Medical student psychological distress and academic performance.

INTRODUCTION: The impact of medical student psychological distress on academic performance has not been systematically examined. This study provided an opportunity to closely examine the potential impacts of workplace and study related stress factors on student's psychological distress and their academic performance during their first clinical year. METHODS: This one-year prospective cohort study was performed at a tertiary hospital based medical school in Melbourne, Australia. Students completed a questionnaire at three time points during the year. The questionnaire included the validated Kessler psychological distress scale (K10) and the General Health Questionnaire-28 (GHQ-28), as well as items about sources of workplace stress. Academic outcome scores were aggregated and correlated with questionnaire results. RESULTS: One hundred and twenty six students participated; 126 (94.7%), 102 (76.7%), and 99 (74.4%) at time points one, two, and three, respectively. 33.1% reported psychological distress at time point one, increasing to 47.4% at time point three. There was no correlation between the K10 scores and academic performance. There was weak negative correlation between the GHQ-28 at time point three and academic performance. Keeping up to date with knowledge, need to do well and fear of negative feedback were the most common workplace stress factors. CONCLUSIONS: Poor correlation was noted between psychological distress and academic performance.

Measuring postnatal demoralisation: adaptation of the Demoralisation Scale-II (DS-II) for postnatal use.

OBJECTIVE: To examine the psychometric properties of the Demoralisation Scale II (DS-II) and adapt it for use with women in the postnatal period. BACKGROUND: Demoralisation is a psychological state characterised by a sense of incompetence and feelings of helplessness and hopelessness in response to a stressful situation. The postnatal period is a life stage of many disruptions. Women may lose their confidence and become demoralised if feeling unprepared for the tasks of motherhood. The DS-II is a 16-item scale developed among cancer patients, but with content that is also relevant postnatally, including items on sense of failure, helplessness, hopelessness, isolation, entrapment and loss of purpose. METHODS: Rasch analysis was used to investigate the psychometric properties of the DS-II and refine the scale for postnatal use. RESULTS: Participants were 209 women admitted with their babies to a residential early parenting programme. A 14-item revised scale was derived, the Postnatal DS-II, showing good psychometric properties, discriminant validity and sensitivity to change, and being well targeted to the sample. CONCLUSION: The Postnatal DS-II could have utility as an assessment tool, helping clinicians to understand better women's postnatal experiences, assess the effectiveness of interventions and communicate with women in a meaningful and non-stigmatising way.

Thiol-reactive drug substrates of human P-glycoprotein label the same sites to activate ATPase activity in membranes or dodecyl maltoside detergent micelles.

P-glycoprotein (P-gp, ABCB1) is an ABC drug pump that is clinically important because it is involved in multidrug resistance. Many studies have used purified P-gp in detergent (n-dodecyl-ß-D-maltoside; DM) micelles to map the locations of the drug-binding sites. A potential problem is that DM could be a substrate and affect binding of drugs to P-gp. To test whether DM was a substrate of P-gp, we used an assay involving drug-rescue of the immature 150 kDa misprocessed P-gp mutant (L1260A) to show that DM is not substrate. By contrast, the detergents Triton X-100 or NP-35 were substrates because they rescued the L1260A P-gp mutant such that the major product was the mature 170 kDa protein. Cross-linking of mutant A80C/R741C in membranes can only be inhibited by the P-gp substrate tariquidar. We show that cross-linking A80C/R741C mutant was also inhibited by tariquidar in the presence of excess DM. This result suggests that the presence of DM did not affect the tariquidar-binding site. Similarly, the presence of DM did not alter the locations of other drug-binding sites since the thiol reactive forms of the substrates verapamil or rhodamine labeled the same sites in transmembrane segments 5 (I306C for verapamil) and 6 (F343C for rhodamine) whether P-gp was in native membranes or in detergent micelles. These results suggest that the presence of DM does not alter the locations of the P-gp drug-binding sites and that the detergent purified protein is suitable for mapping their locations using biochemical or structural assays.

Attachment of a 'molecular spring' restores drug-stimulated ATPase activity to P-glycoprotein lacking both Q loop glutamines.

P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump that is clinically important because it confers multidrug resistance. Drugs bind at the interface between the transmembrane domains to activate ATPase activity at the two nucleotide-binding domains (NBDs). Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. Recently, it was reported that the conserved glutamines residues (Gln475 in NBD1 and Gln1118 in NBD2) in the Q loops of P-gp when mutated to alanine completely inhibited the drug-stimulated ATPase activity. It is unknown why the glutamine residues (Gln475 and Gln1118) in the Q loops of the NBDs of P-gp are required for drug-stimulated ATPase activity. Here we show that introduction of these mutations into the L175C/N820C mutant (L175C/N820C/Q475A/Q1118A) also abolished drug-stimulated ATPase activity. The ATPase activity was restored however, when the L175C/N820C/Q475A/Q1118A mutant was cross-linked with a flexible disulfide cross-linker. These results suggest that both Q-loop glutamines are not required for ATP hydrolysis and they might function as part of a spring-like mechanism in facilitating the open (inactive) to closed (active) conformational change during ATP hydrolysis. The molecular spring-like action of the Q-loop glutamines during drug-stimulated ATPase activity is likely mimicked by the attachment of the flexible cross-linker.

Drugs Modulate Interactions between the First Nucleotide-Binding Domain and the Fourth Cytoplasmic Loop of Human P-Glycoprotein.

Drug substrates stimulate ATPase activity of the P-glycoprotein (P-gp) ATP-binding cassette drug pump by an unknown mechanism. Cross-linking analysis was performed to test if drug substrates stimulate P-gp ATPase activity by altering cross-talk at the first transmission interface linking the drug-binding [intracellular loop 4 (S909C)] and first nucleotide-binding domains [NBD1 (V472C or L443C)]. In the absence of lipid (inactive P-gp), only V472C/S909C showed cross-linking. Drugs blocked V472C/S909C cross-linking. In the presence of lipids (active P-gp), drug substrates promoted only L443C/S909C cross-linking. This suggests that drug substrates stimulate ATPase activity through a conformational change that shifts Ser909 away from Val472 and toward Leu443.

The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein.

P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp.

Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein.

ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.

Refinement and revalidation of the demoralization scale: The DS-II-external validity.

BACKGROUND: The recently refined Demoralization Scale-II (DS-II) is a 16-item, self-report measure of demoralization. Its 2 factors-Meaning and Purpose and Distress and Coping Ability-demonstrate sound internal validity, including item fit, unidimensionality, internal consistency, and test-retest reliability. The convergent and discriminant validity of the DS-II with various measures is reported here. METHODS: Patients who had cancer or other progressive diseases and were receiving palliative care (n = 211) completed a battery of questionnaires, including the DS-II and measures of symptom burden, quality of life, depression, and attitudes toward the end of life. Spearman ρ correlations were determined to assess convergent validity. Mann-Whitney U tests with calculated effect sizes were used to examine discriminant validity and establish the minimal clinically important difference (MCID). Cross-tabulation frequencies with chi-square analyses were used to examine discriminant validity with major depression. RESULTS: The DS-II demonstrated convergent validity with measures of psychological distress, quality of life, and attitudes toward the end of life. It also demonstrated discriminant validity, as the DS-II differentiated patients who had different functional performance levels and high/low symptoms, with a difference of 2 points between groups on the DS-II considered clinically meaningful. Furthermore, discriminant validity was demonstrated, as comorbidity with depression was not observed at moderate levels of demoralization. CONCLUSIONS: The DS-II has sound psychometric properties and is an appropriate measure of demoralization. Given its structural simplicity and brevity, it is likely to be a useful tool in meaning-centered therapies. Cancer 2016;122:2260-7. © 2016 American Cancer Society.

Refinement and revalidation of the demoralization scale: The DS-II-internal validity.

BACKGROUND: The Demoralization Scale (DS) was initially validated in 2004 to enable the measurement of demoralization in patients with advanced cancer. Subsequent shortcomings indicated the need for psychometric strengthening. Here, the authors report on the refinement and revalidation of the DS to form the DS-II, specifically reporting the scale's internal validity. METHODS: Patients with cancer or other progressive diseases who were receiving palliative care (n = 211) completed a revised version of the 24-item DS and a measure of symptom burden (the Memorial Symptom Assessment Scale). Exploratory factor analysis and Rasch modeling were used to evaluate, modify, and revalidate the scale, providing information about dimensionality, suitability of response format, item fit, item bias, and item difficulty. Test-retest reliability was examined for 58 symptomatically stable patients at a 5-day follow-up. RESULTS: Exploratory factor analysis supported a 22-item, 2-component model. Separate Rasch modeling of each component resulted in collapsing the response option categories and removing 3 items from each component. Both final 8-item subscales met Rasch model expectations and were appropriate to sum as a 16-item total score. The DS-II demonstrated internal consistency and test-retest reliability (Meaning and Purpose subscale: α = .84; intraclass correlation [ICC] = 0.68; Distress and Coping Ability subscale: α = .82; ICC = 0.82; total DS: α = .89; ICC = 0.80). CONCLUSIONS: The DS-II is a 3-point response, self-report scale comprising 16 items and 2 subscales. Given its revalidation, psychometric strengthening, and simplification, the DS-II is an improved and more practical measure of demoralization for research and clinical use. External validation of the DS-II will be reported subsequently. Cancer 2016;122:2251-9. © 2016 American Cancer Society.

P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids.

Health Anxiety and Its Relationship to Disability and Service Use: Findings From a Large Epidemiological Survey.

OBJECTIVE: To explore the contribution of health anxiety to disability and use of mental health and medical services, independently of co-occurring mental and physical conditions. METHODS: Data from the Australian National Survey of Mental Health and Wellbeing 2007 were analyzed (n = 8841). Participants were aged 16 to 85 years (mean [standard deviation] = 46.3 [19.0] years) and 54% were women. RESULTS: Health anxiety accounted independently for high disability and service use. People with health anxiety were more likely to use both mental health (for psychiatrists: odds ratio [OR] = 2.1, 95% confidence interval [CI] = 1.2-3.5; for psychologists: OR = 1.9, 95% CI = 1.2-3.3) and specialist medical services (OR = 1.7, 95% CI = 1.2-2.3) than people without health anxiety. However, they were not high-frequency attenders to specialist mental health services (OR = 1.6 [95% CI = 0.9-3.0] and OR = 1.3 [95% CI = 0.6-2.9]) compared with people with other mental disorders (OR = 11.7 [95% CI = 4.3-31.8] and OR = 29.5 [95% CI = 13.5-64.6] for psychiatrists and psychologists, respectively). People with health anxiety were likely to be high-frequency attenders to general practice (OR = 2.0, 95% CI = 1.4-2.8) and specialist medical services (OR = 2.4, 95% CI = 1.7-3.6). CONCLUSIONS: It is important to recognize and treat health anxiety, even when coexisting with other conditions, to prevent high disability burden and excessive service use. The cross-sectional design and self-reported outcomes may have resulted in overestimation of the associations. Future work is needed on actual service use using reviews of medical records.

Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein.

The P-glycoprotein (P-gp) drug pump (ABCB1) has two transmembrane domains and two nucleotide-binding domains (NBDs). Coupling of the drug-binding sites in the transmembrane domains to the NBDs occurs through interaction of the intracellular helices (IHs) with residues in the NBDs (IH1/IH4/NBD1 and IH2/IH3/NBD2). We showed previously that cross-linking of cysteines in IH3 and IH1 with a short cross-linker mimicked drug binding as it activated P-gp ATPase activity. Here we show that residue A259C(IH2) could be directly cross-linked to W803C(IH3). Cross-linking was inhibited by the presence of ATP and adenosine 5'-(ß,γ-imino)triphosphate but not by ADP. Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Because these residues are close to the ball-and-socket joint A266C(IH2)/Phe(1086)(NBD2), we mutated the adjacent Tyr(1087)(NBD2) close to IH3. Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity. Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation. The results suggest that these residues, together with Trp(803) and Phe(804), form a large hydrophobic pocket. The results show that there is an important hydrophobic network at the IH2/IH3/NBD2 transmission interface that is critical for folding and activity of P-gp.

Identification of the distance between the homologous halves of P-glycoprotein that triggers the high/low ATPase activity switch.

P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. Each homologous half contains a transmembrane domain with six transmembrane segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the transmembrane domain and NBDs, respectively. Drug binding activates ATPase activity by an unknown mechanism. There is no high resolution structure of human P-gp, but homology models based on the crystal structures of bacterial, mouse, and Caenorhabditis elegans ATP-binding cassette drug pumps yield both open (NBDs apart) and closed (NBDs together) conformations. Molecular dynamics simulations predict that the NBDs can be separated over a range of distances (over 20 Å). To determine the distance that show high or low ATPase activity, we cross-linked reporter cysteines L175C (N-half) and N820C (C-half) with cross-linkers of various lengths that separated the halves between 6 and 30 Å (α-carbons). We observed that ATPase activity increased over 10-fold when the cysteines were cross-linked at distances between 6 and 19 Å, although cross-linking at distances greater than 20 Å yielded basal levels of activity. The results suggest that the ATPase activation switch appears to be turned on or off when L175C/N820 are clamped at distances less than or greater than 20 Å, respectively. We predict that the high/low ATPase activity switch may occur at a distance where the NBDs are predicted in molecular dynamic simulations to undergo pronounced twisting as they approach each other (Wise, J. G. (2012) Biochemistry 51, 5125-5141).

Cysteines introduced into extracellular loops 1 and 4 of human P-glycoprotein that are close only in the open conformation spontaneously form a disulfide bond that inhibits drug efflux and ATPase activity.

P-glycoprotein (P-gp) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. The protein is organized into two halves. The halves contain a transmembrane domain (TMD) with six transmembrane segments and a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the TMD1/TMD2 and NBD1/NBD2 interfaces, respectively. ATP-dependent drug efflux involves changes between the open inward-facing (NBDs apart, extracellular loops (ECLs) close together) and the closed outward-facing (NBDs close together, ECLs apart) conformations. It is controversial, however, whether the open conformation only exists transiently in intact cells because of the presence of high levels of ATP. To test for the presence of an open conformation in intact cells, reporter cysteines were placed in extracellular loops 1 (A80C, N half) and 4 (R741C, C half). The rationale was that cysteines A80C/R741C would only come close enough to form a disulfide bond in an open conformation (6.9 Å apart) because they are separated widely (30.4 Å apart) in the closed conformation. It was observed that the mutant A80C/R741C cross-linked spontaneously (>90%) when expressed in cells. In contrast to previous reports showing that trapping P-gp in a closed conformation highly activated ATPase activity, here we show that A80C/R741C cross-linking inhibited ATPase activity and drug efflux. Both activities were restored when the cross-linked mutant was treated with a thiol-reducing agent. The results show that an open conformation can be readily detected in cells and that cross-linking of cysteines placed in ECLs 1 and 4 inhibits activity.

Early postnatal demoralisation among primiparous women in the community: measurement, prevalence and associated factors.

BACKGROUND: Demoralisation is a psychological state occurring in stressful life situations where a person feels unable to respond effectively to their circumstances, characterised by feelings of distress, subjective incompetence, helplessness and hopelessness. The period after the birth of a first baby is a time of great changes and disruptions to many aspects of the mother's physical, psychological and social functioning. This can lead to feelings of distress, a sense of incompetence and helplessness. This study aimed to examine: (1) the psychometric properties of the Demoralisation Scale in a community setting; (2) the prevalence of demoralisation symptoms among primiparous women in the community; and (3) factors that are uniquely associated with demoralisation in the early postnatal period. METHODS: Primiparous women attending community maternal health centres (n = 400) were recruited and administered the study's questionnaires through a telephone interview. RESULTS: The Demoralisation Scale was found to be a reliable and valid tool among women in the community who had recently given birth. Higher levels of demoralisation were independently associated with lower confidence on going home from the hospital after birth, lower rating of mother's self-rated global health, more than 3 h of infant crying and fussing in the last 24 h, and a controlling partner, after symptoms of depression and anxiety, and vulnerable personality characteristics were controlled for. CONCLUSIONS: The relevance of demoralisation to postnatal health practitioners in the community is in helping them to better understand women's experiences and to intervene in a way that is more meaningful and less stigmatising to women.

Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface.

The P-glycoprotein drug pump protects us from toxins. Drug-binding sites in the transmembrane (TM) domains (TMDs) are connected to the nucleotide-binding domains (NBDs) by intracellular helices (IHs). TMD-NBD cross-talk is a key step in the transport mechanism because drug binding stimulates ATP hydrolysis followed by drug efflux. Here, we tested whether the IHs are critical for maturation and TMD-NBD coupling by characterizing the effects of mutations to the IH1 and IH2 interfaces. Although IH1 mutations had little effect, most mutations at the IH2-NBD2 interface inhibited maturation or activity. For example, the F1086A mutation at the IH2-NBD2 interface abolished drug-stimulated ATPase activity. The mutant F1086A, however, retained the ability to bind ATP and drug substrates. The mutant was defective in mediating ATP-dependent conformational changes in the TMDs because binding of ATP no longer promoted cross-linking between cysteines located at the extracellular ends of TM segments 6 and 12. Replacement of Phe-1086 (in NBD2) with hydrophobic but not charged residues yielded active mutants. The activity of the F1086A mutant could be restored when the nearby residue Ala-266 (in IH2) was replaced with aromatic residues. These results suggest that Ala-266/Phe-1086 lies in a hydrophobic IH2-NBD2 "ball-and-socket" joint.

The INSPIRED study: a randomised controlled trial of the Whole Person Model of disease self-management for people with type 2 diabetes.

BACKGROUND: The prevalence of type 2 diabetes has increased dramatically in the last decade, and is continuing to rise. It is a chronic condition, often related to obesity, diet and sedentary lifestyles, and can lead to significant health complications, disability and early death. Diabetes is commonly associated with depression, which can impact significantly on a person's ability to manage their illness and, consequently, on disease outcomes. Disease self-management is fundamental in diabetes and requires support from multiple health professionals and the active participation of the person, including in maintaining a healthy lifestyle. The Whole Person Model was developed in order to integrate emotional and behavioural aspects into a self-management program for people with type 2 diabetes. Here we describe a study designed to test the efficacy of the Whole Person Model of disease self-management in type 2 diabetes. METHODS/DESIGN: In a parallel-group randomised trial, 180 people with type 2 diabetes of between 2-10 years duration will be recruited via invitation through the Australian National Diabetes Services Scheme. Participants will undergo baseline assessment, followed by randomisation to either intervention or control condition. Control participants will receive fact sheets containing key information about diabetes self-management. The intervention group will receive the INSPIRED (Individual Support & Resources for Diabetes) Manual and be assigned a Health Coach. The INSPIRED Manual consists of six modules that provide key information about diabetes and disease management using the Whole Person Model. Engagement is facilitated by interactive tasks and contact with a Health Coach over seven weeks--an introductory face-to-face session, and six subsequent contacts by phone following each module. Follow-up assessments occur at 13 weeks (post-intervention) and 26 weeks. Primary outcomes include blood glucose management (HbA1c), weight and mood. Secondary outcomes include level of exercise, confidence to manage diabetes, and psychosocial well-being. DISCUSSION: The Whole Person Model is designed to enable health professionals to address mood disturbance without pathologizing any disorders and, in the context of the chronic illness, empowering behavior change and self-management. If proven effective, this model will strengthen capacity of the healthcare workforce to foster and support effective diabetes self-management. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Register, ACTRN12613000391774.

A salt bridge in intracellular loop 2 is essential for folding of human p-glycoprotein.

There is no high-resolution structure of the human P-glycoprotein (P-gp, ABCB1) drug pump. Homology models based on the crystal structures of mouse and Caenorhabditis elegans P-gps show extensive contacts between intracellular loop 2 (ICL2, in the first transmembrane domain) and the second nucleotide-binding domain. Human P-gp modeled on these P-gp structures yields different ICL2 structures. Only the model based on the C. elegans P-gp structure predicts the presence of a salt bridge. We show that the Glu256-Arg276 salt bridge was critical for P-gp folding.

Drug rescue distinguishes between different structural models of human P-glycoprotein.

There is no high-resolution crystal structure of the human P-glycoprotein (P-gp) drug pump. Homology models of human P-gp based on the crystal structures of mouse or Caenorhabditis elegans P-gps show large differences in the orientation of transmembrane segment 5 (TM5). TM5 is one of the most important transmembrane segments involved in drug-substrate interactions. Drug rescue of P-gp processing mutants containing an arginine at each position in TM5 was used to identify positions facing the lipid or internal aqueous chamber. Only the model based on the C. elegans P-gp structure was compatible with the drug rescue results.

Bithiazole correctors rescue CFTR mutants by two different mechanisms.

Better correctors are needed to repair cystic fibrosis transmembrane conductance regulator (CFTR) processing mutants that cause cystic fibrosis. Determining where the correctors bind to CFTR would aid in the development of new correctors. A recent study reported that the second nucleotide-binding domain (NBD2) was involved in binding of bithiazole correctors. Here, we show that bithiazole correctors could also rescue CFTR mutants that lacked NBD2. These results suggest that bithiazoles rescue CFTR mutants by two different mechanisms.