Lack of evidence for predictive utility from resting state fMRI data for individual exposure-based cognitive behavioral therapy outcomes: A machine learning study in two large multi-site samples in anxiety disorders.

Data-based predictions of individual Cognitive Behavioral Therapy (CBT) treatment response are a fundamental step towards precision medicine. Past studies demonstrated only moderate prediction accuracy (i.e. ability to discriminate between responders and non-responders of a given treatment) when using clinical routine data such as demographic and questionnaire data, while neuroimaging data achieved superior prediction accuracy. However, these studies may be considerably biased due to very limited sample sizes and bias-prone methodology. Adequately powered and cross-validated samples are a prerequisite to evaluate predictive performance and to identify the most promising predictors. We therefore analyzed resting state functional magnet resonance imaging (rs-fMRI) data from two large clinical trials to test whether functional neuroimaging data continues to provide good prediction accuracy in much larger samples. Data came from two distinct German multicenter studies on exposure-based CBT for anxiety disorders, the Protect-AD and SpiderVR studies. We separately and independently preprocessed baseline rs-fMRI data from n = 220 patients (Protect-AD) and n = 190 patients (SpiderVR) and extracted a variety of features, including ROI-to-ROI and edge-functional connectivity, sliding-windows, and graph measures. Including these features in sophisticated machine learning pipelines, we found that predictions of individual outcomes never significantly differed from chance level, even when conducting a range of exploratory post-hoc analyses. Moreover, resting state data never provided prediction accuracy beyond the sociodemographic and clinical data. The analyses were independent of each other in terms of selecting methods to process resting state data for prediction input as well as in the used parameters of the machine learning pipelines, corroborating the external validity of the results. These similar findings in two independent studies, analyzed separately, urge caution regarding the interpretation of promising prediction results based on neuroimaging data from small samples and emphasizes that some of the prediction accuracies from previous studies may result from overestimation due to homogeneous data and weak cross-validation schemes. The promise of resting-state neuroimaging data to play an important role in the prediction of CBT treatment outcomes in patients with anxiety disorders remains yet to be delivered.

Behavioural and functional evidence revealing the role of RBFOX1 variation in multiple psychiatric disorders and traits.

Common variation in the gene encoding the neuron-specific RNA splicing factor RNA Binding Fox-1 Homolog 1 (RBFOX1) has been identified as a risk factor for several psychiatric conditions, and rare genetic variants have been found causal for autism spectrum disorder (ASD). Here, we explored the genetic landscape of RBFOX1 more deeply, integrating evidence from existing and new human studies as well as studies in Rbfox1 knockout mice. Mining existing data from large-scale studies of human common genetic variants, we confirmed gene-based and genome-wide association of RBFOX1 with risk tolerance, major depressive disorder and schizophrenia. Data on six mental disorders revealed copy number losses and gains to be more frequent in ASD cases than in controls. Consistently, RBFOX1 expression appeared decreased in post-mortem frontal and temporal cortices of individuals with ASD and prefrontal cortex of individuals with schizophrenia. Brain-functional MRI studies demonstrated that carriers of a common RBFOX1 variant, rs6500744, displayed increased neural reactivity to emotional stimuli, reduced prefrontal processing during cognitive control, and enhanced fear expression after fear conditioning, going along with increased avoidance behaviour. Investigating Rbfox1 neuron-specific knockout mice allowed us to further specify the role of this gene in behaviour. The model was characterised by pronounced hyperactivity, stereotyped behaviour, impairments in fear acquisition and extinction, reduced social interest, and lack of aggression; it provides excellent construct and face validity as an animal model of ASD. In conclusion, convergent translational evidence shows that common variants in RBFOX1 are associated with a broad spectrum of psychiatric traits and disorders, while rare genetic variation seems to expose to early-onset neurodevelopmental psychiatric disorders with and without developmental delay like ASD, in particular. Studying the pleiotropic nature of RBFOX1 can profoundly enhance our understanding of mental disorder vulnerability.

Establishment of Emotional Memories Is Mediated by Vagal Nerve Activation: Evidence from Noninvasive taVNS.

Emotional memories are better remembered than neutral ones, but the mechanisms leading to this memory bias are not well understood in humans yet. Based on animal research, it is suggested that the memory-enhancing effect of emotion is based on central noradrenergic release, which is triggered by afferent vagal nerve activation. To test the causal link between vagus nerve activation and emotional memory in humans, we applied continuous noninvasive transcutaneous auricular vagus nerve stimulation (taVNS) during exposure to emotional arousing and neutral scenes and tested subsequent, long-term recognition memory after 1 week. We found that taVNS, compared with sham, increased recollection-based memory performance for emotional, but not neutral, material. These findings were complemented by larger recollection-related brain potentials (parietal ERP Old/New effect) during retrieval of emotional scenes encoded under taVNS, compared with sham. Furthermore, brain potentials recorded during encoding also revealed that taVNS facilitated early attentional discrimination between emotional and neutral scenes. Extending animal research, our behavioral and neural findings confirm a modulatory influence of the vagus nerve in emotional memory formation in humans.SIGNIFICANCE STATEMENT Emotionally relevant information elicits stronger and more enduring memories than nonrelevant information. Animal research has shown that this memory-enhancing effect of emotion is related to the noradrenergic activation in the brain, which is triggered by afferent fibers of the vagus nerve (VN). In the current study, we show that noninvasive transcutaneous auricular VN stimulation enhances recollection-based memory formation specifically for emotionally relevant information as indicated by behavioral and electrophysiological indices. These human findings give novel insights into the mechanisms underlying the establishment of emotional episodic memories by confirming the causal link between the VN and memory formation which may help understand the neural mechanisms underlying disorders associated with altered memory functions and develop treatment options.

Neural adaptation of cingulate and insular activity during delayed fear extinction: A replicable pattern across assessment sites and repeated measurements.

Adapting threat-related memories towards changing environments is a fundamental ability of organisms. One central process of fear reduction is suggested to be extinction learning, experimentally modeled by extinction training that is repeated exposure to a previously conditioned stimulus (CS) without providing the expected negative consequence (unconditioned stimulus, US). Although extinction training is well investigated, evidence regarding process-related changes in neural activation over time is still missing. Using optimized delayed extinction training in a multicentric trial we tested whether: 1) extinction training elicited decreasing CS-specific neural activation and subjective ratings, 2) extinguished conditioned fear would return after presentation of the US (reinstatement), and 3) results are comparable across different assessment sites and repeated measures. We included 100 healthy subjects (measured twice, 13-week-interval) from six sites. 24 h after fear acquisition training, extinction training, including a reinstatement test, was applied during fMRI. Alongside, participants had to rate subjective US-expectancy, arousal and valence. In the course of the extinction training, we found decreasing neural activation in the insula and cingulate cortex as well as decreasing US-expectancy, arousal and negative valence towards CS+. Re-exposure to the US after extinction training was associated with a temporary increase in neural activation in the anterior cingulate cortex (exploratory analysis) and changes in US-expectancy and arousal ratings. While ICCs-values were low, findings from small groups suggest highly consistent effects across time-points and sites. Therefore, this delayed extinction fMRI-paradigm provides a solid basis for the investigation of differences in neural fear-related mechanisms as a function of anxiety-pathology and exposure-based treatment.

Does prior traumatization affect the treatment outcome of CBT for panic disorder? The potential role of the MAOA gene and depression symptoms.

Although cognitive behavioral therapy (CBT) is highly effective in the treatment of anxiety disorders, many patients still do not benefit. This study investigates whether a history of traumatic event experience is negatively associated with outcomes of CBT for panic disorder. The moderating role of the monoamine oxidase A (MAOA) gene and depression symptoms as well as the association between trauma history and fear reactivity as a potential mechanism are further analyzed. We conducted a post-hoc analysis of 172 male and 60 female patients with panic disorder treated with CBT in a multi-center study. Treatment outcome was assessed at post-treatment using self-report and clinician rating scales. Fear reactivity before treatment was assessed via heart rate and self-reported anxiety during a behavioral avoidance test. Among females, we did not find any differences in treatment response between traumatized and non-traumatized individuals or any two-way interaction trauma history × MAOA genotype. There was a significant three-way interaction trauma history × MAOA genotype × depression symptoms on all treatment outcomes indicating that in traumatized female patients carrying the low-activity allele, treatment effect sizes decreased with increasing depression symptoms at baseline. No such effects were observed for males. In conclusion, we found no evidence for a differential treatment response in traumatized and non-traumatized individuals. There is preliminary evidence for poorer treatment outcomes in a subgroup of female traumatized individuals carrying the low-active variant of the MAOA gene. These patients also report more symptoms of depression symptomatology and exhibit a dampened fear response before treatment which warrants further investigation.

Active avoidance and attentive freezing in the face of approaching threat.

Defensive behaviors in animals and humans vary dynamically with increasing proximity of a threat and depending upon the behavioral repertoire at hand. The current study investigated physiological and behavioral adjustments and associated brain activation when participants were exposed to dynamically approaching threat that was either inevitable or could be avoided by motor action. When the approaching threat was inevitable, attentive freezing was observed as indicated by fear bradycardia, startle potentiation, and a dynamic increase in activation of the anterior insula and the periaqueductal grey. In preparation for active avoidance a switch in defensive behavior was observed characterized by startle inhibition and heart rate acceleration along with potentiated activation of the amygdala and the periaqueductal grey. Importantly, the modulation of defensive behavior according to threat imminence and the behavioral option at hand was associated with activity changes in the ventromedial prefrontal cortex. These findings improve our understanding of brain mechanisms guiding human behavior during approaching threat depending on available resources.

Acquisition and inhibition of conditioned fear is modulated by individual stimulus fear-relevance.

Inhibitory learning is an important factor for decreasing fear expression. We investigated conditioned inhibition of learned fear responses using conditioned excitors and inhibitors differing in fear-relevance in a sample of 48 healthy female students. To study the effect of stimulus fear-relevance, we used the fear potentiated startle paradigm in an AX+/BX- discrimination learning task with fear-relevant (spider) vs. fear-irrelevant (butterfly) pictures as CS+ (A) and CS- (B), respectively. We found that, during acquisition, participants with elevated fear of spiders showed stronger fear potentiated startle to AX+ compared to BX- when the inhibitor (B) was fear-irrelevant (butterfly) using both median split as well as correlational analyses. In contrast, when the excitor (A) was fear-irrelevant (butterfly), fear potentiated startle to AX+ compared to BX- was reduced for participants with higher fear of spiders. Effects of conditioned inhibition were studied in a summation test, where excitor and inhibitor were presented in compound (AB) and compared to the last four excitor trials during prior acquisition. Conditioned inhibition was stronger for participants with a higher fear of spiders, when the butterfly acted as conditioned inhibitor (B). On the other hand, when the spider served as conditioned inhibitor, effects of conditioned inhibition were weaker for participants with higher fear of spiders. Hence, rather than to a general preparedness our data point to a specific impairment in safety learning for individually fear-relevant stimuli.

When neutral turns significant: brain dynamics of rapidly formed associations between neutral stimuli and emotional contexts.

The ability to associate neutral stimuli with motivationally relevant outcomes is an important survival strategy. In this study, we used event-related potentials (ERPs) to investigate brain dynamics of associative emotional learning when participants were confronted with multiple heterogeneous information. Participants viewed 144 different objects in the context of 144 different emotional and neutral background scenes. During each trial, neutral objects were shown in isolation and then paired with the background scene. All pairings were presented twice to compare ERPs in response to neutral objects before and after single association. After single pairing, neutral objects previously encoded in the context of emotional scenes evoked a larger P100 over occipital electrodes compared to objects that were previously paired with neutral scenes. Likewise, larger late positive potentials (LPPs) were observed over parieto-occipital electrodes (450-750 ms) for objects previously associated with emotional relative to neutral contexts. The LPP - but not P100 - enhancement was also related to subjective object/context binding. Taken together, our ERP data provide evidence for fast emotional associative learning, as reflected by heightened perceptual and sustained elaborative processing for neutral information previously encountered in emotional contexts. These findings could assist in understanding binding mechanisms in stress and anxiety, as well as in addiction and eating-related disorders.

Binding neutral information to emotional contexts: Brain dynamics of long-term recognition memory.

There is abundant evidence in memory research that emotional stimuli are better remembered than neutral stimuli. However, effects of an emotionally charged context on memory for associated neutral elements is also important, particularly in trauma and stress-related disorders, where strong memories are often activated by neutral cues due to their emotional associations. In the present study, we used event-related potentials (ERPs) to investigate long-term recognition memory (1-week delay) for neutral objects that had been paired with emotionally arousing or neutral scenes during encoding. Context effects were clearly evident in the ERPs: An early frontal ERP old/new difference (300-500 ms) was enhanced for objects encoded in unpleasant compared to pleasant and neutral contexts; and a late central-parietal old/new difference (400-700 ms) was observed for objects paired with both pleasant and unpleasant contexts but not for items paired with neutral backgrounds. Interestingly, objects encoded in emotional contexts (and novel objects) also prompted an enhanced frontal early (180-220 ms) positivity compared to objects paired with neutral scenes indicating early perceptual significance. The present data suggest that emotional--particularly unpleasant--backgrounds strengthen memory for items encountered within these contexts and engage automatic and explicit recognition processes. These results could help in understanding binding mechanisms involved in the activation of trauma-related memories by neutral cues.

New learning following reactivation in the human brain: targeting emotional memories through rapid serial visual presentation.

Once reactivated, previously consolidated memories destabilize and have to be reconsolidated to persist, a process that might be altered non-invasively by interfering learning immediately after reactivation. Here, we investigated the influence of interference on brain correlates of reactivated episodic memories for emotional and neutral scenes using event-related potentials (ERPs). To selectively target emotional memories we applied a new reactivation method: rapid serial visual presentation (RSVP). RSVP leads to enhanced implicit processing (pop out) of the most salient memories making them vulnerable to disruption. In line, interference after reactivation of previously encoded pictures disrupted recollection particularly for emotional events. Furthermore, memory impairments were reflected in a reduced centro-parietal ERP old/new difference during retrieval of emotional pictures. These results provide neural evidence that emotional episodic memories in humans can be selectively altered through behavioral interference after reactivation, a finding with further clinical implications for the treatment of anxiety disorders.

When Threat Is Near, Get Out of Here: Dynamics of Defensive Behavior During Freezing and Active Avoidance.

When detecting a threat, humans and other animals engage in defensive behaviors and supporting physiological adjustments that vary with threat imminence and potential response options. In the present study, we shed light on the dynamics of defensive behaviors and associated physiological adjustments in humans using multiple psychophysiological and brain measures. When participants were exposed to a dynamically approaching, uncontrollable threat, attentive freezing was augmented, as indicated by an increase in skin conductance, fear bradycardia, and potentiation of the startle reflex. In contrast, when participants had the opportunity to actively avoid the approaching threat, attention switched to response preparation, as indicated by an inhibition of the startle magnitude and by a sharp drop of the probe-elicited P3 component of the evoked brain potentials. These new findings on the dynamics of defensive behaviors form an important intersection between animal and human research and have important implications for understanding fear and anxiety-related disorders.

Updated meta-analysis of classical fear conditioning in the anxiety disorders.

The aim of the current study was twofold: (1) to systematically examine differences in fear conditioning between anxiety patients and healthy controls using meta-analytic methods, and (2) to examine the extent to which study characteristics may account for the variability in findings across studies. Forty-four studies (published between 1920 and 2013) with data on 963 anxiety disordered patients and 1,222 control subjects were obtained through PubMed and PsycINFO, as well as from a previous meta-analysis on fear conditioning (Lissek et al.). Results demonstrated robustly increased fear responses to conditioned safety cues (CS-) in anxiety patients compared to controls during acquisition. This effect may represent an impaired ability to inhibit fear in the presence of safety cues (CS-) and/or may signify an increased tendency in anxiety disordered patients to generalize fear responses to safe stimuli resembling the conditioned danger cue (CS+). In contrast, during extinction, patients show stronger fear responses to the CS+ and a trend toward increased discrimination learning (differentiation between the CS+ and CS-) compared to controls, indicating delayed and/or reduced extinction of fear in anxiety patients. Finally, none of the included study characteristics, such as the type of fear measure (subjective vs. psychophysiological index of fear), could account significantly for the variance in effect sizes across studies. Further research is needed to investigate the predictive value of fear extinction on treatment outcome, as extinction processes are thought to underlie the beneficial effects of exposure treatment in anxiety disorders.

Encoding and reinstatement of threat: recognition potentials.

On a recognition test, stimuli originally encoded in the context of shock threat show an enhanced late parietal positivity during later recognition compared to stimuli encoded during safety, particularly for emotionally arousing stimuli. The present study investigated whether this ERP old/new effect is further influenced when a threat context is reinstated during the recognition test. ERPs were measured in a yes-no recognition test for words rated high or low in emotional arousal that were encoded and recognized in the context of cues that signaled threat of shock or safety. Correct recognition of words encoded under threat, irrespective of reinstatement, was associated with an enhanced old-new ERP difference (500-700ms; centro-parietal), and this difference was only reliable for emotionally arousing words. Taken together, the data suggest that information processed in a stressful context are associated with better recollection on later recognition, an effect that was not modulated by reinstating the stressful context at retrieval.

Exposure traced in daily life: improvements in ecologically assessed social and physical activity following exposure-based psychotherapy for anxiety disorders.

BACKGROUND: Although exposure-based cognitive-behavioral therapy for anxiety disorders has frequently been proven effective, only few studies examined whether it improves everyday behavioral outcomes such as social and physical activity. METHODS: 126 participants (85 patients with panic disorder, agoraphobia, social anxiety disorder, or specific phobias, and 41 controls without mental disorders) completed smartphone-based ambulatory ratings (activities, social interactions, mood, physical symptoms) and motion sensor-based indices of physical activity (steps, time spent moving, metabolic activity) at baseline, during, and after exposure-based treatment. RESULTS: Prior to treatment, patients showed reduced mood and physical activity relative to healthy controls. Over the course of therapy, mood ratings, interactions with strangers and indices of physical activity improved, while reported physical symptoms decreased. Overall results did not differ between patients with primary panic disorder/agoraphobia and social anxiety disorder. Higher depression scores at baseline were associated with larger changes in reported symptoms and mood ratings, but smaller changes in physical activity CONCLUSIONS: Exposure-based treatment initiates increased physical activity, more frequent interaction with strangers, and improvements in everyday mood. The current approach provides objective and fine-graded process and outcome measures that may help to further improve treatments and possibly reduce relapse.

Cortical and Subcortical Brain Alterations in Specific Phobia and Its Animal and Blood-Injection-Injury Subtypes: A Mega-Analysis From the ENIGMA Anxiety Working Group.

OBJECTIVE: Specific phobia is a common anxiety disorder, but the literature on associated brain structure alterations exhibits substantial gaps. The ENIGMA Anxiety Working Group examined brain structure differences between individuals with specific phobias and healthy control subjects as well as between the animal and blood-injection-injury (BII) subtypes of specific phobia. Additionally, the authors investigated associations of brain structure with symptom severity and age (youths vs. adults). METHODS: Data sets from 31 original studies were combined to create a final sample with 1,452 participants with phobia and 2,991 healthy participants (62.7% female; ages 5-90). Imaging processing and quality control were performed using established ENIGMA protocols. Subcortical volumes as well as cortical surface area and thickness were examined in a preregistered analysis. RESULTS: Compared with the healthy control group, the phobia group showed mostly smaller subcortical volumes, mixed surface differences, and larger cortical thickness across a substantial number of regions. The phobia subgroups also showed differences, including, as hypothesized, larger medial orbitofrontal cortex thickness in BII phobia (N=182) compared with animal phobia (N=739). All findings were driven by adult participants; no significant results were observed in children and adolescents. CONCLUSIONS: Brain alterations associated with specific phobia exceeded those of other anxiety disorders in comparable analyses in extent and effect size and were not limited to reductions in brain structure. Moreover, phenomenological differences between phobia subgroups were reflected in diverging neural underpinnings, including brain areas related to fear processing and higher cognitive processes. The findings implicate brain structure alterations in specific phobia, although subcortical alterations in particular may also relate to broader internalizing psychopathology.

Specificity of homework compliance effects on treatment outcome in CBT: evidence from a controlled trial on panic disorder and agoraphobia.

OBJECTIVES: Although homework assignments are an integral component of cognitive-behavioral therapy (CBT) and relate to positive therapy outcomes, it is unclear whether specific homework types and their completion have specific effects on outcome. METHOD: Data from N = 292 patients (75% female, mean age 36 years) with panic disorder and agoraphobia and treated with standardized CBT were analyzed with homework compliance quality and quantity for different types of homework serving as predictors for different outcome variables. RESULTS: Quality ratings of homework completion were stronger outcome predictors than quantitative compliance ratings. Exposure homework was a better outcome predictor than homework relating to psychoeducation and self-monitoring. CONCLUSION: Different aspects of homework compliance and specific homework types might differentially relate to CBT outcome.

Facilitated extinction but impaired extinction recall by eye movement manipulation in humans - Indications for action mechanisms and the applicability of eye movement desensitization.

Eye movement desensitization and reprocessing (EMDR) therapy utilizes the manipulation of eye movements to reduce affective distress during fear-exposure. Animal research recently suggested a potential neural mechanism underlying these effects, by which increased activity of the superior colliculus (SC), mediating visual attention, increases the inhibition of the basolateral amygdala (BLA), mediating defensive plasticity. We tested such mechanism in forty healthy humans using a multiple-day single-cue fear conditioning and extinction paradigm. The activity of the SC during extinction was experimentally manipulated by eye movements, as half of the participants executed saccadic eye movements (n = 20; major SC involvement), while the other half executed smooth eye pursuits (n = 20; minor SC involvement). Amygdala-mediated fear-potentiated startle responses and fear bradycardia, as well as threat expectancy was analyzed. Saccadic eye movements facilitated the extinction of fear bradycardia and fear-potentiated startle responses. Higher saccadic accuracy and range correlated with reduced fear-potentiated startle. However, during extinction recall, fear-potentiated startle and fear bradycardia resurged and partly reached levels obtained after fear acquisition. Threat expectancy was not affected by different eye movements and was not elevated during extinction recall. Within limitations, results support an inhibitory SC-BLA pathway in humans by which eye movements may reduce low-level defensive responding, but not threat expectancy. Yet, manipulating eye movements during extinction learning seems to impair extinction recall for behavioral and physiological defensive response indices. Thus, increasing SC activity might enhance initial efficacy of exposure treatment, but additional strategies seem necessary for sustained fear attenuation.

Defensive Mobilization During Anticipation of Symptom Provocation: Association With Panic Pathology.

BACKGROUND: Anxious apprehension about feared body symptoms is thought to play a crucial role in the development, chronicity, and treatment of panic disorder (PD). In this study, we therefore aimed to elucidate the role of defensive reactivity to anticipated unpleasant symptoms in PD that can contribute to a better understanding of pathomechanisms of PD as well as identification of potential targets in PD-focused interventions. By measuring amygdala-dependent potentiation of the startle reflex, we aimed to investigate whether 1) patients with PD exhibit a specifically increased defensive reactivity to anticipated unpleasant body symptoms and 2) whether clinical severity of panic symptomatology varies with magnitude of defensive activation. METHODS: Defensive mobilization to anticipated threat was investigated in 73 patients with a primary diagnosis of PD with agoraphobia (PDA) and 52 healthy control subjects. Threat of symptom provocation was established by a standardized hyperventilation task and contrasted to threat of shock to the forearm of the participant. RESULTS: Patients with PDA and healthy control subjects did not differ in their defensive responses during anticipation of shock. In contrast, patients with severe PDA as compared with healthy control subjects exhibited increased defensive response mobilization and reported more anxiety and panic symptoms during anticipation of feared body symptoms. Moreover, startle potentiation during anticipation of hyperventilation covaried with the severity of panic symptomatology. CONCLUSIONS: The present findings suggest that increased defensive mobilization during anticipation of body symptoms is a neurobiological correlate of severe PDA that should be specifically targeted in PD interventions and might be used to monitor treatment success.

Recent advances in studying brain-behavior interactions using functional imaging: The primary startle response pathway and its affective modulation in humans.

The startle response is a cross-species defensive reflex that is considered a key tool for cross-species translational emotion research. While the neural pathway mediating (affective) startle modulation has been extensively studied in rodents, human work on brain-behavior interactions has lagged in the past due to technical challenges, which have only recently been overcome through non-invasive simultaneous EMG-fMRI assessments. We illustrate key paradigms and methodological tools for startle response assessment in rodents and humans and review evidence for primary and modulatory neural circuits underlying startle responses and their affective modulation in humans. Based on this, we suggest a refined and integrative model for primary and modulatory startle response pathways in humans concluding that there is strong evidence from human work on the neurobiological pathway underlying the primary startle response while evidence for the modulatory pathway is still sparse. In addition, we provide methodological considerations to guide future work and provide an outlook on new and exciting perspectives enabled through technical and theoretical advances outlined in this work.