Capturing the catalytic intermediates of parkin ubiquitination.

Parkin is an E3 ubiquitin ligase implicated in early-onset forms of Parkinson's disease. It catalyzes a transthiolation reaction by accepting ubiquitin (Ub) from an E2 conjugating enzyme, forming a short-lived thioester intermediate, and transfers Ub to mitochondrial membrane substrates to signal mitophagy. A major impediment to the development of Parkinsonism therapeutics is the lack of structural and mechanistic detail for the essential, short-lived transthiolation intermediate. It is not known how Ub is recognized by the catalytic Rcat domain in parkin that enables Ub transfer from an E2~Ub conjugate to the catalytic site and the structure of the transthiolation complex is undetermined. Here, we capture the catalytic intermediate for the Rcat domain of parkin in complex with ubiquitin (Rcat-Ub) and determine its structure using NMR-based chemical shift perturbation experiments. We show that a previously unidentified α-helical region near the Rcat domain is unmasked as a recognition motif for Ub and guides the C-terminus of Ub toward the parkin catalytic site. Further, we apply a combination of guided AlphaFold modeling, chemical cross-linking, and single turnover assays to establish and validate a model of full-length parkin in complex with UbcH7, its donor Ub, and phosphoubiquitin, trapped in the process of transthiolation. Identification of this catalytic intermediate and orientation of Ub with respect to the Rcat domain provides important structural insights into Ub transfer by this E3 ligase and explains how the previously enigmatic Parkinson's pathogenic mutation T415N alters parkin activity.

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation.

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early-onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N-terminal ubiquitin-like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho-ubiquitin-loaded C-terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re-modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity.

Implementation and dissemination of an African American popular opinion model to improve lupus awareness: an academic-community partnership.

OBJECTIVE: Lupus is a chronic, autoimmune disease that disproportionately affects African Americans. We adapted the Centers for Disease Control and Prevention's Popular Opinion Leader model to implement an intervention tailored for African American individuals that leverages an academic-community partnership and community-based social networks to disseminate culturally appropriate lupus education. METHODS: Academic rheumatologists, social scientists, and researchers in Boston, MA and Chicago, IL partnered with local lupus support groups, community organizations, and churches in neighborhoods with higher proportions of African Americans to develop curriculum and recruit community leaders with and without lupus (Popular Opinion Leaders; POLs). POLs attended four training sessions focused on lupus education, strategies to educate others, and a review of research methods. POLs disseminated information through their social networks and recorded their impact, which was mapped using a geographic information system framework. RESULTS: We trained 18 POLs in greater Boston and 19 in greater Chicago: 97% were African American, 97% were female; and the mean age was 57 years. Fifty-nine percent of Boston POLs and 68% of Chicago POLs had lupus. POLs at both sites engaged members of their social networks and communities in conversations about lupus, health disparities, and the importance of care. Boston POLs documented 97 encounters with 547 community members reached. Chicago POLs documented 124 encounters with 4083 community members reached. CONCLUSIONS: An adapted, community-based POL model can be used to disseminate lupus education and increase awareness in African American communities. Further research is needed to determine the degree to which this may begin to reduce disparities in access to care and outcomes.

Liquid crystal elastomer foams with elastic properties specifically engineered as biodegradable brain tissue scaffolds.

Tissue regeneration requires 3-dimensional (3D) smart materials as scaffolds to promote transport of nutrients. To mimic mechanical properties of extracellular matrices, biocompatible polymers have been widely studied and a diverse range of 3D scaffolds have been produced. We propose the use of responsive polymeric materials to create dynamic substrates for cell culture, which goes beyond designing only a physical static 3D scaffold. Here, we demonstrated that lactone- and lactide-based star block-copolymers (SBCs), where a liquid crystal (LC) moiety has been attached as a side-group, can be crosslinked to obtain Liquid Crystal Elastomers (LCEs) with a porous architecture using a salt-leaching method to promote cell infiltration. The obtained SmA LCE-based fully interconnected-porous foams exhibit a Young modulus of 0.23 ± 0.07 MPa and a biodegradability rate of around 20% after 15 weeks both of which are optimized to mimic native environments. We present cell culture results showing growth and proliferation of neurons on the scaffold after four weeks. This research provides a new platform to analyse LCE scaffold-cell interactions where the presence of liquid crystal moieties promotes cell alignment paving the way for a stimulated brain-like tissue.

The neuronal metabolite NAA regulates histone H3 methylation in oligodendrocytes and myelin lipid composition.

The neuronal mitochondrial metabolite N-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme N-acetyltransferase-8-like (NAT8L) and broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metabolism of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance thin-layer chromatography (HPTLC) to identify changes in myelin lipid composition in postmortem MS brains and in NAT8L knockout (NAT8L-/-) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amounts of sphingomyelin and sulfatide lipids in the brains of NAT8L-/- mice compared to controls. Metabolomic analysis of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metabolism, and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was associated with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination.

Daily salivary cortisol patterns in midlife women with hot flashes.

OBJECTIVE: Diurnal salivary cortisol patterns in healthy adults are well established but have not been studied in midlife women with hot flashes. We hypothesized that frequent hot flashes are associated with aberrant cortisol patterns similar to sleep-deficient individuals. DESIGN: Cross-sectional. PARTICIPANTS: A total of 306 women, ages 40-62, randomized to a behavioural intervention for hot flashes. MEASUREMENTS: Baseline comparisons of cortisol geometric means (nmol/l) from four daily time points averaged over two consecutive days plus other calculated cortisol measures were made between groups defined by baseline: (i) mean daily hot flash frequency tertile (≤5·5, N = 103; >5·5-8·8, N = 103; >8·8, N = 100) and (ii) selected characteristics. Repeated-measures linear regression models of log-transformed cortisol evaluated group differences, adjusting for covariates. RESULTS: Women were 67% White and 24% African American, with 7·6 (SD 3·9) hot flashes per day. Salivary cortisol geometric means (nmol/l) among all women were as follows: 75·0 (SD 44·8) total, 8·6 (SD 5·6) wake, 10·0 (SD 7·5) wake +30 min, 3·7 (SD 3·3) early afternoon and 1·6 (SD 1·8) bedtime. Wake + 30-minute values showed an 18% median rise from wake values (interquartile range -24 to 96%), and means varied by hot flash frequency tertile, from lowest to highest: 11·4(SD 7·3), 10·3 (SD 6·5) and 8·6 (SD 7·8), respectively, P = 0·003. Beside the early afternoon value (P = 0·02), cortisol values did not vary by hot flash frequency. CONCLUSION: Taken together, these findings suggest that high frequency of moderate-to-severe hot flashes may be associated with subtle abnormalities in cortisol concentrations - a pattern consistent with chronic sleep disturbance.

The mechanoelectrical response of droplet interface bilayer membranes.

Mechanotransduction and interfacial properties in unsupported liquid biomimetic membranes are explored using the droplet-interface bilayer technique. The fluidic monolayer-membrane system afforded by this technique allows for dynamic control over the membrane dimensions and curvature, which under periodic deformations generates capacitive currents (akin to a Kelvin probe), and permits a detailed electrostatic characterization of the boundary layers as well as observation of flexoelectric effects. Both high and low displacement frequency regimes are examined, and the results show that the mechanoelectric signals generated by the membranes may be linked to the membrane electrostatic structure. In addition, we show that periodic membrane bending in a high-frequency regime generates tension sufficient to activate reconstituted mechanosensitive channels.

Field Effect and Strongly Localized Carriers in the Metal-Insulator Transition Material VO(2).

The intrinsic field effect, the change in surface conductance with an applied transverse electric field, of prototypal strongly correlated VO(2) has remained elusive. Here we report its measurement enabled by epitaxial VO(2) and atomic layer deposited high-κ dielectrics. Oxygen migration, joule heating, and the linked field-induced phase transition are precluded. The field effect can be understood in terms of field-induced carriers with densities up to ∼5×10(13) cm(-2) which are trongly localized, as shown by their low, thermally activated mobility (∼1×10(-3) cm(2)/V s at 300 K). These carriers show behavior consistent with that of Holstein polarons and strongly impact the (opto)electronics of VO(2).

Hormone variations associated with quantitative fat measures in the menopausal transition.

OBJECTIVE: Reproductive hormone levels are associated with body size, and the association between estradiol and body size varies over the menopausal transition. This study aims to delineate these relationships using quantitative measures of visceral and subcutaneous fat. METHODS: Early follicular hormones (follicle stimulating hormone (FSH), estradiol, luteinizing hormone, dehydroepiandrosterone sulfate, testosterone) and T-1 weighted abdominal MRI images were obtained in a cross-sectional assessment of 77 women in the Penn Ovarian Aging Study. Fat volume (cm(3)) was quantified using validated software (Amira) and divided into tertiles of visceral and subcutaneous fat volume for analysis. Multivariable linear regression models compared hormone values between tertiles adjusting for race, age, and menopausal status. RESULTS: In adjusted models, estradiol was positively associated with visceral fat tertiles (geometric mean (GM) estradiol (pg/ml): Low 13.0, Mid 17.5, High 26.7, p = 0.006) while FSH was inversely associated with visceral fat tertiles (GM FSH (mIU/ml): Low 42.8, Mid 43.2, High 30.8, p = 0.03). The association of estradiol with visceral and subcutaneous fat tertiles varied by menopausal status (p < 0.001). In the early transition, estradiol was similar across tertiles of fat; postmenopause, estradiol was positively associated with visceral fat. Other hormones were not associated with fat measures. CONCLUSIONS: Estradiol was associated with quantitative measures of visceral fat and varies by menopausal status. This finding suggests that visceral fat may be an important mediator in hormone changes over the menopausal transition.

Decreased NAA in gray matter is correlated with decreased availability of acetate in white matter in postmortem multiple sclerosis cortex.

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS) which leads to progressive neurological disability. Our previous studies have demonstrated mitochondrial involvement in MS cortical pathology and others have documented decreased levels of the neuronal mitochondrial metabolite N-acetyl aspartate (NAA) in the MS brain. While NAA is synthesized in neurons, it is broken down in oligodendrocytes into aspartate and acetate. The resulting acetate is incorporated into myelin lipids, linking neuronal mitochondrial function to oligodendrocyte-mediated elaboration of myelin lipids in the CNS. In the present study we show that treating human SH-SY5Y neuroblastoma cells with the electron transport chain inhibitor antimycin A decreased levels of NAA as measured by HPLC. To better understand the significance of the relationship between mitochondrial function and levels of NAA and its breakdown product acetate on MS pathology we then quantitated the levels of NAA and acetate in MS and control postmortem tissue blocks. Regardless of lesion status, we observed that levels of NAA were decreased 25 and 32 % in gray matter from parietal and motor cortex in MS, respectively, compared to controls. Acetate levels in adjacent white matter mirrored these decreases as evidenced by the 36 and 45 % reduction in acetate obtained from parietal and motor cortices. These data suggest a novel mechanism whereby mitochondrial dysfunction and reduced NAA levels in neurons may result in compromised myelination by oligodendrocytes due to decreased availability of acetate necessary for the synthesis of myelin lipids.

Preventing and treating childhood obesity: time to target fathers.

OBJECTIVE: To examine the long-term effects of having one overweight or obese parent on child weight status and determine whether these effects vary according to parent sex. DESIGN: Prospective study: Longitudinal Study of Australian Children (LSAC). SUBJECTS: Two-parent families (N=3285) from the LSAC were included if height and weight data were available for both parents and their child at the 2004 and 2008 time points. MEASUREMENTS: Child weight status category (healthy, overweight, obese) in 2008 when the child was aged 8-9 years. Regression modelling was used to investigate how self-reported parent weight status in 2004 influenced measured child weight status 4 years later. RESULTS: Parent body mass index (BMI) was significantly correlated with child BMI, but there was no evidence of sex-specific associations between parent and child BMI correlations. The results from the regression analysis showed that having an overweight or obese father, but a healthy weight mother, significantly increased the odds of child obesity (odds ratio: 4.18, 95% confidence interval (CI): 1.01-17.33 and odds ratio: 14.88, 95% CI: 2.61-84.77, respectively), but the reverse scenario (overweight or obese mother with a healthy weight father) was not a significant predictor of child overweight or obesity (odds ratio: 2.52, 95% CI: 0.38-16.71 and odds ratio: 2.56, 95% CI: 0.31-21.26, respectively). CONCLUSIONS: Children with overweight or obese fathers are at a higher risk of becoming obese. This suggests that interventions are urgently required to test the efficacy of treating overweight fathers as a key strategy for childhood obesity prevention and/or treatment.

Transcriptional signatures mediated by acetylation overlap with early-stage Alzheimer's disease.

The mechanisms by which environmental influences lead to the development of complex neurodegenerative diseases are largely unknown. It is known, however, that epigenetic mechanisms can mediate alterations in transcription due to environmental influences. In order to identify genes susceptible to regulation in the adult cortex by one type of epigenetic mechanism, histone, and protein acetylation, we treated mice with the histone deacetylase inhibitor Trichostatin A (TSA). After 1 week of treatment with TSA, RNA was extracted from the brain cortices of mice and gene expression differences were analyzed by microarray profiling. The altered genes were then compared with genes differentially expressed in microarray studies of disease by database and literature searches. Genes regulated by TSA were found to significantly overlap with differentially expressed genes in the Alzheimer's disease (AD) brain. Several TSA-regulated genes involved in chromatin remodeling and epigenetic reprogramming including histone cluster 1, H4 h (Hist1H4 h), methionine adenosyltransferase II, alpha (Mat2a), and 5-methyltetrahydrofolate homocysteine reductase (Mtrr) overlapped with genes altered in early-stage AD in gray matter. We also show that the expression of hemoglobin, which has been shown to be altered in neurons in the AD brain, is regulated by TSA treatment. This analysis suggests involvement of epigenetic mechanisms in neurons in early stages of AD.

The Importance of Understanding Individual Differences of Emotion Regulation Abilities in 22q11.2 Deletion Syndrome.

Chromosome 22q11.2 deletion syndrome (22q11DS) is characterised by a complex behavioural phenotype including anxiety, attention-deficit/hyperactivity disorder and psychosis. In the current study, we aimed at improving our understanding of the heterogeneity of behavioural characteristics in a group of 129 young people (aged 4-22) with a confirmed 22q11.2 microdeletion and 116 age and gender matched typically developing controls. Half the participants with 22q11DS had behaviour characterised by emotion dysregulation. A cluster analyses, of the participants with 22q11DS, revealed four groups characterised by intact emotion regulation; predominantly internalizing problems; both internalizing and externalizing problems; and predominantly externalizing difficulties. Importantly, it was found that young people with 22q11DS whose emotion dysregulation was characterised by externalizing problems had the poorest levels of functioning. As our understanding of 22q11DS improves, it is becoming increasingly clear that we need a better understanding of how individual differences and psychosocial factors contribute to, and interact with one another, to result in the observable individual differences in the 22q11DS behavioural phenotype.

Epidemiology and outcomes of primary sclerosing cholangitis: an Australian multicentre retrospective cohort study.

BACKGROUND AND AIMS: Little is known regarding the epidemiology and outcomes of patients with primary sclerosing cholangitis (PSC) in Australia. We, therefore, evaluated the epidemiology and clinical outcomes of PSC in a large cohort of Australian patients and compared these to the general population. METHODS: We conducted a multicentre, retrospective cohort study of PSC patients at nine tertiary liver centers across three Australian states, including two liver transplant centers. RESULTS: A total of 413 PSC patients with 3,285 person-years of follow-up were included. Three hundred and seventy-one (90%) patients had large duct PSC and 294 (71%) had associated inflammatory bowel disease. A total of 168 (41%) patients developed cirrhosis (including 34 at the time of PSC diagnosis) after a median of 15.8 (95% CI 12.4, NA) years. The composite endpoint of death or liver transplantation occurred in 49 (12%) and 78 (19%) patients, respectively, with a median transplant-free survival of 13.4 (95% CI 12.2-15) years. Compared to the general population, PSC accounted for a 240-fold increased risk of development of cholangiocarcinoma (CCA) and CCA-related death. CCA risk was increased with older age of PSC diagnosis, presence of dominant stricture and colectomy. Compared to same-aged counterparts in the general population, PSC patients who were diagnosed at an older age or with longer disease duration had reduced relative survival. CONCLUSION: In this large retrospective cohort study of PSC patients in Australia, increased age and time from diagnosis was associated with increased mortality and morbidity particularly from CCA and development of cirrhosis, necessitating need for liver transplant.

The responses of professional groups to the use of Section 136 of the Mental Health Act (1983, as amended by the 2007 Act) in Gloucestershire.

BACKGROUND: Section 136 (S136) of the Mental Health Act (1983, as amended by the 2007 Act) empowers the police to detain those suspected of being mentally disordered in a public place and to convey them to a place of safety (POS) for further assessment. Gloucestershire has not had a specialist facility for S136 detentions and individuals were taken to the police cells or occasionally A&E departments for assessment. AIMS: This paper forms one part of three aspects under investigation. Two companion papers by the authors describe the use of S136 using anonymised audit data and the experiences of detainees. The objectives of this paper have been to assess the responses of the different professional groups involved in the process of S136. METHOD: An anonymous postal questionnaire was distributed to eight groups of professionals who were identified as having the potential to be involved in part of the process of a S136 detention. Results were collated and analysed, and formed the basis for a series of follow-up focus groups within groups to explore themes that warranted further investigation. RESULTS: An overall response rate of 59% was achieved. Seventy-four per cent of participants thought that there should be an alternative POS to the police station. A&E was thought to be an unsuitable alternative POS, with a psychiatric hospital being the first choice for 58%. CONCLUSIONS: There is a gap in the expectations of the different agencies involved in the S136 process, which have the potential to be divisive if interagency pathways and agreements are not in place.