Although the molecular mechanisms underlying methylmercury toxicity are not entirely understood, the observed neurotoxicity in early-life is attributed to the covalent binding of methylmercury to sulfhydryl (thiol) groups of proteins and other molecules being able to affect protein post-translational modifications from numerous molecular pathways, such as glutamate signaling, heat-shock chaperones and the antioxidant glutaredoxin/glutathione system. However, for other organomercurials such as ethylmercury or thimerosal, there is not much information available. Therefore, this review critically discusses current knowledge about organomercurials neurotoxicity—both methylmercury and ethylmercury—following intrauterine and childhood exposure, as well as the prospects and future needs for research in this area. Contrasting with the amount of epidemiological evidence available for methylmercury, there are only a few in vivo studies reporting neurotoxic outcomes and mechanisms of toxicity for ethylmercury or thimerosal. There is also a lack of studies on mechanistic approaches to better investigate the pathways involved in the potential neurotoxicity caused by both organomercurials. More impactful follow-up studies, especially following intrauterine and childhood exposure to ethylmercury, are necessary. Childhood vaccination is critically important for controlling infectious diseases; however, the safety of mercury-containing thimerosal and, notably, its effectiveness as preservative in vaccines are still under debate regarding its potential dose-response effects to the central nervous system. M. M101]. Although the postnatal brain might develop some protective mechanisms against prenatal MeHg-induced toxicity, there is growing evidence from animal and human studies about irreversible long-lasting damage of MeHg to the nervous system. Ceccatelli and co-authors provide an excellent overview of these studies . The devThe implication of genetic factors in MeHg-induced neurotoxicity might probably resolve at least some discrepancies between numerous epidemiological studies of human early-life MeHg exposure in fish-consuming populations around the world, including the Faroe Islands , Brazil As discussed above, currently the main source of EtHg exposure is as thimerosal preservative in vaccines, since the EtHg-containing fungicides should have been eliminated from use . It is nThe majority of studies, including large epidemiological studies, try to explore the causal relationship between early-life exposure to Hg from TCVs and ASD . This rAccording to Modabbernia et al., future risk assessment studies of ASD would benefit from a developmental psychopathology approach, prospective design, precise exposure measurement and should take into account the dynamic interplay between gene and environment by using genetically informed designs , since aIn order to solve the discrepancies mentioned above, the analysis of potential genetic or environmental risk factors of susceptibility to EtHg and putative molecular mechanisms of possible EtHg-induced neurotoxicity, experimental non-primate models  could be helpful. Some of the already-documented neurotoxic effects after in utero/perinatal exposure to thimerosal through in vivo experimental studies are shown in SJL/J exhibited profound neurodevelopmental deficits  after thimerosal exposure, compared with strains without autoimmune sensitivity  [ICR mice brain slices, with an IC50 of 5.80 µM for inhibition curves, which is around concentrations previously described for human poisoning [To date, only a few rodent studies examined a potential neurotoxic role of EtHg and there are rare studies aiming to mechanistically investigate the pathways involved in the neurotoxicity of EtHg/thimerosal. One of the first studies that highlighted the importance of genetic profile for EtHg sensitivity showed that the autoimmune disease-sensitive mouse strain 2b mice) . However2b mice) . Later, 2b mice)  and sign2b mice) . This sa2b mice) . Moreove2b mice) . Moreove2b mice) . Howeveroisoning . Althougoisoning .Taken all together, the current knowledge about EtHg-induced neurotoxicity is still controversial perhaps due to the lack of costly genetic assessment of the human populations studied, insufficient data about human subjects involved in the studies , and only a small number of animal studies that could provide molecular mechanisms of EtHg effects on brain development. Childhood vaccination is critically important for control of some infectious diseases; however, the safety of thimerosal and, notably, its effectiveness as a preservative at the used dose, is still under debate.


Traumatic brain injury (TBI) is associated with cognitive, somatomotor, and behavioral dysfunction that may persist indefinitely  and, in Mild TBI (mTBI), which accounts for ~75% of all TBIs, exhibits considerable heterogeneity pertaining to risk of progressive cognitive impairment and AD pathology. Early post-TBI assessment of risk for progressive cognitive decline could inform clinical monitoring, cognitive training and/or other therapeutic interventions. With the recent US Food and Drug Administration approval of aducanumab and with other disease-modifying treatments directed at amyloid and τ that are undergoing clinical trials, this research topic is of considerable interest to federal research agencies, which offer funding initiatives aimed at explicating AD risk after TBI.We have proposed, developed, and tested approaches that combine structural and functional neuroimaging with machine learning to identify persons with mTBI who are at risk for progressive cognitive decline. Structural and functional brain dynamics after TBI are paralleled by older-than-expected  brain ages, which can be estimated using multivariate models of neuroanatomical features derived from magnetic resonance imaging (MRI) . StructuFrom a functional point of view, we have used machine learning to predict network changes based on acute cognitive deficits . We foun


Roux-en-Y gastric bypass (RYGB) leads to beneficial effects on glucose homeostasis, and attenuated hormonal counterregulatory responses to hypoglycemia are likely to contribute. RYGB also induces alterations in neural activity of cortical and subcortical brain regions. We aimed to characterize RYGB-induced changes in resting-state connectivity of specific brain regions of interest for energy homeostasis and behavioral control during hypoglycemia.2 were investigated with brain PET/MR imaging during a hyperinsulinemic normo- and hypoglycemic clamp, before and 4 months after RYGB. Hormonal levels were assessed throughout the clamp. Resting-state (RS) fMRI scans were acquired in the glucose-lowering phase of the clamp, and they were analyzed with a seed-to-voxel approach.Ten patients with BMI > 35 kg/mr = −0.639, p = 0.047) and cortisol  responses. Instead, connectivity between the caudate and the frontal pole after RYGB was reduced and this was associated with less attenuation of glucagon response during the hypoglycemic clamp , smaller reduction in fasting glucose  and less excess weight loss . No other significant associations were found between post-RYGB changes in ROI-to-voxel regional connectivity hormonal responses and metabolic or anthropometric outcomes.RS connectivity during initiation of hypoglycemia was significantly altered after RYGB between nucleus accumbens, thalamus, caudate, hypothalamus and their crosstalk with cortical and subcortical regions. Connectivity between the nucleus accumbens and the frontal pole was increased after RYGB, and this was associated with a reduction of ACTH (RYGB alters brain connectivity during hypoglycemia of several neural pathways involved in reward, inhibitory control, and energy homeostasis. These changes are associated with altered hormonal responses to hypoglycemia and may be involved in the glucometabolic outcome of RYGB. Roux-en-Y gastric bypass (RYGB) is a surgical procedure that induces durable weight loss and has a positive impact on glucose homeostasis, lowering the risk of type 2 diabetes or even leading to diabetes reversal , 2. ThisEnergy metabolism and food intake are finely regulated processes that rely on the hypothalamic integration of both central and peripheral signals . HoweverThe brain reward system is in engaged in the consumption of palatable food , 11, as 18F-FDG-PET in energy balance regions is altered after RYGB [Neuroimaging techniques are versatile, non-invasive informative procedures and have highlighted changes in brain functional connectivity and glucose metabolism upon bariatric intervention. Several studies in the latest years have highlighted that after RYGB, resting-state connectivity in the fasting state is reshaped, especially as to brain regions part of the default-mode network , the prefrontal cortex, and the cerebellum –19. Alsoter RYGB .Our group was the first to perform a combined PET/MRI assessment under a hyperinsulinemic clamp with both a euglycemic and a hypoglycemic phase in patients before and after undergoing RYGB . During We hypothesize that brain connectivity after RYGB is reshaped in order to dampen the homeostatic response to energy deprivation. Therefore, in this study, we focused on RYGB-associated changes in regional brain connectivity during the glucose-lowering phase of the hyperinsulinemic clamp. We targeted brain regions that are of relevance for energy homeostasis and eating behavior according to previous work, including the thalamus, the ventral tegmental area, the basal nuclei, the amygdala, and the hypothalamus. The findings support a role of the brain in the favorable metabolic adaptations brought about by bariatric surgery.2) were recruited at the obesity outpatient facility at Uppsala University Hospital during a routine visit before the scheduled Roux-en-Y gastric bypass (RYGB) operation. Further details of this cohort have been described and published before [Ten non-diabetic individuals with obesity  and a second hypoglycemic phase (2.7 mM). Also, cognitive assessments were performed during both the normoglycemic and the hypoglycemic phase using the Trail Making Test (TMT) and the Digital Symbol Substitution Test (DSST). Hypoglycemic symptoms were evaluated during hypoglycemia via the Edinburgh hypoglycemia symptoms score (EHSS). Blood samples were drawn from an antecubital vein after arterialization and were analyzed at the Department of Clinical Chemistry and the Clinical Diabetes Research Laboratory of Uppsala University Hospital. Neuroimaging was performed using an integrated PET/MR machine , comprising a 3 T MRI scanner . DetailsFor this post-hoc analysis, the CONN toolbox version 21.a  was usedp = 0.05. Because of the exploratory nature of this investigation, correlation analyses were not corrected for FDR, and nominal p-values are given. Statistical analyses were performed using GraphPad Prism 9, and images were created using the CONN toolbox [Data are presented as mean and standard deviation (SD) or median and interquartile range (IQR) as appropriate and specifically indicated. Correlation analyses were performed with Pearson’s method after checking for normal distribution of the data using the Shapiro-Wilk test. Significance level was set at  toolbox  and GrapUsing a ROI-to-voxel approach, we found several voxel clusters that displayed different connectivity after RYGB with brain regions that are relevant to energy homeostasis and feeding behavior Table .Table 1RConnectivity of the nucleus accumbens with voxel clusters located in both left and right frontal poles and the right thalamus was increased Fig. . The caur = −0.639, p = 0.047) and cortisol  during the hypoglycemic clamp  of both ACTH .We have previously shown that cognitive function as assessed by psychometric tests correlated with cerebral blood flow during hypoglycemia, and that both of them were increased after RYGB . HoweverWe found several resting-state neural pathways whose activity was altered after RYGB during the glucose-lowering phase of the hyperinsulinemic clamp, involving both basal nuclei and cortical regions. Some of these changes were also associated with surgery-induced changes in brain glucose metabolism and peripheral counterregulatory responses.In the mesolimbic dopamine pathway, the release of dopamine by neurons projecting from the ventral tegmental area onto dopamine receptors expressed by the GABAergic spiny neurons of the nucleus accumbens promotes the desire for rewarding stimuli, a process known as incentive salience . Both shThe connectivity between the nucleus accumbens and the thalamus, as parts of the reward system, is responsible for the translation of motivation inputs into motor responses . We alsoConnectivity between the caudate nucleus and the frontal pole, which operate together in behavioral inhibitory control  and goalA reduction in the posterior cingulate cortex and the precuneus functional connectivity is known to occur at fasting after gastric bypass –18. BothWe found decreased connectivity of several brain regions, including caudate nucleus, putamen, medial hypothalamus, and thalamus, with the cerebellum, whilst connectivity of both medial and lateral hypothalamus with the cerebellum was increased. The cerebellum is implicated in excessive eating and satiation signals , 34. SimWe found altered connectivity between the thalamus and the nucleus accumbens after RYGB. Paraventricular thalamic projections to the nucleus accumbens are important in motivated behaviors  and promWe also found increased connectivity of the thalamus with the caudate nucleus and the globus pallidus, a pathway implied in cognitive control on behavior .We found increased connectivity between the lateral hypothalamus and the hippocampus. A study carried out on patients with narcolepsy identified decreased connectivity between the lateral hypothalamus and the hippocampus, and speculated that this was a proxy for decreased orexin/hypocretin innervation, a neurotransmitter that is of great importance also in appetite regulation . This ciRecently, it has been shown that connectivity between the lateral hypothalamus and several cerebellar regions was associated with the weight loss outcome in obese subjects undergoing a 3-month diet . AccordiThis study has some limitations. Firstly, it is an exploratory post-hoc investigation, so no formal power calculations were made and some of the analyses might therefore be underpowered. Secondly, this is an observational study, and no conclusions of causality can be made based on the presented results. Thirdly, the lack of a control group does not allow to distinguish between the effects of RYGB and the weight loss per se. However, several studies have highlighted the structural and functional changes following bariatric surgery in the human brain being independent of weight loss . FinallyAltogether, these findings highlight profound changes following RYGB in brain cortical and subcortical connectivity during experimental glucose deprivation. The affected neural pathways are largely involved in reward, inhibitory control or energy homeostasis. Among them, increased connectivity between nucleus accumbens and the frontal pole was associated with attenuated counterregulatory hormonal responses to hypoglycemia. On the other hand, a reduction in caudate nucleus to frontal pole connectivity was associated with a lower magnitude of favorable effects on glucose metabolism and adiposity after RYGB. These findings on the caudate are paradoxical, and might suggest that they occur as an adaptive or compensatory phenomenon, rather than as a primary mediator of metabolic improvement.The findings may support that gastric bypass surgery partly exerts its favorable effects on energy and glucose homeostasis via the central nervous system, where changes in functional connectivity can contribute to modulation of the complex interplay between behavioral responses and neuroendocrine adaptations.Conversely, the highlighted neural pathways may also be of relevance also for the development of obesity, insulin resistance and type 2 diabetes, and further research is warranted, including individuals in different stages of overweight and dysglycemia.


Stroke remains one of the leading causes of various disabilities, including debilitating motor and language impairments. Though various treatments exist, post-stroke impairments frequently become chronic, dramatically reducing daily life quality, and requiring specific rehabilitation. A critical goal of chronic stroke rehabilitation is to induce, usually through behavioral training, experience-dependent plasticity processes in order to promote functional recovery. However, the efficiency of such interventions is typically modest, and very little is known regarding the neural dynamics underpinning recovery processes and possible biomarkers of their efficiency. Some studies have emphasized specific alterations of excitatory–inhibitory balance within distributed neural networks as an important recovery correlate. Neural processes sensitive to these alterations, such as task-dependent oscillatory activity in beta as well as alpha bands, may be candidate biomarkers of chronic stroke functional recovery. In this review, we discuss the results of studies on motor and language recovery with a focus on oscillatory processes centered around the beta band and their modulations during functional recovery in chronic stroke. The discussion is based on a framework where task-dependent modulations of beta and alpha oscillatory activity, generated by the deep cortical excitatory–inhibitory microcircuits, serve as a neural mechanism of domain-general top-down control processes. We discuss the findings, their limitations, and possible directions for future research. Around half of stroke survivors face sustained disabilities that impair their quality of life . Among tDuring the first days and weeks after stroke, functional reorganization of cerebral neural networks takes place . This reTo date, a variety of rehabilitation techniques have been developed to restore motor  and langThis, in turn, entails the need for reliable recovery biomarkers, i.e., measurable parameters of brain activity that could predict clinical and behavioral outcomes  and reflN-methyl-D-aspartate receptors) glutamatergic excitatory activity of pyramidal neurons in the perilesional areas becomes threatening to the preserved tissues—so-called “excitotoxicity” phenomenon  and functional  levels. Particularly, the changes in the neural excitability processes within the surviving neural networks  might reenomenon . Additioenomenon . Furtherenomenon .In a healthy brain, one of the possible mechanisms to control the cortical excitation level across local and global neural networks is based on the synaptic interactions within microcircuits comprised of excitatory glutamatergic pyramidal cells and inhibitory GABAergic interneurons . More prIt therefore appears important to identify such neural measures that could detect excitatory–inhibitory imbalance in functional neural networks using standardized methods. Non-invasive neurophysiological techniques, such as MEG  and EEG , sensitive to postsynaptic processes in cortical microcircuits formed by reciprocally connected glutamatergic pyramidal and GABAergic inhibitory interneurons, theoretically may provide means to track such dynamics indirectly . The priWhereas the mechanisms underpinning beta and alpha neural oscillations are subject of a debate , one widHence, though models of oscillatory activity generation vary, many of them emphasize the role of the GABA-driven inhibitory processes within cortical microcircuits, both for beta and alpha activity. This inhibition controls the firing rate produced by excitatory pyramidal neurons and transferred via their connections, thereby affecting excitability level both locally and in distributed networks. The local generators of beta and alpha rhythms are mostly placed in the deep cortical layers, primarily layer 5 . From thMotor-related oscillatory activity in beta band, registered by EEG or MEG during voluntary movements, shows certain event-related dynamics . This dyA review by Piai and Zheng investigated the roles that different types of oscillatory activity play in language tasks performance . It emphSuch findings demonstrate that beta and alpha oscillatory events might be tightly linked with both voluntary motor and language processes, as well as with the control of complex behaviors more generally. Indeed, the control of motor actions and the domain-general processes  have much in common: both rely on the prediction-driven and context-dependent modulations of the current behavioral output . At the This link between motor and language functions goes well in line with embodied cognition theories postulating that two systems are intrinsically intertwined . Within Still, the exact neural mechanisms driving both motor and language recovery in stroke remain mostly obscure. Particularly, for chronic stroke recovery an important issue is the potential impact of behavioral training interventions on the neural plasticity mechanisms . The stuConsidering the experience-induced plastic reorganization processes in the neural networks supporting domain-general top-down control processes (involved in both motor and language functions), one may suggest that they will involve alterations of the excitatory–inhibitory balance within the neural networks supporting these abilities. Such training-induced alterations might change synaptic efficiency in the microcircuits composed of cortical pyramidal neurons, interneurons, and their connections. Thus, measuring beta and alpha oscillations, produced by these microcircuits, might be a way to probe the experience-induced dynamics of excitation–inhibition balance in motor and language stroke patients as they undergo recovery and rehabilitation. Though several studies have tackled this idea [see To sum up, stroke patients’ studies suggest that functional recovery after stroke depends on the cortical excitation–inhibition balance restoration. This balance might be significantly influenced by the interaction of excitatory pyramidal cells and inhibitory interneurons. The synaptic connections strength in these microcircuits impacts both local and distributed cortical information processing efficiency. Existing models suggest that interactions within such microcircuits might be the source of beta and alpha oscillatory activity. Moreover, beta and alpha activity frequency bands are also considered as a channel of the intracortical communication, providing domain-general top-down control of the working memory processes. Studies of voluntary motor activity and speech processes, in turn, demonstrate that particularly beta activity plays a common role in both of these functional domains, which possibly reflects the involvement of top-down control processes in these complex behavioral and cognitive abilities see . CrucialBelow, in a brief overview of data from several available studies, we examine these suggestions and ask whether modulations of oscillatory activity, particularly in beta and alpha bands, might provide the much-needed biomarkers of functional recovery and behavioral training efficiency in chronic stroke. We start with somewhat better-studied motor stroke recovery before continuing to much less understood PSA. For both systems, we will review selected experimental evidence available for resting state and active task conditions to see if oscillatory processes might be candidate biomarkers of efficient speech and motor recovery in chronic stroke patients and possibly serve as a correlate of experience-induced improvements. Finally, open issues and possible further directions will be discussed.First, we review several studies on the resting state oscillatory activity as a recovery correlate in chronic motor stroke patients. A recent study by Hordacre and colleagues employed resting-state EEG functional connectivity analysis to identify oscillatory patterns associated with better motor functioning in 36 chronic stroke patients compared with 25 healthy controls . TranscrThibaut and colleagues investigated the link between motor abilties in chronic stroke and resting-state oscillations in EEG . OscillaAnother interhemispheric asymmetry measure was employed by Saes and colleagues, who obtained resting-state EEG data from 21 chronic stroke patients with upper-limb paresis and from a matched control sample . They foThe available data on the role of the resting-state oscillations in chronic motor stroke recovery are quite limited and heterogeneous see the . DiffereRelations between motor task performance of chronic stroke patients and task-dependent oscillatory modulations were studied by Shiner and colleagues, who used MEG to record movement-related beta activity (13–30 Hz) in chronic motor stroke patients . The resSuch training effects were investigated by Espenhahn and colleagues, who showed an association between motor skill acquisition through goal-directed training and changes in beta oscillatory power in the sensorimotor cortex in chronic stroke patients . At firsIn turn, Wilson and colleagues found other oscillatory power correlates of training-induced plasticity in motor stroke rehabilitation in a case-series MEG study , which cIn addition to focal effects in the lesioned systems, studies of experience-induced plasticity in motor stroke rehabilitation also address network processes beyond the local lesion or perilesional areas. In an MEG study by Buch and colleagues, movement-related beta and alpha oscillations were analyzed as parts of sensorimotor mu-rhythm . This stIndividual variability of recovery outcomes associated with oscillatory dynamics was inspected using EEG in chronic motor stroke patients who underwent a rehabilitation program based on a robotic orthosis device training guided by self-modulations of sensorimotor rhythm . The resThe studies of neural oscillations in active motor tasks in chronic motor stroke patients show that movement-related modulations (desynchronization and synchronization) of oscillatory activity centered around beta and alpha bands are associated with the more successful recovery of motor functions in chronic stroke see the . The oscFollowing the same logic as above, in this section, we will review a set of studies on resting-state oscillatory activity in relation to chronic stroke recovery, before addressing task-dependent activity in the following section. To link this section with the previous one, we wish to first emphasize the study by Nicolo and colleagues which demonstrated association between oscillatory mechanisms in motor and language recovery . The autDalton and colleagues compared resting-state EEG oscillatory power in different frequency bands  between chronic poststroke aphasia patients and healthy controls . SignifiTraining-induced neural dynamics was also tracked in a single-case study of a chronic PSA patient . In thisMeinzer and colleagues specifically focused on training-induced delta-band modulations . A groupThe limited data on the relations between resting state oscillatory processes and chronic posts-stroke aphasia recovery come from studies with quite heterogeneous designs and different measures of speech abilities see the . Those oBelow, we will overview the available limited findings on the role that beta/alpha oscillatory dynamics may play in language recovery in chronic PSA.Spironelli and colleagues compared high-beta band (21–28 Hz) EEG activity between patients with chronic aphasia and healthy controls in a task with different linguistic demands . SubjectThe MEG technique was used by Meltzer and colleagues to study a group of chronic PSA patients along with controls . The patAnother MEG study  used a dA similar result was found by Piai and colleagues, who used EEG and MEG and compared six chronic aphasics with impaired speech comprehension and six healthy controls on a comprehension task performance. The results showed beta/alpha-ERD (8–25 Hz) after the cue sentence presentation and prior to the target picture stimuli. This effect was left-lateralized in controls and right-lateralized in patients; furthermore, it was absent in patients with the lowest performance scores. The authors hypothesized that this ERD effect might be associated with context-driven retrieval aspects of the working memory, supported by the right hemisphere in a compensatory way.The studies reviewed suggest that task-dependent oscillatory power modulations may show specific power and spatial patterns in chronic posts-stroke aphasia patients and in healthy controls see the . These mAs described in the Introduction, the processes of impairment and recovery in stroke are likely associated with the dynamics of cortical excitation–inhibition balance, which is a product of interactions between excitatory and inhibitory cells . The micIn chronic motor stroke studies, resting-state oscillatory activity correlated with improvements differently, depending on the frequency band. In general, higher-frequency band activity, primarily beta, correlated positively with better recovery, while low-frequency bands (delta and theta) correlated positively with poorer recovery. Similarly, in chronic aphasia studies, resting-state data also showed negative correlations between oscillatory activity and functional improvements for lower (theta and delta) and positive for higher  frequency bands.Active-task studies, in turn, found recovery-associated higher-frequency power modulations during motor tasks related to the primary motor areas, both ipsi- and contralesionally. The modulations include both pre-movement desynchronization and post-movement oscillatory resynchronization, their amplitudes and durations. Further studies show recovery-associated modulations of task-dependent activity to also take place outside the primary motor cortex, involving, e.g., premotor, supplementary motor areas and wider frontoparietal networks. Similarly, in the active-state language studies, task-dependent higher-frequency beta and alpha modulations were observed mostly beyond the primary language perisylvian areas and their contralesional homologs. Furthermore, the available evidence suggests that a stronger decrease (ERD) of beta and alpha power during speech tasks might be associated with more successful recovery. These similar findings point to a distributed network nature of plastic recovery processes common for motor and language abilities, which may become manifest in modulations of rhythmic brain activity.Considering the resting-state findings above, we might speculate that the recovery-related decrease of lower frequencies power and the corresponding increase of the higher-frequency activity (primarily beta) might reflect more successful functional reorganization, taking place before the onset of the stroke’s chronic stage. These higher frequency oscillations are associated with complex cognitive and behavioral processing , in contWhen considering active-state studies, their results generally suggest that task-dependent modulations of high-frequency oscillatory activity  relate more to the greater functional efficiency of wide distributed neural networks during task-related performance. These effects, in contrast to the resting-state patterns, might be observed not only in perilesional, but also in intact extralesional areas. Furthermore, not just the presence of this extralesional activity, but rather its dynamic synchronization and desynchronization taking place online during task execution may imply more efficient excitation/inhibition balance, which enables such rapid network dynamics as the driver of recovery, optimizing cortical information processing .The differences between resting and active state results might be, at least to some extent, related to the neurophysiological processes that underly different, yet not mutually exclusive, functional recovery mechanisms. These are (1) reduction of impairment  and (2) compensation , both asHence, we suggest that the functional role of the oscillatory processes and their recovery-related modulations might differ depending on their origin either in proximal perilesional parts of the respective stroke-affected areas  and their contralateral homologs, or in distal extralesional intact areas. That is, higher-frequency oscillatory modulations observed peri- and extralesionally might be driven by different mechanisms of the excitatory/inhibitory balance change. Perilesional functional recovery might be primarily driven by the release of the GABAergic extra-synaptic tonic inhibition and may be enhanced by an increase in glutamate-driven excitation. This is associated with the increase of the GABAergic synaptic phasic inhibition and synchronization of the higher-frequency oscillatory activity, primarily beta. In turn, compensatory motor and language recovery engaging intact extralesional areas might be primarily driven by the synaptic GABAergic signaling that provides short-living and, importantly, task-dependent phasic inhibition . The incHowever, the exact functional mechanism underpinning compensatory recovery remains obscure. The results of the motor and language studies reviewed might indicate that this compensation could be driven by the involvement of the domain-general neural networks supporting working memory and cognitive control processes. Within the framework of the top-down control mechanism suggested in the Introduction, beta and alpha oscillations are generated by the reciprocally connected excitatory and inhibitory neurons that form microcircuits lying in the deep cortical layers  and sending projections both to more superficial layers and to other cortical areas. Recovery-related modulations of beta and alpha activity may stem from rebalancing the synaptic strengths within the recruited neural microcircuits  which af1. Moreover, within this model, greater beta and alpha desynchronization  might reflect a greater working memory load during top-down controlled retrieval of target representations  need more systematic investigation both in healthy individuals and in stroke patients.training-induced modulations of beta/alpha oscillatory activity within distributed functional neural networks associated with top-down control abilities might also predict motor and language rehabilitation outcomes. However, the compensatory oscillatory dynamics, known from studies of stable patients reviewed above, have not yet been systematically investigated in chronic stroke patients undergoing behavioral training. Some hints can still be drawn from data obtained in healthy controls. It has been shown that oscillatory activity, especially in beta band, is sensitive to training as it reflects experience-dependent plasticity both in sensorimotor and in higher-order cognitive systems  is associated with better recovery, while others show opposite trends. Since beta/alpha oscillatory power modulations are sensitive to training, the observed differences are likely related to plastic reorganization processes linked to re-tuning excitatory–inhibitory balance across distributed cortical areas. Correlations between oscillatory power modulations  with behavioral improvements may vary across patients because they rely on different strategies of motor training and action execution. Clearly, experience-induced oscillatory modulations might be affected by a variety of factors, and substantial further investigations are needed to validate their potential as biomarkers of recovery.There are various limitations of the suggested framework and of the present interpretations of available findings, which will need clarification in future research. First, the present review is mostly focused on the compensatory recovery mechanism driven by the recruitment of the intact extralesional areas and reflected in modulations of their oscillatory activity in beta and alpha bands. However, interactions with other recovery mechanisms were left mostly beyond the scope of this review and remains to be tackled. For instance, substantial research is needed to clarify the relative contributions of the recovery-related dynamics in the primary functional areas vs. associative cortical ones. Moreover, we have emphasized that the recovery driven by the top-down control system might rely on multiple functional and neural mechanisms, different aspect of which need systematic and comprehensive investigation.Also, based on the above findings, it stands to reason that efficient functional recovery of motor and language abilities in chronic stroke is achieved when an optimal task-dependent excitatory–inhibitory balance in the specific neural networks is established as an outcome of interventions . Whereas the available data do not allow for unequivocal confirmation of this suggestion, future studies could pursue this in several ways. For instance, they will need to clarify which particular spatial and temporal patterns of oscillatory modulations may predict more successful recovery. They will also need to take into consideration the input of a variety of individual clinical and demographic variables.training-induced changes in task-dependent oscillatory dynamics in chronic stroke. At present, evidence is particularly lacking for PSA, where more complex metrics of oscillatory activity  are crucial for understanding its functional relevance for recovery. To scrutinize these dynamics, neuroimaging techniques with high spatial and temporal precision, such as MEG with individual MRI-based source analysis  seem optimal.More specifically, studies are needed that could more systematically investigate This future work should consider that the patterns of cortical oscillatory modulations might also vary depending on rehabilitation training techniques and on tasks used to measure behavioral outcomes. Finally, it seems essential to define individual patients’ characteristics affecting the specific dynamics of these processes and their exact associations with better recovery. Ultimately, this knowledge should contribute to better strategies for diagnoses, therapies, and optimal rehabilitation techniques for chronic stroke patients.To sum up, we reviewed the evidence that neural excitation/inhibition balance reflected in oscillatory activity modulations may be a candidate biomarker of functional recovery in chronic stroke. Among different frequency bands, modulations of beta/alpha activity might be most relevant for motor and speech recovery in chronic stroke patients. Based on the limited evidence available, we suggest that this activity might be associated, among others, with domain-general processes of top-down control, such as working memory, selective attention, and executive control, which are involved in both training and performance in motor and language tasks. This interpretation of shared recovery mechanisms and respective biomarkers goes well in line with the established notion of the evolutionary close relationship between neural underpinnings of motor and language functions. Based on this, the following tentative conclusions can be made:•Motor and language beta and alpha activity modulations are associated with chronic post-stroke recovery.•These modulations might reflect the recovery-related dynamics of the excitation–inhibition balance, both in perilesional and, importantly, in extralesional intact cortical areas.•Functionally, the task-dependent modulations in beta and alpha bands may be related to a compensatory involvement of the domain-general top-down control mechanisms, common for motor and language recovery.•Beta and alpha activity modulations in language and motor tasks might thus be a common biomarker of language and motor improvements in chronic stroke.•Beta and alpha oscillatory dynamics might reflect the efficiency of motor and language training in chronic stroke rehabilitation.The latter suggestion requires substantial future research which should both explore the mechanisms underlying these dynamics and validate its potential practical utility as a training-induced recovery biomarker.MU reviewed the literature and prepared an earlier version of the manuscript. MU and YS discussed the review and edited the manuscript. Both authors contributed to the article and approved the submitted version.


However, as the two most common forms of amyloidosis, immunoglobulin light chain (AL) and wild type transthyretin (ATTRwt) amyloidosis do coexist, very careful and stepwise approach needs to be employed in diagnostically evaluating patients who have evidence of amyloid organ deposition with concurrent plasma cell dyscrasia (PCD). The report that describes cardiac amyloid as a presenting feature of multiple myeloma (MM) by Velayutham et al., unfortunately lacks the necessary rigor in verifying the exact etiology of cardiac amyloidosis, and further misidentifies it as a presenting feature of newly diagnosed MM. This can critically influence the choice of appropriate starting therapy.Amyloidosis is a systemic disorder of deposition of more than 30 different types of misfolded precursor proteins in extracellular tissues, leading to variety of organ dysfunctions and subsequent clinical presentations. Cardiac involvement usually results in restrictive cardiomyopathy and diastolic dysfunction leading to congestive heart failure, although conduction system defects and arrhythmias are also very common. Furthermore, IMWG 2014 guidelines do not recommend the use of non-CRAB criteria for the initiation of treatment. While a concurrent presence of MM or increased bone marrow plasmacytosis is very much possible in AL Amyloidosis, and is well described as a high-risk feature in these patients, institution of therapy without the adequate confirmation of amyloid subtype, especially in a patient that otherwise lacks clear myeloma defining events represents a significant clinical risk of mistreatment and potential harm to the patient.Although the 2003 International Myeloma Working Group (IMWG) criteria included non- hypercalcemia, renal insufficiency, anemia, and bone lesions (CRAB) end-organ damage, such as hyperviscosity, AL amyloidosis, and recurrent bacterial infections as sufficient criteria for new diagnosis of MM, the 2014 update regards systemic AL amyloidosis as a distinct PCD, and amyloid presence in a patient with monoclonal gammopathy does not automatically suggest MM nor AL amyloidosis  chain-related via direct examination of the amyloid, ideally using liquid chromatography and mass spectrometry or immunoelectron microscopy . In circ.We now have treatment for both the ATTRwt and hereditary forms of transthyretin amyloid cardiomyopathy, which are associated with reductions in all-cause mortality, cardiovascular-related hospitalizations as well as lower decline in functional capacity and quality of life. Lastly, it should be noted that while lenalidomide therapy has been studied in newly diagnosed AL Amyloidosis, it may be challenging in terms of toxicity, especially in those with renal and cardiac dysfunction. Hence, patients with cardiac amyloidosis should be carefully monitored while on immunomodulatory drugs.Cardiomyopathy represents the leading cause of death in AL amyloidosis, and is unfortunately not rare: 60% of patients have clinically significant cardiac involvement as a presenting manifestation.Current standard for the treatment of systemic light chain amyloidosis includes combination therapy with daratumumab, bortezomib, cyclophosphamide and dexamethasone


Some data implicate microbes in the pathogenesis of Alzheimer’s Disease and Related Dementias (ADRD). Whether the use of antibiotics predisposes to ADRD remains unknown. To investigate the relationship between antibiotics and subsequent cognitive function, we analyzed data from a longitudinal, nationally representative sample of older US adults . The use of antibiotic medications was collected by home interview at baseline. Five years later, cognition was assessed using a survey adapted version of the Montreal Cognitive Assessment (MoCA). Additionally, ADRD status was measured by a report of a physician’s diagnosis (self or proxy). The association between baseline antibiotic use and an interval dementia diagnosis/MoCA scores was tested using multivariate logistic regression, controlling for age, sex, race and ethnicity, education, co-morbidities (modified Charlson Comorbidity Index), and cognition at baseline . Older US adults who used antibiotics had lower MoCA-SA scores at 5-year follow-up . However, the use of antibiotics did not predict a subsequent diagnosis of dementia . Thus, antibiotic use may cause deleterious effects on cognitive function, but does not appear to have a clinical impact in terms of diagnosis of dementia. Further study of the role of microbes and drugs that modulate them may be useful in understanding ADRD.


Cystic fibrosis (CF) is a autosomal recessive, multisystemic disease caused by different mutations in the CFTR gene encoding CF transmembrane conductance regulator. Although symptom management is important to avoid complications, the approval of CFTR modulator drugs in the clinic has demonstrated significant improvements by targeting the primary molecular defect of CF and thereby preventing problems related to CFTR deficiency or dysfunction. CFTR modulator therapies have positively changed the patients’ quality of life, especially for those who start their use at the onset of the disease. Due to early diagnosis with the implementation of newborn screening programs and considerable progress in the treatment options, nowadays pediatric mortality was dramatically reduced. In any case, the main obstacle to treat CF is to predict the drug response of patients due to genetic complexity and heterogeneity. Advances in 3D culture systems have led to the extrapolation of disease modeling and individual drug response in vitro by producing mini organs called “organoids” easily obtained from nasal and rectal mucosa biopsies. In this review, we focus primarily on patient-derived intestinal organoids used as in vitro model for CF disease. Organoids combine high-validity of outcomes with a high throughput, thus enabling CF disease classification, drug development and treatment optimization in a personalized manner. Until a few years ago, treatments for Cystic Fibrosis (CF) were mainly based on relieving symptoms: physiotherapy to enhance airway clearance and combat lung infections and inflammation, nutritional status management and, in case of end-stage lung disease, lung transplantation. The fundamental current standards for CF therapy include pancreatic enzyme supplementation, fat-soluble vitamins and high-calorie ingestion to minimize pancreatic insufficiency and intestinal malabsorption, anti-inflammatory drugs, antibiotics and mucolytics ,2,3. OveThe extensive knowledge obtained in this field has greatly modified the practices of care and outlook for CF pediatric patients. During the last 10 years, the use of newborn bloodspot screening (NBS) for the early diagnosis of children with CF has become widely adopted. Earlier diagnosis and CFTR-targeted therapies have led to efficient improvement of the quality of life. The increasing availability of CFTR-targeted drugs that may halt or severely reduce the disease progression and potentially interrupt the pathological sequences leading to CF organ complications provides the rationale for proposing early treatment (including during pregnancy) to reduce or prevent long-term consequences of the disease. As a result, a larger number of CF-affected children could start a treatment path even a few weeks after the birth, a well before showing symptoms or irreversible organ damage. Indeed, children who had access to earlier diagnosis, which means earlier access to medical management and intervention, show better health outcomes at an older age when compared to those children who had a later CF diagnosis ,6. The eFurthermore, Szczesniak et al.  showed hSince 2018, when the combinations Tezacaftor/Ivacaftor and Tezacaftor/Ivacaftor/Elexavaftor were marketed and introduced into clinical practice, the number of mutations that are responsive to modulators has steadily increased. Alongside conventional therapies, CFTR modulators represent an important advance in the management of CF, as instead of treating the consequences of CFTR dysfunction, they target the underlying cause associated with CFTR mutations .https://cftr2.org/). Only ~20% of them are proven as CF disease-causing variants. For those mutations for which the functional defect has not yet been thoroughly studied prediction of the clinical manifestations is particularly challenging [Nevertheless, the broad range of CFTR mutation classes with various intracellular consequences, epigenetics, modifying genes, individual responsiveness/tolerance to drugs, and still unknown reasons lead to a huge variability in the clinical phenotype of CF. Indeed, CF shows a huge variability between patients, for which several possible explanations can be proposed. Firstly, in the CFTR gene itself, over 2100 CFTR variants have been reported in the Cystic Fibrosis Mutation Database  cells that ectopically express CFTR cDNA are the pre-clinical, high-throughput model that has been mostly used to successfully develop CFTR modulators. The recent FDA approval for label extension of ivacaftor and ivacaftor/tezacaftor/elexacaftor to patients with different mutations was based primarily on laboratory evidence of efficacy in FRT cells ,37. HoweTo evaluate individual CFTR modulators’ responses, several assays using CF patient-derived materials have been implemented and are widely used ,41,42,43Primary HBE cells are typically obtained by invasive procedures (bronchoscopy or lung transplantation) from lungs with advanced/end-stage disease that may or may not reflect cells’ behavior in early disease. They are usually available in a limited number of severely ill patients so cannot be used for large-scale or theratyping studies.HNE cells seem to be a good surrogate for human bronchial epithelial cells. They are collected by minimal-invasive procedures such as nasal brushing or scraping of the lower turbinates. The current gold standard for modeling the primarily affected CF lung epithelium is air–liquid interface (ALI) culture of human nasal epithelial cells . HoweverNevertheless, in the last few years, several research groups have explored several approaches that allow for isolation, expansion, and differentiation of primary nasospheroids ,46,47,48Another tissue representative of CF disease is the gastrointestinal tract which is affected in utero or early after birth by diseases such as meconium ileus and pancreatic insufficiency, the latter featuring typical pancreatic cysts after which the disease was named. Interestingly when nasal and intestinal mucosa were compared as in vivo biomarkers of CFTR function to distinguish people with CF (pwCF) and healthy controls, Intestinal Current Measurement (ICM) was found superior to Nasal Potential Difference (NPD) and ICM demonstrated substantially greater power than NPD to detect low levels of residual CFTR function ,53 suggeIntestinal OGs remain the most advanced three-dimensional in vitro model for CF to date. Other than being a primary target organ in CF it is worth noting that CFTR represents the dominant channel responsible for ion and fluid secretion in gastrointestinal cells, which make intestinal OGs valuable models to investigate CFTR function and modulation ,62,63. MIn vitro human intestinal OGs provide a unique development model that can be applied to study intestinal prematurity diseases such as necrotizing enterocolitis, short bowel syndrome, Hirschsprung’s disease, infectious disease in the intestine and genetic diseases such as CF .Intestinal OGs are robust tools for studying genetic diseases due to their genetic and phenotypic stability such as CF and cancer where genetics can influence disease severity, prognosis, and drug efficacy ,67.Certainly, research in the CF field provides an appropriate example where OG technology has generated a relevant impact. Here, patient-derived intestinal OGs have been used for disease modeling, drug screening, and personalized medicine and have provided advantages in the development of CFTR-modulating drugs. Intestinal OGs have demonstrated high validity and high-throughput potential to predict drug efficacy in individuals with CF ,69.www.hitcf.org) using patient-derived rectal OG to assess cellular responses to various CFTR modulators. This European project aims to develop personalized treatments for PwCF allowing those with rare CFTR mutations access to treatment.The use of OG in high-throughput screening enables pre-clinical testing of many compounds on patient-specific tissues. In vitro tests based on patient-derived rectal OGs can facilitate rapid access to new treatment or can assist in the identification of variants that are currently not registered for treatment but can potentially be responsive to CFTR-targeting drugs. This general approach is underway within the HIT-CF program  and ICM. Interestingly, in cases where SCC and ICM disagreed, FIS appeared to correctly align with the clinical indicators allowing the in vivo residual CFTR function to be accurately estimated for each patient [The potential of intestinal OGs as a pre-clinical model and for personalized medicine has been demonstrated in different studies ,71,72. T patient .Notably, Berkers et al. reported a strong correlation between in vivo and in vitro response of CFTR modulators indicating how OG can play a key role in personalized medicine approach . The preData from Pranke et al., also indicate that in vitro analysis of nasal epithelium may correlate with in vivo outcomes  and airwPredicting individual patient response necessary to optimize the application of a given treatment, based on the use of currently available and future CFTR modulators, require a robust and standardized pre-clinical test .The majority of data describing the readout for quantifying the CFTR function and how it can be recovered by CFTR modulators in intestinal organoids derived from the Forskolin-Induced Swelling test (FIS). This test was described first by Dekkers and collaborators in 2013 . ExposinFIS quantitatively correlates with CFTR function and genotype: healthy control OGs appear already swollen and a further increase in swelling appear marginal due to the presence of liquid in the lumen that cannot accumulate over physiological limits. Absent FIS is present in patients with CFTR null alleles, is decreased in variants classified as mild, and is strongly reduced in OG derived from severe variants ,82.FIS of patient-derived OGs is very helpful to measure patient-specific CFTR activity, comparing CFTR function between individuals presenting with different and even identical CFTR mutations, and quantifying individual CFTR modulator response. Some studies found, however, that in healthy control OG FIS rate is negatively affected by fluid-filled lumens before forskolin treatment leading to an underestimation of CFTR function. The OG initial starting area must be similar to obtain accurate and reliable swelling results. Therefore, to compare healthy donor-derived OG and CF OGs, another test was introduced: the steady-state lumen area (SLA) assay in which lumen surface area is measured as a percentage of total OG size . WhereasFIS and SLA are complementary assays. FIS of CF and healthy controls are not directly comparable therefore it is more suited to only compare CF conditions. SLA facilitates comparison between CF and healthy control OG but has a limited resolution to discriminate at lower CFTR function levels associated with severe CF disease.− and HCO3- transport [- transport compared to HCO3-, suggesting the occurrence of different behavior of rescued CFTR on different genotypes that might differentially affect chloride and bicarbonate transport [Despite these useful features, a limitation of FIS assay is that the selective delivery of compounds to the apical or basolateral compartment in OG growing as 3D structures is complicated. Apical stimulation can be performed in OG by microinjection, but this is especially challenging for CF OGs because of their limited luminal volume. More recent research has described protocols to generate two-dimensional monolayers grown on porous membrane filters from dissociated 3D OG . A monolransport . This isransport ,87. ThisEpithelial monolayers of intestinal OG may be a valuable tool to evaluate ion transport of different channels/transporters in different culture conditions that can be precisely modified for the purpose other than assisting in diagnosis and precision medicine testing: Indeed, CFTR-dependent intestinal epithelial ion transport measured on rectal organoid-derived monolayers of subjects carrying distinct CF genotypes correlates well with donor-matched native ICM and FIS of 3D intestinal OG . The higAnother CFTR bioassay with intestinal OG that can be used as a complementary diagnostic test is rectal organoid morphology analysis (ROMA). ROMA is based on the measurement of two morphological parameters of the organoids: (1) the intensity ratio (IR) that evaluates the presence or absence of a central lumen, and (2) the circularity index (CI) that measures the roundness of the organoids . As outcThe most feasible personalized biomarker platforms for testing CFTR function and CFTR modulator responsiveness in the foreseeable future will likely be intestinal and nasal cells and derived OG as they represent key targets and easily accessible sites for cell procurement, with the former currently representing a more robust and reproducible platform based on available data in the literature . In addiThe common goal is to give new hope to all PwCF by changing the general perception of CF as a lethal disease. Prospects aim to identify new CFTR modulators and to extend eligibility for drugs now adopted for clinical use to patients with CFTR variants without approved treatment so that all CF patients can eventually be treated with a matched therapy. All these new perspectives will hopefully bring existing and novel CFTR modulators faster to patients with rare mutations and in the long run also bring the most efficacious drug to individual patients who carry more common mutations. The possibility to develop and biobank patient’s “avatars”, such as OGs, that can be developed at birth, recovered, and analyzed once new drugs might become available (a highly likely possibility in the foreseeable future) without the need for resampling ensures previously unthinkable opportunities to match the treatment with the individual molecular asset and develop a much needed theratyping approach, especially for those patients whose variants are so rare to require an N-of-1 approach.It remains to establish the relationship between the results obtained from patient-derived materials and the long-term results, but as with any new approach, these data will only be collected as they become available.


Dengue outbreaks have been documented in Singapore since 1901, occurring almost annually in the 1960s and disproportionately affecting the paediatric population. In January 2020, virological surveillance detected a shift from DENV-2, which was the previous dominant strain, to DENV-3. As of 20 September 2022, 27,283 cases have been reported in 2022. Singapore is currently also responding to the COVID-19 pandemic, overcoming another wave of infections with 281,977 cases recorded in the past two months as of 19 September 2022. While Singapore has adopted several policies and interventions to combat dengue, primarily through environmental control but also innovations such as the Wolbachia mosquito programme, there is a need for further efforts to deal with the dual threat of dengue and COVID-19. Drawing lessons from Singapore’s experience, countries facing such dual epidemics should enact clear policy responses, including establishing a multisectoral dengue action committee and action plan prior to potential outbreaks. Key indicators should be agreed upon and tracked at all healthcare levels as part of dengue surveillance and incorporated into the national health information system. Digitizing dengue monitoring systems and implementing telemedicine solutions are innovative measures that would facilitate the response to dengue in the context of restrictions during the COVID-19 pandemic that hinder the detection and response to new cases. There is a need for greater international collaboration in reducing or eradicating dengue in endemic countries. Further research is also required on how best to establish integrated early warning systems and extend our knowledge of the effects of COVID-19 on dengue transmission in affected countries. Dengue is caused by the dengue virus, existing as four different serotypes of single-stranded, positive-sense RNA viruses (DENV-1 to DENV-4), causing over 96 million symptomatic infections per year.1Aedes mosquitoes, the primary vector of dengue, and successfully reduced the incidence of dengue by about 90%.3Dengue outbreaks have been documented in Singapore since 1901, occurring almost annually in the 1960s and disproportionately affecting the paediatric population.Data suggest Singapore experiences dengue epidemics in multi-annual cycles, with larger surges attributed to a switch in the predominant serotype.Bacillus thuringiensis subsp. israelensis (BTi) and Wolbachia bacteria in mosquito breeding hotspots.6Dengue control is spearheaded by National Environment Agency (NEA), collaborating with private, governmental, and academic stakeholders in implementing an integrated vector-control programme.Singapore is currently also responding to the COVID-19 pandemic, overcoming another wave of infections with 281,977 cases recorded in the past two months as of 19 September 2022.Aedes aegypti population and decreasing the number of dengue cases.With dengue endemic, Singapore has adopted long-term policies and interventions to combat dengue, primarily through environmental control but also innovations such as the Wolbachia mosquito programme, as summarised in Furthermore, Singapore has developed a comprehensive response framework that includes legislating dengue environmental control through the Infectious Disease Act (IDA), Control of Vectors and Pesticides Act (CVPA) and the Environmental Public Health Act (EPHA).Aedes mosquitoes as well as faster viral replication within infected mosquitoes.4Research indicates that temperature can influence the transmissibility of dengue as warmer conditions are associated with faster development and increased survivability of Differentiating between COVID-19 and dengue infection is challenging without diagnostic testing, as many initial clinical symptoms overlap. Both present as a fever associated with nonspecific signs and symptoms. Thus, such patients are triaged as potential COVID-19 until proven otherwise via diagnostic testing.17As the world continues to concentrate on controlling the COVID-19 pandemic, persistent measures are still required to mitigate endemic infections and minimise the emergence or re-emergence of other pathogens. The COVID-19 pandemic has coincided with a resurgence of dengue in countries such as Brazil, India, Malaysia, and Pakistan. Drawing lessons from Singapore’s experience, countries facing such dual epidemics should enact clear policy responses, including establishing a multisectoral dengue action committee and action plan prior to potential outbreaks. Key indicators should be agreed upon and tracked at all healthcare levels as part of dengue surveillance and incorporated into the national health information system. Due to potential changes in the dominant serotype, as seen in Singapore’s current dengue epidemic, countries should include the identification of all serotypes. Additionally, affected countries should aim to increase funds for infectious disease control. While COVID-19 mitigation topped most 2020–2022 budgets, funding for other endemic or re-emerging diseases should also increase. A dedicated budget for dengue response, accessible at peripheral and local levels as relevant, can help ensure timely action. Strengthening dengue surveillance, ensuring funding for prevention and response, conducting social mobilisation and vector control, improving diagnostic testing capacities, and health staff training on dengue and COVID-19 in the context of co-epidemics are crucial.18Singapore has relied extensively on individual and community-level environmental management, suggesting social mobilisation campaigns and legislation encouraging individual action and responsibility have a role in reducing breeding sites and increasing knowledge of disease symptoms thus encouraging early health facility attendance and diagnosis. By participating in the Mozzie Wipeout, citizens may help avoid mosquito breeding in their residences, which is an important step in reducing the mosquito count. Homeowners are urged to maintain excellent housekeeping, implement vector control strategies, and take actions including routinely inspecting their dwellings for standing water and discarding any equipment that gathers water.19Public involvement tactics may need to be revised in light of the present COVID-19 scenario and the fallout from the dengue outbreak. In addition to the customary home inspections and dengue leaflets, interacting with stakeholders over social media networks may become the new standard such as nationwide live Facebook updates.20For health systems such as Singapore, which must respond to concurrent epidemics of dengue and COVID-19, following a strict triage algorithm while differentiating the two diseases is essential for infection prevention and control and to avoid coinfection.Implementing telemedicine solutions, particularly in areas with less access to diagnostic facilities or care could aid access and identification of areas requiring additional epidemiological surveillance. Moreover. digitizing dengue monitoring systems should be a top priority for the governments of affected countries. Two cities in the Philippines and one in Indonesia have established mobile-based dengue monitoring systems, exhibiting encouraging findings. For every verified dengue case, these technologies will enable healthcare professionals to promptly alert local and national healthcare authorities.21Finally, Singapore’s response is comprehensive and carefully planned with extensive use of data. This can be challenging for larger poorer countries to emulate. For example, a Wolbachia mosquito release strategy, while potentially effective, may be too expensive for some countries on their own. This suggests a role for international partners in providing technical and financial support.


COVID-19 vaccine clinical development was conducted with unprecedented speed. Immunity measurements were concentrated on the antibody response which left significant gaps in our understanding how robust and long-lasting immune protection develops. Better understanding the cellular immune response will fill those gaps, especially in the elderly and immunocompromised populations which not only have the highest risk for severe infection, but also frequently have inadequate antibody responses. Although cellular immunity measurements are more logistically complex to conduct for clinical trials compared to antibody measurements, the feasibility and benefit of doing them in clinical trials has been demonstrated and so should be more widely adopted. Adding significant cellular response metrics will provide a deeper understanding of the overall immune response to COVID-19 vaccination, which will significantly inform vaccination strategies for the most vulnerable populations. Better monitoring of overall immunity will also substantially benefit other vaccine development efforts, and indeed any therapies that involve the immune system as part of the therapeutic strategy. The historically fast delivery of COVID-19 vaccines has already saved upwards of a million lives globally  have genetic origins, including genetic susceptibility to infectious triggers of autoimmune diseases. The vast majority of these inborn errors of immunity result in defects in antibody production . An extrAbnormalities in antibody production make standard measures of antibody titer difficult to interpret and of limited usefulness in this population. For example, analysis of antibody and cellular immunity responses after a single Pfizer-BioNTech SARS-CoV-2 vaccination in patients with multiple kinds of inborn errors of immunity revealed highly variable antibody responses that did not reach protective levels in a large fraction of subjects . In contIndividuals with cancer generally have suppressed immune function as a result of the disease –24 and oPersons with hematologic cancers, however, that maintained CD8 (cytotoxic) T cell numbers above a defined threshold had better survival rates than those who could not maintain such levels . Thus, sIntentional or collateral immunosuppression may result from therapeutic treatments targeting a wide variety of conditions. Immunosuppressive therapies are a staple for transplant recipients and for those with autoimmune morbidities. Among all immune supressed individuals, transplant recipients produced the weakest response to vaccination , 36, conMany people receive immunosuppressive treatment for a large variety of autoimmune conditions. These include arthritis, inflammatory bowel disease, and multiple sclerosis, among many others , 40. In For example, a study of COVID-19 vaccinated patients with psoriasis receiving immunosuppressant drugs  demonstrAging in healthy individuals is independently associated with a decline of immune responsiveness due to a reduction in the total number of immune cells available as well as a specific reduction in the ability to respond to novel antigens –46. EvenThe effects of aging are not uniform. Recent studies define approximately half the over-55 population as immunologically frail , 55. GreThere are potentially many millions of individuals with at least partial immune deficiency. Approximately 4% of US residents have inherited or acquired immune deficiencies  and apprImmunocompromised persons are a significant segment of the population. They are also the most at risk for severe SARS-CoV-2 infection. By excluding these persons from clinical trials, particularly larger-scale phase 2 and 3 trials represent a potential source of observational bias and has led to large gaps in understanding of the true efficacy of vaccination. By including T cell evaluations in clinical development programs, immunocompromised persons no longer need to be excluded for fear of confounding study data. To the contrary, optimal vaccination strategies can be designed specifically to target these groups.As outlined above, individuals can become immunocompromised by age, disease, or chronic treatment. This reduces their ability to mount robust immune responses to challenges, including vaccination. Better understanding both the cellular and antibody responses in these individuals will inform more effective vaccination strategies. In addition to this generally applicable observation, several COVID-19 specific issues have emerged which further underscore the need for a greater understanding of the overall vaccine-induced immune response.The generation of new viral variants of concern has been discussed extensively elsewhere . Viral vT cell response to vaccination may provide the missing link between experimental testing and what is observed empirically in the real world. As mentioned above, highly infectious variants exhibit mutations that were demonstrated to provide immune escape from neutralizing antibodies induced by either infection with the ancestral strain or vaccination . TherefoBy focusing on antibody testing in vaccine clinical trials, these robust cross-reactive T cell populations remain understudied, limiting our true understanding of vaccine efficacy. As an example, using flow cytometry several recent independent studies have confirmed that the vast majority (>90%) of the T cell response induced by two doses of mRNA vaccines were directed against conserved, “mutation-resistant” sections of the virus , 79. WheLong COVID-19 is defined as sequelae that extend beyond four weeks after initial infection . StatistThe mechanisms leading to long COVID-19 are not fully understood but may involve deficits in cellular immune function. A study that showed no difference in severity of initial infection between individuals with and without post-acute infection sequelae identified lower and more rapidly waning specific T cell responses in the latter population . In subjrd boosters shots for deployment amongst their populations. The argument for comprehensive immune monitoring applies when considering mix-and-matching vaccination are similar to the arguments presented for viral variants or immunocompromised persons. Different SARS-CoV-2 vaccines have been shown to induce T cell responses that can differ in their magnitude, as well as the composition of the T cell response  samples which have more complex handling requirements. These logistical deterrents were not insurmountable as cellular immunity measurements are commonplace in a variety of clinical trials, including examples in CAR-T , immunomIn addition, novel techniques to measure cellular immunity from large groups of participants in non-clinical settings have been implemented , 110. ThELISpot (enzyme-linked immune absorbent spot) and flow cytometry are currently the two main technologies used to conduct cellular immunity measurements in vaccine trials , 112. BoAlthough flow cytometry generates more information per sample, ELISpot is still dominant in vaccine trials, primarily due to cost considerations and perceptions of better standardization for regulatory submissions. Best practice guidelines have been published to guide flow cytometry data evaluation in a standardized manner , 113–117ELISpot tests can be run with fewer cells and fewer reagents than multi-parametric flow cytometry, although ELISpot tests typically require significantly more culture time. However, the principal fixed costs of cellular immunity measurements are derived from the need for living cells, and thus the establishment and execution of the highly qualified sample procurement, preparation, and transport protocols. The costs associated with these activities are the same, regardless of whether ELISpot or flow cytometry would be used. We argue that multi-parametric assessment is not only a better return on that investment, but is essential to the evaluation of vaccine development programs. This is especially true in test populations that lack previous cellular response characterization. This logic would apply equally to the current COVID-19 pandemic as to future vaccination programs. Costs can also be reduced by reserving comprehensive immune response analysis of both antibody and cellular immunity measurements to specific subpopulations, enabling the inclusion of immune deficient and frail elderly subjects into the large vaccine trials. Neglecting to include detailed cellular immunity evaluations in clinical trials now will likely result in collateral costs to public health and healthcare systems that far outweigh the price of these tests.In summary, we advocate for much more holistic assessment of immune status in research cohorts as a prerequisite for advancing both population-based and personalized vaccination strategies. We believe that boarder research testing, including T cell evaluations, will lead to better-informed anti-COVID-19 vaccination strategies which are effective, efficient, and inexpensive. This is particularly important in immunocompromised populations, including those suffering from long COVID-19, where antibody production may be absent or aberrant, and therefore vaccine protection is largely dependent on the T cell-mediated arm of the immune system.The paradigm surrounding immune monitoring for infectious diseases and their corresponding vaccines needs an update. Regulatory bodies, vaccine manufactures, manufactures of immune-suppressive therapeutics, physicians, and others must shift their thinking away from an antibody-only approach towards a more holistic approach including the monitoring of T cell mediated immunity. There is an urgent need to reframe our research paradigm immediately while recruitment of individuals for prospective vaccination studies is still relatively easy. The technologies and logistic channels to perform T cell testing are well established and readily available. These assessments are essential, the cost of not performing them will be paid in financial and operative stress to our healthcare systems, as well as in human morbidity and mortality, particularly amongst immunocompromised groups.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.EuP, SS, EsP, and TC are employed by CellCarta Biosciences Inc., MB is employed by PPD, Inc., JM is employed by Arsenal Capital.The remaining 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.


An appropriate production of mature blood cells, or hematopoiesis, is essential for organismal health and homeostasis. In this developmental cascade, hematopoietic stem cells (HSCs) differentiate into intermediate progenitor types, that subsequently give rise to the many distinct blood cell lineages. Here, we describe tools and methods that permit for temporal and native clonal-level HSC lineage tracing in the mouse, and that can now be combined with emerging single-cell molecular analyses. We integrate new insights derived from such experimental paradigms with past knowledge, which has predominantly been derived from transplantation-based approaches. Finally, we outline current knowledge and novel strategies derived from studies aimed to trace human HSC-derived hematopoiesis. Disruption of hematopoiesis in response to molecular insults or due to environmental stressors can result in benign or malignant hematologic disorders. Therefore, considerable efforts have been devoted to reveal the cellular differentiation pathways of HSCs, which along with self-renewal represents the primary functional traits of HSCs. While HSC biology today represents a well-established research field, recent technological advances have begun to challenge concepts traditionally linked to HSC function. Here, we have tried to describe the key features of such emerging data, and to place it in the context of prior paradigms.in vivo and in some embodiments also to assess HSC self-renewal and has for long been the prevailing method to assess HSC function. This typically involves injection of candidate HSCs into conditioned recipients, followed by monitoring the fate of the transplanted cells. Because a continuous long-term production of mature blood cells is a key property of HSCs, transplantation in its simplest form provides qualitative information on HSC function over time. Furthermore, self-renewal and/or exhaustion can be assessed by serial transplantation. When performed in a competitive setting, transplantation allows quantification of the repopulating activity of the cells evaluated . Based on differential expression of surface markers, long-term (LT) HSCs were identified as a minor fraction of Lineage- Sca1+ cKit+ (LSK) BM cells, that can be further enriched using additional markers such as CD48  . Bulk ap as CD48 , CD150 , where one strain is used as the host (recipient) and the other for the cells to be functionally assessed. This allows evaluation of donor reconstitution in PB leukocyte populations, but precludes determination of chimerism in mature platelets and erythrocytes, which lack expression of CD45. Tracking HSC contribution to these latter lineages was later made possible by the development and implementation of transgenic mouse strains in which HSCs and their progeny ubiquitously express a fluorescent protein  differentiation towards certain lineages. This was first demonstrated for the myeloid and lymphoid lineages , and latAltogether, (single cell) transplantation studies have suggested that individual HSCs are heterogenous as for their lineage output, durability of engraftment, self-renewal potential and responses to external stimuli. Clonal HSC diversity might represent a mechanism that ensures appropriate responses during emergency hematopoiesis. At the same time, heterogeneity of stemness phenotypes is likely relevant also to malignant transformation, in which selective expansion of clones with the most competitive attributes is observed .Although single cell transplantations assay the function of individual HSCs, the methodology associates with several caveats, including laborious and time-consuming analyses and limited data output . This associates with high costs and ethical considerations. Moreover, because of detection limits, somewhat arbitrary reconstitution cutoff values are often applied to define the reconstituted animals. Therefore, alternative lineage tracing techniques based on genetic barcoding have emerged and undergo continuous refinement.In its simplest form, genetic barcoding involves the labeling of individual HSPCs with unique and heritable DNA tags (barcodes), typically delivered by stable-integrating recombinant viral approaches. Next, the barcoded cells are transplanted, which enable the analysis of overlap and the unique distribution of barcodes across different hematopoietic lineages and over time. Given the complexity of these barcoding libraries, they allow for simultaneous analysis of multiple individual clones in a single recipient .via high-throughput sequencing) have allowed for more sensitive detection. While initially used to study T cell biology  to transcriptional signatures (states) . The priin vitro manipulations prior to transplantation might select for specific HSC clones or introduce bias in their lineage output. Therefore, methods to label HSCs in situ and to track their progeny in more native contexts have been developed.While HSCs can regenerate an entire hematopoietic system following transplantation, it is now well recognized that this might reflect poorly of the dynamics and physiology of native hematopoiesis. For instance, the requirement for harsh cytotoxic conditioning —needed to promote HSC engraftment—has a range of systemic effects that are likely to influence the fate of transplanted cells. Specifically, damage to the BM microenvironment/niches may have a non-physiologic impact on the behavior of transplanted HSCs that occupy such a niche. For this reason, alternative conditioning protocols that exploit monoclonal antibodies to specifically deplete cell populations of interest have emerged, and which as such may present a more physiologic setting with less undesired side effects. Using purified antibodies or antibody-drug conjugates, Czechowicz and others demonstrated that antibody-mediated conditioning efficiently depletes endogenous HSPCs and allows for robust and long-term engraftment of transplanted HSCs . Some evIn vivo genetic lineage tracing allows for prospective tracking and quantification of HSC differentiation. They typically involve the use of transgenic or knock-in reporter mouse strains that exploit the activity of enzymes, such as Cre recombinase or Sleeping Beauty transposase, to induce irreversible HSC labeling with fluorescent proteins or DNA barcodes. As these labels are inherited by all progeny of HSCs, they allow for determination of the magnitude and kinetics of HSC differentiation and to infer cellular relationships. Moreover, if the systems are under the control of an inducible element, such as estrogen receptor ERT fused to Cre recombinase or the Tet-ON system, they enable the study of temporal events  of phenotypic HSCs, but that leads to all hematopoietic lineages  acquiring the fluorescent label in the long-term. At the same time, the HSC flux to the immediate short-term HSCs  was extremely rare, suggesting that also ST-HSCs need to self-renew to maintain their numbers and differentiation potential  . This ouotential . Becauseotential .Additional genetic lineage tracing studies further supported an important role for HSCs in native hematopoiesis. Using an Fgd5-based system, we showed that HSCs continuously supply all major hematopoietic lineages throughout life . In linePolyloxExpress model exploits Cre-mediated recombination to create a unique set of DNA barcodes, subsequently transcribed as mRNA  that leads to an absence of functional murine B and T cells. The SCID model was successfully used to engraft human PB leukocytes (Prkdcscid mutation onto the nonobese diabetic (NOD) strain  were generated (Il2rg−/− (NSG) recipients show combined B, T, and NK cell deficiency, with host macrophage tolerance. Importantly, the NSG strain is more permissive for human HSPC engraftment and is resistant to development of thymic lymphomas often emerging in NOD-SCID mice  xenograft systems permissive for human HSPCs engraftment . Pioneerukocytes , fetal tukocytes , and BM ukocytes . Further) strain . NOD micocytosis . Finallyenerated . ConsequCID mice . HumanizCID mice .Via the analysis of spontaneous nuclear DNA mutations, it was estimated that the number of HSCs falls within the range of 50,000–200,000 in an adult individual, with active HSC contributions to the granulocyte and B lymphocyte lineages but to a lesser extent to the T cell lineage  that are passed to daughter cells during differentiation  studies. HSPC-based gene therapy (GT) in combination with transplantation represents a therapeutic avenue to cure inherited genetic blood cell disorders. Analogous to cellular barcoding, GT employs ntiation . Therebyntiation .Initial IS studies in patients with SCID established sustained production of T lymphocytes but with limited long-term engraftment of the myeloid and B lineages , despiteLongitudinal studies in patients with other inherited disorders have provided further insights into the nature of human hematopoiesis. These have demonstrated multilineage output from transplanted HSPCs, as indicated by multiple shared IS that could be found in the myeloid and lymphoid lineages . This asAltogether, existing data imply that human hematopoiesis, similar to the mouse, is maintained by a pool of multipotent HSPCs throughout life. This does not preclude the existence of HSC-like clones with lineage-biased differential potential, nor more long-lived HSPCs, which could serve to more readily respond to specific challenges when needed.in vitro culture systems are likely to provide answers to these questions in a foreseeable future.From novel reporter mouse models and advances in high-throughput sequencing, we know today that both murine and human native hematopoiesis is dynamically orchestrated by a heterogenous pool of primitive HSPCs. Hematopoiesis associates with a low daily input from HSCs but a higher proliferative activity of different intermediate progenitor subsets. Hence, native hematopoiesis is highly polyclonal, which presumably allows for a high flexibility depending on organismal demands. In contrast, hematopoietic stress and external perturbations instantly evoke oligoclonal responses from HSPCs to counteract cell loss and allow for return to homeostasis. In the transplantation setting, this is manifested by distinct phases of reconstitution, which primarily rely on the activity of progenitors downstream of HSCs, and which is followed by the contribution from more long-lived multipotent clones. However, what remains unknown is to what extent the clonal behavior of individual HSPCs is pre-determined at the molecular level, and whether or not it is possible to induce specific HSC programs by genetic or pharmacological interventions. Additional studies using novel lineage tracing tools, advanced molecular profiling techniques and newer