Depression is a highly prevalent condition with devastating personal and public health consequences that often first manifests during adolescence. Though extensively studied, the pathogenesis of depression remains poorly understood, and efforts to stratify risks and identify optimal interventions have proceeded slowly. A major impediment has been the reliance on an all-or-nothing categorical diagnostic scheme based solely on whether a patient endorses an arbitrary number of common symptoms for a sufficiently long period. This approach masks the well-documented heterogeneity of depression, a disorder that is highly variable in presentation, severity, and course between individuals and is frequently comorbid with other psychiatric conditions. In this targeted review, we outline the limitations of traditional diagnosis-based research and instead advocate an alternative approach centered around symptoms as unique dimensions of clinical dysfunction that span across disorders and more closely reflect underlying neurobiological abnormalities. In particular, we highlight anhedonia—the reduced ability to anticipate and experience pleasure—as a specific, quantifiable index of reward dysfunction and an ideal candidate for dimensional investigation. Anhedonia is a core symptom of depression but also a salient feature of numerous other conditions, and its severity varies widely within clinical and even healthy populations. Similarly, reward dysfunction is a hallmark of depression but is evident across many psychiatric conditions. Reward function is especially relevant in adolescence, a period characterized by exaggerated reward-seeking behaviors and rapid maturation of neural reward circuitry. We detail extensive work by our research group and others to investigate the neural and systemic factors contributing to reward dysfunction in youth, including our cumulative findings using multiple neuroimaging and immunological measures to study depressed adolescents but also trans-diagnostic cohorts with diverse psychiatric symptoms. We describe convergent evidence that reward dysfunction: (a) predicts worse clinical outcomes, (b) is associated with functional and chemical abnormalities within and beyond the neural reward circuitry, (c) is linked to elevated peripheral levels of inflammatory biomarkers, and (d) manifests early in the course of illness. Emphasis is placed on high-resolution neuroimaging techniques, comprehensive immunological assays, and data-driven analyses to fully capture and characterize the complex, interconnected nature of these systems and their contributions to adolescent reward dysfunction. Depression in children and adolescents is associated with significant distress, family burden, and functional impairment including academic failure, social dysfunction, and substance use . CriticaEfforts to address this major public health challenge are complicated by the considerable variation in adolescent depression presentation, severity, and especially course. While approximately 40% of depressed youth will fully recover by adulthood , others Although progress has been slow in identifying definite biobehavioral predictors of depression outcomes in youth, mounting evidence has highlighted the prevailing categorical diagnostic framework itself as a major impediment to understanding the neurobiology of depression and other psychiatric conditions. At issue are the heterogeneous and largely arbitrary criteria used to define clusters of symptoms as particular psychiatric diagnoses in the Diagnostic and Statistical Manual of Mental Disorders (DSM)  and simiIn the case of major depressive disorder, current DSM-5 guidelines identify nine core symptoms , many ofThe DSM-5 criteria for diagnosing major depressive disorder in children and adolescents include all the symptoms listed for adults in Accordingly, reliable diagnosis of adolescent depression requires experienced clinicians to accurately discern the presence and severity of numerous symptoms, synthesize potentially conflicting reports from patients and informants, and verify or revise their conclusions through sustained longitudinal observation. These involved procedures represent a serious burden on limited community mental health resources, can delay access to potentially time-sensitive interventions, and perpetuate reductive illness classifications with limited neurobiological validity.vice versa. Another key facet of dimensional research is recruitment of clinically diverse cohorts in order to capture the full range of symptom severity. In marked contrast to the restrictive inclusion criteria used in categorical research to limit variability, dimensional investigations aim to enroll individuals with a variety of psychiatric conditions, including comorbid and sub-threshold symptomatology, resulting in more representative samples and more generalizable findings . Whereas, studies centered around categorical diagnoses treat symptom heterogeneity and comorbidity as limitations to be overcome, the dimensional framework treats inter-individual variability as a strength to be leveraged . Symptomfindings . In recofindings , 29. The1H MRS) to investigate potential neurochemical alterations within the reward system, and diffusion-weighted imaging (DWI) to examine white matter integrity in reward-related tracts. Section Immune Function and Inflammation in Depression and Reward Dysfunction presents findings from our studies of immunological and inflammatory biomarkers using detailed assays of peripheral cytokines and kynurenine pathway metabolites in youth. Throughout, we emphasize how integration of multiple research modalities through data-driven techniques can further enhance understanding of adolescent depression and advance the goals of identifying objective outcome predictors and targets for personalized early interventions.Here, we provide a targeted review of these developing trends in research methodology and their application to the study of reward dysfunction in adolescent depression and beyond. We discuss the emergence of the dimensional investigative approach over the past decade, as exemplified by our group's evolving corpus of work as well as a selection of landmark studies by other researchers. Particularly, we aim to demonstrate how a focus on anhedonia has enabled us to leverage multimodal neuroimaging and biological measures and identify shared mechanisms of reward dysfunction across youth with diverse clinical profiles, including depression but also anxiety and behavioral issues. Section Reward Dysfunction as a Promising Predictor of Adolescent Depression Trajectory highlights anhedonia as a direct reflection of deficits in reward processing and an ideal candidate for the dimensional approach advocated in this review. Section Neuroimaging Reward Dysfunction in Adolescent Depression discusses results from our comprehensive adolescent neuroimaging program, including task-based functional magnetic resonance imaging (fMRI) to probe reward processes directly, resting-state fMRI to study key subcortical reward circuitry as well as whole-brain networks, proton magnetic resonance spectroscopy . Se. Se37). Anhedonia can be readily quantified using a variety of questionnaires, including the 14-item Snaith-Hamilton Pleasure Scale (SHAPS)  and the Several studies have now concluded that anhedonia accounts for a significant proportion of the variance in adolescent depression outcomes. A large cross-sectional analysis by our group examined clinical correlates of two core symptoms, anhedonia and irritability, in a cohort of 90 adolescents  . All parHowever, we found that only anhedonia, not irritability, was associated with poorer clinical outcomes, including greater overall illness severity, longer episode duration, and increased suicidality , as wellMoreover, we recently reported initial results from a follow-up study examining longitudinal outcomes after ~2 years in a mixed group of 29 adolescents with mood and anxiety symptoms as well as 14 healthy controls . In lineSeveral independent lines of evidence further support our conclusion that anhedonia, as an index of reward dysfunction, plays a uniquely predictive role in the trajectory of depression. A multivariate analysis of 334 depressed youth in the Treatment of Resistant Depression in Adolescents (TORDIA) study found that, out of five symptom dimensions, only anhedonia predicted longer remission time and fewer depression-free days over 24 weeks; findings remained significant even when accounting for overall depression severity . A studyTaken together, these findings support the notion that anhedonia reflects key aspects of reward dysfunction across ages and may map particularly well onto underlying neural processes that contribute to the development and maintenance of depression in youth.en route to cortical targets, including the ventromedial prefrontal cortex (PFC), subgenual and pregenual anterior cingulate cortex (ACC), and supplementary motor area  have found that altered neural activation profiles during reward anticipation and attainment in non-depressed adolescents predicted the onset of future depression  to detect neurochemical concentrations in targeted regions, and diffusion-weighted imaging (DWI) to model structural connectivity in white matter. As we seek to demonstrate, combining data from multiple neuroimaging modalities, as well as other biological measures (see section Immune Function and Inflammation in Depression and Reward Dysfunction), within a single cohort substantially enhances our ability to make inferences about brain function and its role in the emergence of psychiatric illness.In this section, we review findings in adolescent depression based on a variety of imaging methods, focusing particularly on advanced MRI techniques employed by our group to examine the neural underpinnings of reward dysfunction. These include task-based and resting-state fMRI to assay neural activity across the brain, proton magnetic resonance spectroscopy  and the habenula . These and other key reward-related structures have limited anatomical tissue contrast and have proven difficult to reliably segment, with the most accepted methods rely on heuristic anatomical landmarks   and desiN = 77, ages 11–13)  . Crucials 11–13) . As detaAs discussed in section Reward Dysfunction as a Promising Predictor of Adolescent Depression Trajectory, reward dysfunction and anhedonia in adolescent depression entail deficits in multiple reward processes. For example, some patients may lack the motivation to pursue potentially pleasurable activities , while others may seek out such activities but find that they experience little or no pleasure as a result . To better delineate neural activity during these distinct phases of reward processing, our group has developed the Reward Flanker Task (RFT). Using the RFT, we are able to directly examine neural responses to reward anticipation (monetary cues) and reward attainment (monetary outcomes), but also a number of important additional constructs related to outcome uncertainty and reward prediction errors.Implementation of the RFT is described extensively in our previous publication . Briefly1) Cue indicating high (50%), low (10%), no (0%), or unknown (?) trial type for 4–6 s2) Flanker stimulus  for 300 ms3) Calibrated response window with a blank display for ≤ 1,700 ms4) Feedback indicating high (50%), low (10%), or no (0%) reward earned for 2 s5) Inter-trial-interval with a blank display for 4–6 s.Unknown cues yield high, low, and no reward outcomes equally. During the response window, participants attempt to identify the unique target letter from the flanker stimulus using button presses. To keep the task challenging and ensure that some errors are made, the amount of time a participant is allotted to make a response is set at 0.8–1.5 times their mean reaction time during a pre-scan practice session. Correct responses to flanker stimuli result in winning a high, low, or no reward outcome based on the trial type. A total of 120 trials (30 of each type) are presented in pseudo-random order across four 4-min fMRI runs.Compared to other reward paradigms such as the Incentive Flanker and Monetary Incentive Delay, the RFT has several novel features. First, cues used to signal the reward value of each trial are presented with varying durations and for sufficient lengths to allow for the separation of brain activity during reward expectancy from attainment despite the slow hemodynamic response. Second, the task uses three levels of reward value  to examine brain activity in response to increasing reward value for both expectancy and attainment. Third, the task includes unknown reward cues, allow us to dissociate responses to certain vs. uncertain reward outcomes. In our studies, the contrast of reward attainment following uncertain vs. certain cues is referred to as positive prediction error. Finally, as cues elicit reward anticipation such that a specific, effortful response is required to obtain the reward, rather than passive anticipation of a definite reward, the RFT is particularly well-suited to probe motivational deficits in anhedonia.pTFCE−FDR < 0.05). We found that reward anticipation activated a large bilateral network including default-mode, salience, and limbic regions important for reward appraisal and conflict resolution. Reward attainment engaged a more restricted network including the hippocampus, medial temporal lobe, dorsal caudate, and occipital areas related to memory and emotion processing but notably not the ventral striatum. Additionally, we found that positive prediction errors yielded widespread cortical activation in many of the same regions identified in during reward anticipation/attainment, including the ventral striatum. Exploratory analyses at a relaxed threshold (uncorrected p < 0.001) further identified several positive correlations between anhedonia severity and RFT activation, specifically in the right angular gyrus during reward anticipation and in the left precuneus during positive prediction errors. These results support the ability to differentiate reward processes and identify links to clinical reward dysfunction in adolescents using the RFT. A follow-up RFT study in an expanded adolescent cohort is currently in preparation, with findings largely corroborating our initial pilot results; additionally, three reward networks derived from this follow-up study served as the basis for our recent graph theory analysis using resting-state fMRI, described in section Using Graph Theory to Bridge Resting-State and Task-Based fMRI Studies below.Following the development of the RFT, we conducted a pilot study to examine brain activation during distinct reward processes in a sample of 22 psychotropic medication-free adolescents, including 16 with primarily mood and anxiety symptoms and 6 healthy controls . ResultspTFCE−FWE < 0.05) predicted future anhedonia severity  yielded largely consistent findings of amygdala hyperactivation to fearful faces and ACC hypoactivation to happy faces relative to low-risk controls (N = 23)  . Using a(N = 23) . A totalWhile evidence supports a negative bias when evaluating the emotional valence of others, excessively negative self-perception is even more characteristic of depression. We therefore conducted a separate 3T fMRI study of self-referential word processing in a group of 23 depressed and 18 healthy control adolescents . SubjectN = 120) combined these approached by having subjects identify their own emotional expressions from photos taken 1–2 weeks prior to the task. Results indicated that, unlike when processing the emotional expressions of others, recognition accuracy among depressed adolescents was highest for happy faces. Analysis of fMRI data further revealed a that elevated amygdala connectivity with regions of the ACC and PFC during the task was associated with increased suicidality and was highest in youth with a history of suicide attempts , which measures correlations in low-frequency BOLD fluctuations  between difference regions. Networks identified using RSFC can reflect not only known anatomical connectivity between regions but also areas with no direct anatomical links, implying that correlated activity is instead driven by shared function –126. SevNumerous studies have now documented resting-state network alterations in adolescent depression and related conditions. Brain structures most frequently implicated in these studies include the anterior cingulate cortex, insula, and especially the default-mode network –130, whia priori reward areas  and data-driven analyses using graph theory (section Using Graph Theory to Bridge Resting-State and Task-Based fMRI Studies).As detailed below, our group has utilized resting-state fMRI extensively to examine reward circuitry in adolescent depression, both through seed-based RSFC analyses of We conducted an early study of striatum-based RSFC in a cohort of 42 psychotropic-medication-free adolescents, 21 with a current major depressive episode and 21 healthy controls with no history of psychiatric illness . Mean ti3 per hemisphere) and limited anatomical contrast of the habenula with nearby thalamic tissue have severely limited in vivo research.While the dopaminergic reward circuit described in section Linking Reward Function to Reward Circuitry is strongly engaged by positive prediction errors , reward-related dopamine signaling is inhibited by negative prediction errors  . Preclinin vivo myelin contrast . At a relaxed exploratory threshold , group contrasts further revealed reduced habenula RSFC with the mid-cingulate, right amygdala, and right anterior insula, regions implicated in mood disorders and aversion processing, in subjects with high subclinical depression scores  in the HCP, 25 with low and 25 with high subclinical depression scores . In linen scores .Building on this work, we developed an optimized method for downsampling anatomical-resolution (~0.8 mm isotropic) habenula segmentations to fMRI resolution (~2 mm isotropic) for use as RSFC seeds. These individual-specific seeds account for tissue probability weights, partial volume effects, and template normalization, significantly improving BOLD sensitivity compared to other seeding methods . In a foReward Anticipation (114 nodes), Reward Attainment (103 nodes), and Reward Prediction Error (117 nodes), reprinted as As described in section The Reward Flanker Task, our task-based fMRI findings using the RFT have identified distinct activation networks engaged by the processes of reward anticipation, reward attainment, and positive prediction error. Building on this work, we conducted a data-driven graph theory analysis to identify resting-state network features associated with psychiatric symptomatology in a sample of 68 clinical adolescents with predominantly mood and anxiety symptoms as well as 19 healthy adolescent controls . This anWhole Brain network (750 nodes) derived from the Cole-Anticevic atlas. Strength Centrality represented the sum of edge weights per node and captured the overall influence of each node within the network. Eigenvector Centrality represented the largest magnitude eigenvector per node and captured the influence of each node over highly influential nodes within the network. Local Efficiency represented the inverse shortest path length between each node and its neighborhood and captured how readily information could propagate to other nodes in the network.Graph theoretical analyses were performed using the Brain Connectivity toolbox  to derivReward Attainment network  is a nuclear magnetic resonance technique closely related to MRI that can distinguish protons from various tissue chemicals, allowing for in vivo measurement of human biochemistry , N-acetylaspartate, and myoinositol. Of particular interest are GABA and glutamate, respectively, the most common inhibitory and excitatory neurotransmitters in the brain. Interest in the role of excitatory and inhibitory neurotransmission in depression has been stimulated by the discovery that ketamine, a glutamatergic NMDA receptor antagonist, exhibits rapid antidepressant activity through an apparently distinct mechanism from classic serotonergic antidepressant drugs   and the whole sample . These findings were the first evidence for an important association between ACC GABA and anhedonia severity in adolescent depression.Our first lescents . Analyse< 0.003) . When we1H MRS study in an expanded cohort, now including 44 depressed and 36 healthy control adolescents (p = 0.003) relative to controls. When depressed subjects were classified based on anhedonia, analyses revealed a significant difference in ACC GABA levels across anhedonic depressed, non-anhedonic depressed, and healthy control adolescents (p = 0.003); as shown in p = 0.002). Similar to our earlier results, anhedonia severity was inversely correlated with ACC GABA levels in the depressed group . These confirmatory findings support our conclusion that deficits in ACC GABA may serve as a biomarker for reward dysfunction in depressed adolescents.Our initial ACC GABA results were subsequently corroborated by a follow-up lescents . Analyseed group , even wh1H MRS study of 20 depressed and 16 healthy control adolescents  using permutation-based non-parametric statistics.In 2013, our group published a pilot investigation into the role of white matter in adolescent depression using DWI . Specifipipeline  to modelRelative to healthy controls, we found that depressed adolescents had altered diffusivity metrics in four clusters: reduced anterior cingulum FA, anterior corona radiata AD, and posterior cingulum RD as well as increased posterior cingulum FA. In line with our other neuroimaging work and supporting our emphasis on dimensional symptom severity analyses, though, we found much more extensive associations with clinical symptomatology.Depression severity correlated with a total of 16 clusters across diffusivity metrics, including negative correlations with FA in the anterior thalamic radiation near the left pallidum, genu of the corpus callosum near the ACC, and anterior cingulum bundle within the left precuneus; positive correlations with RD were observed in several overlapping clusters, including the anterior thalamic radiation bilaterally and the left genu of the corpus callosum.Anhedonia severity was linked to altered diffusivity metric in 14 clusters, including reduced FA in the anterior limb of the internal capsule adjacent to the right basal ganglia; increased MD in the external capsule adjacent to the left putamen and in fibers projecting to the right OFC; and increased RD in the left anterior thalamic radiation, right anterior limb of the internal capsule, and projection fibers to the right OFC.Unlike in our studies of clinical outcomes (section Anhedonia Predicts Worse Adolescent Depression Outcomes) and reward function using the RFT , we also found associations between irritability scores and diffusivity metrics in a total of 14 clusters. These included positive correlations with MD in a cluster spanning the anterior corona radiata and anterior thalamic radiation near the left putamen, with RD in a cluster spanning the anterior limb of the internal capsule and anterior thalamic radiation near the right putamen, and with AD in the anterior corona radiata near the left putamen.While preliminary, these results indicate that reduced white matter integrity in tracts projecting to or through major reward processing regions is associated with adolescent depression symptomatology. Unlike our resting-state and task-based fMRI results, we found that tracts with altered diffusivity metrics partially overlapped between different depression symptoms. This observation suggests that different disease mechanisms may be at work in gray vs. white matter, with the former exhibiting more specific abnormalities linked to reward dysfunction and the latter exhibiting more generalized loss of integrity in adolescent depression.Though the research discussed thus far emphasizes the neural underpinnings of depression and reward dysfunction, substantial evidence also implicates the immune system in these disease processes. Converging data from preclinical and clinical studies document that disturbances in reward circuitry are induced by inflammation –186, pos1H MRS study in adults , an excitotoxic agonist of glutamatergic N-methyl-D-aspartate (NMDA) receptors , which is induced by pro-inflammatory cytokines  and metaeceptors . Alternaeceptors . Thus, iConverging lines of research have indeed linked increased IDO activity to anhedonia and reward dysfunction , 217. InAn earlier series of studies by our group provided the first evidence of kynurenine pathway activation in adolescent depression and specifically in anhedonia. Our first study examined a cohort of 50 adolescents with major depressive disorder and 22 healthy controls, with the depressed cohort divided into 20 melancholic subjects, characterized by more severe illness and prominent anhedonia, and 30 non-melancholic subjects . We founr = 0.42, p = 0.05) and in combination with controls , but this correlation was reduced when the combined sample included medicated depressed subjects  and did not reach significance in the depressed cohort alone when medicated subjects were included . This study was the first to link reward dysfunction to kynurenine pathway induction in youth. Additionally, our findings highlight the important, but frequently underappreciated, impact that treatment can have on biological measures in psychiatric research, suggesting that psychotropic medication may have a normalizing effect on the neurometabolic abnormalities observed in unmedicated participants.Building on these results, we subsequently examined the possible role of the kynurenine pathway in anhedonia, the hallmark symptom of melancholia . In a sarho = 0.93, pBonferroni = 0.03), while peripheral 3-HAA correlated with total choline in the left putamen  only within the melancholic  depressed subgroup. While the small sample of this preliminary investigation is obviously a limitation, the magnitude of the observed correlations was remarkably high, suggesting a possible link between the global TRP metabolism and regional cell membrane metabolism within the striatum. A more recent pilot analysis of resting-state fMRI and kynurenine pathway metabolites in 14 unmedicated adolescents with depression and seven healthy controls similarly found preliminary evidence of associations between several kynurenine pathway metabolites and RSFC within reward- and salience-related neurocircuitry  as well as overall depression severity, body mass index, age, and sex. Even at this stringent threshold, we found that anhedonia was positively correlated (rho = 0.33–0.57) with 19 of the cytokines included in our panel. Cytokines linked to anhedonia were primarily pro-inflammatory and/or hematopoietic growth factors (N = 15), with a small number of chemokines (N = 2) and anti-inflammatory cytokines (N = 2) also identified. In line with our other dimensional work, no significant group differences were observed between depressed adolescents and healthy controls, and no significant association was detected with any other clinical measure . These findings provide strong evidence that inflammatory responses are tied to reward deficits in adolescents regardless of diagnostic status and again showcase the central role of reward dysfunction in early depression symptomatology.To address this gap, initial studies by our group examined plasma levels of major pro-inflammatory cytokines in the context of adolescent depression. We documented significantly higher levels of interferon-gamma (IFN-γ) in medically healthy adolescents with major depressive disorder compared to healthy controls , 231; unhallenge . These challenge , 234. BlNeuroimaging work further documents the specific effects of inflammatory processes within the dopaminergic reward circuitry. Several positron emission tomography (PET) studies in adults have demonstrated changes in striatal glucose metabolism subsequent to immunotherapy , 236. UspFWE < 0.05). Remarkably, 16 of the 19 cytokines found to be associated with anhedonia severity in our earlier dimensional study , resting-state network anomalies, and altered neurochemical profiles. Employing detailed cytokine panels to capture a wide spectrum of immune biomarkers as well as an It is important to note that, while the dimensional approach offers many advantages over categorical diagnostics for understanding disease mechanisms, it also presents its own challenges and limitations. Of particular concern given our focus on reward dysfunction are the nuances of “anhedonia.” While far more specific than “depression,” insofar as anhedonia represents a well-defined clinical phenomenon , efforts to develop reliable anhedonia scales indicate that it is not a monolithic construct. Indeed, the TEPS questionnaire employed in many of our studies is subdivided into items related to reward anticipation  vs. reward consumption . Subsequent work has suggested that two subscales may be inadequate to fully capture anhedonia phenomenology, particularly in cross-cultural settings. The TEPS was originally developed by researchers in the University of California system and validated based on responses of undergraduate volunteers from the Berkeley campus . A team N > 1,000) community profiles across age groups  and HCP Lifespan Initiative, which aims to create large (e groups –249, as e groups . These pe groups . The dynAW, BE, RT, TN, and VG: conceptualization, writing—original draft, and writing—review and editing. BE: visualization. VG, BE, and TN: funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The utility of clinically available antifungals is limited by their narrow spectrum of activity, high toxicity, and emerging resistance. Antifungal drug discovery has always been a challenging area, since fungi and their human host are eukaryotes, making it difficult to identify unique targets for antifungals. Novel antifungals in clinical development include first-in-class agents, new structures for an established target, and formulation modifications to marketed antifungals, in addition to repurposed agents. Membrane interacting peptides and aromatherapy are gaining increased attention in the field. Immunotherapy is another promising treatment option, with antifungal antibodies advancing into clinical trials. Novel targets for antifungal therapy are also being discovered, allowing the design of new promising agents that may overcome the resistance issue. In this mini review, we will summarize the current status of antifungal drug pipelines in clinical stages, and the most recent advancements in preclinical antifungal drug development, with special focus on their chemistry. Candida, Cryptococcus, and Aspergillus are the most common causative organisms of life-threatening human fungal infections -carboxymethyl group is linked by an ester functionality to the triazole nitrogen of isavuconazole. The prodrug is cleaved by human plasma esterases releasing isavuconazole and low levels of cleavage by-product   is a novel echinocandin, another cyclic hexapeptide derivative. It is an analogue of the echinocandin anidulafungin obtained by replacing the hemiaminal region at the C5 ornithine position of anidulafungin with choline ether refer to . This chSuper-bioavailability-itraconazole (SUBA itraconazole) overcame the low bioavailability issue of itraconazole by dispersing itraconazole in a pH-dependent polymer matrix as capsules. This change to the formulation increased the bioavailability of itraconazole by 173% . SUBA it®) -2-yl)-tetrahydropyrimidin-2(1H)-imine  is structurally related to the antifungal abafungin and showed potent in vitro antifungal activity against Aspergillus fumigatus (MIC = 4.7 μM) with a favorable pharmacokinetic profile. In addition, the latter compound exhibited antifungal activity comparable to voriconazole in a murine model of Aspergillus fumigatus infection.On the other hand, Kato et al. prepared thiazoyl guanidine derivatives that inhibit fungal ergosterol biosynthesis . Their hN-hydroxyacetamide  showed excellent in vitro and in vivo synergistic antifungal activity with fluconazole to treat azole-resistant candidiasis.Furthermore, Li and colleagues prepared a series of carboline fungal histone deacetylase (HDAC) inhibitors in an attempt to develop a promising compound for the combinational treatment of azole-resistant candidiasis . Among aCandida albicans with varying levels of FA synthase or desaturase [Candida albicans, Candida auris, and mucormycetes, including activity against azole-resistant Candida and low in vitro cytotoxicity in HepG2 liver cancer cell line.Fungal fatty acid (FA) synthase and desaturase (responsible for introducing double bonds to yield unsaturated FAs) are essential enzymes for the growth and virulence of fungal pathogens . They arsaturase . They id50 = 2.3 ± 0.8 μM) without affecting mammalian cell viability (viability = 101.5 ± 1.4%) and exerted potent in vitro antifungal activity against Candida albicans (MIC = 6.4 ± 2.6 μM).Lastly, Lowes and colleagues designed and synthesized dual inhibitors targeting fungal acetohydroxy-acid synthase and NLRP3 inflammasome . Their dAside from the mentioned small molecules with intracellular targets, membrane-interacting antifungal antimicrobial peptides (AMPs) are another example of a promising class of antifungals. AMPs generally consist of 12 to 54 amino acids with a net positive charge at physiological pH . They arRuta graveolens (REO) [Candida tropicalis and fungistatic activity against Candida albicans by disrupting cellular membrane integrity. REO also showed synergistic activity with amphotericin B [Helichrysum microphyllum Cambess. subsp. tyrrhenicum Bacch., Brullo & Giusso showed potent inhibiting activity on candida growth, making it a promising agent to be used topically in the treatment of candidiasis [Austroeupatorium inulaefolium, which showed strong species-dependent antifungal activity against Penicillium brevicompactum and Fusarium oxysporum [In addition, several studies were conducted to explore the role of essential oils in treating fungal infections. In their recent work, Donadu et al. investigated the antifungal activity of the essential oil of the Colombian rue, ns (REO) . They foericin B . Furtherdidiasis . Anotherxysporum .Repurposing established drugs with possible antifungal activity and pushing them into the antifungal development pipeline saves time and resources, especially given that the pharmacokinetic and pharmacodynamic profiles are already known for such agents. The most promising candidates to be used in antifungal regimens are the selective estrogen receptor modulator tamoxifen (used for breast cancer) and the serotonin reuptake inhibitor sertraline (used for depression) . It binds to the walls of ormation . Mambro ida spp. . The newCandida. It must be noted that efungumab was also investigated in clinical trials as an adjuvant to docetaxel in patients with breast cancer (NCT00217815).Efungumab  is a single-chain variable-fragment antibody that targets heat shock protein 90 (HSP90). Efungumab was tested in combination with amphotericin B in clinical settings, where it showed a reduction in the mortality and an improvement in the survival of patients infected with Cryptococcus neoformans infections [One step beyond antifungal antibodies is radio-immunotherapy, where the antifungal antibody is linked to radioisotopes in order to specifically release fungicidal radiation in fungal cells . This apfections .Their classification as eukaryotes makes fungal infections a more challenging condition to treat in the drug discovery pipeline. It is important to discover unique targets that are present in fungi and not in humans in order to improve the selectivity and subsequently the safety profile of such agents. One interesting approach to discover new antifungals is what Novartis did by screening their chemical dark matter database for potential antifungals. Chemical dark matter libraries include molecules that have no bioactivity in human targets and thus are of increased value in screening for active molecules against other eukaryotes. During their screening campaign, Novartis have identified a novel antifungal agent and a novel fungal target pathway, which is hemebiosynthesis. The identification of molecular targets opened the window for medicinal chemists to design inhibitors following a target-based drug design approach.Candida albicans, Cryptococcus neoformans, and Aspergillus spp. [N-acyltransferase  [Another example of a new, promising fungal pathway for antifungal drug development is a sphingolipid synthesis pathway. Sphingolipid synthesis is highly preserved among eukaryotes; however, a number of vital structures are unique to fungi, making them promising treatment targets. In fungi, sphingolipids such as inositolphosphoryl ceramide (IPC) and glucosylceramide (GlcCer) play an important role in fungal pathogenicity and fungal growth . Structulus spp. ,82. Promifungin) , IPC inhifungin) , GlcCer ifungin) , and Glcifungin) , among oifungin) .Cryptococcus neoformans, as a potential target for antifungal drug development [Cryptococcus neoformans or Cryptococcus gattii in immunocompromised patients with HIV, since such inhibitors are also antiretrovirals. In addition to identifying a new promising target, the authors identified promising inhibitors derived from an N-terminally carboxybenzylated phenylstatine scaffold  secreted from elopment . InhibitWhen comparing older antifungals with the newer ones mentioned in this review, one can sense the influence of the powerful recent advances in structural biology and medicinal chemistry on phenotypic target discovery, drug discovery, and target-based drug design. It must be noted that in order to truly evaluate the efficacy of new antifungals in treating life-threatening infections, infection-specific biomarkers should be relied on rather than death as an endpoint, since such targeted patients have multiple health comorbidities. By doing so, we ensure that no potential hit is lost as a false negative. One important observation from the examples of antifungals in development mentioned earlier in text is the presence of halogen atoms, especially fluorine and chlorine, in their chemical structures. This suggests the significant importance of halogen bonding for the interactions with their corresponding targets.


Volvulus of colon is a very rare phenomenon in post cardiac surgery course, and their predicting factor in most patients is unknown. Between colonic volvulus, splenic flexure is the rarest site for torsion in general population. The main symptoms are vague abdominal pain, vomiting and distension. The primary diagnostic images include plain chest x-ray, CT scan and colonoscopy.We report the case of a 57-year -old opium male addict, who was. Admitted for abdominal pain, nausea, and vomiting five days after off-pump coronary artery bypass surgery (OPCAB). An abdominal x-ray reported a colonic volvulus. Exploratory laparotomy showed acute abdomen resulting from a gangrene of long segment of splenic flexure caused by volvulus.Gastrointestinal complication such as volvulus is an exceedingly rare complication of OPCAB, despite the absence of anatomic abnormalities only complete colonic malrotation as the result of mega colon and constipation, the main pathogenetic causes. This patient was unique because of careful literature search revealed that this case was the first reported volvulus that has been described so far. Splenic colon flexure is a rare site for colonic volvulus and its incidence is even exceedingly rare in OPCAB. However, cardio pulmonary bypass (CPB) is excluded from surgery in OPCAB, but in addicted patients, a specific syndrome named as narcotic bowel syndrome has been reported . In manyThe splenic flexure is a rarest cause of volvulus in general population . The invContrary to many efforts to expand other therapeutics, oral opium (taryak) remains as the basic remedy or auto therapy in many opium-addict patients with pain and to prevent withdrawal syndrome. This report shows a case of splenic flexure volvulus in an addict patient leading to a major gangrene and peritonitis.A 57-year-old man presented in Imam Ali Hospital in 2017 with unstable angina due to severe stenosis of the left anterior descending coronary artery and obtuse marginal, however, the right coronary artery was normal. Laboratory finding including a complete blood count and erythrocyte sedimentation rate and C reactive protein was normal. Chest- ray revealed no abnormal finding. There was not any valular abnormality by preoperative echocardiography. The patient has not had any co-morbid disorder. Left ventricular ejection fraction was reduced (45-40%). The intraoperative course was uneventful. There was no any finding with regard to pericarditis. On physical examination, patient was not in respiratory distress, blood pressure was 130/80 mmHg, heart rate 80 beat per minute, respiratory rate 23/min, neck vein was not distended and without ankle edema. On examination of the cardiovascular system, he had a regular s1 s2 with no gallop. No murmur was appreciated. ECG showed Q wave in precordial leads. The patient underwent uneventful off-pump coronary artery bypass grafting. There was not any finding regarding pericarditis and postoperative ESR and CRP were normal. After hemodynamic stabilization, the patient left the intensive care unit without IABP and inotropic support. The patient presented with distension without nausea and vomiting in the 3rd postoperative day. His past medical history included no history of abdominal pain episodes with abdominal distension, or laparatomy or immune disease, in surgical consultation with an expertise general surgeon, a postoperative ileus was diagnosed. Primary abdominal x-ray revealed only distension of colon. 72 hours after the first consultation, with increasing abdominal distension and poor general condition of patient, abdominal x-ray showed a clear distension of colon suggestive for a volvulus . On thisAbdominal CT scan with contrast revealed distended transverse colon . A colonThe mechanism of splenic flexure volvulus in this specific case has been attributed to displacement, compression and partial obstruction of flexure colon by the chronic constipation. Diagnosis of the condition due to illicit opium use and its analgesic effect is often delayed. The abdominal pain is absent primarily in this patient. The average length of time from the onset of obstructive symptoms until presentation in this patients is 48 hours, because oral opium itself clouds the clinical picture such abdominal pain, nausea, however distension can occur in an otherwise postsurgery periods.In Sever et al.‘s study, large series of patients (1360 adult cardiac surgery) 29 patients with mesenteric ischemia was reported but no case of volvulus has been found . HoweverIn Raht et al.’s study volvulus was confined to the sigmoid 70%), but it may also affect the right (25%) and transverse colon (2-5%) 0%, but i. AtamanaIn conclusion, we think that mega colon caused by chronic constipation induced by oral opium had an important pathogenetic role to elicit the splenic flexure volvulus. Unique finding about this case is related to rarity of volvulus of splenic felcture in cardiac surgery and indeed it was the first case of splenic flexure volvulus that was reported in medical literature in off-pump CABG so far.


The evolution of mutations in SARS-CoV-2 at antigenic sites that impact neutralizing antibody responses in humans poses a risk to immunity developed through vaccination and natural infection. The highly successful RNA-based vaccines have enabled rapid vaccine updates that incorporate mutations from current variants of concern (VOCs). It is therefore important to anticipate future antigenic mutations as the virus navigates the heterogeneous global landscape of host immunity. Toward this goal, we survey epitope-paratope interfaces of anti-SARS-CoV-2 antibodies to map an antigenic space that captures the role of each spike protein residue within the polyclonal antibody response directed against the ACE2-receptor binding domain (RBD) or the N-terminal domain (NTD). In particular, the antigenic space map builds on recently published epitope definitions by annotating epitope overlap and orthogonality at the residue level. We employ the antigenic space map as a framework to understand how mutations on nine major variants contribute to each variant’s evasion of neutralizing antibodies. Further, we identify constellations of mutations that span the orthogonal epitope regions of the RBD and NTD on the variants with the greatest antibody escape. Finally, we apply the antigenic space map to predict which regions of antigenic space—should they mutate—may be most likely to complementarily augment antibody evasion for the most evasive and transmissible VOCs. Since the emergence of the coronavirus SARS-CoV-2 in 2019, SARS-CoV-2 has evolved within the context of a complex global landscape of diverse human host immunity arising from varied infections and vaccinations. The continuing adaptation of the virus has resulted in several variants of concern (VOC) that are more transmissible  and immuAlthough the currently observed SARS-CoV-2 VOCs might not represent true antigenic drift variants, as is observed periodically in the case of influenza viruses that re-infect a substantial portion of individuals with prior exposure to the original antigen ,7,8, SARcontinuous descriptions of RBD antigenic space that capture epitope overlap and orthogonality from the stand point of the polyclonal antibody response and that distill these features down to the residue level for interpretability. Further, these descriptions should be extended to NTD given the significant role that NTD-directed antibodies play in the neutralizing antibody response [The RBD and NTD present numerous epitopes targeted by neutralizing antibodies that are overlapping and also diverse from the standpoint of the mode of engagement of the antibodies with these surfaces. RBD epitopes have thus far been described using a variety of approaches, including the classification of antibodies targeting the RBD into four major “classes” based on structural analysis . Such diresponse . Such anIn this study, we first seek to identify the spike protein sites under antigenic pressure based on computational and structural analyses of over 50 mAb/nanobody-RBD/NTD epitope-paratope interfaces. From this survey we obtain a map of RBD and NTD antigenic space that extends existing epitope definitions by describing epitope overlap and orthogonality at the residue level. We integrate the antigenic space map with an estimate of residue mutability derived from genetic, structural, and functional datasets to highlight potential antigenic drift sites (PADS) that appear both antigenic and mutable. Further, through examination of epitope overlap within our map we find that the VOC mutations with the largest individual escape effects  tend to occur within the overlap of multiple epitopes, suggesting that the escape potency of key variant mutations may be in part due to providing escape from many antibodies targeting overlapping epitopes centered around these sites. We identify NTD residues that play a similar multi-epitope function within the NTD supersite. Examining NTD multi-epitope mutations across variants, we find that the Gamma variant lacks such a mutation potentially explaining its relatively weaker antibody evasion than the mutationally homologous Beta variant which features NTD multi-epitope mutations. We further identify additional RBD multi-epitope escape sites such as R346 (observed to mutate on the Mu variant) within the overlap of RBD epitopes outside of the ACE2 RBS. Finally, through investigation of epitope orthogonality within our map we find that the escape strength of nine prominent variants is correlated with the degree to which the given variant’s mutational fingerprint covers the orthogonal compartments of RBD and NTD antigenic space. This finding suggests that variants with greater degrees of antibody escape leverage mutational complementarity  rather than accumulation of multiple functionally similar mutations  to efficiently escape from the polyclonal antibody response directed against spike. We integrate the findings from this study to present a framework for predicting additional mutations that may complementarily augment antibody evasion for the Beta and Delta variants—the most evasive and transmissible variants identified at the time of this investigation.For each protein or protein-complex, significant interaction network (SIN) scores between every pair of residues within the structure were computed as described previously . BrieflyThe impact of RBD SNPs on epitope-paratope surface complementarity and binding energy (dG) was modeled in PyRosetta  using thGISAID sequences were accessed and downloaded on 7 June 2021 . MutatiomAb-epitope clustering: Direct, Indirect, and Total networking scores for each residue across all mAb and nanobody complexes were plotted using clustermap from the Seaborn statistical data visualization package with the ‘correlation’ distance metric and the ‘average’ linkage method . MutabilThe Antigenic Space Map was constructed from a network model of RBD or NTD, in which nodes represent residues, node size is proportional to estimated antigenicity  given its immunodominance, potent neutralization when targeted, and that key VOC mutations K417N/T, L452R, E484K, and N501Y occur in this ER.From the RBD and NTD epitope-paratope matrices, we derived an antigenic space map that illustrates the relationships between residues as functional components of the polyclonal antibody response. That is, we developed an antigenic space map that seeks to present (1) residue antigenicity, (2) residue mutability, and (3) residue assignment to a component of the antibody response directed against RBD or NTD . We brieThe antigenic space map was constructed as follows. First, we assigned residue antigenicity in our map to node size, where antigenicity is defined as the importance of a given residue as an epitope constituent across the antibodies surveyed. Residue antigenicity was estimated using a principal component analysis describing epitope-paratope networking and epitope-paratope surface complementarity perturbation upon mutation (see Methods). Second, we estimated residue mutability according to genetic, structural, and functional constraints on mutation of a given residue (see Methods). The residues scoring highest in antigenicity and mutability are denoted potential antigenic drift sites (PADS) and identified via node color. Third, we mapped each residue’s function as an epitope constituent in the immune response directed against RBD/NTD by computing the orthogonality between residues in the epitope-paratope interface matrix via cosine similarity. This process assigned functionally similar residues to similar locations in our antigenic space map. That is, a pair of RBD residues interacting with an identical subset of antibodies (and with similar relative strength across Abs) in the epitope-paratope interface matrix would be placed in overlapping positions within the antigenic space map. In contrast, RBD residues interacting with inverse subsets of antibodies serve orthogonal functions as epitope constituents across the polyclonal response directed against RBD and would therefore occupy highly distant positions within the antigenic space map. The antigenic space map thus concisely presents residue antigenicity, mutability, and relationships between epitope residues as overlapping or orthogonal functional epitope constituents.Examining the antigenic space map of RBD ER-2 we find three clusters of residues which suggests three functional groups describe the antigenic space of the ACE2-binding site epitopes (ER-2). Cross-referencing these residue clusters with the roles of the residues in epitope-paratope interfaces , we obseNext, we find a second ER-2 cluster best described as “ER-2B biased” as the sites in this cluster interact most strongly with antibodies assigned to ER-2B in Third, we find a final ER-2 cluster termed “ER-2A biased” consisting largely of residues on the 443–450 loop whose unique location in antigenic space is imparted due to their interaction with antibodies targeting both ER-2 and ER-3 such as REGN-10987. Sites on this loop are estimated to be relatively less antigenic and less mutable than sites within the other two ER-2 clusters. As compared to the other two clusters, the ER-2A biased cluster is relatively low density, with certain residues such as Y449 and N450 spanning this cluster and the ER-2C/D cluster, suggesting that these sites reside within epitope overlap regions and may exhibit certain multi-epitope qualities.Examining the NTD antigenic space map , right wIn addition to observing that well-known VOC mutations with significant contributions to antibody evasion are located in multi-epitope regions of antigenic space describing epitope overlaps , we also note that other key VOC mutations stand out within our computational antigenicity and mutability estimates. In particular, K417 is identified as mutable, has a high estimated antigenicity, and occupies a central position within the ER-2B-biased cluster. Further, K417 is closely associated with residues that have been experimentally measured to have substantial escape effects but that are estimated as immutable due to associated fitness cost such as F456 [Further, we observe multi-epitope roles for P384 and I434 in RBD ER-1 and R346 in RBD ER-3 . R346 isToward understanding how constellations of mutation might explain variant antibody evasion, we annotated the ER-2 + NTD antigenic space map with the mutations of nine major variants whose escape from neutralization had been previously measured experimentally using live virus  Figure . We ordeWe find that variants with weak escape tend to have little coverage of antigenic space while variants with stronger escape feature constellations of mutations with comprehensive coverage of ER-2 + NTD antigenic space. Specifically, we find that variants with weak or mild escape tend to have a single ER-2C/D biased mutation (E484K or L452R) and poor coverage of NTD antigenic space (a single NTD mutation or NTD mutations concentrated within the same NTD cluster). Relative scoring within the weak/mild group is logically explained by antigenic space coverage: Alpha measures the weakest escape and lacks an ER-2C/D biased mutation; Eta/Kappa measure the strongest escape within the weak/mild group and feature at least one ER-2C/D biased mutations in combination with mutations in multiple NTD clusters. The Delta variant is an outlier to this trend with relatively greater mutational coverage than other weakly evading variants, however, we note that Delta features the P681R mutation that has been suggested to enhance susceptibility of live virus to antibody neutralization . MeanwhiThe antigenic space framework provides mechanistic explanations for phenotypic differences between variants. A key open question on relative escape between variants asks why Beta measures stronger antibody evasion than Gamma despite nearly identical RBD mutations and numerous NTD supersite mutations. The antigenic space map illustrates how the constellation of Beta NTD mutations spans the various orthogonal compartments of NTD antigenic space suggesting that the Beta NTD mutations are complementary to each other, while the Gamma NTD mutations are tightly concentrated in a single cluster (Supersite Primary Group) suggesting that the Gamma NTD mutations are functionally redundant with one another in terms of antibody escape. Additionally, Gamma is surprisingly the only variant of the nine examined that lacks a pan-supersite mutation that putatively affects binding of all antibodies targeting the NTD supersite.Given the association between antigenic space coverage and stronger escape from neutralization we sought to present a framework for understanding which mutations on VOC may be more likely to result in complementary escape alongside existing mutations on these VOC. From the set of potential antigenic drift sites (PADS) that we previously estimated to be both mutable and antigenic, we identify a refined set of high-risk sites for the Beta and Delta variants . From thWhile ER-2 is immunodominant and the most potent target of neutralizing antibodies , the polIn this study, we integrated an epitope-paratope interface survey of over 50 RBD/NTD-Ab/nanobody complexes along with experimental datasets describing genetic, structural, and functional constraints on mutation to identify RBD and NTD residues that appear the most mutable and highly targeted by antibodies—the set of potential antigenic drift sites (PADS). The epitope-paratope survey presents a continuous representation of RBD and NTD epitope regions that highlights how antibodies target overlapping or divergent epitopes across RBD and NTD. Further, we find that antibody clustering within the epitope paratope interface matrices captures certain antibody functional qualities, for example distinguishing antibodies that neutralize via avidity  , while other supersite residues only interact with a subset of supersite-directed antibodies. Indeed, we observe that the Gamma variant is the sole variant of the nine variants investigated that lacks a pan-supersite mutation, and propose this distinction from the Beta variant as an explanation for Gamma’s relatively weaker escape from antibodies.Though we do not observe analogous sites within RBD that interact with all antibodies targeting this domain, we find that the RBD ER-2 residues with the largest individual escape effect (E484 and L452) occupy a multi-epitope cluster (ER-2C/D biased) that interacts with a large subset of the antibodies targeting ER-2. Further, E484 is located proximal to the ER-2B biased cluster suggesting that E484 interacts with additional antibodies targeting ER-2B in addition to antibodies targeting ER-2C/D. This finding is supported by examination of the RBD epitope-paratope matrix  in whichFor both RBD and NTD, our study supports the conclusion that residues residing within epitope intersections may be hotspots possessing pan-Ab escape effects. We highlight a number of residues across other RBD and NTD epitopes that may be similar escape hotspots to E484. In particular, we highlight R346 which we find to uniquely serve a pan-epitope function for RBD ER-3  wereWhile predicting VOC emergence and phenotype governed by replication and transmission advantages is difficult or impossible to model and predict, the pandemic’s next phase may be governed by antigenic pressures that are already well-characterized as described in this manuscript. The Antigenic Map framework presented here integrates the wide-ranging data required to chart and interpret the various orthogonal components of RBD and NTD antigenic space. We therefore hope our framework serves as a tool to understand the evolving landscape of antigenic mutations in the RBD and NTD of the spike protein towards guiding vaccine development efforts and public health responses.


Classical Rho GTPases, including RhoA, Rac1, and Cdc42, are members of the Ras small GTPase superfamily and play essential roles in a variety of cellular functions. Rho GTPase signaling can be turned on and off by specific GEFs and GAPs, respectively. These features empower Rho GTPases and their upstream and downstream modulators as targets for scientific research and therapeutic intervention. Specifically, significant therapeutic potential exists for targeting Rho GTPases in neurodegenerative diseases due to their widespread cellular activity and alterations in neural tissues. This study will explore the roles of Rho GTPases in neurodegenerative diseases with focus on the applications of pharmacological modulators in recent discoveries. There have been exciting developments of small molecules, nonsteroidal anti-inflammatory drugs (NSAIDs), and natural products and toxins for each classical Rho GTPase category. A brief overview of each category followed by examples in their applications will be provided. The literature on their roles in various diseases  highlights the unique and broad implications targeting Rho GTPases for potential therapeutic intervention. Clearly, there is increasing knowledge of therapeutic promise from the discovery of pharmacological modulators of Rho GTPases for managing and treating these conditions. The progress is also accompanied by the recognition of complex Rho GTPase modulation where targeting its signaling can improve some aspects of pathogenesis while exacerbating others in the same disease model. Future directions should emphasize the importance of elucidating how different Rho GTPases work in concert and how they produce such widespread yet different cellular responses during neurodegenerative disease progression. Rho GTPases are a subfamily of the Ras superfamily proteins and include the key classical proteins RhoA, Rac1, and Cdc42 of the over 20 that have been identified. In addition to critical functions in the nervous system, Rho GTPases regulate cellular behavior such as morphogenesis, survival, proliferation, membrane trafficking, adhesion, and transcriptional activation  and GTPase-activating proteins (GAPs). Rho proteins are activated in vivo , Rho GTPases have been implicated in nearly all steps of brain development  pathology. One study characterized a direct interaction between a RhoGEF for Rac GTPases Kalirin-7 and synphilin-1 and found that Kalirin-7 expression increased the likelihood of synphilin-1 aggregated Lewy bodies (LB) to be degraded , Parkinson’s disease (PD), Amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), Multiple sclerosis (MS), among others. While it is impossible to cover all regulatory elements of Rho GTPase signaling cascades, this review will present some examples to demonstrate the importance of Rho GTPases in the drug discovery process for neurodegenerative diseases.via the MAPK pathway , RhoA (Ras homolog gene family member A), and Cdc42  and statins have been shown to act likely through the Rho/ROCK signaling pathway directly or indirectly and have been proposed widely as potential therapeutic targets  to activate RhoA  by p75 neurotrophin receptor  in hematopoietic stem and progenitor cells  are produced Additionally, NSAIDs inhibit ADP- and collagen-induced platelet aggregation. It has been discovered that the use of Ibuprofen has led to inhibition of RhoA activation, enhancing axonal regeneration, as well as neuroprotection  is a protein toxin derived from While not a natural toxin, Cucurbitacins—triterpenoid compounds isolated from the Curcurbitacae plants—have been shown to induce actin aggregation and cofilin-actin rod formation —a traditional medicine used for treating CNS diseases in China—that has recently been shown to have anti-inflammatory, anti-HIV, and anti-tumor properties. Treatment with XSE reduced the RhoA/ROCK2 expression in AD model rats as compared to control rats thereby improving memory  all resulting in the cognitive decline associated with the disease. Various studies have shown that cholesterol buildup contributes to Aβ deposit accumulation which, subsequently, led to the discovery that statins (a group of cholesterol reducing drugs) reduce these deposits. Thus, statins show the potential for treatment of AD. Cordle and Landreth  found thAD pathology includes a decrease in dendritic spine density due to the activation of RhoA/ROCK2 in neurons which increases cognitive decline in patients afflicted with AD. The dendritic spine density is reduced when Aβ accumulation activates Caspase-2. Caspase-2 up-regulates RhoA/ROCK2 leading to the relocation of the dendritic spine head and the collapse of neuronal spines. Inhibition of Caspase-2, therefore, represents a potential therapeutic avenue for the treatment of AD to prevent the loss of dendritic spines and ultimately improving learning and memory in AD patients , superoxide dismutase (SOD1), fused in sarcoma (FUS) and ALS2 , both of which exhibit actin dynamic disturbances as a central event associated with motor neuron degeneration. Early targets for SMA/ALS include the synaptic dynamics for proper function and maturation of neuromuscular junctions (NMJs). Treatment with ROCK inhibitor fasudil improves NMJ maturation, and the Rac1-dependent cytoskeletal dysregulation has proven to be a key determinant of motor neuron degeneration  intronic repeat expansion which is often found in ALS patients. Mutating and/or depleting C9ORF72—to mimic the pathogenesis in ALS patients—has been found to enhance phosphorylation of cofilin—inactivating the F-actin assembly function. This mutation/deletion reduces actin dynamics in motor neurons and activates Arf6 which effectively leads to the activation of the downstream effector Rac1  rearrangement of cerebral actin cytoskeleton; (ii) enhanced neurotransmission and synaptic plasticity; and (iii) improved learning and memory  signaling. Therefore, C9ORF72 may function as a dual exchange factor coupling physiological functions such as cytoskeleton modulation and autophagy with endocytosis  neurons. PD pathology consists of characteristic intracellular LB inclusions containing α-Syn aggregates. Familial PD are linked to different mutations in LRRK2, Parkin E3-ubiquitin ligase, and PTEN-induced putative kinase 1 (PINK1), among others  where Miro1 induces Parkin translocation to damaged mitochondria whereas CaMiro is an atypical Rho GTPase and expressed in the outer mitochondrial membrane, anchoring microtubule fiber to healthy mitochondria. Damage to mitochondria results in PINK1 and Parkin relocation in conjunction to affected mitochondria. This relocation effectively leads to a degradation of Miro causing the damaged mitochondria to be removed by lysosomal mitophagy. However, the mutated LRRK2 prevents Miro degradation in damaged mitochondria. A recent discovery of small molecule Miro1 Reducer showed that it can rescue the delayed mitophagy phenotype in PD fibroblasts. Treating patient-derived neurons and fly models with this compound also rescued the locomotor deficits and dopaminergic neurodegeneration  or glucagon-like peptide 1 (GLP-1) was reported to contribute to progressive DA neuron loss in PD. Loss of either of these proteins can lead to an increase of downstream RhoA effectors  causing cell death and subsequent loss of DA neurons. Loganin, one of the best-known iridoid glycosides, is a potential therapeutic treatment that has increased the expression of IGF-1R and GLP-1R. Loganin treatment leads to neurite outgrowth by promoting p-AKT and increasing BDNF, preventing caspase-3 cleavage, and preventing apoptotic cascade activation. In addition, Loganin inhibits RhoA translocation to the membrane down-regulating the downstream RhoA effectors. Thus, Loganin activates many neural protective survival cascades in DA neurons  resulting in loss of function. HD signaling pathways showed highest concentration of dysregulated genes. Primary pathogenic mechanisms of HD link HTT to signaling complexes including Rho GTPases in dynamic organization of neuronal processes and cell-cell contacts  signaling cascades are necessary to effectively evaluate their therapeutic potential. As noted, it is also important to determine the role of Rho GTPase regulators  as such mechanisms have not yet been sufficiently addressed. Even more, crosstalk between Rho GTPases and other molecular regulators underscores the complexity of these pathways. Another obstacle with Rho GTPase modulation In addition, there has been a significant increase in pharmacological tools that have been developed to help effectively screen and target potential therapeutic options. These tools help address the earlier challenges of Rho GTPase drug discovery by enabling exploration of all elements of the Rho GTPase signaling cascade. As such, the elucidation of basic mechanisms, the creation of greater translational models, and the development of more effective screening tools make pharmacologically targeting Rho GTPases ever closer to therapeutic application in neurodegenerative diseases.Rho GTPases serve diverse roles as signaling proteins within cells of the nervous system. Genetic mutations of the genes that encode for Rho GTPases as well as mutations that interrupt the normal functioning of their modulators can lead to many diseases including neurodegenerative disorders. The modulations of Rho GTPases have great translational potential due to Rho GTPase’s strong implications in these human diseases. Meanwhile, recent progresses are also accompanied by the recognition of complex Rho GTPase modulation where targeting its signaling can improve some aspects of pathogenesis while exacerbating others in the same disease model. Therefore. much is still to be elucidated on how different Rho GTPases work in concert and how they produce such widespread yet selective cellular responses during disease progression.WG, AK, CB, and QL wrote the article, held discussions, and finalized article. WG drafted figure and table. CB revised table. QL conceptualized the article and guided the writing project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Multiple field studies provide qualitative accounts of usability barriers experienced by users of wheeled mobility devices on public transit. This study aimed to examine these usability barriers from the theoretical perspective of Environmental Docility by quantifying the relationship between functional capabilities of wheeled mobility device users and ingress–egress performance on accessible fixed-route transit vehicles in an urban setting. Twenty-eight wheeled mobility users each completed three trips on a predetermined route through the local public transit system. Ingress and egress times, user-reported usability ratings and open-ended comments were analyzed. Regression analyses indicated significant interactions between age and minimum parallel-park length on ingress and egress times. Specifically, lower functional capability reflected in older age and less maneuvering ability predicted decreased performance (longer ingress–egress times), indicating less adaptability to environmental demands and agreement with the Environmental Docility Hypothesis. Usability ratings and comments revealed difficulty with negotiating access ramps and turning maneuvers in the vehicle interior and in proximity to other passengers. Despite compliance with accessibility standards, current design of transit vehicles present substantial usability barriers for wheeled mobility users. Environmental Docility provides a theoretical basis to identifying modifiable factors related to person and environment for improving usability of public transit for people aging and/or with mobility impairments. Accessible public transportation is vital to supporting community participation, positive health behaviors, and successful aging among people with disabilities ,2,3. In Users of wheeled mobility devices  in particular face unique design barriers when using fixed-route transit buses—the most prevalent mode of public transportation ,15,16,17Problems related to accessibility for wheeled mobility users on fixed-route transit buses are not unique to the US ,28,29. MThis study was part of a research effort to operationalize relationships between functional capabilities of wheeled mobility device users and environmental barriers to community mobility including the use of public transit. The study was theoretically based in the ecological model of adaptation and aging ,40. CentTo characterize the interaction between person and environment, the ecological model introduces concepts of environmental demand or “press” exerted upon the person , and functional capability or “competence” of the person encountering the environment. The model posits that by achieving a balance or fit between individual competence and environmental demand a user can be productively challenged by the environment and experience a sense of well-being as a consequence of behavior being adaptive . ConversThe aim of this study was to quantify the relationship between functional capabilities of wheeled mobility device users (person component) and task performance  during ingress–egress on accessible low-floor public transit vehicles in a naturalistic fixed-route urban setting . Consistent with the Environmental Docility Hypothesis, we hypothesized that lower functional capabilities would be associated with lower performance  under conditions of a moderately challenging environmental demand. Usability ratings of task difficulty and acceptability and open-ended comments were analyzed to understand and corroborate aggregate performance measures of ingress–egress times.This study used a mixed-method approach consisting of demographic questionnaires and functional capacity assessments in an indoor laboratory, followed by a field-based ride-along technique that had participants independently complete 3 transit bus trips in succession through a predetermined route on the local public transit system. Two researchers shadowed the participant on the trips conducting a time-study and structured observations, and later administered post-trip ratings questionnaires. The study design was quasi-experimental implying that some study aspects were experimentally controlled  or held constant , while other aspects of the study were naturalistic and uncontrolled reflecting real-world conditions .Twenty-eight community-dwelling adults that regularly used wheeled mobility devices were recruited to participate in the study. They included 6 front-wheel drive powered wheelchair users (PWU), 11 mid-wheel drive PWU, 6 manual wheelchair users (MWU), and 5 scooter users (SU). Criteria for study inclusion required that participants rely on a wheeled mobility device as their primary means of mobility, be able to independently maneuver their mobility device, and use access ramps in buildings without assistance. Prior experience using public transit was not necessary, although participants were informed during recruitment that the study would entail riding public transit. In order to obtain a diverse sample, the research team recruited participants through multiple sources, including a local independent living center, the university community, advertisement postings in a local newsletter, and recruitment flyers posted at local senior centers, medical and rehabilitation centers, and public transit terminal. The university’s institutional review board approved the study procedures. All participants provided written informed consent prior to participation. The study was conducted with one participant at a time, and required between 2.5 and 3.0 h to complete with each participants receiving a US$75 honorarium.The study was conducted in Ann Arbor, Michigan—a mid-western city in the US that is relatively flat in topography. The local transit service provider  provides fixed-route and complementary ADA paratransit services to the city and nearby townships. The transit provider has a fixed-route operational fleet consisting of 80 conventional 12 m (40-ft) length low-floor buses . Fares wThe vehicles were compliant with prevailing federal accessibility standards ,21. Key The WMSAs were each equipped with a four-point wheelchair tie-down system for securing the wheeled mobility devices in a forward-facing securement position. These are the most common type of wheeled mobility securement systems used on transit vehicles in the US. While federal standards mandate the provision of wheelchair tie-downs and occupant restraint systems , it permits individual transit service providers to establish their own policies regarding the use of these systems . Hence, This first portion of the study was conducted at a private indoor facility close to the first bus stop. Participants were administered a questionnaire to obtain information about their age, gender, dominant hand, primary medical condition related to use of a wheeled mobility device, type of wheeled mobility device used, years of use, and the type and frequency of different travel modes used. Measurements of occupied mass  were obtained using a platform weighing scale. Occupied and unoccupied device length and width were measured using standard anthropometry calipers and tape measure.Three additional measures of functional capacity were obtained. Power Grip Strength was recorded as the average of three measurements of 3 s maximal power grip exertions measured on the dominant hand using a Jamar grip dynamometer with the elbow flexed at urements ,47. Poweurements ,49,50.Prior task analysis identified three types of wheelchair turns relevant to ingress–egress on transit vehicles, namely, a 90maneuver . This st90∘ turn ,53. A 18ccessful ,53. Use ccessful .Next, each participant completed 3 bus trips in succession through a predefined route on the local transit system. Two researchers accompanied the participant. One researcher was responsible for providing the participant with instructions before the trip , and administered a usability questionnaire after each bus trip . A second researcher performed direct observations and a time-study of the ingress–egress process using a handheld tablet PC. Data recorded for each trip also included: date, time of day, passenger load , number of passengers entering and/or exiting by their mobility status , and stop dwell-time. In order to minimize influence on ingress–egress times and potential observational bias, from the arrival of the bus until egress at the destination, both researchers assumed the role of independent passengers and avoided direct interaction or communication with the participant, i.e., fly-on-the-wall technique . The resThe general sequence of ingress tasks performed by the participant included: ramp ascent at the front doorway, moving to the WMSA, and entering and positioning in the WMSA. If both securement areas were vacant, the participant had the choice of using either one. Fare payment was not performed due to prior paratransit eligibility. All of the participants completed the bus trip while seated in their device and thus had their mobility device secured by the bus driver at the completion of ingress, and securement removed upon arriving at the destination. After securement removal, the sequence of egress tasks performed by the participant included: maneuvering out of the securement area, moving to the front doorway, and descending the access ramp. Participants were instructed to complete the ingress–egress tasks as they normally would, interacting with the bus driver when needed, but without acknowledging or communicating with the researchers.The three bus trips undertaken were in a loop  the participant was administered a questionnaire to rate the difficulty of seven tasks performed on the preceding bus trip using the Difficulty Rating Scale, namely, (1) moving to the vehicle access ramp at the bus stop, (2) ramp ascent, (3) moving to the WMSA, (4) entry and positioning in WMSA during ingress, (5) exiting the WMSA, (6) moving to the front door, and (7) ramp descent during egress. An overall rating of acceptability for the ingress–egress experience was obtained using the Acceptability Rating Scale.The Difficulty and Acceptability Rating Scales are widely used measures of environmental usability and with established psychometric properties ,58,59,60R v.3.6.1 [Statistical analyses were performed using  v.3.6.1 . SummaryIn order to minimize the effects of uncontrollable factors across bus trips  that could influence participant behavior, the task times and user-reported ratings were averaged across the three bus trips for each participant for analysis purposes. One task time each from two participants were considered outliers and excluded from the analysis, namely, (i) Trip-2 ingress for one participant in a mid-wheel powered wheelchair was excessively delayed (267 s) due a different passenger in a wheelchair who was already on-board, and (ii) Trip-1 egress for one scooter user was delayed (263 s) due to the bus driver leaving the vehicle for a short while for reasons unknown to the researchers. Task times for these two participants were obtained by averaging the times from the remaining two trips. Average task times by user group were summarized graphically using box-plots.Linear regression was used to test the study’s main objective, namely, examining the effects of 9 functional capability measures , arthritis (14.3%), and orthopedic impairments (14.3%) . ParticiR = 0.66), occupied mass (R = 0.56), and grip strength (R = −0.56).User groups differed significantly in occupied mass, power grip strength and minimum parallel park length . On averAverage ± SD times for ingress and egress were er group .+39.0 s (+132%) for older users (age: +1 SD);+19.8 s (+67%) for average-aged users (age: 0);Similar ingress times for younger users (age: −1 SD).Regression analyses yielded reduced models with significant interaction effects of standardized age and parallel park length on ingress times R2=0.18) . Figure +23.6 s (+71%) for older users (age: +1 SD);+9.2 s (+28%) for average-aged users (age: 0);A marginal −5.2 s (−16%) decrease in egress times for younger users (age: −1 SD).Likewise, compared to an average egress time of 33.2 s for users of average age and short parallel park length (−1 SD), a +1 SD long increase in parallel park length was associated with an increase in egress time by:p=0.063) . Correspelchairs , howeverelchairs .Comments regarding ramp ascent concerned difficulty with turning at the top of the access ramp due to obstruction from the floor-mounted fare machine , steep ramp slope , limited ramp width (three SU), and limited clearance space on the bus-stop when turning on to the access ramp . Due to concerns about the ramp slope two participants  opted to ascend the ramp facing rear-wards as an adaptive strategy, while one MWU sought assistance from the bus driver with ramp ascent.Remarks concerning moving to the WMSA were about the lack of choice in securement location either due to another wheelchair present on-board or the driver directing the participant to a specific location (two front-wheel drive PWU), limited space due to other passengers on-board with large items, e.g., a walker (one mid-wheel drive PWU), or general anxiety with the task anticipating difficulty with maneuvering in a constrained space (1 SU).The related task of entry and positioning in the WMSA required performing a A few participants also expressed concerns about the device securement attachment and removal process. These comments referred to the inability of the bus driver to locate proper attachment points on the device and efficiently secure the device causing delays  and a perceived uncooperative attitude from the bus driver .Comments regarding exiting the WMSA concerned limited space for turning into the center aisle . Two participants also expressed difficulty reaching the stop cord  to indicate their destination stop. Moving to the exit door was challenged by limited aisle width near the front wheel-wells (one mid-wheel drive PWU) and safety concerns when other passengers are also exiting or entering the vehicle (one SU).Remarks regarding ramp descent pertained to limited space for turning at the top of the ramp due to the obtrusive floor-mounted fare machine , limited ramp width , steep ramp slope (one MWU), and limited runoff space on the bus-stop at the bottom of the ramp . One participant requested verbal cues from the bus driver to help align with ramp edges prior to ramp descent.The Environmental Docility Hypothesis predicts that individuals with lower functional capability are more vulnerable to environmental demands resulting in lower performance compared to individuals with higher functional capability. Study findings support this hypothesis in the specific context of wheeled mobility device users during ingress–egress on low-floor public transit buses in a field setting. Importantly, task performance decreased  for individuals with lower functional capability reflected in increased age and less maneuvering ability . User-reported task ratings and open-ended interview comments revealed difficulty with negotiating turning maneuvers and ramp gradients within the spatially constrained vehicle interior and when in close proximity to other passengers . These cGrounding this study in the Environmental Docility Hypothesis provides a theoretical perspective on the interactive relationship between person  and environment  on behavior and/or performance outcomes. Environmental demand in this study was assumed to be of a moderate intensity—since the transit vehicles and bus stops were compliant with federal accessibility regulations, and a constant factor across participants—since relevant external factors were either similar  or averaged over multiple trips to minimize uncontrolled influences . Future studies could consider more detailed and objective measures to operationalize environmental demand across different types/designs of transit vehicles and bus-stops, transit service provider policies, and/or geographical location ,70.The person component was intentionally operationalized using functional capability measures rather than medical conditions or illnesses . CompareConceptualizing the disablement process as a dynamic interaction between an individual’s capabilities and the environmental context helps to identify modifiable factors for improving accessibility and usability of public transit and outcomes for individuals with disabilities. A consequence of the Environmental Docility Hypothesis is that behaviour and/or performance in activities relevant to daily living can be increased by enhancing functional capacity—the focus of rehabilitation practice, and/or by lowering environmental demand—often the emphasis of engineering design and policy. We discuss a few key implications of this study’s findings to both domains.Knowledge of the relationships between functional capability and environmental demand are important when making decisions about a patients mobility needs. Example applications include when prescribing and customizing a wheeled mobility device to overcome specific functional deficits, when defining individual rehabilitation and functional training goals for wheeled mobility users, for evaluating an individual’s ability to use public transit, and/or documenting their eligibility for special transportation services such as ADA paratransit.The significant interaction between age and maneuverability on task performance suggests a compounding of both these effects resulting in lowest performance for users that were older and had lower maneuvering capabilities. Younger and higher functioning individuals took significantly less time for ingress–egress suggesting greater adaptability to cope with the demands of transit vehicle ingress–egress, compared to their older and lower functioning counterparts. This finding corroborates qualitative studies that report poor mobility skills and lack of mobility confidence among older wheeled mobility users which severely limits their social participation . The infParallel park length as a measure of maneuverability is a combination of multiple modifiable factors related to the occupant and device jointly. Wheeled mobility devices support a broad range of health and functional needs stemming from diverse medical conditions ,74. Manuuverable . Overalluverable ,79. Stud ratings .Clearly, the maneuverability of different mobility device types have implications for usability of the built environment  and overall community mobility. Multiple studies point to the trade-offs when using powered wheelchairs and scooters in terms of their ability to traverse longer distances versus challenges when maneuvering in confined spaces including compromised safety  ,73,80,81ss ramps ,85.Article 9 of the UN Convention on the Rights of Persons with Disabilities requires that governments develop and implement standards to ensure accessibility of public transportation services . Hence, 9.5∘ . Howeverto 15.5∘ . Ramp asto 15.5∘ . Ramp deto 15.5∘ . The intto 15.5∘ ,51. Currto 15.5∘ .The focus on ingress–egress times and its relationship to dwell-time also has operational and economic implications for public transit service providers. Delays in dwell-time and compromised passenger safety adversely impacts service quality for all riders ,57. SpecThis study leveraged aspects of both field and laboratory settings. The study attained ecological validity by assessing task performance in participants’ natural transportation environment. Uncontrollable factors were either treated as a constant  or averaged over multiple trials to minimize effects of factors such as crowding, bus driver, and bus stop conditions. Care was taken to only include task times that pertained to the wheeled mobility user using established procedures ,89. Taskus study . IncorpoFindings from this study also emphasize the value of multi-method approaches to operationalize accessibility and usability in person–environment research. Generally, quantitative measures of task performance offer high validity, granularity (level of detail), and sensitivity to changes in capability and/or task demand. However, performance alone is an insufficient measure of usability ,59. The The present study recruited a convenience sample of wheeled mobility users based on their self-reported ability to independently maneuver and use access ramps without assistance. Thus, the study sample is potentially biased toward more experienced and high functioning users, which may have led to underestimating ingress–egress times and user-reported difficulty relative to the general population of wheeled mobility users. Data on the proportion of all community-dwelling wheeled mobility users that meet these criteria and thus potentially capable of using fixed-route transit  is not readily available.This study’s sample size was relatively modest. Maneuvering capability  and age were identified as significant predictors that explained the most variance in performance. This result should not be interpreted as other functional capability measures as being irrelevant, but only that they explained less model variance in a relative sense. In particular, parallel park length showed a moderate positive correlation with occupied mass and length, and a moderate negative correlation with power grip strength. While a larger sample would be beneficial in terms of statistical power and generalizability, the present sample was sufficient to yield initial evidence supporting the hypothesis and to identify key usability challenges associated with ingress–egress on fixed-route transit.The study was limited to wheeled mobility ingress–egress on fixed-route transit vehicles. Hence, it presently did not include other aspects of the travel chain  that present their own unique set of environmental barriers ,28,29,91This study uniquely operationalized the Environmental Docility Hypothesis in the specific context of wheeled mobility users’ ingress–egress performance on fixed-route transit vehicles. Device users of lower functional capability, reflected in older age and less maneuvering ability, took significantly longer time for ingress and egress, suggesting less adaptability to environmental demands compared to users with higher functional capabilities. User-reported task ratings and open-ended interview comments revealed difficulty with negotiating turning maneuvers and ramp gradients in the vehicle interior and in close proximity to other passengers.Wheeled mobility devices are critical to supporting independence, autonomy, and social participation for people with severe mobility impairments. The ability to use public transit provides wheeled mobility users a cost-effective and reliable means of accessing community resources and successfully age in place. Despite decades of nationwide compliance with federal accessibility regulations in the US, the physical design of current transit vehicles still presents usability barriers for users of contemporary wheeled mobility devices. However, these usability barriers in public transit and the implications of this study are not limited to the US. Conceptualizing usability barriers from an ecological perspective, specifically the Environmental Docility Hypothesis demonstrated in this study, can help identify modifiable factors related to person  and environment  for improving accessibility and usability of public transit for people aging and/or with mobility impairments requiring wheeled mobility devices.