Cholesterol-dependent cytolysins (CDCs) are a large family of bacterial toxins that exhibit a dependence on the presence of membrane cholesterol in forming large pores in cell membranes. Significant changes in the three-dimensional structure of these toxins are necessary to convert the soluble monomeric protein into a membrane pore. We have determined the crystal structure of the archetypical member of the CDC family, streptolysin O (SLO), a virulence factor from Streptococcus pyogenes. The overall fold is similar to previously reported CDC structures, although the C-terminal domain is in a different orientation with respect to the rest of the molecule. Surprisingly, a signature stretch of CDC sequence called the undecapeptide motif, a key region involved in membrane recognition, adopts a very different structure in SLO to that of the well-characterized CDC perfringolysin O (PFO), although the sequences in this region are identical. An analysis reveals that, in PFO, there are complementary interactions between the motif and the rest of domain 4 that are lost in SLO. Molecular dynamics simulations suggest that the loss of a salt bridge in SLO and a cation–pi interaction are determining factors in the extended conformation of the motif, which in turn appears to result in a greater flexibility of the neighboring L1 loop that houses a cholesterol-sensing motif. These differences may explain the differing abilities of SLO and PFO to efficiently penetrate target cell membranes in the first step of toxin insertion into the membrane.
conformational change; membrane insertion; molecular dynamics simulations; pore-forming toxins; X-ray crystallography
Inherited cardiac conditions (ICCs) can lead to sudden cardiac death at any age, yet are often asymptomatic and clinically undetected. Prophylactic interventions are available and cascade testing is recommended to identify family members at risk. When a disease-causing mutation has been identified in a family, pre-symptomatic genetic testing (PSGT) is available. This study explores perceptions of the cascade process, impact of PSGT and attitudes towards direct contact as an alternative to family-mediated dissemination for ICCs. In depth, interviews were conducted with 22 participants eligible for PSGT for Hypertrophic Cardiomyopathy or Long QT syndrome. Data were analysed using an inductive, thematic approach. Risk is perceived to be low pre-test in the absence of symptoms, and participants frequently test with the aim of ruling out risk to self and children. Testing of children is a complex decision; although older participants have concerns about possible adverse effects of genetic testing early in the life course, young participants are pragmatic about their result. The meaning of a positive genetic test result may be difficult to conceptualise in the absence of clinical evidence of disease, and this may deter further dissemination to at-risk family members. A majority of participants see advantages in direct contact from health professionals and support it in principle. Implications for practice include addressing risk perception pre-test, and presenting genetic test information as part of a risk stratification process rather than a binary outcome. Families may require more support or intervention in cascading genetic test information.
HCM; LQTS; genetic; psychosocial; qualitative
To reduce financial barriers to access, and improve access to and use of skilled maternal and newborn healthcare services, the government of Ghana, in 2003, implemented a new maternal healthcare policy that provided free maternity care services in all public and mission healthcare facilities. Although supervised delivery in Ghana has increased from 47% in 2003 to 55% in 2010, strikingly high maternal mortality ratio and low percentage of skilled attendance are still recorded in many parts of the country.
To explore health system factors that inhibit women’s access to and use of skilled maternal and newborn healthcare services in Ghana despite these services being provided free.
We conducted qualitative research with 185 expectant and lactating mothers and 20 healthcare providers in six communities in Ghana between November 2011 and May 2012. We used Attride-Stirling’s thematic network analysis framework to analyze and present our data.
We found that in addition to limited and unequal distribution of skilled maternity care services, women’s experiences of intimidation in healthcare facilities, unfriendly healthcare providers, cultural insensitivity, long waiting time before care is received, limited birthing choices, poor care quality, lack of privacy at healthcare facilities, and difficulties relating to arranging suitable transportation were important health system barriers to increased and equitable access and use of services in Ghana.
Our findings highlight how a focus on patient-side factors can conceal the fact that many health systems and maternity healthcare facilities in low-income settings such as Ghana are still chronically under-resourced and incapable of effectively providing an acceptable minimum quality of care in the event of serious obstetric complications. Efforts to encourage continued use of maternity care services, especially skilled assistance at delivery, should focus on addressing those negative attributes of the healthcare system that discourage access and use.
Maternal and newborn health; Health system barriers; Access; Maternal healthcare; User-fee abolition; Ghana
Broad-spectrum anticonvulsants are of considerable interest as antiepileptic drugs, especially because of their potential for treating refractory patients. Such “neurostabilizers” have also been used to treat other neurological disorders, including migraine, bipolar disorder, and neuropathic pain. We synthesized a series of sulfamide derivatives (4–9, 10a–i, 11a, 11b, 12) and evaluated their anticonvulsant activity. Thus, we identified promising sulfamide 4 (JNJ-26489112) and explored its pharmacological properties. Compound 4 exhibited excellent anticonvulsant activity in rodents against audiogenic, electrically-induced, and chemically-induced seizures. Mechanistically, 4 inhibited voltage-gated Na+ channels and N-type Ca2+ channels, and was effective as a K+ channel opener. The anticonvulsant profile of 4 suggests that it may be useful for treating multiple forms of epilepsy (generalized tonic-clonic, complex partial, absence seizures), including refractory (or pharmacoresistant) epilepsy, at dose levels that confer a good safety margin. On the basis of its pharmacology and other favorable characteristics, 4 was advanced into human clinical studies.
The creation of biobanks depends upon people’s willingness to donate their samples for research purposes and to agree to sample storage. Moreover, biobanks are a public good that requires active participation by all interested stakeholders at every stage of development. Therefore, knowing public’s attitudes towards participation in a biobank and biobank management is important and deserves investigation.
A survey was conducted among family members of patients attending the outpatient department of our institute for a geriatric or neurological visit, documenting their willingness to participate in a biobank and their views on the legal-ethical aspects of biobank management. Information regarding subjects’ attitudes on biomedical research in general and genetic research in particular was also collected. Participants’ data on biobanks were compared with data previously collected from the Italian ethics committees (ECs) to evaluate the extent to which lay people and ethics committees share views and concerns regarding biobanks.
One hundred forty-five subjects took part in the survey. The willingness to give biological samples for the constitution of a biobank set up for research purposes was declared by 86% of subjects and was modulated by subjects’ education. People in favour of providing biological samples for a biobank expressed a more positive view on biomedical research than did people who were not in favour; attitude towards genetic research in dementia was the strongest predictor of participation. Different from ECs that prefer specific consent (52%) and do not choose the option of broad consent (8%) for samples collection in a biobank, participants show a clear preference for broad consent (57%), followed by partially restricted consent (16%), specific consent (15%), and multi-layered consent (12%). Almost all of the subjects available to contribute to a biobank desire to receive both individual research results and research results of general value, while around fifty per cent of ECs require results communication.
Family members showed willingness to participate in a biobank for research and expressed a view on the ethical aspects of a biobank management that differ on several issues from the Italian ECs’ opinion. Laypersons’ views should be taken into account in developing biobank regulations.
Electronic supplementary material
The online version of this article (doi:10.1186/1472-6939-15-81) contains supplementary material, which is available to authorized users.
Public attitudes; Biobanks; Genetic research; Bioethics; Ethical policy
Inequities in accessibility to, and utilisation of maternal healthcare services impede progress towards attainment of the maternal health-related Millennium Development Goals. The objective of this study is to examine the extent to which maternal health services are utilised in Ghana, and whether inequities in accessibility to and utilization of services have been eliminated following the implementation of a user-fee exemption policy, that aims to reduce financial barriers to access, reduce inequities in access, and improve access to and use of birthing services.
We analyzed data from the 2007 Ghana Maternal Health Survey for inequities in access to and utilization of maternal health services. In measuring the inequities, frequency tables and cross-tabulations were used to compare rates of service utilization by region, residence and selected socio-demographic variables.
Findings show marginal increases in accessibility to and utilisation of skilled antenatal, delivery and postnatal care services following the policy implementation (2003–2007). However, large gradients of inequities exist between geographic regions, urban and rural areas, and different socio-demographic, religious and ethnic groupings. More urban women (40%) than rural, 53% more women in the highest wealth quintile than women in the lowest, 38% more women in the best performing region (Central Region) than the worst (Upper East Region), and 48% more women with at least secondary education than those with no formal education, accessed and used all components of skilled maternal health services in the five years preceding the survey. Our findings raise questions about the potential equity and distributional benefits of Ghana’s user-fee exemption policy, and the role of non-financial barriers or considerations.
Exempting user-fees for maternal health services is a promising policy option for improving access to maternal health care, but might be insufficient on its own to secure equitable access to maternal health services in Ghana. Ensuring equity in access will require moving beyond user-fee exemption to addressing wider issues of supply and demand factors and the social determinants of health, including redistributing healthcare resources and services, and redressing the positional vulnerability of women in their communities.
User-fee exemption; Maternal health; Access; Inequity; Ghana
For many decades, access to human biological samples, such as cells, tissues, organs, blood, and sub-cellular materials such as DNA, for use in biomedical research, has been central in understanding the nature and transmission of diseases across the globe. However, the limitations of current ethical and regulatory frameworks in sub-Saharan Africa to govern the collection, export, storage and reuse of these samples have resulted in inconsistencies in practice and a number of ethical concerns for sample donors, researchers and research ethics committees. This paper examines stakeholders’ perspectives of and responses to the ethical issues arising from these research practices.
We employed a qualitative strategy of inquiry for this research including in-depth interviews and focus group discussions with key research stakeholders in Kenya (Nairobi and Kilifi), and Ghana (Accra and Navrongo).
The stakeholders interviewed emphasised the compelling scientific importance of sample export, storage and reuse, and acknowledged the existence of some structures governing these research practices, but they also highlighted the pressing need for a number of practical ethical concerns to be addressed in order to ensure high standards of practice and to maintain public confidence in international research collaborations. These concerns relate to obtaining culturally appropriate consent for sample export and reuse, understanding cultural sensitivities around the use of blood samples, facilitating a degree of local control of samples and sustainable scientific capacity building.
Drawing on these findings and existing literature, we argue that the ethical issues arising in practice need to be understood in the context of the interactions between host research institutions and local communities and between collaborating institutions. We propose a set of ‘key points-to-consider’ for research institutions, ethics committees and funding agencies to address these issues.
Human biological samples; Broad consent; Research collaboration; Sample export; Africa
Mammalian HtrA (high temperature requirement factor A) proteases, comprising 4 multi-domain members HtrA1-4, play important roles in a number of normal cellular processes as well as pathological conditions such as cancer, arthritis, neurodegenerative diseases and pregnancy disorders. However, how HtrA activities are regulated is not well understood, and to date no inhibitors specific to individual HtrA proteins have been identified. Here we investigated five HtrA3 monoclonal antibodies (mAbs) that we have previously produced, and demonstrated that two of them regulated HtrA3 activity in an opposing fashion: one inhibited while the other stimulated. The inhibitory mAb also blocked HtrA3 activity in trophoblast cells and enhanced migration and invasion, confirming its potential in vivo utility. To understand how the binding of these mAbs modulated HtrA3 protease activity, their epitopes were visualized in relation to a 3-dimensional HtrA3 homology model. This model suggests that the inhibitory HtrA3 mAb blocks substrate access to the protease catalytic site, whereas the stimulatory mAb may bind to the PDZ domain alone or in combination with the N-terminal and protease domains. Since HtrA1, HtrA3 and HtrA4 share identical domain organization, our results establish important foundations for developing potential therapeutics to target these HtrA proteins specifically for the treatment of a number of diseases, including cancer and pregnancy disorders.
Yoel Lubell and colleagues consider ethical and economic perspectives on mass drug administration of primaquine to limit transmission of P. falciparum malaria.
Please see later in the article for the Editors' Summary
The practice of making datasets publicly available for use by the wider scientific community has become firmly integrated in genomic science. One significant gap in literature around data sharing concerns how it impacts on scientists’ ability to preserve values and ethical standards that form an essential component of scientific collaborations. We conducted a qualitative sociological study examining the potential for harm to ethnic groups, and implications of such ethical concerns for data sharing. We focused our empirical work on the MalariaGEN Consortium, one of the first international collaborative genomics research projects in Africa.
We conducted a study in three MalariaGEN project sites in Kenya, the Gambia, and the United Kingdom. The study entailed analysis of project documents and 49 semi-structured interviews with fieldworkers, researchers and ethics committee members.
Concerns about how best to address the potential for harm to ethnic groups in MalariaGEN crystallised in discussions about the development of a data sharing policy. Particularly concerning for researchers was how best to manage the sharing of genomic data outside of the original collaboration. Within MalariaGEN, genomic data is accompanied by information about the locations of sample collection, the limitations of consent and ethics approval, and the values and relations that accompanied sample collection. For interviewees, this information and context were of important ethical value in safeguarding against harmful uses of data, but is not customarily shared with secondary data users. This challenged the ability of primary researchers to protect against harmful uses of ‘their’ data.
We identified three protective mechanisms – trust, the existence of a shared morality, and detailed contextual understanding – which together might play an important role in preventing the use of genomic data in ways that could harm the ethnic groups included in the study. We suggest that the current practice of sharing of datasets as isolated objects rather than as embedded within a particular scientific culture, without regard for the normative context within which samples were collected, may cause ethical tensions to emerge that could have been prevented or addressed had the ‘ethical metadata’ that accompanies genomic data also been shared.
Data sharing; Genomics; Africa; MalariaGEN; Ethnic stigmatisation; Secondary use; Ethics; Ethnic groups
Background and Objectives:
Emergency surgery for large bowel obstruction is associated with high morbidity and mortality rates, especially in elderly patients. Colonic self-expanding metal stents may provide temporary relief of obstructions and enable preoperative evaluation. The aim of this retrospective study was to assess the clinical outcomes of emergency stenting in elderly patients with large bowel obstructions.
Between 1997 and 2010, patients presenting with large bowel obstructions were treated predominantly with self-expanding metal stent insertion. Clinical data, including age, site of obstruction, success rate, and surgery and mortality rates, were collected. Patients were divided into 3 groups (I, II, and III) according to age: <69, 70 to 79, or >80 years.
One hundred thirty-two consecutive patients underwent stent implantation, with a mean age of 72.1 years (range, 28–95 years). Similar diversity of sex, indication, and stricture location was found. There were no significant differences in clinical success (88.7%, 73.8%, and 78.4%, P = .16) and stent-related mortality (2.1%, 3.3%, and 3.6%, P = 1.00). Similar rates of stoma creation were also found (59.3%, 46.7%, and 60.0%, P = .76). In contrast, rates of surgery were lower in older patients (50.9%, 38.1%, and 13.5%, P = .0013), and mortality during the same admission was significantly higher in patients >70 years of age (4.0%, 15.0%, and 22.2%, P = .027). Kaplan-Meier 30-day survival curves for the 3 groups showed a trend toward earlier death among patients >70 years of age.
This study demonstrates that stenting provides similar success rates in all age groups but is associated with higher mortality rates in older patients.
Large bowel obstruction; Colonic stenting
The attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however, the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines. Evaluation of the relative importance of different residues positioned −2, −1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the −1 and −2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter the native salt-bridge interactions in Ub and Cdc34, resulting in misplacement of Sic1 lysine 50 in the Cdc34 catalytic cleft. During polyubiquitination, Cdc34 showed a strong preference for Ub lysine 48 (K48), with lower activity towards lysine 11 (K11) and lysine 63 (K63). Mutating the −2, −1, +1 and +2 sites surrounding K11 and K63 to mimic those surrounding K48 did not improve their ubiquitination, indicating that further determinants are important for Ub K48 specificity. Modeling the ternary structure of acceptor Ub with the Cdc34~Ub complex as well as in vitro ubiquitination assays unveiled the importance of K6 and Q62 of acceptor Ub for Ub K48 polyubiquitination. These findings provide molecular and structural insight into substrate lysine and Ub K48 specificity by Cdc34.
ubiquitin; cell cycle; Cdc34; lysine; Sic1; SCF
Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA.
The luminal domain of human endoplasmic reticulum aminopeptidase 2 has been expressed, purified and crystallized. The crystals belonged to the orthorhombic space group P21212 and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house source.
Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a critical enzyme involved in the final processing of MHC class I antigens. Peptide trimming by ERAP2 and the other members of the oxytocinase subfamily is essential to customize longer precursor peptides in order to fit them to the correct length required for presentation on major histocompatibility complex class I molecules. While recent structures of ERAP1 have provided an understanding of the ‘molecular-ruler’ mechanism of substrate selection, little is known about the complementary activities of its homologue ERAP2 despite their sharing 49% sequence identity. In order to gain insights into the structure–function relationship of the oxytocinase subfamily, and in particular ERAP2, the luminal region of human ERAP2 has been crystallized in the presence of the inhibitor bestatin. The crystals belonged to an orthorhombic space group and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house X-ray source. A molecular-replacement solution suggested that the enzyme has adopted the closed state as has been observed in other inhibitor-bound aminopeptidase structures.
aminopeptidases; antigen processing; endoplasmic reticulum aminopeptidase 2; ERAP2; MHC presentation
In order to involve children in the decision-making process about participation in medical research it is widely recommended that the child’s assent be sought in addition to parental consent. However, the concept of assent is fraught with difficulties, resulting in confusion among researchers and ethics committees alike.
In this paper, we outline the current international debate surrounding pediatric consent and assent, and its unique challenges arising in low-income settings. We go on to propose some key requirements for a fit-for-purpose assent model in these difficult settings. The paper recommends that children who are competent, that is, children who are judged to be able to understand and retain relevant information, weigh this information in making a mature judgment, come to a decision and communicate the decision, should be able to consent for themselves. Our proposal is that where the decision about whether to participate in a study is of comparable complexity to the decisions the child is used to making in other aspects of his or her life, it should be made by the child him or herself. The relevant level of complexity should be judged by local standards rather than standards of the developed world. In the paper we explore some of the practical challenges and counter arguments of implementing this proposal. As in high-income settings, we argue that in the case of children who are judged to lack this level of competence both parental consent and assent from the child should be sought and go on to define assent as involving the child to the extent compatible to his or her maturity and with cultural norms and not as obtaining the child’s permission to proceed.
The concept of assent in the current guidelines is confusing. There is an urgent need for clearer guidelines that can be adapted for all types of paediatric research wherever it is to be carried out and an evidence-base concerning good assent/consent practice. This paper argues that a context specific approach should be adopted when assessing whether consent or assent should be sought from children in low-income settings.
Consent; Assent; Paediatric research; Low-income setting; Children
The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs) are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY) receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY) receptors and could apply to many receptors that use large peptidic agonists.
heteropentamer; G-protein heterotrimer; heterodimer; homodimer
Community engagement is increasingly promoted as a marker of good, ethical practice in the context of international collaborative research in low-income countries. There is, however, no widely agreed definition of community engagement or of approaches adopted. Justifications given for its use also vary. Community engagement is, for example, variously seen to be of value in: the development of more effective and appropriate consent processes; improved understanding of the aims and forms of research; higher recruitment rates; the identification of important ethical issues; the building of better relationships between the community and researchers; the obtaining of community permission to approach potential research participants; and, the provision of better health care. Despite these diverse and potentially competing claims made for the importance of community engagement, there is very little published evidence on effective models of engagement or their evaluation.
In this paper, drawing upon interviews with the members of a Community Advisory Board on the Thai-Myanmar border, we describe and critically reflect upon an approach to community engagement which was developed in the context of international collaborative research in the border region.
Results and conclusions
Drawing on our analysis, we identify a number of considerations relevant to the development of an approach to evaluating community engagement in this complex research setting. The paper also identifies a range of important ways in which the Community Advisory Board is in practice understood by its members (and perhaps by community members beyond this) to have morally significant roles and responsibilities beyond those usually associated with the successful and appropriate conduct of research.
Ethics; Evaluation; Community engagement; Community advisory boards; Developing countries; Thailand; Myanmar; Global health; International research
To evaluate and determine the value of monitoring models developed by the Mahidol Oxford Tropical Research Unit and the East African Consortium for Clinical Research, consider how this can be measured and explore monitors’ and investigators’ experiences of and views about the nature, purpose and practice of monitoring.
A case study approach was used within the context of participatory action research because one of the aims was to guide and improve practice. 34 interviews, five focus groups and observations of monitoring practice were conducted.
Setting and participants
Fieldwork occurred in the places where the monitoring models are coordinated and applied in Thailand, Cambodia, Uganda and Kenya. Participants included those coordinating the monitoring schemes, monitors, senior investigators and research staff.
Transcribed textual data from field notes, interviews and focus groups was imported into a qualitative data software program (NVIVO V. 10) and analysed inductively and thematically by a qualitative researcher. The initial coding framework was reviewed internally and two main categories emerged from the subsequent interrogation of the data.
The categories that were identified related to the conceptual framing and nature of monitoring, and the practice of monitoring, including relational factors. Particular emphasis was given to the value of a scientific and cooperative style of monitoring as a means of enhancing data quality, trust and transparency. In terms of practice the primary purpose of monitoring was defined as improving the conduct of health research and increasing the capacity of researchers and trial sites.
The models studied utilise internal and network wide expertise to improve the ethics and quality of clinical research. They demonstrate how monitoring can be a scientific and constructive exercise rather than a threatening process. The value of cooperative relations needs to be given more emphasis in monitoring activities, which seek to ensure that research protects human rights and produces reliable data.
Medical Ethics; Qualitative Research; Statistics & Research Methods; Tropical Medicine
There is no universally accepted definition of what an incidental finding is [Wolf et al., 2008] and broadly speaking this could include variants of known and unknown clinical significance, variants linked to highly penetrant, serious, life-threatening conditions, non-paternity or ancestry data. For the purposes of our study, we have adopted a pragmatic distinction between ‘pertinent’ and ‘incidental’ findings as set out in this text. Whilst in the US definitions of incidental findings are becoming accepted in practice [Green et al., 2013] it is still not known how and whether these also apply elsewhere around the world.
Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a kcat = 22 s-1, KMPYR = 2.55 ± 0.05 mM and KMASA = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (TMapp) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (TMapp = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a KD4→2 = 1.7 nM, which is considerably tighter than its E. coli ortholog (KD4→2 = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å2) compared to its E. coli counterpart (500 Å2). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target.
Uterine proprotein convertase (PC) 6 plays a critical role in embryo implantation and is pivotal for pregnancy establishment. Inhibition of PC6 may provide a novel approach for the development of non-hormonal and female-controlled contraceptives. We investigated a class of five synthetic non-peptidic small molecule compounds that were previously reported as potent inhibitors of furin, another PC member. We examined (i) the potency of these compounds in inhibiting PC6 activity in vitro; (ii) their binding modes in the PC6 active site in silico; (iii) their efficacy in inhibiting PC6-dependent cellular processes essential for embryo implantation using human cell-based models. All five compounds showed potent inhibition of PC6 activity in vitro, and in silico docking demonstrated that these inhibitors could adopt a similar binding mode in the PC6 active site. However, when these compounds were tested for their inhibition of decidualization of primary human endometrial stromal cells, a PC6-dependent cellular process critical for embryo implantation, only one (compound 1o) showed potent inhibition. The lack of activity in the cell-based assay may reflect the inability of the compounds to penetrate the cell membrane. Because compound's lipophilicity is linked to cell penetration, a measurement of lipophilicity (logP) was calculated for each compound. Compound 1o is unique as it appears the most lipophilic among the five compounds. Compound 1o also inhibited another crucial PC6-dependent process, the attachment of human trophoblast spheroids to endometrial epithelial cells (a model for human embryo attachment). We thus identified compound 1o as a potent small molecule PC6 inhibitor with pharmaceutical potential to inhibit embryo implantation. Our findings also highlight that human cell-based functional models are vital to complement the biochemical and in silico analyses in the selection of promising drug candidates. Further investigations for compound 1o are warranted in animal models to test its utility as an implantation-inhibiting contraceptive drug.
The management of misaligned paternity findings raises important controversy worldwide. It has mainly, however, been discussed in the context of high-income countries. Genetic and genomics research, with the potential to show misaligned paternity, are becoming increasingly common in Africa. During a genomics study in Kenya, a dilemma arose over testing and sharing information on paternal sickle cell disease status. This dilemma may be paradigmatic of challenges in sharing misaligned paternity findings in many research and health care settings. Using a deliberative approach to community consultation to inform research practice, we explored residents' views on paternal testing and sharing misaligned paternity information. Between December 2009 and November 2010, 63 residents in Kilifi County were engaged in informed deliberative small group discussions, structured to support normative reflection within the groups, with purposive selection to explore diversity. Analysis was based on a modified framework analysis approach, drawing on relevant social science and bioethics literature.
The methods generated in-depth individual and group reflection on morally important issues and uncovered wide diversity in views and values. Fundamental and conflicting values emerged around the importance of family interests and openness, underpinned by disagreement on the moral implications of marital infidelity and withholding truth. Wider consideration of ethical issues emerging in these debates supports locally-held reasoning that paternal sickle cell testing should not be undertaken in this context, in contrast to views that testing should be done with or without the disclosure of misaligned paternity information. The findings highlight the importance of facilitating wider testing of family members of affected children, contingent on the development and implementation of national policies for the management of this inherited disorder. Their richness also illustrates the potential for the approach adopted in this study to strengthen community consultation.
•Community consultation on misaligned paternity in Kenya highlights morally important diversity.•Openness and family interests are central conflicting values in debates on misaligned paternity.•In research paternal requests for sickle cell disease testing generate prohibitive moral challenges.•Informed deliberative group discussions strengthen ethical outcomes of community consultation.
Kenya; Misaligned paternity; Genetic testing; Genetic and genomics research; Community consultation; Empirical ethics; Sickle cell disease; Africa
This study focuses on the identification of multiple latent trajectories of pain intensity, and it examines how religiousness is related to different classes of pain trajectory. Participants were 720 community-dwelling older adults who were interviewed at four time points over a 3-year period. Overall, intensity of pain decreased over 3 years. Analysis using latent growth mixture modeling (GMM) identified three classes of pain: (1) increasing (n = 47); (2) consistently unchanging (n = 292); and (3) decreasing (n = 381). Higher levels of intrinsic religiousness (IR) at baseline were associated with higher levels of pain at baseline, although it attenuated the slope of pain trajectories in the increasing pain group. Higher service attendance at baseline was associated with a higher probability of being in the decreasing pain group. The increasing pain group and the consistently unchanging group reported more negative physical and mental health outcomes than the decreasing pain group.
pain trajectory; religiousness; latent growth modeling
International health research in malaria-endemic settings may include screening for sickle cell disease, given the relationship between this important genetic condition and resistance to malaria, generating questions about whether and how findings should be disclosed. The literature on disclosing genetic findings in the context of research highlights the role of community consultation in understanding and balancing ethically important issues from participants’ perspectives, including social forms of benefit and harm, and the influence of access to care. To inform research practice locally, and contribute to policy more widely, this study aimed to explore the views of local residents in Kilifi County in coastal Kenya on how researchers should manage study-generated information on sickle cell disease and carrier status.
Between June 2010 and July 2011, we consulted 62 purposively selected Kilifi residents on how researchers should manage study-generated sickle cell disease findings. Methods drew on a series of deliberative informed small group discussions. Data were analysed thematically, using charts, to describe participants’ perceptions of the importance of disclosing findings, including reasoning, difference and underlying values. Themes were derived from the underlying research questions and from issues emerging from discussions. Data interpretation drew on relevant areas of social science and bioethics literature.
Perceived health and social benefits generated strong support for disclosing findings on sickle cell disease, but the balance of social benefits and harms was less clear for sickle cell trait. Many forms of health and social benefits and harms of information-sharing were identified, with important underlying values related to family interests and the importance of openness. The influence of micro and macro level contextual features and prioritization of values led to marked diversity of opinion.
The approach demonstrates a high ethical importance in many malaria endemic low-to-middle income country settings of disclosing sickle cell disease findings generated during research, alongside provision of effective care and locally-informed counselling. Since these services are central to the benefits of disclosure, health researchers whose studies include screening for sickle cell disease should actively promote the development of health policy and services for this condition in situations of unmet need, including through the prior development of collaborative partnerships with government health managers and providers. Community consultation can importantly enrich ethical debate on research practice where in-depth exploration of informed views and the potential for difference are taken into account.
Kenya; Africa; Sickle cell disease; Community consultation; Genetic findings; Genetic and genomics research; Deliberative methods; Empirical ethics
The neuropeptide Y (NPY) Y2 receptor shows a large masked surface population in adherent CHO cells or in forebrain cell aggregates, but not in dispersed cells or in particulates from these sources. This is related to adhesion via acidic motifs in the extracellular N-terminal domain. Masking of the Y2 receptor is lifted by non-permeabilizing mechanical dispersion of cells, which also increases internalization of Y2 agonists. Mechanical dispersion and detachment by EDTA expose the same number of surface sites. As we have already shown, phenylarsine oxide (PAO), a cysteine-bridging agent, and to a lesser extent also the cysteine alkylator N-ethylmaleimide, unmask the surface Y2 sites without cell detachment or permeabilization. We now demonstrate that unmasking by permeabilizing but non-detaching treatment with cholesterol-binding detergents digitonin and edelfosine compares with and overlaps that of PAO. The caveolar/raft cholesterol-targeting macrolide filipin III however produces only partial unmasking. Depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide. These findings indicate presence of a dynamic masked pool including majority of the cell surface Y2 receptors in adherent CHO cells. This compartmentalization is obviously involved in the low internalization of Y2 receptors in these cells.
G-protein-coupled receptor; CHO cells; Receptor compartmentalization; Cysteine redox; Cholesterol complexing