Smoothing a rugged protein folding landscape by sequence-based redesign

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as $\alpha_1$-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create $\textit{conserpin}$, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.BTP is a Medical Research Council Career Development Fellow. AAN and JJH are supported by the Wellcome Trust (grant number WT 095195). SM acknowledges fellowship support from the Australian Research Council (FT100100960). NAB is an Australian Research Council Future Fellow (110100223). GIW is an Australian Research Council Discovery Outstanding Researcher Award Fellow (DP140100087). AMB is a National Health and Medical Research Senior Research Fellow (1022688). JCW is an NHMRC Senior Principal Research fellow and also acknowledges the support of an ARC Federation Fellowship. We thank the Australian Synchrotron for beam-time and technical assistance. This work was supported by the Multi-modal Australian ScienceS Imaging and Visualisation Environment (MASSIVE) (www.massive.org.au). We acknowledge the Monash Protein Production Unit and Monash Macromolecular Crystallization Facilit

Smoothing a rugged protein folding landscape by sequence-based redesign

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics

Neural Activity Patterns in Response to Interspecific and Intraspecific Variation in Mating Calls in the Túngara Frog

During mate choice, individuals must classify potential mates according to species identity and relative attractiveness. In many species, females do so by evaluating variation in the signals produced by males. Male túngara frogs (Physalaemus pustulosus) can produce single note calls (whines) and multi-note calls (whine-chucks). While the whine alone is sufficient for species recognition, females greatly prefer the whine-chuck when given a choice.To better understand how the brain responds to variation in male mating signals, we mapped neural activity patterns evoked by interspecific and intraspecific variation in mating calls in túngara frogs by measuring expression of egr-1. We predicted that egr-1 responses to conspecific calls would identify brain regions that are potentially important for species recognition and that at least some of those brain regions would vary in their egr-1 responses to mating calls that vary in attractiveness. We measured egr-1 in the auditory brainstem and its forebrain targets and found that conspecific whine-chucks elicited greater egr-1 expression than heterospecific whines in all but three regions. We found no evidence that preferred whine-chuck calls elicited greater egr-1 expression than conspecific whines in any of eleven brain regions examined, in contrast to predictions that mating preferences in túngara frogs emerge from greater responses in the auditory system.Although selectivity for species-specific signals is apparent throughout the túngara frog brain, further studies are necessary to elucidate how neural activity patterns vary with the attractiveness of conspecific mating calls

Expanding contraceptive options for PMTCT clients: a mixed methods implementation study in Cape Town, South Africa

Abstract Background Clients of prevention of mother-to-child transmission (PMTCT) services in South Africa who use contraception following childbirth rely primarily on short-acting methods like condoms, pills, and injectables, even when they desire no future pregnancies. Evidence is needed on strategies for expanding contraceptive options for postpartum PMTCT clients to include long-acting and permanent methods. Methods We examined the process of expanding contraceptive options in five health centers in Cape Town providing services to HIV-positive women. Maternal/child health service providers received training and coaching to strengthen contraceptive counseling for postpartum women, including PMTCT clients. Training and supplies were introduced to strengthen intrauterine device (IUD) services, and referral mechanisms for female sterilization were reinforced. We conducted interviews with separate samples of postpartum PMTCT clients (265 pre-intervention and 266 post-intervention) to assess knowledge and behaviors regarding postpartum contraception. The process of implementing the intervention was evaluated through systematic documentation and interpretation using an intervention tracking tool. In-depth interviews with providers who participated in study-sponsored training were conducted to assess their attitudes toward and experiences with promoting voluntary contraceptive services to HIV-positive clients. Results Following the intervention, 6% of interviewed PMTCT clients had the desired knowledge about the IUD and 23% had the desired knowledge about female sterilization. At both pre- and post-intervention, 7% of clients were sterilized and IUD use was negligible; by comparison, 75% of clients used injectables. Intervention tracking and in-depth interviews with providers revealed intervention shortcomings and health system constraints explaining the failure to produce intended effects. Conclusions The intervention failed to improve PMTCT clients’ knowledge about the IUD and sterilization or to increase use of those methods. To address the family planning needs of postpartum PMTCT clients in a way that is consistent with their fertility desires, services must expand the range of contraceptive options to include long-acting and permanent methods. In turn, to ensure consistent access to high quality family planning services that are effectively linked to HIV services, attention must also be focused on resolving underlying health system constraints weakening health service delivery more generally

Mutational analysis of the C-terminal FATC domain of Saccharomyces cerevisiae Tra1

Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the PIKK family, which contain a C-terminal phosphatidylinositol 3-kinase-like (PI3K) domain followed by a 35-residue FATC domain. Single residue changes of L3733A and F3744A, within the FATC domain, resulted in transcriptional changes and phenotypes that were similar but not identical to those caused by mutations in the PI3K domain or deletions of other SAGA or NuA4 components. The distinct nature of the FATC mutations was also apparent from the additive effect of tra1-L3733A with SAGA, NuA4, and tra1 PI3K domain mutations. Tra1-L3733A associates with SAGA and NuA4 components and with the Gal4 activation domain, to the same extent as wild-type Tra1; however, steady-state levels of Tra1-L3733A were reduced. We suggest that decreased stability of Tra1-L3733A accounts for the phenotypes since intragenic suppressors of tra1-L3733A restored Tra1 levels, and reducing wild-type Tra1 led to comparable growth defects. Also supporting a key role for the FATC domain in the structure/function of Tra1, addition of a C-terminal glycine residue resulted in decreased association with Spt7 and Esa1, and loss of cellular viability. These findings demonstrate the regulatory potential of mechanisms targeting the FATC domains of PIKK proteins

Development and psychometric testing of an instrument to compare career choice influences and perceptions of nursing among healthcare students

Background: With the availability of more healthcare courses and an increased intake of nursing students, education institutions are facing challenges to attract school leavers to enter nursing courses. The comparison of career choice influences and perception of nursing among healthcare students can provide information for recruitment strategies. An instrument to compare the influences of healthcare career choice is lacking. The purpose of this study is to develop and evaluate the psychometric properties of an instrument to compare the influences of healthcare career choice with perceptions of nursing as a career choice. Methods: The study was conducted in two phases. In phase one, two sets of scales with parallel items that measure the influences of healthcare career choice and perceptions of nursing as a career choice were developed through an earlier qualitative study, literature review, and expert validation. Phase two involved testing the construct validity, concurrent validity and reliability with a convenience sample of 283 first year healthcare students who were recruited at two education institutions in Singapore. Results: An exploratory factor analysis revealed 35-parallel items in a six-factor solution (personal interest, prior healthcare exposure, self-efficacy, perceived nature of work, job prospects, and social influences) that explained 59 and 64% of the variance for healthcare career choice and nursing as a career choice respectively. A high correlation (r = 0.76, p \u3c 0.001) was obtained with an existing tool, confirming the concurrent validity. The internal consistency was sufficient with Cronbach’s alpha of 0.93 for healthcare career choice and 0.94 for nursing as a career choice. The test-retest reliability was acceptable with an Intraclass Correlation Coefficient of 0.63 for healthcare career choice and 0.60 for nursing as a career choice. Conclusions: The instrument provides opportunities for understanding the differences between influences of healthcare career choice and perceptions of nursing as a career choice. This comparative understanding of career choice influences can guide educator and policy-makers on nursing recruitmen

Renal replacement therapy in acute kidney injury: controversy and consensus

Renal replacement therapies (RRTs) represent a cornerstone in the management of severe acute kidney injury. This area of intensive care and nephrology has undergone significant improvement and evolution in recent years. Continuous RRTs have been a major focus of new technological and treatment strategies. RRT is being used increasingly in the intensive care unit, not only for renal indications but also for other organ-supportive strategies. Several aspects related to RRT are now well established, but others remain controversial. In this review, we review the available RRT modalities, covering technical and clinical aspects. We discuss several controversial issues, provide some practical recommendations, and where possible suggest a research agenda for the future

Regulation of Septin Dynamics by the Saccharomyces cerevisiae Lysine Acetyltransferase NuA4

In the budding yeast Saccharomyces cerevisiae, the lysine acetyltransferase NuA4 has been linked to a host of cellular processes through the acetylation of histone and non-histone targets. To discover proteins regulated by NuA4-dependent acetylation, we performed genome-wide synthetic dosage lethal screens to identify genes whose overexpression is toxic to non-essential NuA4 deletion mutants. The resulting genetic network identified a novel link between NuA4 and septin proteins, a group of highly conserved GTP-binding proteins that function in cytokinesis. We show that acetyltransferase-deficient NuA4 mutants have defects in septin collar formation resulting in the development of elongated buds through the Swe1-dependent morphogenesis checkpoint. We have discovered multiple sites of acetylation on four of the five yeast mitotic septins, Cdc3, Cdc10, Cdc12 and Shs1, and determined that NuA4 can acetylate three of the four in vitro. In vivo we find that acetylation levels of both Shs1 and Cdc10 are reduced in a catalytically inactive esa1 mutant. Finally, we determine that cells expressing a Shs1 protein with decreased acetylation in vivo have defects in septin localization that are similar to those observed in NuA4 mutants. These findings provide the first evidence that yeast septin proteins are acetylated and that NuA4 impacts septin dynamics