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Biological databases and computational biology tools are provided by research groups around the world, and made accessible on the Web. Combining these resources is a common practice in bioinformatics, but integration of heterogeneous and often distributed tools and datasets can be challenging. To date, this challenge has been commonly addressed in a pragmatic way, by tedious and error-prone scripting. Recently however a more reliable technique has been identified and proposed as the platform that would tie together bioinformatics resources, namely Web Services. In the last decade the Web Services have spread wide in bioinformatics, and earned the title of recommended technology. However, in the era of high-throughput experimentation, a major concern regarding Web Services is their ability to handle large-scale data traffic. We propose a stream-like communication pattern for standard SOAP Web Services, that enables efficient flow of large data traffic between a workflow orchestrator and Web Services. We evaluated the data-partitioning strategy by comparing it with typical communication patterns on an example pipeline for genomic sequence annotation. The results show that data-partitioning lowers resource demands of services and increases their throughput, which in consequence allows to execute in-silico experiments on genome-scale, using standard SOAP Web Services and workflows. As a proof-of-principle we annotated an RNA-seq dataset using a plain BPEL workflow engine.

Abstract Background The mammalian brain is divided into distinct regions with structural and neurophysiological differences. As a result, gene expression is likely to vary between regions in relation to their cellular composition and neuronal function. In order to improve our knowledge and understanding of regional patterns of gene expression in the CNS, we have generated a global map of gene expression in selected regions of the adult rat brain (frontomedial-, temporal- and occipital cortex, hippocampus, striatum and cerebellum; both right and left sides) as well as in three major non-neural tissues (spleen, liver and kidney) using the Applied Biosystems Rat Genome Survey Microarray. Results By unsupervised hierarchical clustering, we found that the transcriptome within a region was highly conserved among individual rats and that there were no systematic differences between the two hemispheres (right versus left side). Further, we identified distinct sets of genes showing significant regional enrichment. Functional annotation of each of these gene sets clearly reflected several important physiological features of the region in question, including synaptic transmission within the cortex, neurogenesis in hippocampus and G-protein-mediated signalling in striatum. In addition, we were able to reveal potentially new regional features, such as mRNA transcription- and neurogenesis-annotated activities in cerebellum and differential use of glutamate signalling between regions. Finally, we determined a set of 'CNS-signature' genes that uncover characteristics of several common neuronal processes in the CNS, with marked over-representation of specific features of synaptic transmission, ion transport and cell communication, as well as numerous novel unclassified genes. Conclusion We have generated a global map of gene expression in the rat brain and used this to determine functional processes and pathways that have a regional preference or ubiquitous distribution within the CNS, respectively. The existence of shared specialised neuronal activities in CNS is interesting in a context of potential functional redundancy, and future studies should further explore the overall characteristics of CNS-specific versus region-specific gene profiles in the brain.

Abstract Background Sharing of microarray data within the research community has been greatly facilitated by the development of the disclosure and communication standards MIAME and MAGE-ML by the MGED Society. However, the complexity of the MAGE-ML format has made its use impractical for laboratories lacking dedicated bioinformatics support. Results We propose a simple tab-delimited, spreadsheet-based format, MAGE-TAB, which will become a part of the MAGE microarray data standard and can be used for annotating and communicating microarray data in a MIAME compliant fashion. Conclusion MAGE-TAB will enable laboratories without bioinformatics experience or support to manage, exchange and submit well-annotated microarray data in a standard format using a spreadsheet. The MAGE-TAB format is self-contained, and does not require an understanding of MAGE-ML or XML.

Background: Understanding wellbeing in adolescents and within education settings is crucial to supporting young people. However, research defining and exploring wellbeing has typically taken a focus on subjective, psychological, social and emotional domains and has failed to incorporate aspects of physical health and wellbeing. This study aimed to explore how both physical and subjective and psychological wellbeing can be combined to generate different profiles of wellbeing in adolescents, and to understand the characteristics associated with this profile membership., Methods: 366 adolescents aged 11-16yrs (mean age 12.75) from three mainstream secondary schools across England completed an online survey capturing demographic characteristics, physical, subjective and psychological wellbeing, physical activity, emotional literacy, school belonging, and perceptions of learning ability. Latent profile analysis used a data driven approach to explore profiles of wellbeing using physical wellbeing and positive emotional state and positive outlook as predictors of profile membership. To understand profile characteristics demographics, physical activity and educational variables were added as co-variates., Results: Three profiles were identified, (1) low wellbeing (n = 68, 19%) displaying low scores across physical wellbeing, positive emotional state and positive outlook, (2) moderate wellbeing (n = 168, 46%) characterised by average levels across physical wellbeing, positive emotional state and positive outlook, and (3) high wellbeing (n = 128, 35%) showing high score across physical wellbeing, positive emotional state and positive outlook. Compared to the high wellbeing profile, the moderate and low profiles membership was characterised by being older, being a girl, lower perceived socio-economic status, fewer hours of physical activity a week, and lower emotional literacy, school belonging and perceptions of learning., Conclusions: The results evidence that physical, subjective and psychological wellbeing are closely inter-related, this finding coupled with increased physical activity in the higher wellbeing group signify physical health and activity are important components of overall wellbeing and should form part of a holistic approach to school wellbeing curriculums., Competing Interests: Declarations. Ethics approval and consent to participate: Ethical approval was granted by the University of Manchester University Research Ethics Committee 2 (Ref: 2022-14607-24837). All procedures on ethical working and taking consent with young people was in accordance with the British Psychological Society ethical guidelines 2021 and the University of Manchester Research Governance, Ethics and Integrity protocols. Informed consent was obtained for all participants, as participants were under the age of 16 parental/carer informed consent was obtained alongside young person assent. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Impulsivity is a behavioral trait that is elevated in many neuropsychiatric disorders. Parkinson's disease (PD) patients can exhibit a specific pattern of reward-seeking impulsive-compulsive behaviors (ICBs), as well as more subtle changes to generalized trait impulsivity. Prior studies in healthy controls (HCs) suggest that trait impulsivity is regulated by D 2/3 autoreceptors in mesocorticolimbic circuits. While altered D 2/3 binding is noted in ICB + PD patients, there is limited prior assessment of the trait impulsivity-D 2/3 relationship in PD, and no prior direct comparison with patterns in HCs. We examined 54 PD (36 M; 18 F) and 31 sex- and age-matched HC (21 M; 10 F) subjects using [ 18 F]fallypride, a high-affinity D 2/3 receptor ligand, to measure striatal and extrastriatal D 2/3 nondisplaceable binding potential (BP ND ). Subcortical and cortical assessment exclusively used ROI or exploratory-voxelwise methods, respectively. All completed the Barratt Impulsiveness Scale, a measure of trait impulsivity. Subcortical ROI analyses indicated a negative relationship between trait impulsivity and D 2/3 BP ND in the ventral striatum and amygdala of HCs but not in PD. By contrast, voxelwise methods demonstrated a positive trait impulsivity-D 2/3 BP ND correlation in ventral frontal olfactocentric-paralimbic cortex of subjects with PD but not HCs. Subscale analysis also highlighted different aspects of impulsivity, with significant interactions between group and motor impulsivity in the ventral striatum, and attentional impulsivity in the amygdala and frontal paralimbic cortex. These results suggest that dopamine functioning in distinct regions of the mesocorticolimbic circuit influence aspects of impulsivity, with the relative importance of regional dopamine functions shifting in the neuropharmacological context of PD. SIGNIFICANCE STATEMENT The biological determinants of impulsivity have broad clinical relevance, from addiction to neurodegenerative disorders. Here, we address biomolecular distinctions in Parkinson's disease. This is the first study to evaluate a large cohort of Parkinson's disease patients and age-matched healthy controls with a measure of trait impulsivity and concurrent [ 18 F]fallypride PET, a method that allows quantification of D2/3 receptors throughout the mesocorticolimbic network. We demonstrate widespread differences in the trait impulsivity-dopamine relationship, including (1) loss of subcortical relationships present in the healthy brain and (2) emergence of a new relationship in a limbic cortical area. This illustrates the loss of mechanisms of behavioral regulation present in the healthy brain while suggesting a potential compensatory response and target for future investigation., (Copyright © 2023 the authors.)

Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, the proper functioning of Myo6 relies on precise spatiotemporal control of motor activity via a poorly defined off-state and interactions with partners. Our structural, functional, and cellular studies reveal key features of myosin regulation and indicate that not all partners can activate Myo6. TOM1 and Dab2 cannot bind the off-state, while GIPC1 binds Myo6, releases its auto-inhibition and triggers proximal dimerization. Myo6 partners thus differentially recruit Myo6. We solved a crystal structure of the proximal dimerization domain, and show that its disruption compromises endocytosis in HeLa cells, emphasizing the importance of Myo6 dimerization. Finally, we show that the L926Q deafness mutation disrupts Myo6 auto-inhibition and indirectly impairs proximal dimerization. Our study thus demonstrates the importance of partners in the control of Myo6 auto-inhibition, localization, and activation., (© 2023. Springer Nature Limited.)

Background: Persons with HIV (PWH) have both more frequent depression and higher levels of plasma inflammatory biomarkers compared to persons without HIV (PWoH). Inflammation and depressive symptoms are linked, including in PWH; however, it is unclear whether these associations differ by HIV serostatus and biological sex., Methods: Six plasma inflammatory biomarkers were assessed using samples from PWH and PWoH who participated in six NIH-funded studies through the UCSD HIV Neurobehavioral Research Program (HNRP) from 2011 to 2019. Factor analysis was performed to identify intercorrelated groups of biomarkers. Factors and their components were then examined for relationships with Beck Depression Inventory-II (BDI-II) and modifying effects of sex or HIV serostatus using multivariable linear regression, adjusting for demographics, substance use diagnoses, and relevant co-morbidities., Results: Participants included 150 PWH (age = 48.3 ± 13.1 yr; 88% biologically male) and 138 PWoH (age = 46.3 ± 15.9; 56% male). Two inflammatory factors were identified: Factor 1 loaded on interleukin-6 (IL-6), C-reactive protein (CRP), and D-dimer; Factor 2 loaded on interleukin-8, chemokine C-C ligand 2 (CCL2), and chemokine C-X-C ligand 10 (CXCL10). Sex modified the effect of Factor 1 on BDI-II, with a more positive association for men than women ( p  = 0.04). No significant association between Factor 2 and BDI-II was found. Of the biomarkers in Factor 1, only IL-6 was significantly associated with BDI-II and was modified by sex ( p  = 0.003). In sex-stratified analysis, a positive association was found for men (β = 5.42; 95% confidence interval = [1.32, 9.52]) but not women (β = -3.88; 95% C.I. = [-11.02, 3.26]). No HIV-related interactions were detected., Interpretation: We identified a depression-associated inflammatory factor present in both PWH and PWoH, consistent with prior studies of PWH only. The association was driven by a correlation between IL-6 and depression exclusively in men, suggesting that the depression-inflammation link differs by sex. Future studies of depression etiology or treatment, including those on persons with HIV, should consider the impact of biological sex in both design and analysis., Competing Interests: None., (© 2023 The Authors. Published by Elsevier Inc.)

Background: Combination antiretroviral therapy (cART) has allowed for viral load (VL) suppression and increased life expectancy for persons with HIV (PWH). Altered brain integrity, measured by neuropsychological (NP) performance and neuroimaging, is still prevalent among virally suppressed PWH. Age-related conditions such as cardiovascular disease may also affect brain integrity. This study investigated the effects of cardiovascular risk, VL, and HIV serostatus on cerebral blood flow (CBF), brain volumetrics, and cognitive function in PWH and persons without HIV (PWoH)., Methods: Ten-year cardiovascular risk, using the Framingham Heart Study criteria, was calculated in PWH (n = 164) on cART with undetectable (≤20 copies/mL; n = 134) or detectable (>20 copies/mL; n = 30) VL and PWoH (n = 66). The effects of cardiovascular risk on brain integrity (CBF, volume, and cognition) were compared for PWH (undetectable and detectable VL) and PWoH., Results: PWH had smaller brain volumes and worse NP scores than PWoH. PWH with detectable and undetectable VL had similar brain integrity measures. Higher cardiovascular risk was associated with smaller volumes and lower CBF in multiple brain regions for PWH and PWoH. Significant interactions between HIV serostatus and cardiovascular risk on brain volumes were observed in frontal, orbitofrontal, and motor regions. Cardiovascular risk was not associated with cognition for PWH or PWoH., Conclusions: Neuroimaging, but not cognitive measures, was associated with elevated cardiovascular risk. HIV serostatus was associated with diminished brain volumes and worse cognition while CBF remained unchanged, reflecting potential protective effects of cART. Neuroimaging measures of structure (volume) and function (CBF) may identify contributions of comorbidities, but future longitudinal studies are needed., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: The orbitofrontal cortex (OFC) is involved in diverse cognitive and behavioral processes including incentive valuation, decision-making, and reinforcement learning. Anatomic and cytoarchitectonic studies divide the OFC along both medial-lateral and rostral-caudal axes. OFC regions diverge in structure and function, assessed in vivo using white matter tractography and blood oxygenation level-dependent (BOLD) MRI, respectively. However, interpretation of T 2 *-weighted BOLD is limited by susceptibility artifacts in the inferior frontal lobes, with the spatial pattern of these artifacts frequently assuming the geometry of OFC organization. Here, we utilize a novel perfusion-weighted arterial spin labeling (ASL) functional connectivity approach, which is minimally susceptibility-weighted, to test the hypothesis that OFC topology reflects correlated temporal hemodynamic activity., Methods: In healthy participants (n = 20; age = 29.5 ± 7.3), 3D ASL scans were acquired (TR/TE = 3,900/13 ms; spatial resolution = 3.8 mm isotropic). To evaluate reproducibility, follow-up scanning on a separate day was performed on a participant subset (n = 8). ASL-based connectivity was modeled for gray matter OFC voxels, and k-means clustering (k = 2-8) applied to correlation statistics., Results: These approaches revealed both medial-lateral and rostral-caudal OFC divisions, confirming our hypothesis. Longitudinal reproducibility testing revealed 84% voxel clustering agreement between sessions for the k = 2 solution., Conclusion: To our knowledge, this constitutes the first in vivo cortical parcellation based on perfusion fluctuations. Our approach confirms functional OFC subdivisions predicted from anatomy using a less susceptibility-sensitive method than the conventional approach., (© 2021 The Authors. Brain and Behavior published by Wiley Periodicals LLC.)

Objective: Lipedema is characterized by pain, fatigue, and excessive adipose tissue and sodium accumulation of the lower extremities. This case-control study aims to determine whether sodium or vascular dysfunction is present in the central nervous system., Methods: Brain magnetic resonance imaging was performed at 3 T in patients with lipedema (n = 15) and control (n = 18) participants matched for sex, age, race, and BMI. Standard anatomical imaging and intracranial angiography were applied to evaluate brain volume and vasculopathy, respectively; arterial spin labeling and sodium magnetic resonance imaging were applied to quantify cerebral blood flow (CBF) (milliliters per 100 grams of tissue/minute) and brain tissue sodium content (millimoles per liter), respectively. A Mann-Whitney U test (significance criteria P < 0.05) was applied to evaluate group differences., Results: No differences in tissue volume, white matter hyperintensities, intracranial vasculopathy, or tissue sodium content were observed between groups. Gray matter CBF was elevated (P = 0.03) in patients with lipedema (57.2 ± 9.6 mL per 100 g/min) versus control participants (49.8 ± 9.1 mL per 100 g/min)., Conclusions: Findings provide evidence that brain sodium and tissue fractions are similar between patients with lipedema and control participants and that patients with lipedema do not exhibit abnormal radiological indicators of intracranial vasculopathy or ischemic injury. Potential explanations for elevated CBF are discussed in the context of the growing literature on lipedema symptomatology and vascular dysfunction., (© 2020 The Obesity Society.)

Objective: The aim of this study is to compare tissue sodium and fat content in the upper and lower extremities of participants with lipedema versus controls using magnetic resonance imaging (MRI)., Methods: MRI was performed at 3.0 T in females with lipedema (n = 15, age = 43.2 ± 10.0 years, BMI = 30.3 ± 4.4 kg/m 2 ) and controls without lipedema (n = 14, age = 42.8 ± 13.2 years, BMI = 28.8 ± 4.4 kg/m 2 ). Participants were assessed for pain and disease stage. Sodium MRI was performed in the forearm and calf to quantify regional tissue sodium content (TSC, mmol/L). Chemical-shift-encoded water-fat MRI was performed in identical regions for measurement of fat/water (ratio)., Results: In the calf, skin TSC (16.3 ± 2.6 vs. 14.4 ± 2.2 mmol/L, P = 0.04), muscle TSC (20.3 ± 3.0 vs. 18.3 ± 1.7 mmol/L, P = 0.03), and fat/water (1.03 ± 0.37 vs. 0.56 ± 0.21 ratio, P < 0.001) were significantly higher in participants with lipedema versus control participants. In the forearm, skin TSC (13.4 ± 3.3 vs. 12.0 ± 2.3 mmol/L, P = 0.2, Cohen's d = 0.50) and fat/water (0.65 ± 0.24 vs. 0.48 ± 0.24 ratio, P = 0.07, Cohen's d = 0.68) demonstrated moderate effect sizes in participants with lipedema versus control participants. Calf skin TSC was significantly correlated with pain (Spearman's rho = 0.55, P = 0.03) and disease stage (Spearman's rho = 0.82, P < 0.001) among participants with lipedema., Conclusions: MRI-measured tissue sodium and fat content are significantly higher in the lower extremities, but not upper extremities, of patients with lipedema compared with BMI-matched controls., (© 2020 The Obesity Society.)

The dentate nucleus (DN) is a gray matter structure deep in the cerebellum involved in motor coordination, sensory input integration, executive planning, language, and visuospatial function. The DN is an emerging biomarker of disease, informing studies that advance pathophysiologic understanding of neurodegenerative and related disorders. The main challenge in defining the DN radiologically is that, like many deep gray matter structures, it has poor contrast in T1-weighted magnetic resonance (MR) images and therefore requires specialized MR acquisitions for visualization. Manual tracing of the DN across multiple acquisitions is resource-intensive and does not scale well to large datasets. We describe a technique that automatically segments the DN using deep learning (DL) on common imaging sequences, such as T1-weighted, T2-weighted, and diffusion MR imaging. We trained a DL algorithm that can automatically delineate the DN and provide an estimate of its volume. The automatic segmentation achieved higher agreement to the manual labels compared to template registration, which is the current common practice in DN segmentation or multiatlas segmentation of manual labels. Across all sequences, the FA maps achieved the highest mean Dice similarity coefficient (DSC) of 0.83 compared to T1 imaging ( DSC = 0.76 ), T2 imaging ( DSC = 0.79 ), or a multisequence approach ( DSC = 0.80 ). A single atlas registration approach using the spatially unbiased atlas template of the cerebellum and brainstem template achieved a DSC of 0.23, and multi-atlas segmentation achieved a DSC of 0.33. Overall, we propose a method of delineating the DN on clinical imaging that can reproduce manual labels with higher accuracy than current atlas-based tools., (© 2019 Society of Photo-Optical Instrumentation Engineers (SPIE).)

Background: Latent class analysis (LCA) can be used to identify subgroups of children with similar patterns of mental health symptoms and/or strengths. The method is becoming more commonly used in child mental health research, but there are reservations about the replicability, reliability, and validity of findings. Objective: A systematic literature review was conducted to investigate the extent to which LCA has been used to study population mental health in children, and whether replicable, reliable and valid findings have been demonstrated. Methods: Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed. A search of literature, published between January 1998 and December 2017, was carried out using MEDLINE, EMBASE, PsycInfo, Scopus, ERIC, ASSIA, and Google Scholar. A total of 2,748 studies were initially identified, of which 23 were eligible for review. The review examined the methods which studies had used to choose the number of mental health classes, the classes that they found, and whether there was evidence for the validity and reliability of the classes. Results: Reviewed studies used LCA to investigate both disparate mental health symptoms, and those associated with specific disorders. The corpus of studies using similar indicators was small. Differences in the criteria used to select the final LCA model were found between studies. All studies found meaningful or useful subgroups, but there were differences in the extent to which the validity and reliability of classes were explicitly demonstrated. Conclusions : LCA is a useful tool for studying and classifying child mental health at the population level. Recommendations are made to improve the application and reporting of LCA and to increase confidence in findings in the future, including use of a range of indices and criteria when enumerating classes, clear reporting of methods for replicability, and making efforts to establish the validity and reliability of identified classes.

Vesicular and tubular transport intermediates regulate organellar cargo dynamics. Transport carrier release involves local and profound membrane remodeling before fission. Pinching the neck of a budding tubule or vesicle requires mechanical forces, likely exerted by the action of molecular motors on the cytoskeleton. Here, we show that myosin VI, together with branched actin filaments, constricts the membrane of tubular carriers that are then released from melanosomes, the pigment containing lysosome-related organelles of melanocytes. By combining superresolution fluorescence microscopy, correlative light and electron microscopy, and biochemical analyses, we find that myosin VI motor activity mediates severing by constricting the neck of the tubule at specific melanosomal subdomains. Pinching of the tubules involves the cooperation of the myosin adaptor optineurin and the activity of actin nucleation machineries, including the WASH and Arp2/3 complexes. The fission and release of these tubules allows for the export of components from melanosomes, such as the SNARE VAMP7, and promotes melanosome maturation and transfer to keratinocytes. Our data reveal a new myosin VI- and actin-dependent membrane fission mechanism required for organelle function., (© 2018 Ripoll et al.)

The nigrostriatal and mesocorticolimbic dopamine networks regulate reward-driven behavior. Regional alterations to mesolimbic dopamine D 2/3 receptor expression are described in drug-seeking and addiction disorders. Parkinson's disease (PD) patients are frequently prescribed D 2 -like dopamine agonist (DAgonist) therapy for motor symptoms, yet a proportion develop clinically significant behavioral addictions characterized by impulsive and compulsive behaviors (ICBs). Until now, changes in D 2/3 receptor binding in both striatal and extrastriatal regions have not been concurrently quantified in this population. We identified 35 human PD patients (both male and female) receiving DAgonist therapy, with ( n = 17) and without ( n = 18) ICBs, matched for age, disease duration, disease severity, and dose of dopamine therapy. In the off-dopamine state, all completed PET imaging with [ 18 F]fallypride, a high affinity D 2 -like receptor ligand that can measure striatal and extrastriatal D 2/3 nondisplaceable binding potential (BP ND ). Striatal differences between ICB+/ICB- patients localized to the ventral striatum and putamen, where ICB+ subjects had reduced BP ND In this group, self-reported severity of ICB symptoms positively correlated with midbrain D 2/3 receptor BP ND Group differences in regional D 2/3 BP ND relationships were also notable: ICB+ (but not ICB-) patients expressed positive correlations between midbrain and caudate, putamen, globus pallidus, and amygdala BP ND s. These findings support the hypothesis that compulsive behaviors in PD are associated with reduced ventral and dorsal striatal D 2/3 expression, similar to changes in comparable behavioral disorders. The data also suggest that relatively preserved ventral midbrain dopaminergic projections throughout nigrostriatal and mesolimbic networks are characteristic of ICB+ patients, and may account for differential DAgonist therapeutic response. SIGNIFICANCE STATEMENT The biologic determinants of compulsive reward-based behaviors have broad clinical relevance, from addiction to neurodegenerative disorders. Here, we address biomolecular distinctions in Parkinson's disease patients with impulsive compulsive behaviors (ICBs). This is the first study to image a large cohort of ICB+ patients using positron emission tomography with [18F]fallypride, allowing quantification of D 2/3 receptors throughout the mesocorticolimbic network. We demonstrate widespread differences in dopaminergic networks, including (1) D2-like receptor distinctions in the ventral striatum and putamen, and (2) a preservation of widespread dopaminergic projections emerging from the midbrain, which is associated with the severity of compulsive behaviors. This clearly illustrates the roles of D 2/3 receptors and medication effects in maladaptive behaviors, and localizes them specifically to nigrostriatal and extrastriatal regions., (Copyright © 2018 the authors 0270-6474/18/383231-10$15.00/0.)

Parkinson's disease (PD) is characterized by widespread degeneration of monoaminergic (especially dopaminergic) networks, manifesting with a number of both motor and non-motor symptoms. Regional alterations to dopamine D 2/3 receptors in PD patients are documented in striatal and some extrastriatal areas, and medications that target D 2/3 receptors can improve motor and non-motor symptoms. However, data regarding the combined pattern of D 2/3 receptor binding in both striatal and extrastriatal regions in PD are limited. We studied 35 PD patients off-medication and 31 age- and sex-matched healthy controls (HCs) using PET imaging with [ 18 F]fallypride, a high affinity D 2/3 receptor ligand, to measure striatal and extrastriatal D 2/3 nondisplaceable binding potential (BP ND ). PD patients completed PET imaging in the off medication state, and motor severity was concurrently assessed. Voxel-wise evaluation between groups revealed significant BP ND reductions in PD patients in striatal and several extrastriatal regions, including the locus coeruleus and mesotemporal cortex. A region-of-interest (ROI) based approach quantified differences in dopamine D 2/3 receptors, where reduced BP ND was noted in the globus pallidus, caudate, amygdala, hippocampus, ventral midbrain, and thalamus of PD patients relative to HC subjects. Motor severity positively correlated with D 2/3 receptor density in the putamen and globus pallidus. These findings support the hypothesis that abnormal D 2/3 expression occurs in regions related to both the motor and non-motor symptoms of PD, including areas richly invested with noradrenergic neurons.

The formation of filopodia in Metazoa and Amoebozoa requires the activity of myosin 10 (Myo10) in mammalian cells and of Dictyostelium unconventional myosin 7 (DdMyo7) in the social amoeba Dictyostelium However, the exact roles of these MyTH4-FERM myosins (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in the initiation and elongation of filopodia are not well defined and may reflect conserved functions among phylogenetically diverse MF myosins. Phylogenetic analysis of MF myosin domains suggests that a single ancestral MF myosin existed with a structure similar to DdMyo7, which has two MF domains, and that subsequent duplications in the metazoan lineage produced its functional homolog Myo10. The essential functional features of the DdMyo7 myosin were identified using quantitative live-cell imaging to characterize the ability of various mutants to rescue filopod formation in myo7-null cells. The two MF domains were found to function redundantly in filopod formation with the C-terminal FERM domain regulating both the number of filopodia and their elongation velocity. DdMyo7 mutants consisting solely of the motor plus a single MyTH4 domain were found to be capable of rescuing the formation of filopodia, establishing the minimal elements necessary for the function of this myosin. Interestingly, a chimeric myosin with the Myo10 MF domain fused to the DdMyo7 motor also was capable of rescuing filopod formation in the myo7-null mutant, supporting fundamental functional conservation between these two distant myosins. Together, these findings reveal that MF myosins have an ancient and conserved role in filopod formation., Competing Interests: The authors declare no conflict of interest.

Protein kinase C α (PKCα) is a nodal regulator in several intracellular signaling networks. PKCα is composed of modular domains that interact with each other to dynamically regulate spatial-temporal function. We find that PKCα specifically, rapidly and reversibly self-assembles in the presence of calcium in vitro. This phenomenon is dependent on, and can be modulated by an intramolecular interaction between the C1a and C2 protein domains of PKCα. Next, we monitor self-assembly of PKC-mCitrine fusion proteins using time-resolved and steady-state homoFRET. HomoFRET between full-length PKCα molecules is observed when in solution with both calcium and liposomes containing either diacylglycerol (DAG) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Surprisingly, the C2 domain is sufficient to cluster on liposomes containing PI(4,5)P2, indicating the C1a domain is not required for self-assembly in this context. We conclude that three distinct clustered states of PKCα can be formed depending on what combination of cofactors are bound, but Ca2+ is minimally required and sufficient for clustering., Competing Interests: The authors have declared that no competing interests exist.

Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the "static" images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis.

We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prepower stroke state of the relay helix and a straight postpower stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient time-resolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphate-binding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postpower stroke state, preventing reversal of the power stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production.