Minimal clinically important differences in patient-reported outcomes after coronary artery bypass surgery in the arterial revascularization trial.

OBJECTIVES: This article identifies minimal clinically important differences (MCIDs) in quality of life (QoL) measures among patients who had coronary artery bypass grafting (CABG) and were enrolled in the arterial revascularization trial (ART). METHODS AND RESULTS: The European Quality of Life-5 Dimensions (EQ-5D) and the Short Form Health Survey 36-Item (SF-36) physical component (PC) and mental component (MC) scores were recorded at baseline, 5 years and 10 years in ART. The MCIDs were calculated as changes in QoL scores anchored to 1-class improvement in the New York Heart Association functional class and Canadian Cardiovascular Society scale at 5 years. Cox proportional hazard models were used to evaluate associations between MCIDs and mortality. Patient cohorts were examined for the SF-36 PC (N = 2671), SF-36 MC (N = 2815) and EQ-5D (N = 2943) measures, respectively. All QoL scores significantly improved after CABG compared to baseline. When anchored to the New York Heart Association, the MCID at 5 years was 17 (95% confidence interval: 17-20) for SF-36 PC, 14 (14-17) for the SF-36 MC and 0.12 (0.12-0.15) for EQ-5D. Using the Canadian Cardiovascular Society scale as an anchor, the MCID at 5 years was 15 (15-17) for the SF-36 PC, 12 (13-15) for the SF-36 MC and 0.12 (0.11-0.14) for the EQ-5D. The MCIDs for SF-36 PC and EQ-5D at 5 years were associated with a lower risk of mortality at the 10-year follow-up point after surgery. CONCLUSIONS: MCIDs for CABG patients have been identified. These thresholds may have direct clinical applications in monitoring patients during follow-up and in designing new trials that include QoL as a primary study outcome. CLINICAL TRIAL REGISTRATION NUMBER: ISRCTN46552265.

Bortezomib Inhibits Open Configurations of the 20S Proteasome.

Bortezomib, a small dipeptide-like molecule, is a proteasome inhibitor used widely in the treatment of myeloma and lymphoma. This molecule reacts with threonine side chains near the center of the 20S proteasome and disrupts proteostasis by blocking enzymatic sites that are responsible for protein degradation. In this work, we use novel mass-spectrometry-based techniques to examine the influence of bortezomib on the structures and stabilities of the 20S core particle. These studies indicate that bortezomib binding dramatically favors compact 20S structures (in which the axial gate is closed) over larger structures (in which the axial gate is open)─suppressing gate opening by factors of at least ∼400 to 1300 over the temperature range that is studied. Thus, bortezomib may also restrict degradation in the 20S proteasome by preventing substrates from entering the catalytic pore. That bortezomib influences structures at the entrance region of the pore at such a long distance (∼65 to 75 Å) from its binding sites raises a number of interesting biophysical issues.

Transcriptome-Based Prognostic and Predictive Biomarker Analysis of ENACT: A Randomized Controlled Trial of Enzalutamide in Men Undergoing Active Surveillance.

PURPOSE: Few studies have explored the potential for pharmacological interventions to delay disease progression in patients undergoing active surveillance (AS). This preplanned transcriptomic analysis of patient samples from the ENACT trial aims to identify biomarkers in patients on AS who are at increased risk for disease progression or who may derive the greatest benefit from enzalutamide treatment. PATIENTS AND METHODS: In the phase II ENACT (ClinicalTrials.gov identifier: NCT02799745) trial, patients on AS were randomly assigned 1:1 to 160 mg orally once daily enzalutamide monotherapy or continued AS for 1 year. Transcriptional analyses were conducted on biopsies collected at trial screening, year 1, and year 2. Three gene expression signatures were evaluated in samples collected at screening and in available samples from patients on AS at any time during surveillance (expanded cohort): Decipher genomic classifier, androgen receptor activity (AR-A) score, and Prediction Analysis of Microarray 50 (PAM50) cell subtype signature. RESULTS: The Decipher genomic classifier score was prognostic; higher scores were associated with disease progression in the expanded cohort and AS arm of the expanded cohort. Patients with higher Decipher scores had greater positive treatment effect from enzalutamide as measured by time to secondary rise in prostate-specific antigen >25% above baseline. In patients treated with enzalutamide, higher AR-A scores and PAM50 luminal subtypes were associated with a greater likelihood of negative biopsy incidence at year 2. CONCLUSION: This analysis suggests that the Decipher genomic classifier may be prognostic for disease progression in AS patients with low- to intermediate-risk prostate cancer. Higher Decipher and AR-A scores, as well as PAM50 luminal subtypes, may also serve as biomarkers for treatment response.

2024 Australia-New Zealand Expert Consensus Statement on Cardiac Amyloidosis.

Over the past 5 years, early diagnosis of and new treatments for cardiac amyloidosis (CA) have emerged that hold promise for early intervention. These include non-invasive diagnostic tests and disease modifying therapies. Recently, CA has been one of the first types of cardiomyopathy to be treated with gene editing techniques. Although these therapies are not yet widely available to patients in Australia and New Zealand, this may change in the near future. Given the rapid pace with which this field is evolving, it is important to view these advances within the Australian and New Zealand context. This Consensus Statement aims to update the Australian and New Zealand general physician and cardiologist with regards to the diagnosis, investigations, and management of CA.

Emerging racial disparities among Medicare beneficiaries and Veterans with metastatic castration-sensitive prostate cancer.

BACKGROUND: Previous studies have shown that Black men receive worse prostate cancer care than White men. This has not been explored in metastatic castration-sensitive prostate cancer (mCSPC) in the current treatment era. METHODS: We evaluated treatment intensification (TI) and overall survival (OS) in Medicare (2015-2018) and Veterans Health Administration (VHA; 2015-2019) patients with mCSPC, classifying first-line mCSPC treatment as androgen deprivation therapy (ADT) + novel hormonal therapy; ADT + docetaxel; ADT + first-generation nonsteroidal antiandrogen; or ADT alone. RESULTS: We analyzed 2226 Black and 16,071 White Medicare, and 1020 Black and 2364 White VHA patients. TI was significantly lower for Black vs White Medicare patients overall (adjusted odds ratio [OR] 0.68; 95% confidence interval [CI] 0.58-0.81) and without Medicaid (adjusted OR 0.70; 95% CI 0.57-0.87). Medicaid patients had less TI irrespective of race. OS was worse for Black vs White Medicare patients overall (adjusted hazard ratio [HR] 1.20; 95% CI 1.09-1.31) and without Medicaid (adjusted HR 1.13; 95% CI 1.01-1.27). OS was worse in Medicaid vs without Medicaid, with no significant OS difference between races. TI was significantly lower for Black vs White VHA patients (adjusted OR 0.75; 95% CI 0.61-0.92), with no significant OS difference between races. CONCLUSIONS: Guideline-recommended TI was low for all patients with mCSPC, with less TI in Black patients in both Medicare and the VHA. Black race was associated with worse OS in Medicare but not the VHA. Medicaid patients had less TI and worse OS than those without Medicaid, suggesting poverty and race are associated with care and outcomes.

BRD4354 Is a Potent Covalent Inhibitor against the SARS-CoV-2 Main Protease.

Numerous organic molecules are known to inhibit the main protease (MPro) of SARS-CoV-2, the pathogen of Coronavirus Disease 2019 (COVID-19). Guided by previous research on zinc-ligand inhibitors of MPro and zinc-dependent histone deacetylases (HDACs), we identified BRD4354 as a potent inhibitor of MPro. The in vitro protease activity assays show that BRD4354 displays time-dependent inhibition against MPro with an IC50 (concentration that inhibits activity by 50%) of 0.72 ± 0.04 µM after 60 min of incubation. Inactivation follows a two-step process with an initial rapid binding step with a KI of 1.9 ± 0.5 µM followed by a second slow inactivation step, kinact,max of 0.040 ± 0.002 min-1. Native mass spectrometry studies indicate that a covalent intermediate is formed where the ortho-quinone methide fragment of BRD4354 forms a covalent bond with the catalytic cysteine C145 of MPro. Based on these data, a Michael-addition reaction mechanism between MPro C145 and BRD4354 was proposed. These results suggest that both preclinical testing of BRD4354 and structure-activity relationship studies based on BRD4354 are warranted to develop more effective anti-COVID therapeutics.

ARL5b inhibits human rhinovirus 16 propagation and impairs macrophage-mediated bacterial clearance.

Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.

Double and triple thermodynamic mutant cycles reveal the basis for specific MsbA-lipid interactions.

Structural and functional studies of the ATP-binding cassette transporter MsbA have revealed two distinct lipopolysaccharide (LPS) binding sites: one located in the central cavity and the other at a membrane-facing, exterior site. Although these binding sites are known to be important for MsbA function, the thermodynamic basis for these specific MsbA-LPS interactions is not well understood. Here, we use native mass spectrometry to determine the thermodynamics of MsbA interacting with the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). The binding of KDL is solely driven by entropy, despite the transporter adopting an inward-facing conformation or trapped in an outward-facing conformation with adenosine 5'-diphosphate and vanadate. An extension of the mutant cycle approach is employed to probe basic residues that interact with KDL. We find the molecular recognition of KDL is driven by a positive coupling entropy (as large as -100 kJ/mol at 298 K) that outweighs unfavorable coupling enthalpy. These findings indicate that alterations in solvent reorganization and conformational entropy can contribute significantly to the free energy of protein-lipid association. The results presented herein showcase the advantage of native MS to obtain thermodynamic insight into protein-lipid interactions that would otherwise be intractable using traditional approaches, and this enabling technology will be instrumental in the life sciences and drug discovery.

The intersocietal IWGDF, ESVS, SVS guidelines on peripheral artery disease in people with diabetes and a foot ulcer.

Diabetes related foot complications have become a major cause of morbidity and are implicated in most major and minor amputations globally. Approximately 50% of people with diabetes and a foot ulcer have peripheral artery disease (PAD) and the presence of PAD significantly increases the risk of adverse limb and cardiovascular events. The International Working Group on the Diabetic Foot (IWGDF) has published evidence based guidelines on the management and prevention of diabetes related foot complications since 1999. This guideline is an update of the 2019 IWGDF guideline on the diagnosis, prognosis and management of peripheral artery disease in people with diabetes mellitus and a foot ulcer. For this guideline the IWGDF, the European Society for Vascular Surgery and the Society for Vascular Surgery decided to collaborate to develop a consistent suite of recommendations relevant to clinicians in all countries. This guideline is based on three new systematic reviews. Using the Grading of Recommendations, Assessment, Development, and Evaluation framework clinically relevant questions were formulated, and the literature was systematically reviewed. After assessing the certainty of the evidence, recommendations were formulated which were weighed against the balance of benefits and harms, patient values, feasibility, acceptability, equity, resources required, and when available, costs. Through this process five recommendations were developed for diagnosing PAD in a person with diabetes, with and without a foot ulcer or gangrene. Five recommendations were developed for prognosis relating to estimating likelihood of healing and amputation outcomes in a person with diabetes and a foot ulcer or gangrene. Fifteen recommendations were developed related to PAD treatment encompassing prioritisation of people for revascularisation, the choice of a procedure and post-surgical care. In addition, the Writing Committee has highlighted key research questions where current evidence is lacking. The Writing Committee believes that following these recommendations will help healthcare professionals to provide better care and will reduce the burden of diabetes related foot complications.

Targeted photodynamic therapy for breast cancer: the potential of glyconanoparticles.

Photodynamic therapy (PDT) uses a non-toxic light sensitive molecule, a photosensitiser, that releases cytotoxic reactive oxygen species upon activation with light of a specific wavelength. Here, glycan-modified 16 nm gold nanoparticles (glycoAuNPs) were explored for their use in targeted PDT, where the photosensitiser was localised to the target cell through selective glycan-lectin interactions. Polyacrylamide (PAA)-glycans were chosen to assess glycan binding to the cell lines. These PAA-glycans indicated the selective uptake of a galactose-derivative PAA by two breast cancer cell lines, SK-BR-3 and MDA-MD-231. Subsequently, AuNPs were modified with a galactose-derivative ligand and an amine derivate of the photosensitiser chlorin e6 was incorporated to the nanoparticle surface via amide bond formation using EDC/NHS coupling chemistry. The dual modified nanoparticles were investigated for the targeted cell killing of breast cancer cells, demonstrating the versatility of using glycoAuNPs for selective binding to different cancer cells and their potential use for targeted PDT.

The Intersocietal IWGDF, ESVS, SVS Guidelines on Peripheral Artery Disease in People With Diabetes Mellitus and a Foot Ulcer.

Diabetes related foot complications have become a major cause of morbidity and are implicated in most major and minor amputations globally. Approximately 50% of people with diabetes and a foot ulcer have peripheral artery disease (PAD) and the presence of PAD significantly increases the risk of adverse limb and cardiovascular events. The International Working Group on the Diabetic Foot (IWGDF) has published evidence based guidelines on the management and prevention of diabetes related foot complications since 1999. This guideline is an update of the 2019 IWGDF guideline on the diagnosis, prognosis, and management of peripheral artery disease in people with diabetes mellitus and a foot ulcer. For this updated guideline, the IWGDF, the European Society for Vascular Surgery, and the Society for Vascular Surgery decided to collaborate to develop a consistent suite of recommendations relevant to clinicians in all countries. This guideline is based on three new systematic reviews. Using the Grading of Recommendations, Assessment, Development and Evaluation framework clinically relevant questions were formulated, and the literature was systematically reviewed. After assessing the certainty of the evidence, recommendations were formulated which were weighed against the balance of benefits and harms, patient values, feasibility, acceptability, equity, resources required, and when available, costs. Through this process five recommendations were developed for diagnosing PAD in a person with diabetes, with and without a foot ulcer or gangrene. Five recommendations were developed for prognosis relating to estimating likelihood of healing and amputation outcomes in a person with diabetes and a foot ulcer or gangrene. Fifteen recommendations were developed related to PAD treatment encompassing prioritisation of people for revascularisation, the choice of a procedure and post-surgical care. In addition, the Writing Committee has highlighted key research questions where current evidence is lacking. The Writing Committee believes that following these recommendations will help healthcare professionals to provide better care and will reduce the burden of diabetes related foot complications.

The intersocietal IWGDF, ESVS, SVS guidelines on peripheral artery disease in people with diabetes mellitus and a foot ulcer.

Diabetes related foot complications have become a major cause of morbidity and are implicated in most major and minor amputations globally. Approximately 50% of people with diabetes and a foot ulcer have peripheral artery disease (PAD) and the presence of PAD significantly increases the risk of adverse limb and cardiovascular events. The International Working Group on the Diabetic Foot (IWGDF) has published evidence based guidelines on the management and prevention of diabetes related foot complications since 1999. This guideline is an update of the 2019 IWGDF guideline on the diagnosis, prognosis, and management of peripheral artery disease in people with diabetes mellitus and a foot ulcer. For this updated guideline, the IWGDF, the European Society for Vascular Surgery, and the Society for Vascular Surgery decided to collaborate to develop a consistent suite of recommendations relevant to clinicians in all countries. This guideline is based on three new systematic reviews. Using the Grading of Recommendations, Assessment, Development and Evaluation framework clinically relevant questions were formulated, and the literature was systematically reviewed. After assessing the certainty of the evidence, recommendations were formulated which were weighed against the balance of benefits and harms, patient values, feasibility, acceptability, equity, resources required, and when available, costs. Through this process five recommendations were developed for diagnosing PAD in a person with diabetes, with and without a foot ulcer or gangrene. Five recommendations were developed for prognosis relating to estimating likelihood of healing and amputation outcomes in a person with diabetes and a foot ulcer or gangrene. Fifteen recommendations were developed related to PAD treatment encompassing prioritisation of people for revascularisation, the choice of a procedure and post-surgical care. In addition, the Writing Committee has highlighted key research questions where current evidence is lacking. The Writing Committee believes that following these recommendations will help healthcare professionals to provide better care and will reduce the burden of diabetes related foot complications.

Giving information strategically and transparently: A pilot trial of the Oncolo-GIST intervention to promote patients' prognostic understanding.

PURPOSE: Most patients with cancer lack the prognostic understanding necessary to make informed decisions. We tested the feasibility and acceptability of the Oncolo-GIST ("Giving Information Strategically and Transparently, GIST") intervention and explored its associations with patients' improved prognostic understanding. METHODS: The Oncolo-GIST intervention distills prognostic discussions into easy-to-understand talking points. Patients with metastatic cancers that progressed on ≥1 line of chemotherapy and not expected to survive 12 months (n = 31) were recruited from October 2020 through November 2022. We compared patients who discussed their progressive scans with an oncologist trained in the GIST technique or not (i.e., usual care). A primary outcome was prognostic understanding (e.g., patients reporting a life-expectancy of months) assessed within a week of the scan discussion visit. RESULTS: Oncologists (n = 4) appeared receptive to the Oncolo-GIST intervention and scored nearly perfectly on post-training tests of material mastery after a < 2-h tutorial. Post-scan discussion visit, 100% of patients who met with an Oncolo-GIST-trained clinician understood that their cancer was considered incurable (a 31% improvement from pre-visit) compared with 91% of patients meeting with usual care oncologists (an 18% improvement); 33% of patients who met with an Oncolo-GIST-trained oncologist understood that they likely had months, not years, compared to 18% in the usual care group. No statistically significant differences emerged for these changes, nor for therapeutic alliance, anxiety, or depression scores between groups. CONCLUSION: Oncolo-GIST appears to be an easily learned approach to improve prognostic understanding that neither undermines therapeutic alliances nor increases patients' anxiety or depressive symptoms. Efficacy testing in a larger trial is warranted.

Double and triple thermodynamic mutant cycles reveal the basis for specific MsbA-lipid interactions.

Structural and functional studies of the ATP-binding cassette transporter MsbA have revealed two distinct lipopolysaccharide (LPS) binding sites: one located in the central cavity and the other at a membrane-facing, exterior site. Although these binding sites are known to be important for MsbA function, the thermodynamic basis for these specific MsbA-LPS interactions is not well understood. Here, we use native mass spectrometry to determine the thermodynamics of MsbA interacting with the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). The binding of KDL is solely driven by entropy, despite the transporter adopting an inward-facing conformation or trapped in an outward-facing conformation with adenosine 5'-diphosphate and vanadate. An extension of the mutant cycle approach is employed to probe basic residues that interact with KDL. We find the molecular recognition of KDL is driven by a positive coupling entropy (as large as -100 kJ/mol at 298K) that outweighs unfavorable coupling enthalpy. These findings indicate that alterations in solvent reorganization and conformational entropy can contribute significantly to the free energy of protein-lipid association. The results presented herein showcase the advantage of native MS to obtain thermodynamic insight into protein-lipid interactions that would otherwise be intractable using traditional approaches, and this enabling technology will be instrumental in the life sciences and drug discovery.

T1Gd is reduced in bone marrow lesions overlying cartilage in the hip.

OBJECTIVE: Bone Marrow Lesions (BMLs) are areas in bone with high fluid signal on MRI associated with painful and progressive OA. While cartilage near BMLs in the knee has been shown to be degenerated, this relationship has not been investigated in the hip. RESEARCH QUESTION: is T1Gd lower in areas of cartilage overlying BMLs in the hip? DESIGN: 128 participants were recruited from a population-based study of hip pain in 20-49-year-olds. Proton-density weighted fat-suppressed and delayed Gadolinium Enhanced MR Imaging of Cartilage (dGEMRIC) images were acquired to locate BMLs and quantify hip cartilage health. BML and cartilage images were registered and cartilage was separated into BML overlying and surrounding regions. Mean T1Gd was measured in 32 participants with BMLs in both cartilage regions and in matched regions in 32 age- and sex-matched controls. Mean T1Gd in the overlying cartilage was compared using linear mixed-effects models between BML and control groups for acetabular and femoral BMLs, and between cystic and non-cystic BML groups. RESULTS: Mean T1Gd of overlying cartilage was lower in the BML group compared to the control group (acetabular: -105 ms; 95% CI: -175, -35; femoral: -8 ms; 95% CI: -141, 124). Mean T1Gd in overlying cartilage was lower in cystic compared to non-cystic BML subjects, but the confidence interval is too large to provide certainty in this difference (-3 [95% CI: -126, 121]). CONCLUSIONS: T1Gd is reduced in overlying cartilage in hips from a population-based sample of adults aged 20-49, which suggests BMLs are associated with local cartilage degeneration in hips.

Mechanism of rotenone binding to respiratory complex I depends on ligand flexibility.

Respiratory complex I is a major cellular energy transducer located in the inner mitochondrial membrane. Its inhibition by rotenone, a natural isoflavonoid, has been used for centuries by indigenous peoples to aid in fishing and, more recently, as a broad-spectrum pesticide or even a possible anticancer therapeutic. Unraveling the molecular mechanism of rotenone action will help to design tuned derivatives and to understand the still mysterious catalytic mechanism of complex I. Although composed of five fused rings, rotenone is a flexible molecule and populates two conformers, bent and straight. Here, a rotenone derivative locked in the straight form was synthesized and found to inhibit complex I with 600-fold less potency than natural rotenone. Large-scale molecular dynamics and free energy simulations of the pathway for ligand binding to complex I show that rotenone is more stable in the bent conformer, either free in the membrane or bound to the redox active site in the substrate-binding Q-channel. However, the straight conformer is necessary for passage from the membrane through the narrow entrance of the channel. The less potent inhibition of the synthesized derivative is therefore due to its lack of internal flexibility, and interconversion between bent and straight forms is required to enable efficient kinetics and high stability for rotenone binding. The ligand also induces reconfiguration of protein loops and side-chains inside the Q-channel similar to structural changes that occur in the open to closed conformational transition of complex I. Detailed understanding of ligand flexibility and interactions that determine rotenone binding may now be exploited to tune the properties of synthetic derivatives for specific applications.

The ability of Interleukin-10 to negate haemozoin-related pro-inflammatory effects has the potential to restore impaired macrophage function associated with malaria infection.

BACKGROUND: Although pro-inflammatory cytokines are involved in the clearance of Plasmodium falciparum during the early stages of the infection, increased levels of these cytokines have been implicated in the pathogenesis of severe malaria. Amongst various parasite-derived inducers of inflammation, the malarial pigment haemozoin (Hz), which accumulates in monocytes, macrophages and other immune cells during infection, has been shown to significantly contribute to dysregulation of the normal inflammatory cascades. METHODS: The direct effect of Hz-loading on cytokine production by monocytes and the indirect effect of Hz on cytokine production by myeloid cells was investigated during acute malaria and convalescence using archived plasma samples from studies investigating P. falciparum malaria pathogenesis in Malawian subjects. Further, the possible inhibitory effect of IL-10 on Hz-loaded cells was examined, and the proportion of cytokine-producing T-cells and monocytes during acute malaria and in convalescence was characterized. RESULTS: Hz contributed towards an increase in the production of inflammatory cytokines, such as Interferon Gamma (IFN-γ), Tumor Necrosis Factor (TNF) and Interleukin 2 (IL-2) by various cells. In contrast, the cytokine IL-10 was observed to have a dose-dependent suppressive effect on the production of TNF among other cytokines. Cerebral malaria (CM) was characterized by impaired monocyte functions, which normalized in convalescence. CM was also characterized by reduced levels of IFN-γ-producing T cell subsets, and reduced expression of immune recognition receptors HLA-DR and CD 86, which also normalized in convalescence. However, CM and other clinical malaria groups were characterized by significantly higher plasma levels of pro-inflammatory cytokines than healthy controls, implicating anti-inflammatory cytokines in balancing the immune response. CONCLUSIONS: Acute CM was characterized by elevated plasma levels of pro-inflammatory cytokines and chemokines but lower proportions of cytokine-producing T-cells and monocytes that normalize during convalescence. IL-10 is also shown to have the potential to indirectly prevent excessive inflammation. Cytokine production dysregulated by the accumulation of Hz appears to impair the balance of the immune response to malaria and exacerbates pathology.

An ion mobility-mass spectrometry study of copper-metallothionein-2A: binding sites and stabilities of Cu-MT and mixed metal Cu-Ag and Cu-Cd complexes.

The presence of Cu, a highly redox active metal, is known to damage DNA as well as other cellular components, but the adverse effects of cellular Cu can be mitigated by metallothioneins (MT), small cysteine rich proteins that are known to bind to a broad range of metal ions. While metal ion binding has been shown to involve the cysteine thiol groups, the specific ion binding sites are controversial as are the overall structure and stability of the Cu-MT complexes. Here, we report results obtained using nano-electrospray ionization mass spectrometry and ion mobility-mass spectrometry for several Cu-MT complexes and compare our results with those previously reported for Ag-MT complexes. The data include determination of the stoichiometries of the complex (Cui-MT, i = 1-19), and Cu+ ion binding sites for complexes where i = 4, 6, and 10 using bottom-up and top-down proteomics. The results show that Cu+ ions first bind to the ß-domain to form Cu4MT then Cu6MT, followed by addition of four Cu+ ions to the α-domain to form a Cu10-MT complex. Stabilities of the Cui-MT (i = 4, 6 and 10) obtained using collision-induced unfolding (CIU) are reported and compared with previously reported CIU data for Ag-MT complexes. We also compare CIU data for mixed metal complexes (CuiAgj-MT, where i + j = 4 and 6 and CuiCdj, where i + j = 4 and 7). Lastly, higher order Cui-MT complexes, where i = 11-19, were also detected at higher concentrations of Cu+ ions, and the metalated product distributions observed are compared to previously reported results for Cu-MT-1A (Scheller et al., Metallomics, 2017, 9, 447-462).

Mechanistic Insights into IscU Conformation Regulation for Fe-S Cluster Biogenesis Revealed by Variable Temperature Electrospray Ionization Native Ion Mobility Mass Spectrometry.

Iron-sulfur (Fe-S) cluster (ISC) cofactors are required for the function of many critical cellular processes. In the ISC Fe-S cluster biosynthetic pathway, IscU assembles Fe-S cluster intermediates from iron, electrons, and inorganic sulfur, which is provided by the cysteine desulfurase enzyme IscS. IscU also binds to Zn, which mimics and competes for binding with the Fe-S cluster. Crystallographic and nuclear magnetic resonance spectroscopic studies reveal that IscU is a metamorphic protein that exists in multiple conformational states, which include at least a structured form and a disordered form. The structured form of IscU is favored by metal binding and is stable in a narrow temperature range, undergoing both cold and hot denaturation. Interestingly, the form of IscU that binds IscS and functions in Fe-S cluster assembly remains controversial. Here, results from variable temperature electrospray ionization (vT-ESI) native ion mobility mass spectrometry (nIM-MS) establish that IscU exists in structured, intermediate, and disordered forms that rearrange to more extended conformations at higher temperatures. A comparison of Zn-IscU and apo-IscU reveals that Zn(II) binding attenuates the cold/heat denaturation of IscU, promotes refolding of IscU, favors the structured and intermediate conformations, and inhibits the disordered high charge states. Overall, these findings provide a structural rationalization for the role of Zn(II) in stabilizing IscU conformations and IscS in altering the IscU active site to prepare for Zn(II) release and cluster synthesis. This work highlights how vT-ESI-nIM-MS can be applied as a powerful tool in mechanistic enzymology by providing details of relationships among temperature, protein conformations, and ligand/protein binding.