from the most commonly used radiotracer 18F-FDG. This makes it available at virtually every PET practice with access to, or supply by, a cyclotron facility 18F-FDS, an analogue of sorbitol, may have similar kinetics as inulin, with free filtration at the renal glomerulus and no tubular reabsorption.18F-FDS as a renal PET radiotracer, basic biodistribution properties and clearance through the renal collecting system pathway were explored in healthy rats. Dynamic PET imaging showed rapid accumulation in the kidneys followed by a time-dependent transition to the collecting system. There was almost no accumulation of background, such as liver, intestines and spleen, suggesting low hepatobiliary clearance and exclusive renal filtration of 18F-FDS. PET renography showed cortical accumulation in the initial blood flow phase and subsequent rapid parenchyma and transfer of the radiotracer to the collecting system. Split renal function assessment demonstrated a normal renogram pattern. Accumulation in the cortex in the early blood flow phase was confirmed by ex vivo autoradiography. Comparable renal distribution of 18F-FDS and 99mTc-DTPA was achieved in the identical animal after dual radiotracer administration. Postmortem tissue analysis showed that 18F-FDS had the highest accumulation in the kidneys at all imaging time points. Radio-thin-layer chromatography analysis also demonstrated that no radioactive metabolites were detected in blood and urine 35 minutes after radiotracer administration. Furthermore, in vivo serum protein binding of 18F-FDS was less than 0.1% at 35 minutes after radiotracer administration 99mTc-DTPA has been reported to range from 2% to 10% 18F-FDS indicate that 18F-FDS is not only an excellent GFR substance with excellent pharmacokinetics similar to the gold standard inulin, but also has a substantially higher stability in the blood relative to its SPECT counterpart.To explore the potential of 18F-FDS as a functional renal imaging agent has been investigated using two different rat models of acute renal failure (ARF) and unilateral ureteral obstruction (UUO). On PET images, ARF rats, induced by intramuscular injection of glycerol, had significantly less uptake in the renal cortex and slower excretion into the bladder than controls. Furthermore, the renogram presented a non-functional pattern. In UUO rats, obtained by complete ligation of the left ureter near the renal pelvis, lower and delayed uptake was observed on the obstructed left side, with no transition into the collecting system, whereas the contralateral side showed results similar to those obtained in control rats. Renograms showed that the UUO collecting system presented a typical obstructed pattern 18F-FDS as a radiotracer providing a non-invasive read-out of the urinary tract that may also allow for precise GFR measurements.Based on these preclinical results, the potential of 18F-FDS PET was performed in two healthy human volunteers Figure 3). The kidneys had the highest radiotracer accumulation after the initial blood phase. Due to the higher resolution of the PET images, it was possible to contour three-dimensional volumes of interest on both cortical and medullary segments and obtain time activity curves  18F has a higher positron yield  and lower positron energy  which increases diagnostic yield relative to 68Ga Dynamic 68Ga-labeled prostate-specific membrane antigen (PSMA) PET imaging agent. When compared to 99mTc-MAG3 in 97 prostate cancer patients, high correlation indices for assessing split renal function between both techniques could be achieved Recently, split renal function has been evaluated by a 68Ga-Ga-HBED-CC-DiAsp benzyl]-ethylenediamine-N,N'-diacetic acid) is a kit-based radiotracer, that potentially exhibits a higher stability when compared to 68Ga-EDTA, rendering it as a potential diagnostic agent to assess GFR in-vivo. In a rat study, biodistribution and dynamic PET/CT imaging studies in rats revealed a rapid clearance 18F-PFH reflects ERPF and may predict the longterm prognosis of the kidney as demonstrated in animal studies 18F-PFH is similar to that of p-aminohippurate, a gold standard for the measurement of ERPF. Awasthi et al. reported the synthesis and initial in vivo evaluation of 18F-PFH in a rat model. Dynamic PET revealed a rapid clearance of 18F-PFH through the renal-urinary pathway 18F-PFH with 125I-OIH as a gold standard, and with a clinically used agent (99mTc-MAG3), in healthy rats. The results showed no significant difference in time-to-peak, but in the visual analysis of renograms, 18F-PFH seemed to be closer to 125I-OIH compared to 99mTc-MAG3. In addition, the quality of the renogram and the images obtained with the dynamic 18F-PFH PET study were remarkably better than those obtained with the 99mTc-MAG3 dynamic planar imaging study 18F-PFH PET renography to predict future progression of PKD in Han:SPRD rats with slowly progressive autosomal dominant PKD. They obtained T2 and T20 values, which represent the percentage of the injected dose of 18F-PFH in kidneys at 2 and 20 min after injection, from the imaging data and assessed T20/T2 ratio as a prognostic marker. T20/T2 values at 6 weeks correlated with serum creatinine, serum urea nitrogen, and kidney weight per body weight at 26 weeks. These results demonstrated 18F-PFH could be a surrogate marker, e.g. for progression of autosomal dominant polycycstic kidney disease 18F-FEDA) is an ERPF agent, and can be labeled with either 99mTc or 18F, depending on the application. Lipowska et al. demonstrated that 99mTc-Re(CO)3(FEDA) had comparable pharmacokinetics to 131I-OIH in rats 18F-labeled counterpart exhibited a high specificity for the kidneys along with rapid renal excretion and high in vitro and in vivo stability comparable to those of its 99mTc-labeled analogue Re(CO)3 18F-FDG, 11C-acetate PET/CT may also provide human glomerular filtration rate based on compartmental modeling Attempts to calculate renal function using 215O, which is freely diffusible between tissue and blood, has been investigated in patients with renal dysfunction demonstrating significantly lower flow values when normalized to 100 g of tissue measured with H215O when compared to healthy individuals  82Rubidium (82Rb) has a high first pass extraction and slow renal washout rendering it as an ideal imaging agent to measure RBF. Tamaki et al investigated 82Rb in dogs using a steady-state kinetic model with various degrees of kidney injury and concluded that 82Rb allows for serial quantitative assessment of RBF 82Rb is feasible, allowing for excellent image quality, high resolution and contrast 82Rb PET 13N-ammonia, which is widely utilized for myocardial perfusion imaging 13N-ammonia demonstrated an excellent correlation when compared to the reference standard H215O Several radiotracers have been introduced for assessing renal blood flow (RBF). For instance, H18F-FDG for autosomal dominant polycycstic kidney disease in 30 individuals and revealed a high diagnostic accuracy in detecting renal or hepatic cyst infections. Highlighting the potential of guiding therapy, baseline 18F-FDG PET/CT led to change in antiobiotics use in up to 63% of the patients 18F-FDG, however, is rather nonspecific for inflammation imaging. 68Ga-Pentixafor targeting the C-X-C motif chemokine receptor 4 (overexpressed on leucocytes) has been investigated in patients with complicated urinary tract infections in combination with rMRI. Leukocyte infiltration was identified by areas of CXCR4 upregulation compared with unaffected parenchyma in PET, corresponding to areas with increased cell density in rMRI, which was further validated by ex vivo analysis. As such, combined PET/rMRI data revealed the potential of non-invasive detection of leukocytes in renal allografts Figure 6).A recent study evaluated the value of 18F-FDG or somatostatin receptor-targeting 68Ga-DOTA-D-Phe-Tyr3-octreotide (DOTATOC) or 68Ga-DOTA-D-Phe-Tyr3-octreotate (DOTATATE) 18F]-flurocyclobutane-1-carboxylic acid (anti-18F-FACBC) assessing the upregulation of increased amino acid metabolism, however, has almost no renal excretion. Spearheaded by Schuster et al, six patients afflicted with renal lesions were investigated and correlation with histology revealed that amino acid transport compared with renal cortex is elevated in renal papillary cell carcinoma but not in clear cell renal cell carcinoma (ccRCC) 18F-DCFPyL demonstrating an accurate detection of sites of metastatic ccRCC 18F-DCFPyL 68Ga-labeled imaging agent PSMA-11 has received FDA approval just recently, which makes PSMA PET imaging more widely available for potential use in RCC. Nonetheless, such radiopharmaceuticals have been primarily investigated for assessing tumor burden in patients afflicted with RCC, whereas their use for renal functional imaging is rather limited Assessment of primary renal tumors is challenging, mainly due the high physiological uptake seen with imaging agents such as Although the use of PET can be limited by the installed base and high synthesis and maintenance costs, there are definite clinical applications of new renal PET radiotracers. For example, the low radiation exposures may lead to use in pediatrics, and the intrinsic advantages of PET may make these agents useful for those settings where an accurate renal functional assessment is required. Measurement of renal function in children can be difficult with noninvasive techniques because changes in body size and creatinine excretion can make creatinine clearance less reliable. In addition, children often have ureteral obstruction and anatomic abnormalities which may not be well evaluated by convetional scintigraphy 68Ga-EDTA in adult patients, which results in approximately 1.6 mSv from the PET component and equates to approximately the exposure of 320 MBq of injected 99mTc-DTPA 11C-PABA was 1.6-fold lower than that of 99mTc-MAG3 in a murine data-based human dosimetry analysis. The CT component of the PET/CT would add additional radiation exposure to the patient, but this can be as low as 0.25 mSv with an ultralow dose CT and the limited field-of-view required for renal imaging In pediatric nuclear medicine, nuclear medicine specialists are facing the dilemma of reducing both radiation exposure and scan time. According to an IAEA study of 133 institutions from 62 countries on pediatric renal scintigraphy, most of the institutions would in fact administer a higher activity as recommended. The authors noted that the sites may have prioritized a shorter scan time  rather than reducing radiation exposure 99mTc-DTPA 99mTc-MAG3 reflecting tubular function could not identify high-risk patients with a late onset of renal failure 223Ra and 225At) are now being used clinically, and their higher mean energy deposition and shorter range in tissue relative to beta radiation may allow for increased therapeutic efficacy with minimal side effects in normal tissue 225Ac-PSMA  225Ac and 213Bi in preclinical studies 213Bi-DOTATATE for treatment of neuroendocrine tumors has also been reported 177Lu-prostate-specific membrane antigen (PSMA) also develop renal decline 18F-FEDA) and 18F-PFH, which reflect proximal tubular secretion, may also provide a surrogate marker of short- and long-term renal damage under chemotherapeutic regimens Recent years have also seen an expanded use of renal radionuclide SPECT imaging to identify patients which may have renal functional decline after multiple cycles of endoradiotherapies. There has been an expansion of the use of receptor radionuclide therapy (PRRT) in neuroendocrine tumors using somatostatin analogs labeled with beta-emitting radionuclides 68Ga-Pentixafor may identify the most suitable time-point for treatment initiation, which in turn may increase therapeutic efficacy . In addition, the baseline-derived radiotracer signal may be used as a predictor of outcome with defined endpoints such as major adverse renal events (MARE).The theranostic concept in oncology is based on diagnostic molecular imaging, followed by an individually tailored treatment decision using the identical or similar therapeutic radiotracer In uro-oncology, PSMA-PET/CT assists in identifying prostate cancer patients which are suitable for PSMA-based radioligand therapy (RLT). This theranostic concept is currently penetrating the clinical arena, mainly due favorable results of recent major prospective trials. First, the phase 2 LuPSMA trial reported on progressive metastatic castration-resistant prostate cancer patients that have been previously treated with antiandrogens and taxane-based chemotherapy. RLT demonstrated high response rates and low toxic effects with grade 3 or 4 thrombocytopenia in 13% of the patients 68Ga-labeled PSMA PET/CT and 75% of the patients demonstrated avid primary lesions, with the majority of ccRCC subtype. Of note, in 42% of the cases, 68Ga-PSMA PET/CT led to change in management, due to the identification of new sites of suspected metastases and synchronous primaries 68Ga/18F-PSMA imaging followed by 177Lu-PSMA treatment As alluded to earlier, PSMA-targeting imaging agents have also been investigated for potential use in RCC. In this regard, 16 patients with suspicion for ccRCC have been scanned with Figure 7) Apart from applying theranostics in nephrology and urology, interactions with remote organs, such as the vessels or the spleen, could be also pursued by whole-body molecular imaging, potentially allowing for non-invasive analyses of kidney-organ networks 18F-FDS can be synthesized from 18F-FDG in a one-step synthesis and thus, 18F-FDS would be available at virtually every PET practice with access to a cyclotron. In addition, the relatively long half-life of 18F renders it as a potential agent for clinical trials, in particular in pediatric populations as it has significantly less radiation exposure compared to SPECT equivalents Despite the development of CT, MRI, and ultrasound techniques for evaluating the kidney, renal scintigraphy is still frequently requested in clinical practice. In a manner similar to brain, cardiac, and tumor molecular imaging, renal radionuclide imaging is beginning a shift from SPECT to PET. The GFR-reflecting radiotracer


We rMost prediction models have concordance statistics (C-statistics) of 0.65–0.80 indicating only fair accuracy . Can we Recent studies suggest data regarding the bone marrow micro-environment, immune system and leukaemia cell metabolism might improve prediction accuracy. These data are reported to independently predict outcomes such as complete remission rate, cumulative incidence of relapse, event-, relapse- and leukaemia-free survivals  and/or survival in multi-variable analyses. Moreover, these co-variates are reported to improve the accuracy of more widely-used models such as the 2017 ELN model. We discuss these models below.low CD8+ T-cells correlates with fewer complete remissions (CR) and worse survival [Considerable data indicate cells in the bone marrow microenvironment, including immune, endothelial and stromal cells, the composition of the extracellular matrix and soluble factors such as cytokines, hepatocyte growth factor, vascular endothelial growth factor and angiopoietins are important in leukaemia development and progression . The impsurvival .lo regulatory T-cells (Tregs) in blood and bone marrow were associated to poor prognosis [+ T-cells is increased in persons with AML and correlates with induction chemotherapy failure and with posttransplant relapse [Zhang et al. reported frequencies of CD4+CD25+CD127rognosis . Han et rognosis . Kong et relapse . Several relapse , 20. Inc relapse .−LAG3− helper T-cells [There are several predictive models of AML using data of immune cells identified by multi-parameter flow cytometry and/or NGS with bio-informatics. We reported an immune risk score derived from public datasets from Gene Expression Omnibus where we estimated proportions of immune cells in bone marrow samples using CIBERSORTx . Data of T-cells . Dong et T-cells . Zhu et  T-cells . Cytokin T-cells . Also, t T-cells –29. In cAnother approach to improving prediction accuracy in AML involves leukaemia cell metabolism. Mutations in genes with metabolically active gene products such as isocitrate dehydrogenase isoform-1 (IDH1) and IDH2 are associated with changes in cell metabolism and possibly leukaemia initiation , 31 ChenSerum metabolomic profiling has also been used to identify metabolites associated with outcomes of children with AML receiving chemotherapy . Higher Two recent studies in older persons with AML using predominately conventional subject-related co-variates reported C-statistics of 0.72–0.74 similar to the C-statistic of the 2017 ELN risk classification , 38. PreAccurate prediction can improve therapy decisions in persons with AML . IncreasAccurate prediction is fundamental to optimising AML therapy Fig. . Data weNone of current or newer prediction models we cite included data of MRD-testing at the end of therapy as an outcome predictor in model building. Whether current or new pre-therapy predictive models are better than results of post-therapy MRD-testing in predicting post-remission therapy outcomes is uncertain. Elsewhere we discuss the advantages and limitations of post-therapy MRD-testing as a predictor of subsequent outcomes in persons with AML . PresentAnother issue is why and when do we want to determine prognosis or predict outcomes. Is it to identify the best initial therapy, say intensive therapy with cytarabine and daunorubicin, less intensive chemotherapy say with azacitidine with venetoclax, or a targeted therapy, say with enasideinib or ivosidenib? This decision is driven not only by the co-variates we identify but also by co-variates less often considered such as co-morbidities, access to medical care, expertise of the treating team, sophistication of supportive care, patient preference and economics. Or is our goal to predict what intervention should be given next. Obviously, this will be largely driven by outcome of the initial therapy and co-variates we cite above. For example, if the goal of an older person with substantial co-morbidities is to achieve the longest interval of high quality-of-life, achieving a complete remission may not be the appropriate therapy objective. In contrast, a young, otherwise healthy person may be willing to accept substantial adverse events for a chance, however small, of cure. In this instance a co-variate such as post-therapy MRD-testing may be the best predictive biomarker. What is important is that physicians and patients acknowledge our inaccuracy and imprecision in predicting outcomes of an intervention. What level of accuracy and precision is acceptable to drive a therapy decision is obviously subjective with no correct answer?The new prediction models we discuss need optimisation and external validation in large datasets of uniformly-treated persons. To know if they are prognostic rather than predictive, they need to be tested in studies of diverse therapies. If validated they could be introduced into clinical practice and help with therapy decision-making.In summary we show adding micro-environment-, immune- and metabolism-related and other co-variates improves prediction accuracy in newly-diagnosed persons with AML, predominately young people receiving intensive induction chemotherapy. Whether these co-variates are similarly useful in other therapy settings in unknown such as less intensive or targeted and immune. Because initial intensive therapy of AML is relatively uniform these co-variates are presently best regarded as predictive rather than prognostic. In appropriate therapy settings using prediction models which include micro-environment-, immune- and metabolism-related co-variates may be clinically useful. However, we need validation and optimisation in large prospective dataset of AML receiving diverse therapies such as cytarabine and daunorubicin versus azacitidine and venetoclax or enasideinib. When these are accomplished these new models be clinically-useful to predict outcomes and choose therapy(ies).