Self-regulation enables individuals to guide their thoughts, feelings, and behaviors in a purposeful manner. Self-regulation is thus crucial for goal-directed behavior and contributes to many consequential outcomes in life including physical health, psychological well-being, ethical decision making, and strong interpersonal relationships. Neuroscientific research has revealed that the prefrontal cortex plays a central role in self-regulation, specifically by exerting top-down control over subcortical regions involved in reward  and emotion . To orient readers, we first offer a methodological overview of tDCS and then review experiments using non-invasive brain stimulation techniques  to target prefrontal brain regions implicated in self-regulation. We focus on brain stimulation studies of self-regulatory behavior across three broad domains of response: persistence, delay behavior, and impulse control. We suggest that stimulating the prefrontal cortex promotes successful self-regulation by altering the balance in activity between the prefrontal cortex and subcortical regions involved in emotion and reward processing. At least since Walter Mischel’s seminal work on delay of gratification , the prapersistence, or the sustained performance of aversive behavior ; delay behavior, which refers to choices that favor more long-term investments at the expense of short-term gains; and impulse control, which involves the purposeful inhibition of emotive response tendencies. Below we focus on these three paradigmatic forms of self-regulation and review experiments that have used brain stimulation techniques to try to improve them. We then consider inconsistencies in findings, address unresolved questions, and point to new directions for future research.One possible answer is by stimulating the brain with electrical current. Self-regulation is typically assessed with laboratory analogs of common challenges encountered in daily life, such as persisting at difficult tasks, choosing between immediate versus more delayed rewards, and managing emotional impulses. These examples represent paradigmatic forms of self-regulation, namely Convergent evidence from social, cognitive, and affective neuroscience research reveals that the interplay between the prefrontal cortex and subcortical threat and reward processing is crucial for self-regulation  during a task requiring inhibitory control as adults. In contrast, children who were unsuccessful at delaying gratification showed preferential recruitment of the ventral striatum in a delay task as adults . These fSeveral models of emotion regulation similarly emphasize the balance in activity between prefrontal and subcortical brain regions. For example, Another relevant model of emotion regulation highlighted the key role of a cortico-subcortical network consisting in a dorsal system and a ventral system . The dorConsistent with and prior to these models of emotion regulation, Botvinick and colleagues  discusseWe propose that non-invasive brain stimulation targeting the prefrontal cortex holds promise for improving human self-regulation. To orient the reader to neuromodulation, we first provide a methodological overview of transcranial direct current stimulation (tDCS) – one of the most popular brain stimulation techniques in psychological science. Then we review behavioral evidence that non-invasive brain stimulation targeting the PFC may enhance three paradigmatic forms of self-regulation: persistence, delay behavior, and impulse control  to increase conductivity, reduce resistance, and improve the homogeneity of the electric field under the electrodes.Transcranial direct current stimulation differs from other brain stimulation techniques, such as transcranial electrical stimulation (TES) and transcranial magnetic stimulation (TMS), because tDCS does not induce action potentials in neuronal membrane. Instead, tDCS transiently modifies spontaneous neuronal excitability by depolarizing or hyperpolarizing neurons’ resting membrane potentials, producing ionic concentration shifts within the extracellular fluid . Anodal N-methyl-D-aspartate) receptors, GABAergic activity, glutamatergic activity, intracellular CA2+ concentration, brain-derived neurotrophic factor (BDNF) secretion, and tropomyosin-related kinase B (TrkB) activation  and long-term depression (LTD) mechanisms at the synaptic level, affecting hyper-communicative activity through the anode and hypo-communicative activity through the cathode. These tDCS-driven changes in LTP and LTD may be mediated by a number of synaptic mechanisms including: NMDA . Current density determines the delivered electrical field strength  and electrode size  does not induce long-lasting effects, whereas tDCS stimulations of about 10 min (up to 30 min) typically do elicit aftereffects .The orientation of the electric field normally depends on electrodes’ polarity and position. As already described, tDCS produces polarity-dependent effects whereby anodal stimulation increases the activity of the stimulated area, whereas cathodal stimulation decreases it. Several studies of both the primary motor and visual cortices have found that different electrode positions modulate different neuronal groups and elicit different evoked potentials  suggestiIndeed, not only may electrode position affect the amount of current delivered to the brain and the direction of current flow, but also it may determine effects on the targeted brain region, due to electrical field interactions associated with neuronal geometry . WhereasAnother important tDCS parameter is the induced electric field focality. Generally, large electrodes and bipolar scalp electrode arrangements limit tDCS focality , in partStudies using fMRI to assess online and offline tDCS effects have found that anodal and cathodal stimulation elicit, respectively, an increment and a decrement of perfusion in a wide set of brain areas, including cortical and subcortical structures, even at some distance from the target area e.g., . SimilarRegarding its practical applications, tDCS, allows for more effective placebo stimulation-controlled studies compared to TMS where notable issues with placebo effects and limited blinding success . PlaceboAnother practical consideration concerns individual differences. Following seminal work in the tDCS literature e.g.,  we recomTranscranial direct current stimulation is a safe and non-invasive neuromodulatory technique. Several neuroimaging and EEG studies have demonstrated that tDCS does not cause adverse effects on the brain . The mosTo summarize, tDCS is a safe, inexpensive, well-tolerated, and easy to use. Its physiological consequences have led to an increasing interest in using tDCS to alter psychological processes. With an appreciation for the history and mechanics of tDCS, the next section will explore the consequences of prefrontal tDCS psychological processes in the domain of self-regulation.Self-regulation enables individuals to guide their thoughts, feelings, and behaviors in a goal-directed fashion. Self-regulation can be aversive both physically and psychologically. Goal directed behavior often entails persistence, including the sustained performance of aversive behavior. For example, physical exercise sometimes elicits short-term pain or physical discomfort but also brings more long-term appearance- and health-related benefits. Individuals who are better able to endure the discomfort presumably exercise longer and more frequently. As a result, these same individuals may be more successful at achieving their fitness goals. Other situations may require individuals to endure psychologically aversive states in the interest of accomplishing one’s goals. For example, academic and occupational successes may entail many failed attempts to solve complex problems. The ability to endure these aversive states seems likely to facilitate goal-directed behavior and thus may constitute an important facet of self-regulation.Research suggests that the DLPFC is a key brain region for various aspect of pain, including pain tolerance see . Consistno pain to 10 = severe pain. Ratings were obtained after the first 7 min of tDCS in each testing block and immediately after the cold pressor and breath holding tasks. Stimulation did not influence performance on the cold pressor task as measured by threshold, tolerance, or endurance, nor did it influence breath holding time. However, anodal stimulation over the left DLPFC decreased the experience of pain as measured by the DVPRS. This study suggests that excitatory stimulation of the DLPFC may influence pain perception.More recently, a study by Another relevant pilot study by Delay behavior refers to choices that favor more long-term investments at the expense of short-term gains. To the extent that individuals stifle or subdue their immediate urges, the better they are in striving toward their long-term goals.Often one’s end goals are situated far in the future, and pursuing such goals comes at the cost of satisfying more immediate desires. Saving for the future comes at the cost of spending today. Maintaining a healthy physique comes at the expense of delicious desserts. Academic achievement often comes at the expense of fraternization. k) reflects a stronger preference for smaller-but-immediate rewards. A less steep slope  reflects a stronger preference for larger-but-delayed rewards. Typically, a preference for smaller-but-immediate rewards is thought to reflect impulsivity (poor self-regulation) whereas a preference for larger-but-delayed rewards is thought to reflect self-control (good self-regulation).Delay discounting refers to the reduction in the present value of a reward with delayed receipt. The basic idea is that the valuation of rewards degrades over time. So, for example, gaining $10 today would typically be valued more than gaining $10 in a month from today. For each unit of time increase in the delay to receipt, the value of a reward decreases (or is discounted) by a non-fixed proportion. In other words, the effect of delay on value is not the same across the range of delays. At short delays value decreases less steeply, whereas at longer delays value degrades more steeply. A hyperbolic discounting function captures the pattern that at shorter delays reward valuation degrades less than it does at longer delay periods. The steepness of the slope within this hyperbolic model reflects the extent to which people prefer smaller-but-immediate (compared to larger-but-delayed) rewards. A steep slope . Because of this, the study by Nejati and colleagues differs in a critical way from the discounting studies reviewed above as those studies speak moreso to the role of hemispheric asymmetry in discounting behavior. Thus, the majority of evidence here suggests that stimulation to shift the balance in neural activity toward the left prefrontal cortex increases delay discounting in a manner that suggests poorer self-regulation.Food choice and eating entail delay behavior insofar as choosing nutritious foods  over tempting ones  represents a choice favoring long-term investments in health at the expense of a short-term hedonic gain. More recently, a study by Inspired by the evidence linking excitatory right DLPFC stimulation to reductions in food cravings, Why does tDCS seem more likely to reduce craving but not consumption as the studies above suggest? It may be that cravings or subjective responses are easier to modulate with tDCS (and other interventions) than consumption or behavior. The fact that more of the studies reviewed above find craving effects than consumption effects is consistent with this view.enough to reduce eating. Additional consumption without craving (or low levels of craving) may reflect a form of dysregulation suggesting more automatic/habitual factors may influence consumption independent of craving . These reasons suggest that many contextual factors may make detecting links between impulse and consumption more difficult.In the case of studies where cravings are reduced but not reduced consumption, in these studies it may be the case that craving was not reduced Impulse control involves the inhibition of emotive response tendencies. It plays a key role in diverse behavioral domains including emotion regulation, prosocial behavior, and risk taking.Emotions are not always functional. For example, emotions may work against one’s goals when they are expressed at the wrong time or at an inappropriate level of intensity . EmotionCurrent neural models of emotion regulation e.g.,  further Evidence from fMRI research regarding the contributions of VLPFC and DLPFC to emotion regulation paved the way for the application of non-invasive brain stimulation to these regions to investigate stimulation effects on both explicit and implicit control strategies. For instance, one study tested the hypothesis that a top-down form of antecedent-focused emotion regulation – cognitive reappraisal – involves DLPFC. Indeed, anodal tDCS over the right DLPFC combined with a cognitive reappraisal task reduced negative emotions (but not positive emotions or craving) compared to anodal tDCS over the left hemisphere and sham stimulation . This paAnger and aggression are associated with the activation of the behavioral approach system . The behOne difference between the results of One common response to negative emotions, like anger, is rumination, which is an automatic cognitive process characterized by repetitive and distressful thoughts . ParalleA study by A follow-up study further explored the consequences of excitatory tDCS over the right DLPFC for inhibitory processing. A meta-analysis of neuroimaging studies using a Go/No-Go task found a broad pattern of right frontal cortical activation during response inhibition . A separThe results revealed that anodal stimulation over the right frontal cortex facilitates the inhibition of both approach-incongruent and avoidance-incongruent responding. These results are not readily explained by an increase in avoidance motivation. Indeed, an increase in avoidance motivation would have slowed the enactment of an avoidance-incongruent response. But Several studies have concentrated on testing tDCS efficacy in sadness regulation, which has important implications for considering tDCS as a treatment device for major depression. Indeed, it has been widely observed that major depression is associated with an asymmetry in prefrontal cortical activity, specifically hypoactivity in the left DLPFC and hyperactivity in the right DLPFC. For instance, one study found that five sessions of bilateral, twice-a-day tDCS over the DLFC (anode left/cathode right) decreased self-reported sadness in a group of patients with severe, drug-resistant major depression. These improvements lasted up to a month after the end of the treatment . These fAnother major, consequential negative emotion is fear. Fear plays a crucial role in the onset and maintenance of chronic pain  and seveOn the other hand, a recent tDCS study on the consolidation of fear memories observed that greater right frontal cortical activity may enhance fear-related responding. Social pain refers to the hurt feelings caused by rejection or ostracism from the group. These aversive social experiences generally cause negative emotions and result in a loss of sense of belonging, control, and self-esteem . Some stThese findings have lead researchers to test the effects of tDCS over brain regions involved in social pain regulation, mainly the right VLPFC. For example, participants in a study by In a similar study, the same research group  tasted tSimilar results were obtained by Collectively, these studies highlight the effects of tDCS over the prefrontal cortex on regulating responses to social exclusion. This work may have clinical implications for disorders characterized by maladaptive responses to social exclusion . Future research should test the extent to which the increased negative affect and autonomic arousal  and inapRecently, some research has observed that personal physical suffering and empathy for the pain of others share the same neural network, involving prefrontal cortex as well as somatosensory cortex, anterior cingulate cortex, amygdala, and anterior insula . To inveThese findings were expanded by a similar study comparing emotional reactions to negative, positive, and neutral human pictures . This stA similar finding was reported also by In addition to emotion and mood manipulations, another way researchers have explored the consequences of prefrontal brain stimulation on emotion regulation is by studying risk taking. Risk taking involves the possibility of punishment or potential harm in the pursuit of rewards or goal-relevant actions. From an emotion-regulation perspective, risk-taking involves managing the emotions associated with the anticipation of winning and losing. In a seminal tDCS study, These findings have been partially replicated by Another study probed the underlying neurocircuitry that may be driving the changes in risk taking. In addition to laboratory risk-taking paradigms, an innovative study paired prefrontal tDCS with a more ecologically valid form of risk taking: driving behavior. In a driving simulator study, In summary, numerous studies have observed enhanced self-regulation after anodal tDCS over right DLPFC . Improved self-regulation manifested in a variety of ways including greater pain tolerance, healthier food choices, less impulsive decision-making, improved emotion regulation, reduced anger and aggression, less sadness and fear, more empathy, and less risk taking. Collectively, these results anodal tDCS over right DLPFC may be one tool that shows promise in helping individuals live happier, healthier lives.We have reviewed evidence suggesting that non-invasive brain stimulation over the prefrontal cortex may improve human self-regulation. First, we detailed a methodological review of tDCS highlighting that tDCS is a safe, inexpensive, and easy to use technique that can be used to study higher-order cognition, emotion, and clinical phenomena. We also reviewed stimulation parameters that may help or hinder tDCS efficacy including current density, stimulation duration, and the orientation and focality of the electrical field. Second, we reviewed findings from experiments observing that non-invasive brain stimulation over the PFC can enhance three paradigmatic forms of self-regulation: persistence, delay behavior, and impulse control. Although the current review focused predominantly on tDCS, other techniques such as low energy neurofeedback e.g.,  and tranAlthough much of this review highlighted the ability of brain stimulation to promote self-regulation, under the right circumstances brain stimulation may also undermine self-regulation. For example, we highlighted studies wherein excitatory stimulation over the right DLPFC enhances inhibition e.g.,  whereas The efficacy of eliciting changes in brain activity using tDCS depends on several physical parameters including current density, stimulation duration, and the orientation and focality of the electrical field. These parameters constitute the tDCS dosage. As we noted above, different electrode positions modulate different neuronal groups and elicit different evoked potentials in the case of primary motor cortex and primary visual cortex stimulation, respectively , and theWhereas the coupling of anodal-excitatory and cathodal-inhibitory effects is well established in the sensorimotor domain, the evidence pertaining to higher cognitive functions is more controversial . Indeed,To illustrate this point, consider the studies above on physical pain tolerance. These studies found increased pain tolerance as the result of excitatory stimulation of the right DLPFC , excitatMore broadly, however, the findings from the pain studies are congruent with the findings from studies of other self-regulatory domains in suggesting that stimulation over right DLPFC facilitates self-regulation. Whether such stimulation is most profitably paired with inhibitory  stimulation over left DLPFC remains to be seen. And the effects of excitatory  stimulation over left DLPFC on self-regulation are even more uncertain, with some studies finding better and some finding worse self-regulation after excitatory stimulation over left DLPFC.Another inconsistency in past research concerns individual differences. Despite seminal work suggesting that participants should be matched on individual differences like sex or age that can influence tDCS efficacy , many ofIn addition to demographic factors, which may subtly influence tDCS efficacy, many of the studies we reviewed also did not consider the extent to which participants are responsive to tDCS. Previous research has found that between 50 and 64 percent of participants are responsive to tDCS protocols, which leaves a substantial proportion of individuals who are not particularly responsive to tDCS . StudiesAnother inconsistency in the research reviewed above concerns whether changes in emotive responding extend to changes in behavior. We summarized evidence that tDCS may alter food craving without altering food consumption  well liked, such as chips, cookies, and chocolate. This may be problematic insofar as some participants may not prefer any of the food options offered by the experimenter. Future studies using more ecologically valid measures of food consumption and others types of regulatory behavior  may prove useful in clarifying the extent to which tDCS-induced changes in emotions/impulses influence subsequent behavior. However, we must also consider the possibility that cravings/subjective states are more readily influenced by tDCS, whereas behaviors may be harder to change as they have a multiple of determinates above and beyond the preceding subjective state.The path from impulse to behavior entails multiple determinants, and thus impulses and behaviors may diverge for several reasons. Practical considerations including variability in stimulation dosage may explain why tDCS sometimes does and sometimes does not influence behavior. For example, one important aspect of the tDCS dosage is electrode size. The study by The effects of tDCS on underlying brain activity are not well understood and subject to ongoing debate. It remains to be seen how tDCS affects brain activity and how these changes relate to changes in self-regulation. One way to advance research on this topic is to pair concurrent measures of brain function with electrical stimulation. Candidate neural processes mediating links between tDCS and self-regulation include changes in prefrontal EEG alpha and functional connectivity between the prefrontal cortex and subcortical regions.One potential mediator driving many of the effects reported in the current review is EEG alpha activity. Specifically, lateralized patterns of alpha activity may reflect a person’s motivational orientation with left lateralized EEG alpha activity reflecting approach-motivation  and righIndirect support for the role of EEG alpha activity in tDCS effects comes from parallel findings using EEG and tDCS, respectively, to study the emotion of jealousy. In an innovative new study, The self-regulation findings we reviewed may also be mediated by frontal cortical-subcortical interactions. Due to its high spatial resolution, functional MRI is perhaps best suited to probe such possibilities. Research along these lines has already begun. Potential neural aftereffects of tDCS, such as changes in prefrontal alpha asymmetry or functional connectivity, may mediate the relationship between tDCS and regulatory behavior. What remains to be established is the extent to which alpha asymmetry or functional connectivity carry the causal effects of tDCS on self-regulation, or whether they merely correlate with observed changes in regulatory behavior.Imagine a person who, when offered a chocolate treat, decides not to eat it. Does foregoing chocolate reflect the application of control to resist chocolate consumption, a low desire for chocolate, or some combination of both things? Social psychologists have traditionally viewed self-regulatory success as the consequence of applying self-regulatory strength see , 2007. IIn addition to limited understanding of the effects of tDCS on underlying neural processes, we also have a limited understanding of the long-term effects of tDCS. Most of the studies we reviewed involved just a single session of tDCS. Although some studies have involved the administration of tDCS once a day for multiple days , those sSelf-regulation research touches on alcohol and drug addiction, personal debt, obesity, and a variety of other consequential outcomes. It is important to acknowledge that these outcomes are not the result of one decision or behavior but instead results from multiple patterns of behavior unfolding over long periods of time. Few studies have examined long-term downstream effects of stimulation. One notable exception was a study by Self-regulation is crucial for goal-directed behavior and contributes to many consequential outcomes in life including physical and mental health, decision making, and interpersonal functioning. Likewise, failure at self-regulation is thought to contribute to alcohol and drug addiction, personal debt, obesity, and other outcomes that carry both personal and societal costs. Based on the evidence reviewed here, we conclude that brain stimulation techniques hold promise for improving self-regulation across three broad domains of response: persistence, delay behavior, and impulse control. To maximize the utility of tDCS in future research, we encourage researchers be especially mindful of issues such as dosage and individual differences – issues that have been commonly been neglected in past research and have likely contributed to inconsistencies in past findings. Future research that takes these concerns into account and incorporates concurrent measures of brain activity will bring the promise of tDCS into sharper focus and help to reveal underlying neural mechanisms.All authors contributed to the conception, writing, and revising of this 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.


Phosphotyrosine signaling is regulated by the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Here we discuss the potential of vanadium derivatives as PTP enzyme inhibitors and metallotherapeutics. We describe how vanadate in the V oxidized state is thought to inhibit PTPs, thus acting as a pan-inhibitor of this enzyme superfamily. We discuss recent developments in the biological and biochemical actions of more complex vanadium derivatives, including decavanadate and in particular the growing number of oxidovanadium compounds with organic ligands. Pre-clinical studies involving these compounds are discussed in the anti-diabetic and anti-cancer contexts. Although in many cases PTP inhibition has been implicated, it is also clear that many such compounds have further biochemical effects in cells. There also remain concerns surrounding off-target toxicities and long-term use of vanadium compounds in vivo in humans, hindering their progress through clinical trials. Despite these current misgivings, interest in these chemicals continues and many believe they could still have therapeutic potential. If so, we argue that this field would benefit from greater focus on improving the delivery and tissue targeting of vanadium compounds in order to minimize off-target toxicities. This may then harness their full therapeutic potential. In this review, we will focus on vanadium-derived chemicals as broad-specificity PTP inhibitors. We will discuss their use in pre-clinical models and look at their therapeutic potential in diseases such as cancer and diabetes. We also highlight a range of associated caveats. For those readers interested in more wide-ranging reviews of vanadium chemistry or comprehensive reviews of the growing multitude of vanadium compounds, these have been recently discussed in some other excellent reviews ,2,3,4.The successful existence of multicellular organisms relies on the ability of cells within them to communicate with one another through biochemical signalling, thus regulating responses to both intra- and extracellular stimuli. Reversible phosphorylation of tyrosine residues is a critical component of this process, and one that is itself heavily regulated. The addition and removal of phosphate groups is catalysed by two classes of enzymes, the protein tyrosine kinases (PTKs) for phosphorylation and the protein tyrosine phosphatases (PTPs) for dephosphorylation. Given the pivotal role of PTKs and PTPs in cell signalling, it is unsurprising that disruption to their normal regulation results in abnormal proliferation, survival, motility, differentiation and metabolism, underlying the pathogenesis of a range of human diseases and rendering these enzymes as promising therapeutic targets .PTPs are characterised by their active site cysteine residues, which act as critical nucleophiles during catalysis ,6. The PThe roles of PTKs and PTPs in the pathogenesis of a wide range of human disease has long been appreciated and there has been significant progress in the field of kinase inhibition in the last few decades. There are over two dozen kinase inhibitors already on the market, as well as many therapeutic antibodies directed at kinases . HoweverAs aluded to above, a common experimental strategy for PTP inhibition has been to use classes of molecules that act as pan-inhibitors of the whole enzyme family. Vanadium-derived compounds are the clearest examples of these . VanadiuVanadium exists in oxidation states ranging from −III to +V, however, the majority of vanadium in aqueous solutions is in the form of tetravalent vanadyl cations (IV) or pentavalent vanadate ions (V). The orthovanadate ion has a tetra-coordinated structure and a negative charge, features that it shares with the phosphate group of PTP substrates  and vanadate (V) redox states of the vanadium ion occur in the cell via Fenton-like reactions, generating reactive oxygen species (ROS) ,22. ThesGiven that PTPs share a conserved consensus sequence at the catalytic site, including the critical cysteine residue, it is reasonable to assume that vanadium compounds can inhibit all members of the PTP superfamily . For exaEarly examples of studies using vanadium were focussed around simple inorganic vanadium salts, whereas more recently larger, more complex compounds with organic ligands, as well as vanadium oligomers, namely decavanadate , several further studies have shown that vanadium compounds dissociate in vitro and in circulation to release the ligating molecules, or modified forms thereof. This introduces complexity in defining how oxidovanadium complexes work in vivo, in particular if the ligands themselves have cytotoxic properties . For exaThis demonstrates the importance in considering the effect of all parts of vanadium complexes in cells before prematurely assigning phenotypes as vanadium- or PTP-induced responses. In some cases therefore, the differences in biological responses to the different classes of vanadium compounds may actually be largely due to free systemic ligand effects . NeverthOverall, oxidovanadium in ligand complexes can be effective at increasing vanadium bioavailability, but caution is advised concerning the nature of the ligands used and the interpretation of biological responses.Given its potential for modulating a wide range of signalling networks, vanadium is an attractive candidate metallotherapeutic for the treatment of a range of human disorders. In fact there has been interest in the medicinal properties of vanadium for many decades. The first reported case of vanadium use in man was by Lyonnet et al. as far back as 1899. Sixty patients, three of whom were diabetic, were given sodium metavanadate. A modest reduction in blood glucose was described for two out of the three diabetics, and no adverse effects were reported . This geIn the second half of the 20th century the insulin mimetic/enhancing activity of vanadium ions began to be characterised, leading to suggestions again that vanadium compounds may indeed be able to replace insulin injections, allowing patients to be treated with less invasive oral formulations . This haInsulin signalling is critical for the clearance of glucose from the blood and to promote energy storage in fat cells. High levels of blood glucose trigger insulin release from the beta cells of the pancreas. Insulin binds to its cell surface receptor, inducing receptor autophosphorylation on specific tyrosine residues. These phosphotyrosines become docking sites for Src homology 2 (SH2) domain containing proteins including insulin receptor substrate 1 (IRS-1), in turn initiating signalling cascades ultimately leading to translocation of glucose transporter type 4 (GLUT-4) channels to the plasma membrane and increased glucose uptake. Insulin signalling can also promote glycogenesis, lipogenesis, and protein synthesis, whilst inhibiting lipid breakdown and gluconeogenesis. Phosphotyrosine signalling is heavily implicated throughout these signalling cascades, thus PTPs are highly relevant.Normal insulin signalling is perturbed in diabetic patients, where either insulin can’t be produced (type 1 diabetes) or the cellular response to insulin doesn’t function correctly (type 2 diabetes). Vanadium-based compounds have resulted in reduced diabetic symptoms in animal models of type 1 and 2 diabetes, and even in some human patients ,47,48,49With an increasing bank of preclinical data in favour of vanadium-based diabetes therapeutics, randomized clinical trials were carried out. Initially inorganic vanadium compounds such as vanadium sulphate and ammonium metavanadate were used ,54,55,56Despite these apparently positive results, a systematic review published in 2008 stated that based on data from current clinical trials, the use of vanadium to treat diabetes could not be recommended since efficacy was too variable and side effects were consistently reported . AdditioPhosphotyrosine signalling is heavily implicated in virtually all aspects of cancer biology due to its widespread influence over cell signalling. Alterations to normal phosphotyrosine signalling brought about by mutations in relevant signalling molecules can drive the initiation and progression of many different tumour types . Therefopromote cell survival, making them potential cancer drug targets [PTPs were formally discovered in the late 1980s and at that point were still thought to function as fairly unregulated housekeeping enzymes, negatively regulating phosphotyrosine signalling. They were therefore labelled as candidate tumour suppressors. Some PTPs are indeed tumour suppressors, including PTEN. PTEN is nevertheless quite specific in negatively regulating AKT signalling and it is mutated in many human malignancies ,60. It i targets . A numbe targets ,63,64. I targets . Other P targets ,67,68.With the above in mind, there is now increasing interest in the development and use of PTP inhibitors for anti-cancer therapeutics. Vanadium-based chemicals may represent one source of these.Vanadium has long been of interest in cancer biology, with the first report of its anticancer activity in 1965 . Since tMany publications to date have described in vivo chemoprevention by vanadium-supplemented drinking water in chemically induced cancer models of breast, colon and liver ,102,107.A plethora of studies using cell culture models for various tumour types have corroborated these findings and have begun to dissect the intracellular targets of vanadium compounds . These sClearly there is a large bank of preclinical data supporting anti-cancer activities of vanadium-based compounds. Whilst many of these studies do not directly explore PTP inhibition as a mechanism driving these properties, it is likely that many of the effects described are driven by PTP inhibition, both directly by competitive inhibition by vanadate, and via ROS driven active site cysteine oxidation. If this is correct, then it is worth pointing out that the beneficial effects of vanadium compounds must be the net effect of inhibiting many PTPs.2+ ATPase, but in this case through a distinct, allosteric, non-competitive mechanism by binding to a site distinct from the ATP-binding pocket [b [Although vanadium compounds do inhibit PTPs , they alg pocket ,26,27). ocket [b ,28,29. Tocket [b ,116.As discussed previously, conversion from vanadyl to vanadate generates ROS . This inWhen administered orally, vanadium enters the circulation via absorption from the GI tract. Once in the bloodstream, vanadium compounds undergo ligand exchange, and can become bound to metabolites such as lactate and citrate, and proteins, predominantly transferrin ,119. VanAlthough vanadium has not been classified by the International Agency for Research on Cancer (IARC) as a carcinogen, there have been some reports that vanadium compounds can induce tumourigenesis, potentially due to increased ROS production . A studyVanadium-based compounds have undoubtedly displayed promising properties relating to the treatment of diabetes and cancer, as well as some other diseases . HoweverOne way to potentially overcome issues surrounding the off-target toxicity of ligands and oxidovanadium itself, is to package oxidovanadium compounds into nanoparticles or nanocomposites that can be passively or actively targeted to cells. For example, packaging of vanadate with chitosan into nanocomposite carriers has been demonstrated, and these have been used for diabetes studies ,124, wheIn summary, although there are significant caveats hindering the use of current vanadium-based therapeutics, there are good reasons why this field should maintain its efforts. With new technologies for drug delivery constantly becoming available, several of the less advantageous effects of vanadium could become avoidable, raising the opportunity for development of truly effective and safe drugs based on this transition metal and its attractive, flexible chemistry.


Gut microbiota is widely considered to be one of the most important components to maintain balanced homeostasis. Looking forward, probiotic bacteria have been shown to play a significant role in immunomodulation and display antitumour properties. Bacterial strains could be responsible for detection and degradation of potential carcinogens and production of short-chain fatty acids, which affect cell death and proliferation and are known as signaling molecules in the immune system. Lactic acid bacteria present in the gut has been shown to have a role in regression of carcinogenesis due to their influence on immunomodulation, which can stand as a proof of interaction between bacterial metabolites and immune and epithelial cells. Probiotic bacteria have the ability to both increase and decrease the production of anti-inflammatory cytokines which play an important role in prevention of carcinogenesis. They are also capable of activating phagocytes in order to eliminate early-stage cancer cells. Application of heat-killed probiotic bacteria coupled with radiation had a positive influence on enhancing immunological recognition of cancer cells. In the absence of active microbiota, murine immunity to carcinogens has been decreased. There are numerous cohort studies showing the correlation between ingestion of dairy products and the risk of colon and colorectal cancer. An idea of using probiotic bacteria as vectors to administer drugs has emerged lately as several papers presenting successful results have been revealed. Within the next few years, probiotic bacteria as well as gut microbiota are likely to become an important component in cancer prevention and treatment. Bifidobacterium, or Lactobacillus, widely present in commonly consumed fermented milk products, are known to have various beneficial effects on health. To date, there is a plethora of studies investigating the correlation between intestinal microbiota and carcinogenesis which have been evaluated in this article. A growing body of research has been analyzed and reviewed for the potential application of probiotics strains in prevention and treatment of cancer.Cancer is considered as one of the most significant causes of death. The treatment of tumors has received much attention in the last years; however, the number of people suffering neoplastic syndrome is still increasing. Thus, researchers are trying to face this process searching for innovative therapies and prophylaxis. Despite the fact that cancer risk indisputably depends on genetic factors, immunological condition of the organism plays a considerable role in it, that being closely associated with probiotic bacteria and commensal bacterial flora presented mainly in the digestive tract. Probiotic strains, inter alia Goldin and Gorbach  were amoL. acidophilus and B. bifidum have been demonstrated to be a promising tool in cancer prevention [A specific mechanism associated with antitumor properties of probiotics remains unclear. Gut microbiota is engaged in a variety of pathways, which are considered to play a central role in that process. Primarily, probiotic bacteria play an essential role in the preservation of homeostasis, maintaining sustainable physicochemical conditions in the colon. Reduced pH caused inter alia by the excessive presence of bile acids in feces may be a direct cytotoxic factor affecting colonic epithelium leading to colon carcinogenesis , 28. Regevention , 29, 30.Escherichia coli and Clostridium perfringens naturally present in the gut, has been proven to be involved in production of carcinogenic compounds using enzymes like b-glucuronidase, azoreductase, and nitroreductase. Some preliminary research conducted by Goldin and Gorbach in the late 1970s have proven consumption of milk fermentation products to have a beneficial effect on the increase in the number of L. acidophilus in rat’s gut, which subsequently resulted in a reduction of putrefactive bacteria and decrease in the level of harmful enzymes [Probiotic strains are also responsible for maintaining the balance between the quantity of other participants of natural intestinal microflora and their metabolic activity. Putrefactive bacteria, such as  enzymes . Several enzymes , 33 and  enzymes , 34–38.  enzymes .Lactobacillus and Bifidobacillus strains, could be linked to the binding and degradation of potential carcinogens. Mutagenic compounds associated with the increased risk of colon cancer are commonly found in unhealthy food, especially fried meat. Ingestion of Lactobacillus strain by human volunteers alleviated the mutagenic effect of diet rich in cooked meat, which resulted in a decreased urinary and fecal excretion of heterocyclic aromatic amines (HAAs) [Another cancer-preventing strategy involving probiotic bacteria, chiefly s (HAAs) , 41. Sups (HAAs) . Furthers (HAAs) –51 or mes (HAAs) , 47, 49 s (HAAs) , nitrosas (HAAs) , 49, afls (HAAs) , and oths (HAAs) . In somes (HAAs) , 53. It s (HAAs) .Many beneficial compounds produced and metabolized by gut microbiota have been demonstrated to play an essential role in maintaining homeostasis and suppressing carcinogenesis. Specific population of gut microbiota are dedicated to production of short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate as a result of the fermentation of fiber-rich prebiotics. Except for their principal function as an energy source, SCFA have also been proven to act as signaling molecules affecting the immune system, cell death, and proliferation  as well Bifidobacteria and Lactobacilli can modulate the gut microbiota composition and consequently affect the production of SCFA [Firmicutes families  [Butyrivibrio fibrisolvens MDT-1, (known for their high production of butyrate) in mouse model of colon cancer, inhibited progression of tumor development, affecting also the reduction of β-glucuronidase and increasing the immune response [Although lactic acid bacteria are not directly involved in SCFA production, certain probiotic strains of  of SCFA . Butyratdiaceae)  has beendiaceae)  and remadiaceae) . Adminisresponse .B. lactis and resistant starch in rat-azoxymethane model has been proven to protect against the development of CRC, which was correlated with increased SCFA production [B. lactis nor prebiotic were sufficient to achieve that effect alone. This and some previous assays suggest that prebiotic activity of fiber-enriched diet, projecting on the level of beneficial bacteria, is promising strategy to prevent CRC.More recent evidence suggests modulation of SCFA-producing bacteria by dietary intervention with fermentable fibers as a possible colorectal cancer treatment. A more recent study on mice demonstrated amelioration of polyposis in CRC  after increasing SCFA-producing bacteria after introduction of probiotic diet. Previously investigated application of synbiotic combination of oduction . InteresNature summarizing the mechanisms engaging microbiota in immune homeostasis and disease [Lactic acid bacteria have been receiving much attention due to its contribution to immunomodulation correlated with either suppression or regression of carcinogenesis. This phenomenon is the result of multidimensional activity involving interaction between the bacteria or their metabolites with the immune and epithelial cells , 61–63.  disease .H. pylori infections [It has been demonstrated that some probiotics strains of Lactobacilli have been proven to suppress gastric-cancer-related fections –67. Anotfections .L. casei strains (LC9018) and improved the induction of immune response mechanisms against cancer cells thereby enhancing tumor regression in patients with carcinoma of the uterine cervix [B. infantis administration in CRC rat model demonstrated a considerable attenuation of chemotherapy-induced intestinal mucositis correlated with decreased level on proinflammatory cytokines  and increased CD4+ CD25+ Foxp3+ T regulatory cell response [In recent years, there has been growing interest in the possible application of probiotics as a part of combination therapy with conventional treatment of cancer. An early but controlled and comparative study on 223 patients carried out in 1993 showed that combination therapy including radiation and treatment with heat-killed e cervix . Researce cervix . Furtherresponse .Science highlighted the significant role played by gut microbiota in the immune response to cancer treatment. Disruption of the microbiota in mice was made evident by a decreased immune response and thereby tumor resistance for either cyclophosphamide [Over and above that, two seminal papers published in sphamide  or oxalisphamide . As a resphamide , 74–76.Bacteroides and Bifidobacterium) in anti-PD-L1 (Programmed death-ligand 1) and anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) therapies [Bifidobacterium treatment alone compared to anti–PD-L1 therapy, whereas combination of both strategies was sufficient to nearly eliminate tumor outgrowth. These groundbreaking results indicate that administration of probiotics appears to be a promising strategy in maximizing the efficiency of cancer immunotherapy.The most recent experiments on mice have illustrated the key role of gut microbiota  and a decreased colorectal cancer risk . AnotherA key problem with the majority of the cohort studies mentioned above is that they covered general dairy intake, including high-fat components such as cream and cheese, suspected of carcinogenic properties due to their ability to increase bile acid levels in the colon , 87. MorUtilization of the recombinant probiotic strains as a delivery system for various therapeutic molecules such as drugs, as well as cytokines, enzymes, or even DNA , 90 is qLactobacillus lactis expressing anti-inflammatory compounds  has been shown to ameliorate the intestinal inflammation and demonstrated cytoprotective effect [Lactococcus lactis expressing catalase has been proven to decrease the production of reactive oxygen species (ROS) such as H2O2, reducing colonic damage, and inflammation, consequently projecting on tumor invasion and proliferation [In several independent studies on rodents, intragastric application of recombinant strains of e effect –94. In aferation .More recent study investigating multiple strategies of inhibition of the inflammatory-related carcinogenesis with different combination of probiotic vectors expressing antioxidant enzymes  or IL-10 (produced as cDNA or in expression system inducible by stress—SICE) has shown these strains as agents causing significant changes of the immune response as well as pre-neoplastic lesions or even causing the entire inhibition of tumor development  for det.Table 4CA plethora of studies reported potential application of the probiotic expression systems as vaccines, demonstrating stimulation of the adaptive immune system response against the pathogens –99. A nuLactobacillus lactis capable of expressing oncoprotein E7 and immunostimulatory compounds, such as interleukin-12 [Studies on TC-1 tumor murine model have shown that therapeutic effect can be enhanced by co-administration of eukin-12 , 102. Preukin-12  survivaleukin-12 , 102.Bifidobacterium expressing Wilms’ tumor 1 (WT1) protein [Most recent evidence proposes the utilization of probiotics in the delivery of tumor-associated antigens (TAAs) as an orally administrated vaccine, based on a recently reported prosperous approach with  protein .Bifidobacteria as a delivery vehicle for specific drugs such as cytosine deaminase [Occurrence of hypoxic and neurotic areas among solid cancer tissues gives rise to the opportunity of utilization of a specific tendency of certain probiotic strains for selective localization and proliferation in anaerobic environment –109. Thieaminase  or angioeaminase , 112 or eaminase .The most important limitation of abovementioned strategies lies in the fact that genes for antibiotic resistance, commonly used as selective marker in the procedure of cloning, could be potentially transferred to resident intestinal microbiota by probiotic delivery vectors. Finding an alternative, secure selection marker for cloning in therapeutic strains still remains a challenging area in this field .This paper has given an account of the role played by gut microbiota in cancer prevention and treatment. It is noteworthy that until now most of these innovative methods mentioned above have only been investigated in animal models. Clinical tests of this strategy are expected to raise a possibility of utilizing probiotic bacteria as comprehensive drug-delivery vectors for non-invasive cancer treatment in humans. Taken together, a growing body of literature had highlighted a role of probiotic balance in maintenance of widely understood homeostasis, projecting on successful cancer therapy. The evidence from latest studies points towards the idea of possible implementation of probiotics in cutting-edge cancer therapies. Future investigations on the current topic are therefore necessary in order to validate these findings and establish therapeutic strategies. This could conceivably lead to a breakthrough in various fields of medicine not only supporting immunotherapy in cancer treatment or elaboration and production of an innovative vaccines, but also improving drug delivery in other bowel diseases while preventing and mitigating inflammation at the same time.


Physical inactivity and excessive postprandial hyperglycemia are two major independent risk factors for type 2 diabetes and cardiovascular-related mortality. Current health policy guidelines recommend at least 150 min of physical activity per week coupled with reduced daily sedentary behavior by interrupting prolonged sitting with bouts of light activity every 30-min. This evidence-based strategy promotes health and quality of life. Since modern lifestyle enforces physical inactivity through motorized transportation and seated office working environments, this review examines the practical strategies  for reducing sitting time and increasing activity during the workday. Furthermore, since postprandial hyperglycemia poses the greatest relative risk for developing type 2 diabetes and its cardiovascular complications, this review examines a novel hypothesis that interrupting sitting time would be best focused on the postprandial period in order to optimize blood glucose control and maximize cardiometabolic health. In doing so, we aim to identify the science gaps which urgently need filling if we are to optimize healthcare policy in this critical area. Since Plato wrote, “And is not the bodily habit spoiled by rest and idleness, but preserved for a long time by motion and exercise?” , it is nDiabetes is a major health problem because it causes blindness, kidney failure, and lower limb amputations, and between 1980 and 2014 global prevalence doubled to 8.5%, equivalent to 422 million people . UltimatAlthough physical activity guidelines have long been available, the addition of inactivity guidelines to manage sedentary behavior by reducing sitting time is relatively new. For example, the recommendation to interrupt prolonged sitting was added to the American Diabetes Association's standards of care for diabetes in 2017 . More reFurther studies have examined the effects of simply standing to interrupt prolonged sitting. Epidemiological observations indicate that increased standing to break up sitting is protective against cardiovascular  and all-From the evidence presented above, it is clear that interruption of prolonged sitting with walking or cycling or even just standing up may be effective for improving postprandial blood glucose control, an independent cardiometabolic risk factor. However, as of 2018, no long-term large-scale experimental study has determined the effect of interrupting sitting time on diabetes risk and/or hard endpoints like subsequent cardiovascular complications and mortality.Epidemiological and experimental studies show that the degree to which blood glucose is elevated 1–2-h following a meal is associated with increased cardiovascular disease risk . FurtherThe studies described in the previous section provide evidence that interruption of prolonged sitting with physical activity may improve postprandial blood glucose control. However, such studies did not examine the timing between physical activity and meals and, as of 2018, no long-term randomized, controlled physical activity or exercise intervention study with a primary focus on blood glucose control has reported activity-meal timing. Whether activity/exercise is completed in the fed or fasted state is seldom reported in training studies and as authors of prior studies in this field, we too are guilty. Sometimes this information is not known since activity interventions are self-administered, but activity-meal timing information is even lacking in studies where bouts of activity have been fully-supervised.Postprandial blood glucose levels are determined by several factors, such as the total caloric value of a meal, macronutrient composition, and carbohydrate quality , all of which may be monitored and controlled. However, multiple other factors are more complex because they cannot be controlled and are variable between individuals. These include gastric emptying rate, intestinal absorption rate, enteroendocrine incretin secretion, incretin sensitivity, pancreatic beta-cell insulin secretory function, hepatic insulin extraction, hepatic glucose production, glucose effectiveness, glucose uptake in all tissues , insulin sensitivity, and renal glucose reabsorption. An abundance of studies (far too many to cite) also show that the above factors are also altered by a single bout of exercise and/or changed following a prolonged period of increased physical activity or structured exercise training. That said, large inter-individual variability exists in the hyperglycemia-lowering effect of physical activity in individuals with prediabetes or type 2 diabetes , 43, 44.2max and/or fat oxidation for optimizing athletes' performance in hundreds of publications. A large number of studies have also studied the interactions between exercise-timing, insulin dosing, and carbohydrate intake for managing blood glucose and preventing hypoglycemia in patients with type 1 diabetes. However, there is a relative paucity of data comparing fed vs. fasted physical activity in relation to type 2 diabetes and cardiometabolic risk. One example from 2001 found that in 10 middle-aged men with type 2 diabetes blood glucose levels were significantly lower when 60-min of moderate-intensity bicycle ergometry were completed 2-h after breakfast, rather than before breakfast  is the best time for lowering postprandial hyperglycemia . This sureakfast . While areakfast . Howeverreakfast . That sareakfast . Similarreakfast . Moreovereakfast . In addireakfast . Pre- anRetrospective analyses of patient food and activity logs from a 12-week training study including three supervised 60-min sessions/week of moderate intensity bicycle ergometry, in 19 middle-aged, overweight men with type 2 diabetes, provided insight into the optimal activity-meal timing . Again, The above-described literature addressing activity-meal timing is indeed scant with small sample sizes and dichotomous outcomes Table . Some stRecent data shows that physical inactivity accounts for more CVD-related deaths (37%) than smoking (19%), and hypertension (13%) combined, and that 15–17% of all premature deaths is attributable to low fitness , 60. ThePrevious works by Levine extensively examine the ability of different types of simple physical activities to increase daily energy expenditure above resting levels , 63. NotA number of additional practical approaches to reducing sedentary time exist. Levine's work showed that greater levels of energy expenditure are induced by walk-commuting vs. drive-commuting . This woDelivery drivers and public transportation drivers should also develop strategies to take regular breaks from sitting. Comparisons between seated workers (bus drivers) and active workers  in the 1950s showed increased cardiovascular morbidity in the former , 74, andThere is a clear disconnect between the knowledge of the detriments of inactivity and actual implementation of physical activity at multiple levels. Health care services have a responsibility to formulate and disseminate policy. The media have a responsibility to facilitate this dissemination to the public, with accuracy. The public also have a responsibility to empower themselves to maximize their own health. Despite these responsibilities, overwhelming evidence that scientific knowledge fails to inform public implementation emerges from objective epidemiological assessments of activity levels, where people on average are far from meeting public health activity guidelines , 5. Howeone-size-fits-all approach for interrupting sedentary time.To win the war on physical inactivity it is necessary to understand the health psychology of physical activity in addition to the physiology of the optimal timing between meals and physical activity. Health psychology is critical to create the optimal societal environment in which the physical activity guidelines can be achieved. As such, a huge societal shift in physical environment is required to implement physical activity into daily routines. For instance, incredible architectural foresight in major cities in Scandinavia and the Netherlands has had great impact on daily physical activity by making a commute via bicycle simple, affordable, safe, and enjoyable. In the 1970s, the Danish Government set out to develop the infrastructure needed to increase cycling as a means to reduce traffic accidents, reduce pollution, an improve health. In 2002, this culminated with a long-term “cycling policy” being established in the city of Copenhagen to increase commuting via bicycle . The resBesides the built environment, psychological barriers to energy expenditure also exist. Minimization of energy costs is biologically advantageous and is a strategy that is evolutionarily conserved . For exaTo change behavior, health education informing individuals about the risks of prolonged sitting is key , 90. To It is not a new message that physical activity helps optimize blood glucose control, improve cardiovascular health, and reduce cardiovascular-related mortality, but increased awareness of the dangers of inactivity and refinement of physical activity advice is essential. Experimental evidence demonstrates that interrupting inactivity (sitting time) with physical activity breaks is a useful approach for managing blood glucose levels but recommendations concerning the optimal time to interrupt sitting do not yet exist. Since postprandial hyperglycemia is an independent cardiovascular risk factor and since we spend many hours each day in a postprandial state, timing the interruption of sitting with physical activity to minimize postprandial fluctuations in blood glucose is a sensible approach to maximize cardiometabolic health. However, randomized controlled trials determining the optimal timing between meals and activity are required and studies of this nature must examine hard endpoints like cardiovascular-related mortality. It would also be prudent for such studies to examine other cardiovascular risk factors, such as postprandial lipemia. As scientists acquire such knowledge, health psychology interventions exploring the behavioral and environmental barriers that prevent people interrupting their sedentary time must be developed. This would include consideration of the environmental barriers that influence the practicality of different activities and thus minimize the distraction from work tasks  or hobbies . Furthermore, greater focus is required for increasing employers' awareness of the long-term benefits to their work force by allowing activity breaks and creating a work environment that facilitates and encourages an active workday. Such approaches will help curb the ever-increasing incidence of diabetes and thereby improve cardiovascular health, longevity, and quality of life for the increasingly inactive global population.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Flaviviruses are the most medically relevant group of arboviruses causing a wide range of diseases in humans and are associated with high mortality and morbidity, as such posing a major health concern. Viruses belonging to this family can be endemic , but can also cause fulminant outbreaks . Intense research efforts in the past decades uncovered shared fundamental strategies used by flaviviruses to successfully replicate in their respective hosts. However, the distinct features contributing to the specific host and tissue tropism as well as the pathological outcomes unique to each individual flavivirus are still largely elusive. The profound footprint of individual viruses on their respective hosts can be investigated using novel technologies in the field of proteomics that have rapidly developed over the last decade. An unprecedented sensitivity and throughput of mass spectrometers, combined with the development of new sample preparation and bioinformatics analysis methods, have made the systematic investigation of virus–host interactions possible. Furthermore, the ability to assess dynamic alterations in protein abundances, protein turnover rates and post-translational modifications occurring in infected cells now offer the unique possibility to unravel complex viral perturbations induced in the infected host. In this review, we discuss the most recent contributions of mass spectrometry–based proteomic approaches in flavivirus biology with a special focus on Zika virus, and their basic and translational potential and implications in understanding and characterizing host responses to arboviral infections. Viruses are highly adapted intracellular pathogens that co-evolved with their hosts over millions of years. This resulted in a sophisticated adaptation that allowed viruses to engross cellular machineries for their own replication, with the benefit to encode their genetic information with high efficiency. However, this strict dependency on cellular factors comes with the obligation to replicate intracellularly and the requirement of intimate interactions between viral and cellular proteins. Moreover, most pathogens are preferentially replicating in specific organs and cause virus-associated pathologies. Technological developments in the fields of genomics and proteomics became available in recent years, allowing to systematically map interactions between viruses and their hosts in an unbiased manner. Such interactions are of particular interest especially when uncharacterized pathogens strike in a naïve population.Flaviviridae family, causing the first large unexpected outbreak in Asia and French Polynesia in 2013 [Flaviviridae family like Dengue (DENV), Yellow fever (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), ZIKV is also transmitted by mosquitoes. Furthermore, analogously to JEV, WNV and tick-borne encephalitis virus (TBEV), Zika virus infections are associated with neurological diseases, although ZIKV is the only Flaviviridae member with documented ability to cross the placental barrier and be sexually transmitted [One such pathogen is the Zika virus, a positive stranded RNA virus belonging to the  in 2013 ,5. Follo in 2013 ,7. Howevnsmitted .Technical advancement in mass-spectrometry analysis and the availability of replication competent epitope-tagged viruses and individual viral proteins have prompted several studies to investigate persistent modulation of protein abundances, post-translational modifications and protein–protein interactions on a global scale and with unprecedented sensitivity and quantitation ,11,12,13The recent technological revolution allows acquiring knowledge on biological processes at incredible speed. Several systematic and unbiased genetic approaches to identify host factors involved in flavivirus replication have been conducted to date ,19,20,21Development of sample preparation methodologies, highly sensitive mass analyzers as well as tailored bioinformatics processing pipelines (comprehensively summarized in ) have caFlaviviridae family are considered highly pathogenic infectious agents and knowledge on their interactions is pivotal to understand these pathogens in order to identify new forms of therapy. This is particularly true for epidemic viruses such as Zika virus, which since 2016 has left a devastating impact on large populations in southern America. In an effort to understand the pathogenicity of this virus, a number of laboratories employed systems analysis to elucidate the functions of individual proteins and the virus as a whole.Some members of the Systematic studies are of paramount importance to identify virus–host protein–protein interactions (PPIs), since they provide an unbiased view of molecular complexes and therefore insights into viral mechanisms of replication, host resources exploitation and immune evasion strategies ,12,31,32Among the other novel capsid interactors, additional work by the Ott and the Ramage groups identified the nonsense-mediated decay (NMD) pathway and the exon-junction complex (EJC) as targets of the WNV, DENV and ZIKA virus capsids ,46. WhilIn addition to proteins recruited by several flavivirus capsids, the ZIKV capsid is the only one reported to specifically associate with cellular factors that are involved in neuronal development or neurological disorders . These pAltogether, flavivirus capsids evidently evolved evasive mechanisms through binding/sequestration of ZC3HAV1 and other RNA-binding proteins . Capsid proteins are apparently exploiting their versatile subcellular localization (both nuclear and cytoplasmic) and their intrinsic hydrophobic character to simultaneously highjack and diverge regulation or expression of specific transcripts.Another interesting ZIKV protein that appears to have both conserved and unique cellular interactors across flaviviruses and plays a central role in virus–host adaptation, is the non-structural protein NS4B. Indeed, ZIKV-NS4B associates with subunits of the ATP synthase , voltage-dependent anion selective channel proteins  and calcium-binding mitochondrial proteins , as well as several components of the cytochrome C oxidase complex  c. These NS4B also appeared to associate with cellular proteins that could be associated with the whole spectrum of ZIKV-associated pathogenesis including neurodegenerative disorders and retinal degeneration , neuronal differentiation defects  and axonal dysfunction  . While tIn addition to capsid and NS4B, several novel ZIKV NS5 binders were recently identified in these proteomics surveys. Among these, multiple members of the PAF1C complex , confirming similar observations previously made on DENV-NS5 by the Gamarnik group  and receFurthermore, among the novel cellular targets identified in the ZIKV NS4A interactome is ANKLE2, a protein previously shown to cause autosomal recessive microcephaly in humans . Interesbona fide interactors in diverse cellular backgrounds as a selection criterion to pinpoint targets relevant for in vivo infections. This study identified a strong enrichment of the CCT complex (cytosolic chaperonin-containing T complex), and subunits of the OST (oligosaccharyl transferase) complex such as DDOST, STT3A, STT3B and RPN2, in the NS1 interactome. Previous reports identified some of these proteins as host-dependency or druggable targets in genetic [An important aspect is that AP-LC-MS/MS often identifies protein complexes rather than binary protein–protein interactions. This is also illustrated in an interactome study of DENV-NS1  in the c genetic ,56 and d genetic , and the genetic ,40,41,42 genetic ). Likewi genetic ) and NS4 genetic ). ProteoIn addition to PPI, flaviviruses were also shown to exploit their own genomic RNA (gRNA) or subgenomic derivatives, such as the subgenomic flavivirus RNA (sfRNA), to “sponge” critical RNA-binding host proteins out of the cellular pool, or to specifically inhibit or redirect their homeostatic regulatory functions ,62,63. IOnly one study to date reported the systematic analysis of ZIKV and DENV gRNA-associated proteins, using the ChIRP-MS (comprehensive identification of RNA-binding proteins by mass spectrometry) method. In this method, target RNA and associated proteins are retrieved after formaldehyde-based cross-linking using hybridization with antisense biotinylated oligos and streptavidin-conjugated beads, and protein complexes eluted via incubation with biotin . This apSeveral studies systematically investigated the impact of flavivirus infections on protein abundance on a global scale ,69. GarcA similar unbiased study investigating the proteome of ZIKV-infected iPSC-derived human neuronal progenitors, confirmed specific downregulation of neuronal factors upon ZIKV infection, as well as activation of general antiviral response pathways, such as upregulation of type-I interferon stimulated genes  . InteresRecently, additional reports investigated further the impact of ZIKV infection on human mesenchymal stem cells using the MudPIT method . This identified a profound downregulation of PI3K/AKT and mTOR, as well as dysregulation of several components of the phosphatidylinositol pathway . AlternaA unifying theme crystallizing from proteomic and transcriptomic analysis of ZIKA virus infected neuronal cells is that ZIKV infection has a profound effect on expression of proteins involved in neurogenesis or on proteins that are markers of neuronal differentiation. At this stage it is not clear whether this effect is limited to ZIKA virus or whether other viruses elicit similar effects in a neuronal background. Along these lines would be important to test whether neuropathic flaviviruses share certain binding patterns that may be etiologically linked to their pathology. Similarly, it would be of great interest to investigate the differential proteomes of diverse flaviviruses from primary biopsies of infected patients.Initial studies by the Cao group focused on the global protein phosphorylation status in WNV- and JEV-infected cells ,75. In tIn the case of ZIKV, only one study so far reported a time-resolved phosphoproteomic analysis of ZIKV-infected cells . SimilarFurthermore, a profound upregulation of the ATM (ataxia-telangiectasia mutated)/ATR (ATM- and Rad3-related) DNA-damage pathway, was observed at multiple levels  including a number of proteins involved in cell-cycle regulation and DNA damage checkpoint . Altogether, these effects provide possible explanations for the observed ZIKV-induced cell-cycle arrest and apoptosis  and p53 Another important PTM is ubiquitination, which becomes more and more accessible to proteomics analysis and has recently been studied in the context of flavivirus infections . For insCollectively, the analysis of PTMs in flavivirus-infected cells starts to unravel unappreciated cellular activities. Development of techniques to study PTMs as well as development in bioinformatic tools allowing better interpretation of PTM activities, hold promise to dramatically accelerate our understanding of virus–host interactions and to appreciate additional important pathways engaged during virus infections.Large-scale approaches allowing the accurate assessment of virus–host interactions have revolutionized the way we investigate and deconvolute the complex cellular changes triggered by virus infections. While central linear pathways were the main research focus in the past, it becomes more evident that innate immune signaling, and cellular responses in general, engage many interconnected pathways in parallel. The knowledge of these pathways is paramount to holistically understand how viral pathogens interact with their hosts. Certainly more work on viral–host interactions, protein turnover as well as post-translational modifications is required to further expand our understanding of the intracellular landscape of flavivirus-infected cells. This requires well-controlled comparative studies in relevant cell culture systems of multiple members of this highly diverse arbovirus family. Similarly, rigorous validation methods by alternative approaches , robust and transparent cut-off criteria, and functional assays on selected candidates are increasingly needed to pinpoint physiologically relevant host factors. In this respect, further efforts are required to integrate and standardize data generated by mass spectrometry based surveys in easy-to-mine databases, ideally providing critical annotations on virus isolates and cell culture systems as well as integration with other -omics data . Integra


Overestimators also scored significantly lower on the communication (p = .012) and responsibility (p = .021) LSP subscales compared to underestimators, and the total LSP score for overestimators was significantly lower compared to accurate raters (p = .036) and underestimators (p = .009). However, underestimators displayed significantly higher confusion on the titular POMS subscale than did overestimators (p = .021). These findings indicate that, among the three groups, overestimators scored lowest for objectively rated functioning. In contrast, underestimators attained the highest functioning; however, they were also confused. Clinical staff should examine how patients' self-perceptions deviate from the perceptions of staff and implement an appropriate approach considering the patient characteristics revealed from the results of this study.In Japan, long-term hospitalization of patients with schizophrenia is still prevalent, even though the focus of psychiatric care is shifting from hospitals to the community. Difficulties in discharge planning often arise because clinical staff members' functional assessment differs from that of patients' self-assessment. Therefore, we attempted to identify characteristics related to these perceptual differences to promote the development of more effective approaches toward the discharge and societal reintegration of patients with schizophrenia undergoing prolonged hospitalization. Forty-eight long-term inpatients (23 men and 25 women with a mean age of 60.72 years) with schizophrenia were examined using the Schizophrenia Cognition Rating Scale Japanese version (SCoRS-J), Life Skills Profile (LSP), and Profile of Mood States- (POMS-) Brief Form. Differences between patients' self-ratings and clinical staff members' ratings on the SCoRS-J were used to divide patients into overestimators, underestimators, and accurate raters. These groups were then comparatively analyzed. Accordingly, overestimators displayed significantly severe cognitive dysfunction on the SCoRS-J objective ratings ( Psychiatric care in Japan has long been centered around hospitalization . ConsequIn 2004, the Japanese government formulated a vision for reforming mental health care and welfare based on the concept of moving “from a hospital-centric to a community-centric” model of care and began promoting rehabilitation interventions and community transition and integration from an early stage . HoweverA wide range of factors make it difficult to plan the discharge of long-term inpatients with schizophrenia, including patients' personal issues, family problems, hospital environment, and community environment such as insufficient community-level assistance . Poor inOverconfidence resulting from unawareness of illness and poor insight or, conversely, low confidence even though objectively rated to have adequate capability suggest a divergence between patients' self-ratings and objective ratings by others concerning functional capacity or performance. This circumstance is encountered frequently in clinical settings and has been documented in many studies –20. ThisFurthermore, it has been reported that misestimation of ability is the strongest predictive factor for real-world functioning . Harvey Incidentally, this divergence in perception can be divided into two patterns: one where self-ratings are lower and the other where self-ratings are higher than the ratings by others. Differences in the divergence patterns and the presence or absence of divergence may be influenced by patients' characteristics. Bowie and colleagues  investigConsistently, several other studies examined outpatients , 19; howPatients who met the following criteria from six psychiatric hospitals in A Prefecture were included in the present study: (1) aged ≥ 20 years, (2) received a diagnosis of schizophrenia, (3) been hospitalized for ≥1 year, (4) presented with stable psychiatric symptoms (no restlessness) as confirmed by an attending physician, and (5) able to communicate verbally and consented to participate. Patients were excluded if they were judged by the attending physician as unable to understand the purpose of the study, questionnaire contents, or instructions owing to significant unawareness of their illness or reduced intellectual function.In addition, for each patient, members of the attending clinical staff  who best understood the patient's daily life were recruited as informants. Informant selection was left to the hospital staff.To assess cognitive functioning, everyday life skills, and mood states, several measures were administered to patients fulfilling the criteria stated above and to their attending clinical staff. All patients and informants were interviewed by the first author using the Japanese version of the Schizophrenia Cognition Rating Scale (SCoRS-J)  to evaluDeveloped by Keefe and colleagues , the SCoIn the present study, the difference between total objective and subjective values on the SCoRS-J (objective severity-subjective difficulty) was designated as the perceptual discrepancy score for disability in cognitive functioning. Perceptual discrepancy scores approaching 0 imply that patient and informant ratings are consistent, and patients can accurately self-assess their own cognitive functioning. Greater negative scores represent less self-estimation and suggest that patients feel more difficulties than the clinical staff members' ratings reveal, while greater positive scores represent more excessive self-estimation and suggest that the patient does not feel as impaired as implied by staff members' objective severity.Developed by Rosen and colleagues , the LSPDeveloped by McNair and colleagues , 41, theAfter calculating basic statistical values from collected data, the Wilcoxon signed-rank test was applied to compare total SCoRS-J scores for objective severity and subjective difficulty and identify disparities in patients' and clinical staff members' ratings of disabilities in cognitive functioning. In addition, participants were divided into three groups based on perceptual discrepancy scores quantified by the differential between objective severity and subjective difficulty values on the SCoRS-J. Those with scores within ±0.5 standard deviations (SDs) of objective severity were designated as accurate raters, those with scores diverging from 0.5 SD of objective severity in the positive direction were designated as overestimators, and those with scores diverging greatly from 0.5 SD of objective severity in the negative direction were designated as underestimators. A one-way analysis of variance or the Kruskal-Wallis test was applied to detect differences among the three groups in each measured item. Items showing significant differences were then subject to multiple comparison analyses. Furthermore, a chi-squared test was used to examine differences in sex, marital status, and general work experience among the three groups. Statistical analyses were conducted with IBM SPSS Statistics for Windows, version 24 , and the two-tailed level of significance was set at 5%.The present study was conducted with approval from the Ethical Review Board of the Graduate School of Biomedical and Health Sciences, Hiroshima University.The first author provided all participants with individual explanations of the purpose, method, and ethical considerations of this study. Written consent to participate in this study was obtained from the participants themselves.Forty-eight patients  participated.p = .046). Mean scores and SDs for perceptual discrepancy  were −4.0 and 13.7, respectively. The least perceptual discrepancy was ±1. The largest positive perceptual discrepancy where objective severity surpassed subjective difficulty was +39, while the largest negative perceptual discrepancy where objective severity fell below subjective difficulty was −35.Regarding SCoRS-J results, the Wilcoxon signed-rank test detected a significant disparity between clinical staff members' ratings of objective severity and patients' self-ratings of subjective difficulty (The number of accurate raters with subjective difficulty within ±0.5 SD of objective difficulty was 14, while the number of overestimators and underestimators was 11 and 23, respectively. Regarding performance on instrument items, significant differences among the three groups were detected for the POMS subscale of confusion, the LSP subscales of communication and responsibility, and total LSP score. Multiple comparisons revealed that underestimators scored significantly lower than did overestimators on the individual items and that total LSP scores for overestimators were also significantly lower than those of either accurate raters or underestimators .When comparing the three groups regarding the differential between objective severity and subjective difficulty values on the SCoRS-J, objective severity ratings for underestimators were lower than those for either accurate raters or overestimators. In other words, although underestimators were objectively seen to experience disabilities in cognitive functioning to a lesser degree, their own ratings of subjective difficulty were much higher and significantly different from those of overestimators .No significant differences were found among the three groups regarding sex, work experience, or marital history.The current results corroborate the findings of prior research that patients' self-ratings and clinical staff members' objective ratings of cognitive functioning diverge in many cases of schizophrenia. However, although previous studies encountered high rates of overestimators , 17, 19,These patients tended to be older and more educated and had the highest onset age compared to the other two groups, albeit not to a level of statistical significance. They also showed the lowest frequency of hospitalization. However, their chlorpromazine equivalent dosage and scores on the tension-anxiety subscale of the POMS were greater than were those of the other two groups.Compared to the other groups, they also achieved the best performance on the nonturbulence and social contact subscales of the LSP, suggesting that they were highly sociable. Such sociability may have been enhanced by their older age and longer years of education. This outcome supports the observations of previous studies by Bowie and colleagues  that accFurther, their psychiatric symptoms were not inconsequential, as evidenced by their higher dosage of chlorpromazine equivalents; however, their symptom profile was believed to be unrelated to their competence in self-assessment, and they could maintain their social skills despite their condition. In contrast to their high social functioning, accurate raters experienced greater tension and anxiety, which insinuates that they feel stressed when they act socially or attempt to do so even though they experience psychiatric symptoms.Although rates for hospitalization frequency and duration were lower compared to the other two groups, the present results alone cannot resolve the question of whether high social functioning mitigates hospitalization or vice versa. That said, it may be possible that patients who are adept at properly perceiving themselves are more likely to be discharged because they can cultivate their own personal motivations and goals.This group included patients who reported difficulties in cognitive functioning due to cognitive disability to a greater degree than rated by clinical staff. Among the three groups, underestimators achieved the lowest objective ratings of disability in cognitive functioning and displayed high everyday living skills. They were evaluated the best on the self-care, communication, and responsibility LSP subscales, gaining significantly better results than did overestimators on the latter two subscales.The communication subscale of the LSP measures verbal and nonverbal exchanges through factors such as starting or responding to conversation, refraining from intrusion into others' conversation, making eye contact, being difficult to understand because of disordered speech, talking about odd or strange ideas, and making expressions and gestures appropriate to the atmosphere. The responsibility subscale measures treatment compliance in general through factors such as being able to manage medication usage, proactively taking prescribed medications, and cooperating with health services by clinical staff. In other words, underestimators seem to have superior life skills particularly because they can take care of themselves and readily engage in daily conversations and because they are cooperative in treatment and diligently obey clinical staff members' instructions concerning proper use of medication and other matters. The present results suggest that although staff members may view such patients as capable individuals who merely lack confidence or motivation, it is possible that underestimators experience difficulties in everyday functioning due to cognitive disability to a greater degree than presumed by clinical staff.Bowie and colleagues  reportedOverestimators are unconcerned with their issues although they are objectively seen to have many problems. Such patients are regarded by clinical staff to be typical cases of poor reality testing. Among the three groups, overestimators exhibited the most severe disability in cognitive functioning and the lowest level of everyday living skills. Nevertheless, they were unaffected by mental confusion and led serene lives because they were unperturbed by the fact that they were objectively unable to accomplish many everyday tasks.It is believed that self-awareness among overestimators was further reduced because, in addition to being affected by poor reality testing, they had become accustomed to long-term hospitalization and were living an existence where they did not have to face any daily inconveniences or difficulties. It is necessary to adopt an approach that provides such patients with opportunities to gradually acquire various experiences at a reasonable pace so that they can improve their reality testing through practical engagement. However, as illustrated by Sasaki and Yamada , long-teFurthermore, factors that make it difficult to discharge long-term inpatients include not only personal factors, such as cognitive functioning and everyday living skills, but also social factors, such as family problems and inadequate community support. Therefore, it is essential to consider these social factors in addition to patients' personal issues when assisting long-term inpatients with discharge. Although the present study did not investigate in detail the social factors that resulted in long-term hospitalization of individual patients or those of patients without long-term hospitalization, in order to assist discharge more effectively for long-term hospitalized patients, it will be necessary to investigate their individual social factors and to take concrete measures to address them.The present study had several limitations. First, regarding determining cognitive functioning, we relied solely on the SCoRS-J, an interview-based coprimary measure, and no neuropsychological or performance-based tests were used. Such tests were excluded considering the burden they would place on the sizable proportion of elderly long-term inpatients who were examined. Since clinician assessments with the SCoRS-J have been confirmed to correlate with total scores on the Japanese version of the BACS, a primary rating scale of cognitive function, objective severity values generated by clinical staff are believed to reflect patients' degree of cognitive disability. However, for greater specificity, future research should consider employing objective tests and measurements that deliver a more detailed picture of cognitive levels and profiles.Second, psychiatric symptoms and intellectual functioning could have influenced perceptual discrepancy. We included patients whose psychiatric symptoms were determined to be stable by a physician and excluded those with significant unawareness of illness and reduced intellectual capacity. However, it may be possible that diverse psychiatric symptoms, unawareness of illness, and intellectual capacity—even though they met the eligibility criteria—influenced perceptual discrepancies in some manner. Since we did not conduct assessments with instruments for psychiatric symptoms, administer intellectual tests, or collect data on unawareness of illness, the nature and degree of such influences cannot be identified. Moving forward, research should consider these issues when examining factors related to perceptual discrepancy in more detail.Third, since the sample size of this study was small for comparing three groups, in future studies, it will be necessary to analyze a larger sample size.The present study revealed that approximately 70% of participants had a divergence between patients' self-ratings and clinical staff members' objective ratings of cognitive functioning. Two-thirds of the participants who had a divergence were underestimators, and one-third were overestimators. We detected a higher rate of underestimators, which contrasted with previous studies on outpatients.Furthermore, the psychological factor determining the characteristics of the group with different divergence patterns between self and objective perception was confusion rather than depression. This is another new finding different from previous study results. In order to provide more effective support for discharge, it is imperative that clinical staff and patients share rehabilitation goals. To that end, clinical staffs need to examine how their perceptions differ from patients' self-perceptions. Furthermore, better support should be provided by adopting approaches that recognize the key characteristic differences identified in the present study.