Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides.

Dampening functional levels of the mitochondrial deubiquitylating enzyme Ubiquitin-specific protease 30 (USP30) has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinson's Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria, which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide-containing compound, USP30inh. Activity-based protein profiling mass spectrometry confirmed target engagement, high selectivity, and potency of USP30inh for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of USP30inh enzyme kinetics inferred slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with USP30inh, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that USP30inh binds to this thumb-palm cleft, which guides the ubiquitin C terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.

Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications.

Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localization of dynamic changes. Consequently, the HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase the data quality by providing an orthogonal mode of peptide ion separation in the gas phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multipass IM separations. Using one-pass and multipass cIM routines, we use the recently commercialized SELECT SERIES Cyclic IM spectrometer for HDX-MS analyses of four detergent solubilized IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multipass cIM data. Interestingly, use of one-pass and multipass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM that has the potential to enable the analysis of more complex systems with greater accuracy and speed.

Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein-Lipid Assemblies.

Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins due to their presence of impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Herein, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium-labeled protein-lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein-lipid nanodisc─both empty and loaded with the ∼115 kDa transmembrane protein AcrB─proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO2-coated and TiO2 beads and describe how solution conditions can be optimized to maximize not only the performance of our online but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even native cell membranes.

Validation of ion mobility spectrometry - mass spectrometry as a screening tool to identify type II kinase inhibitors of FGFR1 kinase.

RATIONALE: The protein kinase FGFR1 regulates cellular processes in human development. As over-activity of FGFR1 is implicated with cancer, effective inhibitors are in demand. Type I inhibitors, which bind to the active form of FGFR1, are less effective than type II inhibitors, which bind to the inactive form. Screening to distinguish between type I and type II inhibitors is required. METHODS: X-ray crystallography was used to indicate whether a range of potential inhibitors bind to the active or inactive FGFR1 kinase conformation. The binding affinity of each ligand to FGFR1 was measured using biochemical methods. Electrospray ionisation - ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) in conjunction with collision-induced protein unfolding generated a conformational profile of each FGFR1-ligand complex. The results indicate that the protein's conformational profile depends on whether the inhibitor is type I or type II. RESULTS: X-ray crystallography confirmed which of the kinase inhibitors bind to the active or inactive form of FGFR1 kinase. Collision-induced unfolding combined with ESI-IMS-MS showed distinct differences in the FGFR1 folding landscape for type I and type II inhibitors. Biochemical studies indicated a similar range of FGFR1 affinities for both types of inhibitors, thus providing confidence that the conformational variations detected using ESI-IMS-MS can be interpretated unequivocally and that this is an effective screening method. CONCLUSIONS: A robust ESI-IMS-MS method has been implemented to distinguish between the binding mode of type I and type II inhibitors by monitoring the conformational unfolding profile of FGFR1. This rapid method requires low sample concentrations and could be used as a high-throughput screening technique for the characterisation of novel kinase inhibitors.

Testing a Model of Resilience in Family Members of Relatives with Traumatic Brain Injury vs Spinal Cord Injury: Multigroup Analysis.

OBJECTIVE: To test a model comprising explanatory (neurologic impairment, coping, personality) and mediating (resilience, self-efficacy, hope, social support) variables on psychological adjustment and burden among family caregivers of individuals with traumatic brain injury (TBI) vs spinal cord injury (SCI). DESIGN: Structural equation modeling with multigroup analysis. SETTING: Six rehabilitation centers across New South Wales and Queensland, Australia. PARTICIPANTS: A total of 181 family members (N=181; 131 TBI, 50 SCI). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Connor-Davidson Resilience Scale, Eysenck Personality Questionnaire, Ways of Coping Questionnaire, General Self-Efficacy Scale, Herth Hope Scale, Medical Outcome Study Social Support Survey; and 4 measures of psychological adjustment including: Caregiver Burden Scale, Medical Outcomes Survey Short Form-36 (SF-36), General Health Questionnaire-28, and Positive and Negative Affect Scale. RESULTS: The model for the aggregated sample demonstrated a very good model fit (χ2=47.42, df=39, ρ=0.167, normed fit index=.962, incremental fit index=.993, Tucker-Lewis index=.985, comparative fit index=.993, root-mean-squared error of approximation=.035). Multi-group analysis found significant commonalities in the pattern of relationships among variables across the 2 groups. In the only differences found, neuroticism was significantly more influential on burden in family members supporting individuals with TBI than family members of individuals with SCI. Furthermore, problem-focused coping was statistically more influential on positive affect in family members of individuals with TBI when compared with family members of individuals with SCI. CONCLUSIONS: The study found significant similarities in the patterns of resilience and psychological adjustment among family caregivers of individuals with TBI and SCI.

Neoadjuvant chemotherapy in non-metastatic breast cancer: a study on practice trends in a regional cancer treatment service.

BACKGROUND: Neoadjuvant chemotherapy (NACT) is increasingly used for managing locally advanced and high risk non-metastatic breast cancer. AIMS: To describe trends in NACT use, assess compliance to best practice recommendations and determine treatment response rates in a regional cancer treatment service. METHODS: In this retrospective cross- sectional study, electronic records of patients who underwent NACT in centres covered by the MidCentral Regional Cancer Treatment Service in 2013 and 2017 were reviewed. Data pertaining to patient demographics, disease status, compliance to best practice recommendations and treatment outcomes were extracted and analysed. RESULTS: Of a total of 502 referrals for non-metastatic breast cancer, 34 underwent NACT with the estimated NACT rate rising from 3.85% (2013) to 9.92% (2017). Compliance to practice recommendations improved in all domains (pre-treatment tumour and axillary evaluation, marker placement, multidisciplinary discussion). Overall, NACT was well tolerated with only three patients experiencing treatment limiting toxicity. Response rates mirror published data (complete response: 29.4%, partial: 61.8%) with higher responses registered in HER2 positive and triple negative subtypes. Discordance between radiological and pathological response was 28%, with imaging overestimating response in five out of seven cases. Of the 11 (32%) patients who initially underwent breast conserving surgery, six required a second surgery. CONCLUSION: NACT is increasingly used in the Regional Cancer Treatment Service, with improving compliance to practice recommendations. These results are reassuring and can be used to help patients develop a realistic expectation towards NACT.

A predictive model of resilience among family caregivers supporting relatives with traumatic brain injury (TBI): A structural equation modelling approach.

Constructs from positive psychology were employed to create an explicit model of caregiver resilience. Predictive and mediating relationships among resilience and related variables (personality, coping, self-efficacy, hope, social support) were then tested for their association with burden and psychological adjustment among family members caring for relatives with severe TBI. Family participants (n = 131) from six rehabilitation units from New South Wales and Queensland completed assessments which elicited explanatory (Eysenck Personality Questionnaire, Ways of Coping Questionnaire), mediating (Connor-Davidson Resilience Scale, General Self-Efficacy Scale, Herth Hope Scale, Medical Outcome Study Social Support Survey), and caregiver outcome (Caregiver Burden Scale, Mental Health sub-Scale-SF36, General Health Questionnaire, and Positive and Negative Affect Scale) variables. Structural Equation Modeling (SEM) showed that resilience had a direct effect on positive affect in caregivers. Resilience also played a protective role in relation to two variables associated with caregiver vulnerability: an indirect association with caregiver burden mediated through social support; a direct effect on hope, which, in turn, was associated with positive mental health. Positive mental health then played a buffering role in relation to psychological distress and negative affect. Resilience, in combination with other psychological attributes, was associated with reduced morbidity among family caregivers after severe TBI.

A study of whether individual and dyadic relations between spirituality and resilience contribute to psychological adjustment among individuals with spinal cord injuries and their family members.

OBJECTIVE: To test a model of spiritual well-being and resilience among individuals with spinal cord injuries and their family members. DESIGN: Prospective cross-sectional observational data were analyzed by structural equation modelling. SETTING: Inpatient and community services at one rehabilitation hospital. SUBJECTS: Individual with spinal cord injury (n = 50) and family member (n = 50) dyads. INTERVENTIONS: Standard rehabilitation, both inpatient and community. MAIN MEASURE(S): Functional assessment of chronic illness therapy - spiritual well-being scale - expanded, Connor-Davidson resilience scale, positive and negative affect scale, depression anxiety and stress scale-21, satisfaction with life scale. RESULTS: Median time post-injury was 8.95 months (IQR (interquartile range) = 14.15). Individuals with spinal cord injury and family members reported high scores for both spiritual well-being (66.06 ± 14.89; 68.42 ± 13.75) and resilience (76.68 ± 13.88; 76.64 ± 11.75), respectively. Analysis found the model had acceptable fit (e.g. chi-square goodness of fit statistic = 38.789; P = .263). For individuals with spinal cord injury, spiritual well-being was positively associated with resilience which, in turn, was associated with increasing positive affect and satisfaction with life. Among family members, spiritual well-being was positively associated with resilience. Resilience was then associated with lowered levels of depression and mediated the impact of depression on satisfaction with life. Limited evidence was found for mutual dyadic links, with the only significant pathway finding that resilience in the individual with spinal cord injury was associated with increased satisfaction with life among family members. CONCLUSION: Increased spirituality and resilience make a significant contribution (both independently and in combination) to positive psychological outcomes for both individuals with spinal cord injury and their family members.

Direct evidence for conformational dynamics in major histocompatibility complex class I molecules.

Major histocompatibility complex class I molecules (MHC I) help protect jawed vertebrates by binding and presenting immunogenic peptides to cytotoxic T lymphocytes. Peptides are selected from a large diversity present in the endoplasmic reticulum. However, only a limited number of peptides complement the polymorphic MHC specificity determining pockets in a way that leads to high-affinity peptide binding and efficient antigen presentation. MHC I molecules possess an intrinsic ability to discriminate between peptides, which varies in efficiency between allotypes, but the mechanism of selection is unknown. Elucidation of the selection mechanism is likely to benefit future immune-modulatory therapies. Evidence suggests peptide selection involves transient adoption of alternative, presumably higher energy conformations than native peptide-MHC complexes. However, the instability of peptide-receptive MHC molecules has hindered characterization of such conformational plasticity. To investigate the dynamic nature of MHC, we refolded MHC proteins with peptides that can be hydrolyzed by UV light and thus released. We compared the resultant peptide-receptive MHC molecules with non-hydrolyzed peptide-loaded MHC complexes by monitoring the exchange of hydrogen for deuterium in solution. We found differences in hydrogen-deuterium exchange between peptide-loaded and peptide-receptive molecules that were negated by the addition of peptide to peptide-receptive MHC molecules. Peptide hydrolysis caused significant increases in hydrogen-deuterium exchange in sub-regions of the peptide-binding domain and smaller increases elsewhere, including in the α3 domain and the non-covalently associated ß2-microglobulin molecule, demonstrating long-range dynamic communication. Comparing two MHC allotypes revealed allotype-specific differences in hydrogen-deuterium exchange, consistent with the notion that MHC I plasticity underpins peptide selection.

Enhanced Detection of Low-Abundance Host Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry Coupled with Multidimensional Liquid Chromatography.

The enormous dynamic range of proteinaceous species present in protein biotherapeutics poses a significant challenge for current mass spectrometry (MS)-based methods to detect low-abundance HCP impurities. Previously, an HCP assay based on two-dimensional chromatographic separation (high pH/low pH) coupled to high-resolution quadrupole time-of-flight (QTOF) mass spectrometry and developed in the author's laboratory has been shown to achieve a detection limit of about 50 ppm (parts per milion) for the identification and quantification of HCPs present in monoclonal antibodies following Protein A purification.1 To improve the HCP detection limit we have explored the utility of several new analytical techniques for HCP analysis and thereby developed an improved liquid chromatography-mass spectrometry (LC-MS) methodology for enhanced detection of HCPs. The new method includes (1) the use of a new charge-surface-modified (CSH) C18 stationary phase to mitigate the challenges of column saturation, peak tailing, and distortion that are commonly observed in the HCP analysis; (2) the incorporation of traveling-wave ion mobility (TWIM) separation of coeluting peptide precursors, and (3) the improvement of fragmentation efficiency of low-abundance HCP peptides by correlating the collision energy used for precursor fragmentation with their mobility drift time. As a result of these improvements, the detection limit of the new methodology was greatly improved, and HCPs present at a concentration as low as 1 ppm (1 ng HCP/mg mAb) were successfully identified and quantified. The newly developed method was applied to analyze two high-purity mAbs (NIST mAb and Infliximab) expressed in a murine cell line. For both samples, low-abundance HCPs (down to 1 ppm) were confidently identified, and the identities of the HCPs were further confirmed by targeted MS/MS experiments. In addition, the performance of the assay was evaluated by an interlaboratory study in which three independent laboratories performed the same HCP assay on the mAb sample. The reproducibility of this assay is also discussed.

Chapter 7 the relationship between coping and psychological adjustment in family caregivers of individuals with traumatic brain injury: a systematic review.

A systematic review was conducted to evaluate the association between coping (as measured by the Ways of Coping Questionnaire [WOCQ]) and psychological adjustment in caregivers of individuals with traumatic brain injury (TBI). A search conducted using the CINAHL, Medline, and PsycINFO databases yielded 201 citations between 1974 and 2014. A total of seven articles met the inclusion criteria; namely, the respondents who completed the WOCQ were family caregivers of individuals with TBI (including 66-item, 42-item, or 21-item versions). Reviews were conducted in accordance with the American Academy of Neurology guidelines (2011) for classifying evidence. The results found no Class 1 or Class II studies but only four Class III and three Class IV studies. The major finding across the better-rated Class III studies was that the use of emotion-focused coping and problem-focused coping was possibly associated with psychological adjustment in caregivers. The Class IV studies were determined to be inadequate or conflicting in determining the association between coping and psychological adjustment. Future studies need to employ carefully crafted designs, adhere to statistical procedure, apply advanced analytic techniques, and employ explicit models of coping, which will increase the accuracy and generalizability of the findings.

Erlotinib or Gefitinib for Treating Advanced Epidermal Growth Factor Receptor Mutation-Positive Lung Cancer in Aotearoa New Zealand: Protocol for a National Whole-of-Patient-Population Retrospective Cohort Study and Results of a Validation Substudy.

BACKGROUND: Starting in 2010, the epidermal growth factor receptor (EGFR) kinase inhibitors erlotinib and gefitinib were introduced into routine use in Aotearoa New Zealand (NZ) for treating advanced lung cancer, but their impact in this setting is unknown. OBJECTIVE: The study described in this protocol aims to understand the effectiveness and safety of these new personalized lung cancer treatments and the contributions made by concomitant medicines and other factors to adverse outcomes in the general NZ patient population. A substudy aimed to validate national electronic health databases as the data source and the methods for determining patient eligibility and identifying outcomes and variables. METHODS: This study will include all NZ patients with advanced EGFR mutation-positive lung cancer who were first dispensed erlotinib or gefitinib before October 1, 2020, and followed until death or for at least 1 year. Routinely collected health administrative and clinical data will be collated from national electronic cancer registration, hospital discharge, mortality registration, and pharmaceutical dispensing databases by deterministic data linkage using National Health Index numbers. The primary effectiveness and safety outcomes will be time to treatment discontinuation and serious adverse events, respectively. The primary variable will be high-risk concomitant medicines use with erlotinib or gefitinib. For the validation substudy (n=100), data from clinical records were compared to those from national electronic health databases and analyzed by agreement analysis for categorical data and by paired 2-tailed t tests for numerical data. RESULTS: In the validation substudy, national electronic health databases and clinical records agreed in determining patient eligibility and for identifying serious adverse events, high-risk concomitant medicines use, and other categorical data with overall agreement and κ statistic of >90% and >0.8000, respectively; for example, for the determination of patient eligibility, the comparison of proxy and standard eligibility criteria applied to national electronic health databases and clinical records, respectively, showed overall agreement and κ statistic of 96% and 0.8936, respectively. Dates for estimating time to treatment discontinuation and other numerical variables and outcomes showed small differences, mostly with nonsignificant P values and 95% CIs overlapping with zero difference; for example, for the dates of the first dispensing of erlotinib or gefitinib, national electronic health databases and clinical records differed on average by approximately 4 days with a nonsignificant P value of .33 and 95% CIs overlapping with zero difference. As of May 2024, the main study is ongoing. CONCLUSIONS: A protocol is presented for a national whole-of-patient-population retrospective cohort study designed to describe the safety and effectiveness of erlotinib and gefitinib during their first decade of routine use in NZ for treating EGFR mutation-positive lung cancer. The validation substudy demonstrated the feasibility and validity of using national electronic health databases and the methods for determining patient eligibility and identifying the study outcomes and variables proposed in the study protocol. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12615000998549; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=368928. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/51381.

The impact of neurobehavioral impairment on family functioning and the psychological well-being of male versus female caregivers of relatives with severe traumatic brain injury: multigroup analysis.

OBJECTIVE: To examine the differential effect of neurobehavioral impairments (cognitive, behavioral, and social) on family functioning, family roles, and psychological distress in male versus female caregivers of relatives with severe traumatic brain injury (TBI). DESIGN: Structural equation modeling with multigroup analysis conducted in a cross-sectional sample to test an established theoretical model. PARTICIPANTS: An aggregated sample of 122 caregivers (46 male, 76 female) of people with severe TBI. The sample comprised 64 spouses and 58 parents (29 parental couples) of 93 persons with TBI. MEASURES: Neurobehavioral Problem Checklist; Family Assessment Device; and Brief Symptom Inventory. RESULTS: Structural equation modeling showed that the proposed model had acceptable fit indices for the combined sample. Multigroup analysis indicated that both male and female caregivers (i) responded similarly to the neurobehavioral impairments experienced by the injured relative and (ii) reported behavior having a direct effect on family functioning, which, in turn, increased psychological distress. However, the effect of disrupted family functioning was more influential on the level of distress in male caregivers than in female caregivers. CONCLUSION: Evidence was found for gender-specific pathways underlying the psychological distress of male versus female caregivers. Such findings can assist in tailoring family support strategies so that they cater for caregivers of both genders.

Testing the stability of a family resilience model at 2 and 5 years after traumatic brain injury or spinal cord injury: A longitudinal study.

BACKGROUND: Recent studies have tested models of resilience and caregiver adjustment in individuals with traumatic brain injury (TBI) or spinal cord injury (SCI). Few studies have examined the role of adaptive variables over time. OBJECTIVE: Conduct a longitudinal study to test a model of caregiver resilience with caregiver outcomes at 2- and 5-years post-injury. METHOD: Caregivers of relatives with TBI or SCI were surveyed at 2 years (Time 1) and 5 years (Time 2) post-injury. Stability of the resilience model across the 2 time-points was tested using structural equation modeling with multi-group analysis. Measures included resilience related variables (Connor-Davidson Resilience Scale, General Self-Efficacy Scale, Herth Hope Scale, Social Support Survey) and outcome variables (Caregiver Burden Scale, General Health Questionnaire-28, Medical Outcome Study Short Form -36 [SF-36] and Positive and Negative Affect Scale). RESULTS: In total, 100 caregivers were surveyed at both 2 and 5 years (TBI =77, SCI =23). Scores for resilience (Time 1, 75.9 SD 10.6; Time 2, 71.5 SD 12.6) and self-efficacy (Time 1, 32.51 SD 3.85; Time 2, 31.66 SD 4.28) showed significant minor declines, with other variables remaining stable. The resilience model for the pooled responses (Time 1+ Time 2) demonstrated a good fit (Goodness of Fit Index [GFI] = 0.971; Incremental Fit Index [IFI] = 0.986; Tucker-Lewis Index [TLI] = 0.971; Comparative Fit Index [CFI] = 0.985 and Root Mean Square Error of Approximation [RMSEA] = 0.051). Multi-group analysis then compared Time 1 to Time 2 responses and found that a variant (compared to invariant) model best fitted the data, with social support having stronger associations with mental health and positive affect at Time 2 than Time 1. Hope reduced from Time 1 to Time 2. CONCLUSIONS: The model suggests that resilience-related variables can play an important role in positive caregiver adjustment over time.

Nurse Executives' Perceptions of the Executive Training for Research Application (EXTRA) Program.

BACKGROUND: To improve the overall quality and effectiveness of the Canadian health care system through better decisions supported by research-based evidence (RBE), the Canadian Health Services Research Foundation (CHSRF) and partners have created the Executive Training for Research Application (EXTRA) program. OBJECTIVES: To evaluate how nurse executive fellows perceive changes in their levels of knowledge of RBE and in their level of use of RBE following participation in the EXTRA program. METHODS: Nurse executives in the first four cohorts of the program (2004-2007) completed a survey during their 2-year fellowship period. RESULTS: Statistically significant improvements were observed regarding nurse executives' perceived knowledge and use of RBE. According to the participants, the EXTRA fellowship contributes to their role and function in their organization by providing tools, learning, and access to resources and networking, which contributes to their credibility, leadership, and knowledge transfer skills. CONCLUSIONS: The EXTRA program has been structured to reduce barriers and to enhance the facilitators found in the literature on the implementation of evidence-based practices (EBP) in health care settings. Overall, nurse executives perceived that the benefits of participating in the EXTRA program were both individual and organizational.

Ligand binding and conformational dynamics of the E. coli nicotinamide nucleotide transhydrogenase revealed by hydrogen/deuterium exchange mass spectrometry.

Nicotinamide nucleotide transhydrogenases are integral membrane proteins that utilizes the proton motive force to reduce NADP+ to NADPH while converting NADH to NAD+. Atomic structures of various transhydrogenases in different ligand-bound states have become available, and it is clear that the molecular mechanism involves major conformational changes. Here we utilized hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map ligand binding sites and analyzed the structural dynamics of E. coli transhydrogenase. We found different allosteric effects on the protein depending on the bound ligand (NAD+, NADH, NADP+, NADPH). The binding of either NADP+ or NADPH to domain III had pronounced effects on the transmembrane helices comprising the proton-conducting channel in domain II. We also made use of cyclic ion mobility separation mass spectrometry (cyclic IMS-MS) to maximize coverage and sensitivity in the transmembrane domain, showing for the first time that this technique can be used for HDX-MS studies. Using cyclic IMS-MS, we increased sequence coverage from 68 % to 73 % in the transmembrane segments. Taken together, our results provide important new insights into the transhydrogenase reaction cycle and demonstrate the benefit of this new technique for HDX-MS to study ligand binding and conformational dynamics in membrane proteins.

A prospective audit on the use of prophylactic antiemetics and rates of CINV in patients receiving carboplatin AUC≥4 or combination anthracycline-cyclophosphamide.

BACKGROUND: Chemotherapy-induced nausea and vomiting (CINV) are two of the most frequently experienced and distressing side effects of cancer treatment. Recent updates by ESMO/MASCC and ASCO on guidelines for prevention of CINV have recommended the addition of a neurokinin-1 receptor antagonist to antiemetic regimens for patients receiving carboplatin-based chemotherapy area under the curve (AUC) ≥4 mg/mL per minute, and an addition of olanzapine for those receiving combination anthracycline/cyclophosphamide chemotherapy. AIMS: To assess current use of prophylactic antiemetics and rates of CINV in patients under the care of MidCentral Regional Cancer Treatment Service (MRCTS) receiving carboplatin AUC≥4 or combination anthracycline/cyclophosphamide. METHODS: Data was prospectively collected on patients under the care of MRCTS receiving carboplatin AUC≥4 or combination anthracycline/cyclophosphamide chemotherapy, including breast cancer patients receiving 5-fluorouracil, epirubicin and cyclophosphamide (FEC) and lymphoma patients receiving rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Questionnaires were given to eligible patients to be completed daily from day two to day six of first cycle of chemotherapy only. Data on each patient's gender, age, types of chemotherapies, types of malignancies, presence of nausea or vomiting, number of dry retching or vomiting episodes and anti-emetics were recorded. RESULTS: From 15 September 2018 to 10 August 2019, a total of 44 patients receiving carboplatin-based chemotherapy AUC≥4 and 30 patients receiving combination anthracycline/cyclophosphamide were included. Twenty-two patients (50%) had either emesis or significant nausea in the overall and delayed phase when treated with carboplatin AUC≥4, and only three (7%) in the acute phase. Fourteen patients (56%) had either emesis or significant nausea in the overall phase when treated with FEC chemotherapy, mostly in the acute phase (13 patients) rather than in the delayed phase (9 patients). CONCLUSION: The rates of CINV are high with the existing antiemetic regimens used at MidCentral Regional Cancer Treatment Service. Therefore, in accordance with international guidelines, we will add a neurokinin-1 antagonist to the antiemetic regimens for patients receiving carboplatin-based chemotherapy AUC≥4, and olanzapine for those receiving combination anthracycline/cyclophosphamide chemotherapy, in an attempt to improve the rates of CINV in these groups. Repeating this audit post-implementation of above recommendations will be important to assess for any improvement.

Affinity-based, biophysical methods to detect and analyze ligand binding to recombinant proteins: matching high information content with high throughput.

Affinity-based technologies have become impactful tools to detect, monitor and characterize molecular interactions using recombinant target proteins. This can aid the understanding of biological function by revealing mechanistic details, and even more importantly, enables the identification of new improved ligands that can modulate the biological activity of those targets in a desired fashion. The selection of the appropriate technology is a key step in that process, as each one of the currently available technologies offers a characteristic type of biophysical information about the ligand-binding event. Alongside the indisputable advantages of each of those technologies they naturally display diverse restrictions that are quite frequently related to the target system to be studied but also to the affinity, solubility and molecular size of the ligands. This paper discusses some of the theoretical and experimental aspects of the most common affinity-based methods, what type of information can be gained from each one of those approaches, and what requirements as well as limitations are expected from working with recombinant proteins on those platforms and how those can be optimally addressed.

The efficacy of a programme of landslide risk reduction in areas of unplanned housing in the Eastern Caribbean.

Poor countries are disproportionately affected by the cost of disasters. Yet there is evidence of the benefits of seeking to mitigate the impact of a disaster, compared with the costs incurred in 'making good' after a major event has occurred. This article reviews a programme of landslide risk reduction in unplanned communities in the Eastern Caribbean. The construction of appropriate surface water management measures, based on the application of scientific and engineering principles, has been demonstrated to reduce the hazard from rainfall-triggered landslides. Adopting a community-based approach additionally delivers social and environmental benefits relating to employment generation, improvements in the environmental conditions within the community, and improvements slope management practices. The sustained implementation of the community-based projects has provided the necessary evidence-base for these practices to influence Government policy and practice, and gain recognition from regional development agencies. The strategic and incremental uptake of the community-based methodology is demonstrated to be an effective means for delivering physical landslide risk reduction measures in the most 'at risk' areas of unplanned housing.

Getting the right information to the table: using technology to support evidence-based decision making.

Healthcare executives report that it is difficult to access the research literature and once found, it is frequently not relevant. A study was conducted to explore ways in which healthcare executives, enrolled in the EXTRA program, used a virtual desktop environment. Despite some design and function limitations, the desktop was perceived positively by most participants and was effective in supporting evidence-informed practice and decision making.