Mass spectrometry of B. subtilis CopZ: Cu(I)-binding and interactions with bacillithiol

CopZ from Bacillus subtilis is a well-studied member of the highly conserved family of Atx1-like copper chaperones. It was previously shown via solution and crystallographic studies to undergo Cu(I)-mediated dimerisation, where the CopZ dimer can bind between one and four Cu(I) ions. However, these studies could not provide information about the changing distribution of species at increasing Cu(I) levels. To address this, electrospray ionisation mass spectrometry using soft ionisation was applied to CopZ under native conditions. Data revealed folded, monomeric CopZ in apo- and Cu(I)-bound forms, along with Cu(I)-bound dimeric forms of CopZ at higher Cu(I) loading. Cu4(CopZ)2 was the major dimeric species at loadings >1 Cu(I)/CopZ, indicating the cooperative formation of the tetranuclear Cu(I)-bound species. As the principal low molecular weight thiol in B. subtilis, bacillithiol (BSH) may play a role in copper homeostasis. Mass spectrometry showed that increasing BSH led to a reduction in Cu(I)-bound dimeric forms, and the formation of S-bacillithiolated apo-CopZ and BSH adducts of Cu(I)-bound forms of CopZ, where BSH likely acts as a Cu(I) ligand. These data, along with the high affinity of BSH for Cu(I), determined here to be β2(BSH) = ∼4 × 1017 M−2, are consistent with a role for BSH alongside CopZ in buffering cellular Cu(I) levels. Here, mass spectrometry provides a high resolution overview of CopZ–Cu(I) speciation that cannot be obtained from less discriminating solution-phase methods, thus illustrating the potential for the wider application of this technique to studies of metal–protein interactions

The Two-State Prehensile Tail of the Antibacterial Toxin Colicin N

Intrinsically disordered regions within proteins are critical elements in many biomolecular interactions and signaling pathways. Antibacterial toxins of the colicin family, which could provide new antibiotic functions against resistant bacteria, contain disordered N-terminal translocation domains (T-domains) that are essential for receptor binding and the penetration of the Escherichia coli outer membrane. Here we investigate the conformational behavior of the T-domain of colicin N (ColN-T) to understand why such domains are widespread in toxins that target Gram-negative bacteria. Like some other intrinsically disordered proteins in the solution state of the protein, ColN-T shows dual recognition, initially interacting with other domains of the same colicin N molecule and later, during cell killing, binding to two different receptors, OmpF and TolA, in the target bacterium. ColN-T is invisible in the high-resolution x-ray model and yet accounts for 90 of the toxin’s 387 amino acid residues. To reveal its solution structure that underlies such a dynamic and complex system, we carried out mutagenic, biochemical, hydrodynamic and structural studies using analytical ultracentrifugation, NMR, and small-angle x-ray scattering on full-length ColN and its fragments. The structure was accurately modeled from small-angle x-ray scattering data by treating ColN as a flexible system, namely by the ensemble optimization method, which enables a distribution of conformations to be included in the final model. The results reveal, to our knowledge, for the first time the dynamic structure of a colicin T-domain. ColN-T is in dynamic equilibrium between a compact form, showing specific self-recognition and resistance to proteolysis, and an extended form, which most likely allows for effective receptor binding

Predicting beneficial effects of atomoxetine and citalopram on response inhibition in Parkinson's disease with clinical and neuroimaging measures.

Recent studies indicate that selective noradrenergic (atomoxetine) and serotonergic (citalopram) reuptake inhibitors may improve response inhibition in selected patients with Parkinson's disease, restoring behavioral performance and brain activity. We reassessed the behavioral efficacy of these drugs in a larger cohort and developed predictive models to identify patient responders. We used a double-blind randomized three-way crossover design to investigate stopping efficiency in 34 patients with idiopathic Parkinson's disease after 40 mg atomoxetine, 30 mg citalopram, or placebo. Diffusion-weighted and functional imaging measured microstructural properties and regional brain activations, respectively. We confirmed that Parkinson's disease impairs response inhibition. Overall, drug effects on response inhibition varied substantially across patients at both behavioral and brain activity levels. We therefore built binary classifiers with leave-one-out cross-validation (LOOCV) to predict patients' responses in terms of improved stopping efficiency. We identified two optimal models: (1) a "clinical" model that predicted the response of an individual patient with 77-79% accuracy for atomoxetine and citalopram, using clinically available information including age, cognitive status, and levodopa equivalent dose, and a simple diffusion-weighted imaging scan; and (2) a "mechanistic" model that explained the behavioral response with 85% accuracy for each drug, using drug-induced changes of brain activations in the striatum and presupplementary motor area from functional imaging. These data support growing evidence for the role of noradrenaline and serotonin in inhibitory control. Although noradrenergic and serotonergic drugs have highly variable effects in patients with Parkinson's disease, the individual patient's response to each drug can be predicted using a pattern of clinical and neuroimaging features.The BCNI is supported by the Wellcome Trust and Medical Research Council. We are grateful to Dr Gordon Logan for advice on stop-signal reaction time estimation and to Dr Marta Correia for advice on diffusion-weighted imaging data analysis. Conflict of interest: Prof. Sahakian has received grants from Janssen/J&J, personal fees from Cambridge Cognition, personal fees from Lundbeck, and personal fees from Servier, outside the submitted work. Prof. Robbins has received personal fees and royalties from Cambridge Cognition, personal fees and grants from Eli Lilly Inc, personal fees and grants from Lundbeck, grants from GSK, personal fees from Teva Pharmaceuticals, personal fees from Shire Pharmaceuticals, grants from Medical Research Council, editorial honorarium from Springer Verlag Germany, and personal fees from Chempartners, outside the submitted work. Prof. Rowe has received grant funding from AZ-Medimmune unrelated to the current work. Dr Housden is an employee of Cambridge Cognition. Other authors reported no biomedical financial interests or potential conflict of interest.This is the final version of the article. It was first available from Wiley via http://dx.doi.org/10.1002/hbm.2308

Robotic-assisted ultrasound for fetal imaging: Evolution from single-arm to dual-arm system

© Springer Nature Switzerland AG 2019. The development of robotic-assisted extracorporeal ultrasound systems has a long history and a number of projects have been proposed since the 1990s focusing on different technical aspects. These aim to resolve the deficiencies of on-site manual manipulation of hand-held ultrasound probes. This paper presents the recent ongoing developments of a series of bespoke robotic systems, including both single-arm and dual-arm versions, for a project known as intelligent Fetal Imaging and Diagnosis (iFIND). After a brief review of the development history of the extracorporeal ultrasound robotic system used for fetal and abdominal examinations, the specific aim of the iFIND robots, the design evolution, the implementation details of each version, and the initial clinical feedback of the iFIND robot series are presented. Based on the preliminary testing of these newly-proposed robots on 42 volunteers, the successful and reliable working of the mechatronic systems were validated. Analysis of a participant questionnaire indicates a comfortable scanning experience for the volunteers and a good acceptance rate to being scanned by the robots

Investigation of low 5-year relative survival for breast cancer in a London cancer network

BACKGROUND: Breast cancer 5-year relative survival is low in the North East London Cancer Network (NELCN). METHODS: We compared breast cancer that was diagnosed during 2001-2005 with that in the rest of London. RESULTS: North East London Cancer Network women more often lived in socioeconomic quintile 5 (42 vs 21%) and presented with advanced disease (11 vs 7%). Cox regression analysis showed the survival difference (hazard ratio: 1.27, 95% confidence interval (CI): 1.15-1.41) reduced to 1.00 (95% CI: 0.89-1.11) after adjustment for age, stage, socioeconomic deprivation, ethnicity and treatment. Major drivers were stage and deprivation. Excess mortality was in the first year. CONCLUSION: Late diagnosis occurs in NELCN

An efficient auxin-inducible degron system with low basal degradation in human cells

Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron minilAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes.Peer reviewe

Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling

The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems

Effect of Levodopa on Reward and Impulsivity in a Rat Model of Parkinson's Disease

The use of dopamine replacement therapies (DRT) in the treatment of Parkinson's disease (PD) can lead to the development of dopamine dysregulation syndrome (DDS) and impulse control disorders (ICD), behavioral disturbances characterized by compulsive DRT self-medication and development of impulsive behaviors. However, the mechanisms behind these disturbances are poorly understood. In animal models of PD, the assessment of the rewarding properties of levodopa (LD), one of the most common drugs used in PD, has produced conflicting results, and its ability to promote increased impulsivity is still understudied. Moreover, it is unclear whether acute and chronic LD therapy differently affects reward and impulsivity. In this study we aimed at assessing, in an animal model of PD with bilateral mesostriatal and mesocorticolimbic degeneration, the behavioral effects of LD therapy regarding reward and impulsivity. Animals with either sham or 6-hydroxydopamine (6-OHDA)-induced bilateral lesions in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) were exposed to acute and chronic LD treatment. We used the conditioned place preference (CPP) paradigm to evaluate the rewarding effects of LD, whereas impulsive behavior was measured with the variable delay-to-signal (VDS) task. Correlation analyses between behavioral measurements of reward or impulsivity and lesion extent in SNc/VTA were performed to pinpoint possible anatomical links of LD-induced behavioral changes. We show that LD, particularly when administered chronically, caused the development of impulsive-like behaviors in 6-OHDA-lesioned animals in the VDS. However, neither acute or chronic LD administration had rewarding effects in 6-OHDA-lesioned animals in the CPP. Our results show that in a bilateral rat model of PD, LD leads to the development of impulsive behaviors, strengthening the association between DRT and DDS/ICD in PD.Portuguese Foundation for Science and Technology: Ciência 2007 Program and IF Development Grant (IF/00111/2013) to AJS, Portuguese Foundation for Science and Technology PhD scholarships attributed to MMC (SFRH/BD/51061/2010), FLC (SFRH/BD/47311/2008) and CS-C (SFRH/BD/51992/2012), and Post-Doctoral Fellowship to HL-A (SFRH/BPD/80118/2011). Neurochemical analysis was funded from ELKE/UOA: 11650. This article has been developed under the scope of the project NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038info:eu-repo/semantics/publishedVersio