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3.  Aboriginal health research in the remote Kimberley: an exploration of perceptions, attitudes and concerns of stakeholders 
Background
For decades Indigenous peoples have argued for health research reform claiming methods used and results obtained often reflect the exploitative history of colonisation. In 2006 the Kimberley Aboriginal Health Planning Forum (KAHPF) Research Subcommittee (hereafter, the Subcommittee) was formed to improve research processes in the remote Kimberley region of north Western Australia. This paper explores the major perceptions, attitudes and concerns of stakeholders in the Subcommittee.
Methods
Qualitative analysis was carried out on data retrospectively collected from multiple evidentiary sources linked to the Subcommittee i.e. database, documents, interviews, review forms and emails from 1 January 2007 to 31 October 2013.
Results
From 1 January 2007 to 30 June 2013 the Subcommittee received 95 proposals, 57 (60%) driven by researchers based outside the region. Local stakeholders (22 from 12 different Kimberley organisations) raised concerns about 36 (38%) projects, 30 (83%) of which were driven by external researchers. Major concerns of local stakeholders were inadequate community consultation and engagement; burden of research on the region; negative impact of research practices; lack of demonstrable community benefit; and power and control of research. Major themes identified by external stakeholders (25 external researchers who completed the review form) were unanticipated difficulties with consultation processes; barriers to travel; perceiving research as a competing priority for health services and time-consuming ethics processes. External stakeholders also identified strategies for improving research practices in the Kimberley: importance of community support in building good relationships; employing local people; flexibility in research approaches; and importance of allocating sufficient time for consultation and data collection.
Conclusions
Health research in the Kimberley has improved in recent years, however significant problems remain. Prioritising research addressing genuine local needs is essential in closing the gap in Indigenous life expectancy. The long-term aim is for local health service connected researchers to identify priorities, lead, conduct and participate in the majority of local health research. For this to occur, a more radical move involving reconceptualising the research process is needed. Changes to institutional timeframes and funding processes could improve Indigenous and community-based research.
doi:10.1186/s12913-014-0517-1
PMCID: PMC4213490  PMID: 25343849
Aboriginal and Torres Strait Islander research; Ethics; Kimberley Aboriginal Health Planning Forum Research Subcommittee; Community consultation; Research processes; Decolonising research
4.  Quality improvement in practice: improving diabetes care and patient outcomes in Aboriginal Community Controlled Health Services 
Background
Management of chronic disease, including diabetes, is a central focus of most Aboriginal Community Controlled Health Services (ACCHSs) in Australia. We have previously demonstrated that diabetes monitoring and outcomes can be improved and maintained over a 10-year period at Derby Aboriginal Health Service (DAHS). While continuous quality improvement (CQI) has been shown to improve service delivery rates and clinical outcome measures, the process of interpreting audit results and developing strategies for improvement is less well described. This paper describes the evaluation of care of patients with type 2 diabetes mellitus (T2DM) and features of effective CQI in ACCHSs in the remote Kimberley region of north Western Australia.
Methods
Retrospective audit of records for Aboriginal and Torres Strait Islander primary care patients aged ≥15 years with a confirmed diagnosis of T2DM at four Kimberley ACCHSs from 1 July 2011 to 30 June 2012. Interviews with health service staff and focus group discussions with patients post audit. Main outcome measures: diabetes care related activities, clinical outcome measures and factors influencing good diabetes related care and effective CQI.
Results
A total of 348 patients from the four ACCHSs were included in the study. Clinical care activities were generally high across three of the four health services (at least 71% of patients had cholesterol recorded, 89% blood pressure, 84% HbA1c). Patients from DAHS had lower median cholesterol levels (4.4 mmol/L) and the highest proportion of patients meeting clinical targets for HbA1c (31% v 16% ACCHS-3; P = 0.02). Features that facilitated good care included clearly defined staff roles for diabetes management, support and involvement of Aboriginal Health Workers, efficient recall systems, and well-coordinated allied health services. Effective CQI features included seamless and timely data collection, local ownership of the process, openness to admitting deficiencies and willingness to embrace change.
Conclusions
Well-designed health care delivery and CQI systems, with a strong sense of ownership over diabetes management led to increased service delivery rates and improved clinical outcome measures in ACCHSs. Locally run CQI processes may be more responsive to individual health services and more sustainable than externally driven systems.
doi:10.1186/1472-6963-14-481
PMCID: PMC4282197  PMID: 25288282
Indigenous; Aboriginal; Torres Strait Islander; Diabetes; Quality improvement cycles; Primary health care
5.  Dual-Phase Cardiac Diffusion Tensor Imaging with Strain Correction 
PLoS ONE  2014;9(9):e107159.
Purpose
In this work we present a dual-phase diffusion tensor imaging (DTI) technique that incorporates a correction scheme for the cardiac material strain, based on 3D myocardial tagging.
Methods
In vivo dual-phase cardiac DTI with a stimulated echo approach and 3D tagging was performed in 10 healthy volunteers. The time course of material strain was estimated from the tagging data and used to correct for strain effects in the diffusion weighted acquisition. Mean diffusivity, fractional anisotropy, helix, transverse and sheet angles were calculated and compared between systole and diastole, with and without strain correction. Data acquired at the systolic sweet spot, where the effects of strain are eliminated, served as a reference.
Results
The impact of strain correction on helix angle was small. However, large differences were observed in the transverse and sheet angle values, with and without strain correction. The standard deviation of systolic transverse angles was significantly reduced from 35.9±3.9° to 27.8°±3.5° (p<0.001) upon strain-correction indicating more coherent fiber tracks after correction. Myocyte aggregate structure was aligned more longitudinally in systole compared to diastole as reflected by an increased transmural range of helix angles (71.8°±3.9° systole vs. 55.6°±5.6°, p<0.001 diastole). While diastolic sheet angle histograms had dominant counts at high sheet angle values, systolic histograms showed lower sheet angle values indicating a reorientation of myocyte sheets during contraction.
Conclusion
An approach for dual-phase cardiac DTI with correction for material strain has been successfully implemented. This technique allows assessing dynamic changes in myofiber architecture between systole and diastole, and emphasizes the need for strain correction when sheet architecture in the heart is imaged with a stimulated echo approach.
doi:10.1371/journal.pone.0107159
PMCID: PMC4156436  PMID: 25191900
6.  Clinical trials in a remote Aboriginal setting: lessons from the BOABS smoking cessation study 
BMC Public Health  2014;14:579.
Background
There is limited evidence regarding the best approaches to helping Indigenous Australians to stop smoking. The composite analysis of the only two smoking cessation randomised controlled trials (RCTs) investigating this suggests that one-on-one extra support delivered by and provided to Indigenous Australians in a primary health care setting appears to be more effective than usual care in encouraging smoking cessation. This paper describes the lessons learnt from one of these studies, the Be Our Ally Beat Smoking (BOABS) Study, and how to develop and implement an integrated smoking cessation program.
Methods
Qualitative study using data collected from multiple documentary sources related to the BOABS Study. As the project neared completion the research team participated in four workshops to review and conduct thematic analyses of these documents.
Results
Challenges we encountered during the relatively complex BOABS Study included recruiting sufficient number of participants; managing the project in two distant locations and ensuring high quality work across both sites; providing appropriate training and support to Aboriginal researchers; significant staff absences, staff shortages and high workforce turnover; determining where and how the project fitted in the clinics and consequent siloing of the Aboriginal researchers relating to the requirements of RCTs; resistance to change, and maintaining organisational commitment and priority for the project. The results of this study also demonstrated the importance of local Aboriginal ownership, commitment, participation and control. This included knowledge of local communities, the flexibility to adapt interventions to local settings and circumstances, and taking sufficient time to allow this to occur.
Conclusions
The keys to the success of the BOABS Study were local development, ownership and participation, worker professional development and support, and operating within a framework of cultural safety. There were difficulties associated with the BOABS Study being an RCT, and many of these are shared with stand-alone programs. Interventions targeted at particular health problems are best integrated with usual primary health care. Research to investigate complex interventions in Indigenous health should not be limited to randomised clinical trials and funding needs to reflect the additional, but necessary, cost of providing for local control of planning and implementation.
doi:10.1186/1471-2458-14-579
PMCID: PMC4064520  PMID: 24912949
Indigenous; Aboriginal; Torres Strait Islander; Smoking cessation; Be Our Ally Beat Smoking (BOABS) study; Qualitative; Randomised controlled trial
7.  The Kimberley Assessment of Depression of Older Indigenous Australians: Prevalence of Depressive Disorders, Risk Factors and Validation of the KICA-dep Scale 
PLoS ONE  2014;9(4):e94983.
Objective
This study aimed to develop a culturally acceptable and valid scale to assess depressive symptoms in older Indigenous Australians, to determine the prevalence of depressive disorders in the older Kimberley community, and to investigate the sociodemographic, lifestyle and clinical factors associated with depression in this population.
Methods
Cross-sectional survey of adults aged 45 years or over from six remote Indigenous communities in the Kimberley and 30% of those living in Derby, Western Australia. The 11 linguistic and culturally sensitive items of the Kimberley Indigenous Cognitive Assessment of Depression (KICA-dep) scale were derived from the signs and symptoms required to establish the diagnosis of a depressive episode according to the DSM-IV-TR and ICD-10 criteria, and their frequency was rated on a 4-point scale ranging from ‘never’ to ‘all the time’ (range of scores: 0 to 33). The diagnosis of depressive disorder was established after a face-to-face assessment with a consultant psychiatrist. Other measures included sociodemographic and lifestyle factors, and clinical history.
Results
The study included 250 participants aged 46 to 89 years (mean±SD = 60.9±10.7), of whom 143 (57.2%) were women. The internal reliability of the KICA-dep was 0.88 and the cut-point 7/8 (non-case/case) was associated with 78% sensitivity and 82% specificity for the diagnosis of a depressive disorder. The point-prevalence of a depressive disorder in this population was 7.7%; 4.0% for men and 10.4% for women. Heart problems were associated with increased odds of depression (odds ratio = 3.3, 95% confidence interval = 1.2,8.8).
Conclusions
The KICA-dep has robust psychometric properties and can be used with confidence as a screening tool for depression among older Indigenous Australians. Depressive disorders are common in this population, possibly because of increased stressors and health morbidities.
doi:10.1371/journal.pone.0094983
PMCID: PMC3989269  PMID: 24740098
8.  USING FLUORODEOXYTHYMIDINE TO MONITOR ANTI-EGFR INHIBITOR THERAPY IN SQUAMOUS CELL CARCINOMA XENOGRAFTS 
Head & neck  2008;30(6):790-799.
Background
3′-18F-fluoro-3′-deoxy-fluorothymidine (18F-FLT), a nucleoside analog, could monitor effects of molecularly targeted therapeutics on tumor proliferation.
Methods
We tested whether 18F-FLT positron emission tomography (PET) uptake changes are associated with antitumor effects of erlotinib in A431 xenografts or cetuximab in SCC1 xenografts.
Results
Compared with pretreatment FLT PET scans, 3 days of erlotinib in A431 reduced the standardized uptake value (SUV) by 18%, whereas placebo increased SUV by 1% (p = .005). One week of cetuximab in SCC1 reduced SUV by 62%, whereas placebo reduced SUV by 16% (p = .005). FLT uptake suppression following anti–epidermal growth factor receptor (EGFR) treatment was associated with reduced tumor thymidine kinase-1 (TK1) activity. In vitro TK1 knockdown studies confirmed the importance of TK1 activity on intracellular FLT accumulation suppression.
Conclusions
18F-FLT PET imaging detects tumor responses to EGFR-inhibitors within days of starting therapy. This technique may identify patients likely to benefit from EGFR-inhibitors early in their treatment course.
doi:10.1002/hed.20770
PMCID: PMC3942889  PMID: 18286491
fluorodeoxythymidine (FLT); anti-EGFR inhibitor therapy; squamous cell carcinoma xenografts; cetuximab; erlotinib
10.  The Be Our Ally Beat Smoking (BOABS) study, a randomised controlled trial of an intensive smoking cessation intervention in a remote aboriginal Australian health care setting 
BMC Public Health  2014;14:32.
Background
Australian Aboriginal and Torres Strait Islander peoples (Indigenous Australians) smoke at much higher rates than non-Indigenous people and smoking is an important contributor to increased disease, hospital admissions and deaths in Indigenous Australian populations. Smoking cessation programs in Australia have not had the same impact on Indigenous smokers as on non-Indigenous smokers. This paper describes the outcome of a study that aimed to test the efficacy of a locally-tailored, intensive, multidimensional smoking cessation program.
Methods
A randomised controlled trial of Aboriginal researcher delivered tailored smoking cessation counselling during face-to-face visits, aiming for weekly for the first four weeks, monthly to six months and two monthly to 12 months. The control (“usual care”) group received routine care relating to smoking cessation at their local primary health care service. Data collection occurred at enrolment, six and 12 months. The primary outcome was self-reported smoking cessation with urinary cotinine confirmation at final follow-up (median 13 (interquartile range 12–15) months after enrolment).
Results
Participants in the intervention (n = 55) and usual care (n = 108) groups were similar in baseline characteristics, except the intervention group was slightly older. At final follow-up the smoking cessation rate for participants assigned to the intervention group (n = 6; 11%), while not statistically significant, was double that of usual care (n = 5; 5%; p = 0.131). A meta-analysis of these findings and a similarly underpowered but comparable study of pregnant Indigenous Australian women showed that Indigenous Australian participants assigned to the intervention groups were 2.4 times (95% CI, 1.01-5.5) as likely to quit as participants assigned to usual care.
Conclusions
Culturally appropriate, multi-dimensional Indigenous quit smoking programs can be successfully implemented in remote primary health care. Intensive one-on-one interventions with substantial involvement from Aboriginal and Torres Strait Islander workers are likely to be effective in these settings.
Trial registration
Australian New Zealand Clinical Trials Registry (ACTRN12608000604303).
doi:10.1186/1471-2458-14-32
PMCID: PMC3905726  PMID: 24418597
Indigenous; Aboriginal; Torres Strait Islander; Randomised controlled trial; Smoking cessation; Be Our Ally Beat Smoking (BOABS) Study
11.  Rheumatic Fever Follow-Up Study (RhFFUS) protocol: a cohort study investigating the significance of minor echocardiographic abnormalities in Aboriginal Australian and Torres Strait Islander children 
Background
In Australia, rheumatic heart disease (RHD) is almost exclusively restricted to Aboriginal Australian and Torres Strait Islander people with children being at highest risk. International criteria for echocardiographic diagnosis of RHD have been developed but the significance of minor heart valve abnormalities which do not reach these criteria remains unclear. The Rheumatic Fever Follow-Up Study (RhFFUS) aims to clarify this question in children and adolescents at high risk of RHD.
Methods/design
RhFFUS is a cohort study of Aboriginal and/or Torres Strait Islander children and adolescents aged 8–17 years residing in 32 remote Australian communities. Cases are people with non-specific heart valve abnormalities detected on prior screening echocardiography. Controls (two per case) are age, gender, community and ethnicity-matched to cases and had a prior normal screening echocardiogram. Participants will have echocardiography about 3 years after initial screening echocardiogram and enhanced surveillance for any history suggestive of acute rheumatic fever (ARF). It will then be determined if cases are at higher risk of (1) ARF or (2) developing progressive echocardiography-detected valve changes consistent with RHD.
The occurrence and timing of episodes of ARF will be assessed retrospectively for 5 years from the time of the RhFFUS echocardiogram. Episodes of ARF will be identified through regional surveillance and notification databases, carer/subject interviews, primary healthcare history reviews, and hospital separation diagnoses.
Progression of valvular abnormalities will be assessed prospectively using transthoracic echocardiography and standardized operating and reporting procedures. Progression of valve lesions will be determined by specialist cardiologist readers who will assess the initial screening and subsequent RhFFUS screening echocardiogram for each participant. The readers will be blinded to the initial assessment and temporal order of the two echocardiograms.
Discussion
RhFFUS will determine if subtle changes on echocardiography represent the earliest changes of RHD or mere variations of normal heart anatomy. In turn it will inform criteria to be used in determining whether secondary antibiotic prophylaxis should be utilized in individuals with no clear history of ARF and minor abnormalities on echocardiography. RhFFUS will also inform the ongoing debate regarding the potential role of screening echocardiography for the detection of RHD in this setting.
doi:10.1186/1471-2261-12-111
PMCID: PMC3536578  PMID: 23186515
Rheumatic heart disease; Acute rheumatic fever; Screening; Aboriginal; Torres Strait Islander; Indigenous; Diagnosis; Prevention; Australia; Echocardiography
12.  The protocol for the Be Our Ally Beat Smoking (BOABS) study, a randomised controlled trial of an intensive smoking cessation intervention in a remote Aboriginal Australian health care setting 
BMC Public Health  2012;12:232.
Background
Australian Aboriginal peoples and Torres Strait Islanders (Indigenous Australians) smoke at much higher rates than non-Indigenous people and smoking is an important contributor to increased disease, hospital admissions and deaths in Indigenous Australian populations. Smoking cessation programs in Australia have not had the same impact on Indigenous smokers as on non-Indigenous smokers. This paper describes the protocol for a study that aims to test the efficacy of a locally-tailored, intensive, multidimensional smoking cessation program.
Methods/Design
This study is a parallel, randomised, controlled trial. Participants are Aboriginal and Torres Strait Islander smokers aged 16 years and over, who are randomly allocated to a 'control' or 'intervention' group in a 2:1 ratio. Those assigned to the 'intervention' group receive smoking cessation counselling at face-to-face visits, weekly for the first four weeks, monthly to six months and two monthly to 12 months. They are also encouraged to attend a monthly smoking cessation support group. The 'control' group receive 'usual care' (i.e. they do not receive the smoking cessation program). Aboriginal researchers deliver the intervention, the goal of which is to help Aboriginal peoples and Torres Strait Islanders quit smoking. Data collection occurs at baseline (when they enrol) and at six and 12 months after enrolling. The primary outcome is self-reported smoking cessation with urinary cotinine confirmation at 12 months.
Discussion
Stopping smoking has been described as the single most important individual change Aboriginal and Torres Strait Islander smokers could make to improve their health. Smoking cessation programs are a major priority in Aboriginal and Torres Strait Islander health and evidence for effective approaches is essential for policy development and resourcing. A range of strategies have been used to encourage Aboriginal peoples and Torres Strait Islanders to quit smoking however there have been few good quality studies that show what approaches work best. More evidence of strategies that could work more widely in Indigenous primary health care settings is needed if effective policy is to be developed and implemented. Our project will make an important contribution in this area.
Trial Registration
Australian New Zealand Clinical Trials Registry (ACTRN12608000604303)
doi:10.1186/1471-2458-12-232
PMCID: PMC3349500  PMID: 22439653
Indigenous; Aboriginal; Torres Strait Islander; Randomised controlled trial; Smoking cessation; Study protocol; Be Our Ally Beat Smoking (BOABS) Study
17.  Enhancing the Contrast of ApoB to Locate the Surface Components in the 3D Density Map of Human LDL 
Journal of molecular biology  2010;405(1):274-283.
A 26Å resolution map of the structure of human LDL was obtained from cryo-EM and single particle image reconstruction. The structure showed a discoidal shaped LDL particle with high-density regions mainly distributed at the edge of the particle and low-density regions at the flat surface that covers the core region. To determine the chemical components that correspond to these density regions and to delineate the distribution of protein and phospholipid located at the particle surface at the resolution of the map, we used Mono-Sulfo-NHS-Undecagold labeling to increase preferentially the contrast of the apoB protein component on the LDL particle. In the 3D maps from the image reconstruction of the undecagold labeled LDL particles, the high-density region from the undecagold label was distributed mainly at the edge of the particle and lower density regions were found at the flat surfaces that cover the neutral lipid core. This suggests that apoB mainly encircles LDL at the edge of the particle and the phospholipid monolayers are located at the flat surfaces, which are parallel to the cholesterol ester layers in the core and may interact with the core lipid layers through the acyl-chains.
doi:10.1016/j.jmb.2010.10.034
PMCID: PMC3006490  PMID: 21029740
LDL structure; apoB; atherosclerosis; 3D image reconstruction; undecagold labeling
19.  Reconstituting Initial Events during the Assembly of ApoB-containing Lipoproteins in a Cell-free System 
Journal of molecular biology  2008;383(5):1181-1194.
Summary
The synthesis of apolipoprotein B (apoB) dictates the formation of chylomicrons and very low density lipoproteins (VLDL), two major lipoprotein precursors in the human plasma. Despite its biological significance, the mechanism of the assembly of these apoB-containing lipoproteins remains elusive. An essential obstacle is the lack of systems that allow fine dissection of key components during assembly, including nascent apoB peptide, lipids in defined forms, chaperones, and microsomal triglyceride transfer protein (MTP). In this study, we use a prokaryotic cell-free expression system to reconstitute early events in the assembly of apoB-containing lipoprotein that involve the N-terminal domains of apoB. Our study shows that the N-terminal domains larger than 20.5% of apoB (B20.5) have an intrinsic ability to remodel vesicular phospholipid bilayers into discrete protein-lipid complexes. The presence of appropriate lipid substrates during apoB translation plays a pivotal role for successful lipid recruitment, and similar lipid recruitment fails to occur if the lipids are added posttranslationally. Cotranslational presence of MTP can dramatically promote the folding of B6.4–20.5 and B6.4–22. Furthermore, apoB translated in the presence of MTP retains its phospholipid recruitment capability posttranslationally. Our data suggest that during the synthesis of apoB, the N-terminal domain has a short window for intrinsic phospholipid recruitment, the timeframe of which is predetermined by the environment where apoB synthesis occurs. The presence of MTP prolongs this window of time by acting as a chaperone. The absence of either proper lipid substrate or MTP may result in the improper folding of apoB and consequently its degradation.
doi:10.1016/j.jmb.2008.09.006
PMCID: PMC2637522  PMID: 18804479
apolipoprotein B; microsomal triglyceride transfer protein; cell free; cotranslational; low density lipoprotein
20.  Biophysical Properties of Apolipoprotein E4 Variants: Implications in Molecular Mechanisms of Correction of Hypertriglyceridemiaχ 
Biochemistry  2008;47(47):12644-12654.
In humans and animal models, high plasma concentrations of apolipoprotein (apo) E are associated with hypertriglyceridemia. It has been shown that overexpression of human wild-type (WT) apoE4 in apoE-deficient mice induces hypertriglyceridemia. In contrast, overexpression of an apoE4 variant, apoE4-mut1 (apoE4(L261A, W264A, F265A, L268A, V269A)), does not induce hypertriglyceridemia and corrects hypercholesterolemia. Furthermore, overexpression of another variant, apoE4-mut2 (apoE4(W276A, L279A, V280A, V283A)), induces mild hypertriglyceridemia and does not correct hypercholesterolemia. To better understand how these mutations improve the function of apoE4, we investigated the conformation and stability of apoE4-mut1 and apoE4-mut2 and their binding to dimyristoyl phosphatidylcholine (DMPC) vesicles and to triglyceride (TG)-rich emulsion particles. We found that the mutations introduced in apoE4-mut1 lead to a more stable and compactly folded conformation of apoE4. These structural changes are associated with a slower rate of solubilization of DMPC vesicles by apoE4-mut1 and reduced binding of the protein to emulsion particles as compared to WT apoE4. Under conditions of apoE4 overexpression, the reduced binding of apoE4-mut1 to TG-rich lipoprotein particles may facilitate the lipolysis of these particles and may alter the conformation of the lipoprotein-bound apoE in a way that favors the efficient clearance of the lipoprotein remnants. Mutations introduced in apoE4-mut2 result in smaller structural alterations compared to those observed in apoE4-mut1. The slightly altered structural properties of apoE4-mut2 are associated with slightly reduced binding of this protein to TG-rich lipoprotein particles and milder hypertriglyceridemia as compared to WT apoE4.
doi:10.1021/bi8015857
PMCID: PMC2748909  PMID: 18959431
21.  Structure and Stability of Apolipoprotein A-I in Solution and in Discoidal High Density Lipoprotein Probed by Double Charge Ablation and Deletion Mutationχ 
Biochemistry  2006;45(4):1242-1254.
To identify residues and segments in the central region of apolipoprotein A-I (apoA-I) that are important for the protein structure and stability, we studied the effects of four double charge ablations, D102A/D103A, E110A/E111A, R116V/K118A, and R160V/H162A, and two deletion mutations, Δ(61-78) and Δ(121-142), on the conformation and stability of apoA-I in the lipid-free state and in reconstituted discoidal phospholipid:cholesterol:apoA-I particles (rHDL). The findings suggest that D102/D103 and E110/E111 located in helix 4, and segment(s) between residues 61 and 78 are involved in maintenance of the conformation and stability of apoA-I in both the lipid-free state and in rHDL. R116/K118 located in helix 4 are essential for the conformation and stabilization of apoA-I in rHDL, but not vital for the lipid-free state of the protein. The R160V/H162A substitutions in helix 6 lead to a less compact tertiary structure of lipid-free apoA-I without notable effects on the lipid-free or lipid-bound secondary conformation suggesting involvement of R160/H162 in important inter-helical interactions. The results on the Δ(121-142) mutant, together with our earlier findings, suggest disordered structure of a major segment between residues 121 and 143, likely including residues 131-143, in lipid-free apoA-I. Our findings provide the first experimental evidence for stabilization of rHDL by electrostatic inter-helical interactions, in agreement with the double belt model. The effects of alterations in the conformation and stability of the apoA-I mutants on in vitro and in vivo functions of apoA-I and lipid homeostasis are discussed.
doi:10.1021/bi051669r
PMCID: PMC2532493  PMID: 16430220
22.  Conformation and Lipid Binding of a C-Terminal (198-243) Peptide of Human Apolipoprotein A-I (apoA-I)† 
Biochemistry  2007;46(6):1624-1634.
Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on [1-44]apoA-I demonstrated that this N-terminal region is unstructured and folds into ~ 60% α-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46 residue peptide, [198-243]apoA-I, encompassing putative helix repeats 10, 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far ultraviolet circular dichroism spectra show that [198-243] apoA-I is also unfolded in aqueous solution. However, self-association induces ~ 50% α-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid mimicking detergents, above their CMC, ~ 60% α-helix was induced in the peptide. In contrast, SDS, an anionic lipid mimicking detergent, induced helical folding in the peptide at a concentration of ~ 0.003% (~ 100 μM), ~ 70 fold below its typical CMC (0.17–0.23% or 6–8 mM). Both monomeric and tetrameric peptide can solublize dimyristoyl phosphatidyl choline (DMPC) liposomes and fold into ~ 60% α-helix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy, demonstrated that the peptide binds to DMPC with high affinity to form at least two sizes of relatively homogenous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide w/w, diameter and density) of both complexes are similar to those of plasma A-I/DMPC formed under similar conditions: small discoidal complexes (~ 3:1 w/w, ~ 110Å and ~ 1.10g/cm3) formed at initial 1:1 w/w ratio and larger discoidal complexes ( ~ 4.6:1 w/w, ~ 165 Å and ~ 1.085g/cm3) formed at initial 4:1 w/w ratio. The cross-linking of the peptide on the two sizes of disks is consistent with the calculated peptide numbers per particle, which result in sufficient helix to surround the lipid bilayer twice. Thus, our data provide direct evidence that this C-terminal region of apoA-I is responsible for the self-association of apoA-I, and this C-terminal peptide model can mimic the interaction with phospholipid of plasma apoA-I to form two sizes of homogenous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma.
doi:10.1021/bi061721z
PMCID: PMC2518689  PMID: 17279626
23.  Protists decrease in size linearly with temperature: ca. 2.5% degrees C(-1). 
An inverse relationship between organism size and rearing temperature is widely observed in ectotherms ('the temperature-size rule', TSR). This has rarely been quantified for related taxa, and its applicability to protists also required testing. Here, we quantify the relationship between temperature and mean cell volume within the protists by a meta-analysis of published data covering marine, brackish water and freshwater autotrophs and heterotrophs. In each of 44 datasets, a linear relationship between temperature and size could not be rejected, and a negative trend was found in 32 cases (20 gave significant negative regressions, p < 0.05). By combining 65 datasets, we revealed, for each 1 degrees C increase, a cell-size reduction of 2.5% (95% CI of 1.7-3.3%) of the volume observed at 15 degrees C. The value did not differ across taxa (amoebae, ciliates, diatoms, dinoflagellates, flagellates), habitats, modes of nutrition or combinations of these. The data are consistent with two hypotheses that are capable of explaining the TSR in ectotherms generally: (i) resource, especially respiratory gas, limitation; and (ii) fitness gains from dividing earlier as population growth increases. Using the above relationship we show how changes in cell numbers with temperature can be estimated from changes in biomass and vice versa; ignoring this relationship would produce a systematic error.
doi:10.1098/rspb.2003.2538
PMCID: PMC1691543  PMID: 14728784
25.  Studies on the Structure of Low Density Lipoproteins Isolated from Macaca Fascicularis Fed an Atherogenic Diet 
Journal of Clinical Investigation  1978;62(6):1354-1363.
Cynomolgus monkeys, Macaca fascicularis, fed cholesterol-containing saturated-fat diets develop increased levels of high molecular weight plasma low density lipoproteins (LDL), associated with accelerated atherosclerosis. To study the composition and structure of these abnormal particles, LDL from monkeys, fed atherogenic and control diets, were characterized chemically and examined by differential scanning calorimetry and low-angle X-ray scattering. LDL from animals on the experimental diet showed an increase in molecular weight (4.0 to 7.0 × 106, experimental diet compared with 3.0 to 3.7 × 106, control diet) associated with a large increase in cholesterol ester content and concomitant smaller increases in protein, phospholipid, and free cholesterol. There was a strong positive correlation between molecular weight and the number of saturated and monounsaturated cholesterol esters in the particle. In contrast, particle content of polyunsaturated cholesterol esters remained constant despite large changes in total particle cholesterol esters.
When examined by calorimetry and X-ray scattering, LDL from monkeys on both diets diplayed a reversible transition of cholesterol esters from an ordered smeticlike (layered) structure to a more disordered state. For all animals on the experimental diet, the peak temperature of the cholesterol-ester transition (42-48°C) was above body temperature (39°C), but below body temperature on the control diet (34-38.5°C). In the experimental group, the transition temperature was correlated with the LDL molecular weight. However, after thermal disruption of LDL, liquid-crystalline transitions of LDL cholesterol esters were observed in the same temperature range as in the intact lipoprotein, which shows that changes in particle size had little effect on the cholesterol-ester transition temperature. Rather, the transition temperature was determined by the degree of saturation of the LDL cholesterol ester fatty acids and the LDL cholesterol ester: triglyceride ratio, both of which correlated with increased LDL molecular weight.
The existence of smectic-like cholesterol ester in LDL at body temperature was clearly a discriminating feature between monkeys on control and experimental diets. Diet-induced changes in the lipid composition of precursor lipoproteins of LDL appeared to lead to the existence of smectic-like cholesterol ester in LDL above body temperature. The altered composition and structure of the core lipids of high molecular weight LDL probably account, in part, for the previously documented correlation between increased LDL molecular weight and atherosclerosis in this species.
PMCID: PMC371901  PMID: 219029

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