Studies of copper trafficking proteins from Bacillus subtilis by native mass spectrometry

Copper is essential for life, but potentially toxic due to its ability to redox cycle and displace metal cofactors. Therefore, ubiquitous protein networks exist to safely handle and deliver copper. Copper is removed from the cell via an integral membrane P1B-ATPase, characterised by its soluble metal-binding domains (MBDs) which receive copper from Atx1-like copper chaperones in the cytoplasm. The mechanism of copper removal is unknown. Low molecular weight thiol species (LMWT) may play a role in cellular copper trafficking. The metallochaperone (CopZ) and soluble domains of the ATPase (CopAab) from Bacillus subtilis each bind Cu(I) with high affinity/specificity and form higher-order assemblies. Native electrospray ionisation mass spectrometry (ESI-MS) revealed formation of multiple copper-bound species with increasing Cu(I) level; cooperative formation of Cu4(CopZ)2 and Cu6(CopAab)2 was observed. The affinity for Cu(I) of bacillithiol (BSH), the primary LMWT in the B.subtilis cytoplasm, was determined to be b2 = 4.1 x 1017 M-2. ESI-MS revealed reduced intensity of dimeric forms of CopZ and CopAab in the presence of BSH (and other LMWT) due to copper competition; bacillithiolation of CopZ was observed. Higher order copper-bound complexes were observed for metallochaperones from Streptomyces lividans and Saccharomyces cerevisiae, and their decrease in the presence of LMWT. Rapid and reversible copper transfer between CopZ and CopAab was observed with similar rate constants at 25°C for forward (247 ± 2.2 s-1) and reverse (258 ± 2.6 s-1) directions of transfer. The rate constant was independent of concentration, suggesting the rate-limiting step is first-order, and likely to be protein complex formation. Kinetic studies demonstrated that protein complex formation resulted in a 7-fold increase in the rate of copper transfer; a copper-bound CopZ:CopAab complex was observed via ESI-MS. Bidirectional copper transfer between CopZ and CopAab consistent with a regulatory role for the MBDs

Therapeutic-yoga after stroke : effect on walking recovery

Indiana University-Purdue University Indianapolis (IUPUI)Stroke is a sudden and devastating medical condition. People who experience a stroke tend to have long-term physical limitations including impaired walking as part of the ongoing consequences of stroke. While a variety of rehabilitation interventions have demonstrated efficacy for improving walking after stroke, none of the interventions have emerged as superior, and prior to this study, therapeutic-yoga had not been tested as an intervention to improve walking recovery after stroke. METHODS: This study was a secondary data analysis of group therapeutic-yoga on walking recovery measures including walking speed, walking distance, and spatiotemporal step parameter symmetry. The walking recovery measures were collected as secondary outcomes in a sub-sample (n=12) in a pilot randomized controlled study (n=47) designed to test the efficacy of 8-weeks of group therapeutic-yoga on balance and fear of falling. Participants in the current study completed 12-weeks of group therapeutic yoga with outcome assessments at baseline, 8-weeks, and 12-weeks. The main analysis was repeated measures ANOVA to assess the main effect of time with additional analyses including effect sizes, percent of participants achieving change greater than or equal to minimal detectable change (MDC), and mean change score comparisons between baseline and 8-weeks, 8-weeks and 12-weeks, and baseline and 12-weeks. RESULTS: Twelve people with chronic stroke enrolled in the study with 9 completing the intervention and all 3 assessments. No significant main effect of time was found on any of the variables of interest. Walking distance demonstrated a trend toward significant change (p=0.064) and step length symmetry demonstrated significant change (p=0.05) between baseline and 12-weeks. Several spatiotemporal step parameter symmetry ratios demonstrated small to medium effect sizes with the majority (91%) being a negative effect. CONCLUSION: Twelve weeks of group therapeutic-yoga appears to be feasible in a population of people with chronic stroke. Walking distance and step parameter symmetry should be tested in a larger sample. An improved understanding of the impact, progression, and remediation of walking asymmetry is needed

Adherence With Home Exercise Programs 1-6 Months After Discharge From Physical Therapy By Individuals Post-Stroke

Indiana University-Purdue University Indianapolis (IUPUI

Mass spectrometric studies of Cu(I)-binding to the N-terminal domains of B. subtilis CopA and influence of bacillithiol

CopA is a Cu(I)-exporting transmembrane P1B-type ATPase from Bacillus subtilis. It contains two N-terminal cytoplasmic domains, CopAab, which bind Cu(I) with high affinity and to form higher-order complexes with multiple Cu(I) ions. To determine the precise nature of these species, electrospray ionisation mass spectrometry (ESI-MS) under non-denaturing conditions was employed. Up to 1 Cu per CopAab resulted in Cu coordination to one or both CopAab domains. At >1 Cu/CopAab, two distinct dimeric charge state envelopes were observed, corresponding to distinct conformations, each with Cu6(CopAab)2 as its major form. The influence of the physiologically relevant low molecular weight thiol bacillithiol (BSH) on Cu(I)-binding to CopAab was assessed. Dimeric CopAab persisted in the presence of BSH, with previously undetected Cu7(CopAab)2 and Cu6(CopAab)2(BSH) forms apparent

Contribution of Berry Polyphenols to the Human Metabolome

Diets rich in berries provide health benefits, however, the contribution of berry phytochemicals to the human metabolome is largely unknown. The present study aimed to establish the impact of berry phytochemicals on the human metabolome. A "systematic review strategy" was utilized to characterize the phytochemical composition of the berries most commonly consumed in the USA; (poly)phenols, primarily anthocyanins, comprised the majority of reported plant secondary metabolites. A reference standard library and tandem mass spectrometry (MS/MS) quantitative metabolomics methodology were developed and applied to serum/plasma samples from a blueberry and a strawberry intervention, revealing a diversity of benzoic, cinnamic, phenylacetic, 3-(phenyl)propanoic and hippuric acids, and benzyldehydes. 3-Phenylpropanoic, 2-hydroxybenzoic, and hippuric acid were highly abundant (mean > 1 µM). Few metabolites at concentrations above 100 nM changed significantly in either intervention. Significant intervention effects (P < 0.05) were observed for plasma/serum 2-hydroxybenzoic acid and hippuric acid in the blueberry intervention, and for 3-methoxyphenylacetic acid and 4-hydroxyphenylacetic acid in the strawberry intervention. However, significant within-group effects for change from baseline were prevalent, suggesting that high inter-individual variability precluded significant treatment effects. Berry consumption in general appears to cause a fluctuation in the pools of small molecule metabolites already present at baseline, rather than the appearance of unique berry-derived metabolites, which likely reflects the ubiquitous nature of (poly)phenols in the background diet

The CDM-Net Project: The Development, Implementation and Evaluation of a Broadband-Based Network for Managing Chronic Disease

Background. In Australia most chronic disease management is funded by Medicare Australia through General Practitioner Management Plans (GPMPs) and Team Care Arrangements (TCAs). Identified barriers may be reduced effectively using a broadband-based network known as the Chronic Disease Management Service (CDMS). Aims. To measure the uptake and adherence to CDMS, test CDMS, and assess the adherence of health providers and patients to GPMPs and TCAs generated through CDMS. Methods. A single cohort before and after study. Results. GPMPs and TCAs increased. There was no change to prescribed medicines or psychological quality of life. Attendance at allied health professionals increased, but decreased at pharmacies. Overall satisfaction with CDMS was high among GPs, allied health professionals, and patients. Conclusion. This study demonstrates proof of concept, but replication or continuation of the study is desirable to enable the impact of CDMS on diabetes outcomes to be determined

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