Characterisation of neonatal Staphylococcus capitis NRCS-A isolates compared with non NRCS-A Staphylococcus capitis from neonates and adults.

Staphylococcus capitis is a frequent cause of late-onset sepsis in neonates admitted to Neonatal Intensive Care Units (NICU). One clone of S. capitis, NRCS-A has been isolated from NICUs globally although the reasons for the global success of this clone are not well understood.We analysed a collection of S. capitis colonising babies admitted to two NICUs, one in the UK and one in Germany as well as corresponding pathological clinical isolates. Genome analysis identified a population structure of three groups; non-NRCS-A isolates, NRCS-A isolates, and a group of 'proto NRCS-A' - isolates closely related to NRCS-A but not associated with neonatal infection. All bloodstream isolates belonged to the NRCS-A group and were indistinguishable from strains carried on the skin or in the gut. NRCS-A isolates showed increased tolerance to chlorhexidine and antibiotics relative to the other S. capitis as well as enhanced ability to grow at higher pH values. Analysis of the pangenome of 138 isolates identified characteristic nsr and tarJ genes in both the NRCS-A and proto groups. A CRISPR-cas system was only seen in NRCS-A isolates which also showed enrichment of genes for metal acquisition and transport.We found evidence for transmission of S. capitis NRCS-A within NICU, with related isolates shared between babies and multiple acquisitions by some babies. Our data show NRCS-A strains commonly colonise uninfected babies in NICU representing a potential reservoir for potential infection. This work provides more evidence that adaptation to survive in the gut and on skin facilitates spread of NRCS-A, and that metal acquisition and tolerance may be important to the biology of NRCS-A. Understanding how NRCS-A survives in NICUs can help develop infection control procedures against this clone.

Collective memory and autobiographical memory: Perspectives from the humanities and cognitive sciences.

The current overview provides an interdisciplinary synthesis of autobiographical and collective memory studies, focusing on history and cognitive psychology, to help other scholars bridge the disciplinary gap. We describe the various interpretative frameworks used to build theoretical knowledge on how autobiographical memory and collective memory are intertwined. We expose how research exploring self, social and directive functions of autobiographical memory echoes three main functions that can be identified for collective memory, that is, social identity, social schemata, and means for actions, or a political decision tool of research in these fields. In doing so, we hope to stimulate opportunities for more interdisciplinary research. This article is categorized under: Psychology > Memory Psychology > Theory and Methods.

Evaluating nursing opinion and perception of maggot therapy for hard-to-heal wound management.

OBJECTIVE: Maggot therapy (MT) or larval debridement therapy is a recognised, effective but underutilised treatment for the management of hard-to-heal wounds and infected ulcers. It is available on NHS prescription in the UK, where wound management is predominantly nurse-led. Anecdotal reports and published literature suggest that nurses may be reluctant to utilise the therapy. The aim of this study was to evaluate the feelings and opinions of nurses regarding the use of MT. METHOD: The first stage of this mixed-methods study was a focus group held to discuss MT and opinions of specialist nurse clinicians. Next, an anonymised web-based online survey was launched through the Nursing Times journal and distributed through social media targeting all nurses. Finally, in-depth interviews were held with specialist and generalist nurses. RESULTS: Awareness of MT among all nurses was extremely high. A breakdown of results showed that MT was much more highly regarded by wound specialist nurses than non-wound specialist nurses. The latter exhibited a greater level of reluctance to administer the therapy, with almost one-third of these nurses surveyed saying they found maggots disgusting and that the idea of MT made their skin crawl. In-depth interviews revealed that a lack of knowledge about MT was a prime concern. CONCLUSION: Wound specialist nurses are more likely to embrace MT than non-wound nurse specialists, who report a varying degree of wariness to MT. Our study highlights a need for better education and training in MT for all nurses, to address issues with acceptance and willingness to treat or help treat patients with hard-to-heal wounds which are suitable for MT.

An exploration of public perceptions and attitudes towards maggot therapy.

OBJECTIVE: The aim of the study was to explore public opinion and perceptions of maggot therapy (larval therapy), a treatment option for hard-to-heal wounds. METHOD: The study used a mixed-method approach to obtain quantitative and qualitative data. A focus group was convened to explore opinions and views of maggot therapy with a small group of members of the public. Analysis of emerging themes from the focus group was used to design an anonymised web-based survey, which was made available online to members of the public through email and social media. RESULTS: The focus group participants identified four key themes concerning the acceptability of maggot therapy. The subsequent online survey was completed by 412 participants, analysis of which revealed some worries and fears. Only 36% of survey participants agreed that they would accept maggot therapy as a first choice for a hypothetical painful wound, although this number increased with wound severity. The most predominant concerns regarding maggot therapy were sensation and a feeling of disgust associated with the therapy. However, participants could see some benefits to maggot therapy. CONCLUSION: Our study showed that public perception of maggot therapy is varied. Survey participants expressed worries and fears associated with its use. However, positive relationships existed between knowledge scores and potential acceptability of maggot therapy, suggesting that information dissemination and education may be an important factor in public perception and acceptability of maggot therapy.

Dynamic internalization and recycling of a metal ion transporter: Cu homeostasis and CTR1, the human Cu⁺ uptake system.

Cu ion (Cu) entry into human cells is mediated by CTR1 (also known as SLC31A1), the high-affinity Cu transporter. When extracellular Cu is raised, the cell is protected against excess accumulation by rapid internalization of the transporter. When Cu is lowered, the transporter returns to the membrane. We show in HEK293 cells overexpressing CTR1 that expression of either the C-terminal domain of AP180 (also known as SNAP91), a clathrin-coat assembly protein that sequesters clathrin, or a dominant-negative mutant of dynamin, decreases Cu-induced endocytosis of CTR1, as does a dynamin inhibitor and clathrin knockdown using siRNA. Utilizing imaging, siRNA techniques and a new high-throughput assay for endocytosis employing CLIP-tag methodology, we show that internalized CTR1 accumulates in early sorting endosomes and recycling compartments (containing Rab5 and EEA1), but not in late endosomes or lysosomal pathways. Using live cell fluorescence, we find that upon extracellular Cu removal CTR1 recycles to the cell surface through the slower-recycling Rab11-mediated pathway. These processes enable cells to dynamically alter transporter levels at the plasma membrane and acutely modulate entry as a safeguard against excess cellular Cu.

Human breast tumor cells are more resistant to cardiac glycoside toxicity than non-tumorigenic breast cells.

Cardiotonic steroids (CTS), specific inhibitors of Na,K-ATPase activity, have been widely used for treating cardiac insufficiency. Recent studies suggest that low levels of endogenous CTS do not inhibit Na,K-ATPase activity but play a role in regulating blood pressure, inducing cellular kinase activity, and promoting cell viability. Higher CTS concentrations inhibit Na,K-ATPase activity and can induce reactive oxygen species, growth arrest, and cell death. CTS are being considered as potential novel therapies in cancer treatment, as they have been shown to limit tumor cell growth. However, there is a lack of information on the relative toxicity of tumor cells and comparable non-tumor cells. We have investigated the effects of CTS compounds, ouabain, digitoxin, and bufalin, on cell growth and survival in cell lines exhibiting the full spectrum of non-cancerous to malignant phenotypes. We show that CTS inhibit membrane Na,K-ATPase activity equally well in all cell lines tested regardless of metastatic potential. In contrast, the cellular responses to the drugs are different in non-tumor and tumor cells. Ouabain causes greater inhibition of proliferation and more extensive apoptosis in non-tumor breast cells compared to malignant or oncogene-transfected cells. In tumor cells, the effects of ouabain are accompanied by activation of anti-apoptotic ERK1/2. However, ERK1/2 or Src inhibition does not sensitize tumor cells to CTS cytotoxicity, suggesting that other mechanisms provide protection to the tumor cells. Reduced CTS-sensitivity in breast tumor cells compared to non-tumor cells indicates that CTS are not good candidates as cancer therapies.

Hyperplasia of pancreatic beta cells and improved glucose tolerance in mice deficient in the FXYD2 subunit of Na,K-ATPase.

Restoration of the functional potency of pancreatic islets either through enhanced proliferation (hyperplasia) or increase in size (hypertrophy) of beta cells is a major objective for intervention in diabetes. We have obtained experimental evidence that global knock-out of a small, single-span regulatory subunit of Na,K-ATPase, FXYD2, alters glucose control. Adult Fxyd2(-/-) mice showed significantly lower blood glucose levels, no signs of peripheral insulin resistance, and improved glucose tolerance compared with their littermate controls. Strikingly, there was a substantial hyperplasia in pancreatic beta cells from the Fxyd2(-/-) mice compared with the wild type littermates, compatible with an observed increase in the level of circulating insulin. No changes were seen in the exocrine compartment of the pancreas, and the mice had only a mild, well-adapted renal phenotype. Morphometric analysis revealed an increase in beta cell mass in KO compared with WT mice. This appears to explain a phenotype of hyperinsulinemia. By RT-PCR, Western blot, and immunocytochemistry we showed the FXYD2b splice variant in pancreatic beta cells from wild type mice. Phosphorylation of Akt kinase was significantly higher under basal conditions in freshly isolated islets from Fxyd2(-/-) mice compared with their WT littermates. Inducible expression of FXYD2 in INS 832/13 cells produced a reduction in the phosphorylation level of Akt, and phosphorylation was restored in parallel with degradation of FXYD2. Thus we suggest that in pancreatic beta cells FXYD2 plays a role in Akt signaling pathways associated with cell growth and proliferation.

Subunit isoform selectivity in assembly of Na,K-ATPase α-ß heterodimers.

To catalyze ion transport, the Na,K-ATPase must contain one α and one ß subunit. When expressed by transfection in various expression systems, each of the four α subunit isoforms can assemble with each of the three ß subunit isoforms and form an active enzyme, suggesting the absence of selective α-ß isoform assembly. However, it is unknown whether in vivo conditions the α-ß assembly is random or isoform-specific. The α(2)-ß(2) complex was selectively immunoprecipitated by both anti-α(2) and anti-ß(2) antibodies from extracts of mouse brain, which contains cells co-expressing multiple Na,K-ATPase isoforms. Neither α(1)-ß(2) nor α(2)-ß(1) complexes were detected in the immunoprecipitates. Furthermore, in MDCK cells co-expressing α(1), ß(1), and ß(2) isoforms, a greater fraction of the ß(2) subunits was unassembled with α(1) as compared with that of the ß(1) subunits, indicating preferential association of the α(1) isoform with the ß(1) isoform. In addition, the α(1)-ß(2) complex was less resistant to various detergents than the α(1)-ß(1) complex isolated from MDCK cells or the α(2)-ß(2) complex isolated from mouse brain. Therefore, the diversity of the α-ß Na,K-ATPase heterodimers in vivo is determined not only by cell-specific co-expression of particular isoforms, but also by selective association of the α and ß subunit isoforms.

Regulation of Na,K-ATPase subunit abundance by translational repression.

The Na,K-ATPase is an alphabeta heterodimer responsible for maintaining fluid and electrolyte homeostasis in mammalian cells. We engineered Madin-Darby canine kidney cell lines expressing alpha(1)FLAG, beta(1)FLAG, or beta(2)MYC subunits via a tetracycline-regulated promoter and a line expressing both stable beta(1)MYC and tetracycline-regulated beta(1)FLAG to examine regulatory mechanisms of sodium pump subunit expression. When overexpression of exogenous beta(1)FLAG increased total beta subunit levels by >200% without changes in alpha subunit abundance, endogenous beta(1) subunit (beta(1)E) abundance decreased. beta(1)E down-regulation did not occur during beta(2)MYC overexpression, indicating isoform specificity of the repression mechanism. Measurements of RNA stability and content indicated that decreased beta subunit expression was not accompanied by any change in mRNA levels. In addition, the degradation rate of beta subunits was not altered by beta(1)FLAG overexpression. Cells stably expressing beta(1)MYC, when induced to express beta(1)FLAG subunits, showed reduced beta(1)MYC and beta(1)E subunit abundance, indicating that these effects occur via the coding sequences of the down-regulated polypeptides. In a similar way, Madin-Darby canine kidney cells overexpressing exogenous alpha(1)FLAG subunits exhibited a reduction of endogenous alpha(1) subunits (alpha(1)E) with no change in alpha mRNA levels or beta subunits. The reduction in alpha(1)E compensated for alpha(1)FLAG subunit expression, resulting in unchanged total alpha subunit abundance. Thus, regulation of alpha subunit expression maintained its native level, whereas beta subunit was not as tightly regulated and its abundance could increase substantially over native levels. These effects also occurred in human embryonic kidney cells. These data are the first indication that cellular sodium pump subunit abundance is modulated by translational repression. This mechanism represents a novel, potentially important mechanism for regulation of Na,K-ATPase expression.

What do patients choose to tell their doctors? Qualitative analysis of potential barriers to reattributing medically unexplained symptoms.

BACKGROUND: Despite both parties often expressing dissatisfaction with consultations, patients with medically unexplained symptoms (MUS) prefer to consult their general practitioners (GPs) rather than any other health professional. Training GPs to explain how symptoms can relate to psychosocial problems (reattribution) improves the quality of doctor-patient communication, though not necessarily patient health. OBJECTIVE: To examine patient experiences of GPs' attempts to reattribute MUS in order to identify potential barriers to primary care management of MUS and improvement in outcome. DESIGN: Qualitative study. PARTICIPANTS: Patients consulting with MUS whose GPs had been trained in reattribution. A secondary sample of patients of control GPs was also interviewed to ascertain if barriers identified were specific to reattribution or common to consultations about MUS in general. APPROACH: Thematic analysis of in-depth interviews. RESULTS: Potential barriers include the complexity of patients' problems and patients' judgements about how to manage their presentation of this complexity. Many did not trust doctors with discussion of emotional aspects of their problems and chose not to present them. The same barriers were seen amongst patients whose GPs were not trained, suggesting the barriers are not particular to reattribution. CONCLUSIONS: Improving GP explanation of unexplained symptoms is insufficient to reduce patients' concerns. GPs need to (1) help patients to make sense of the complex nature of their presenting problems, (2) communicate that attention to psychosocial factors will not preclude vigilance to physical disease and (3) ensure a quality of doctor-patient relationship in which patients can perceive psychosocial enquiry as appropriate.

beta-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells.

In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the Na-K-ATPase alpha- and beta-subunits led us to question whether this was alterable and thus if some form of regulation was involved. We have examined the consequences of overexpressing Na-K-ATPase beta1-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged beta1-subunits (beta1flag) or Myc-tagged beta1-subunits (beta1myc) under the control of a tetracycline-dependent promoter. The induction of beta1flag subunit synthesis in MDCK cells, which increases beta1-subunit expression at the plasma membrane by more than twofold, while maintaining stable alpha1 expression levels, revealed that all mature beta1-subunits associate with alpha1-subunits, and no evidence of "free" beta1-subunits was obtained. Consequently, the ratio of assembled beta1- to alpha1-subunits is significantly increased when "extra" beta-subunits are expressed. An increased beta1/alpha1 stoicheometry is also observed in cells treated with tunicamycin, suggesting that the protein-protein interactions involved in these complexes are not dependent on glycosylation. Confocal images of cocultured beta1myc-expressing and beta1flag-expressing MDCK cells show colocalization of beta1myc and beta1flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the beta-subunits. Immunoprecipitation using MDCK cells constitutively expressing beta1myc and tetracycline-regulated beta1flag subunits confirmed beta-beta-subunit interactions. These results demonstrate that the equimolar ratio of assembled beta1/alpha1-subunits of the Na-K-ATPase in kidney cells is not fixed by the inherent properties of the interacting subunits. It is likely that cellular mechanisms are present that regulate the individual Na-K-ATPase subunit abundance.

Cluster randomised controlled trial of training practices in reattribution for medically unexplained symptoms.

BACKGROUND: Reattribution is frequently taught to general practitioners (GPs) as a structured consultation that provides a psychological explanation for medically unexplained symptoms. AIMS: To determine if practice-based training of GPs in reattribution changes doctor-patient communication, thereby improving outcomes in patients with medically unexplained symptoms of 3 months' duration. METHOD: Cluster randomised controlled trial in 16 practices, 74 GPs and 141 patients with medically unexplained symptoms of 6 hours of reattribution training v. treatment as usual. RESULTS: With training, the proportion of consultations mostly consistent with reattribution increased (31 v. 2%, P=0.002). Training was associated with decreased quality of life (health thermometer difference -0.9, 95% CI -1.6 to -0.1; P=0.027) with no other effects on patient outcome or health contacts. CONCLUSIONS: Practice-based training in reattribution changed doctor-patient communication without improving outcome of patients with medically unexplained symptoms.

Peering through the barriers in GPs' explanations for declining to participate in research: the role of professional autonomy and the economy of time.

BACKGROUND: The level of participation in research by GPs is low internationally. Previous reports of the reasons why practitioners decline opportunities for research participation have tended to recount the barriers that they describe as if they are objective accounts. OBJECTIVE: By theoretical sampling of practitioners who had declined to participate in a research trial, we sought to interpret the functional significance and interrelationship of the barriers that they reported. METHODS: Twenty-three GPs who had declined to participate in a trial of training to manage medically unexplained symptoms were interviewed and their accounts analysed interpretatively. RESULTS: The practitioners described general practice and research as alien fields. Research lacked intrinsic, clinical or professional value and was linked to evidence-based medicine which they rejected as incompatible with person-centered care. Every doctor described a lack of time for research, but time was an elastic resource that payment could release from the reservoir of their 'own time'. CONCLUSION: The findings should inform the design and interpretation of future quantitative surveys to identify how common the attitudes that we report are. Doctors with the attitudes of those whom we interviewed will not be drawn into research by measures predicated on the assumption that it is intrinsically, clinically or professionally valuable. If they cannot be convinced of its utility, value could be conferred by payment for participation.

Why do general practitioners decline training to improve management of medically unexplained symptoms?

BACKGROUND: General practitioners' (GPs) communication with patients presenting medically unexplained symptoms (MUS) has the potential to somatize patients' problems and intensify dependence on medical care. Several reports indicate that GPs have negative attitudes about patients with MUS. If these attitudes deter participation in training or other methods to improve communication, practitioners who most need help will not receive it. OBJECTIVE: To identify how GPs' attitudes to patients with MUS might inhibit their participation with training to improve management. DESIGN: Qualitative study. PARTICIPANTS: GPs (N = 33) who had declined or accepted training in reattribution techniques in the context of a research trial. APPROACH: GPs were interviewed and their accounts analysed qualitatively. RESULTS: Although attitudes that devalued patients with MUS were common in practitioners who had declined training, these coexisted, in the same practitioners, with evidence of intuitive and elaborate psychological work with these patients. However, these practitioners devalued their psychological skills. GPs who had accepted training also described working psychologically with MUS but devalued neither patients with MUS nor their own psychological skills. CONCLUSIONS: GPs' attitudes that suggested disengagement from patients with MUS belied their pursuit of psychological objectives. We therefore suggest that, whereas negative attitudes to patients have previously been regarded as the main barrier to involvement in measures to improve patient management, GPs devaluing of their own psychological skills with these patients may be more important.

Selective basolateral localization of overexpressed Na-K-ATPase beta1- and beta2- subunits is disrupted by butryate treatment of MDCK cells.

The exclusive basolateral localization of the Na-K-ATPase in kidney epithelium is a critical aspect of nephron function. It has been suggested that mislocalized delivery of the Na-K-ATPase to the apical surface in autosomal dominant polycystic kidney disease (ADPKD) is due to the inappropriate expression of an alternative isoform of the beta-subunit, the beta(2)-isoform. It has been reported that heterologous expression of this beta(2)-isoform in Madin-Darby canine kidney (MDCK) cells results in apical delivery of the Na-K-ATPase. We created a MDCK cell line containing a tetracycline-inducible promoter and expressed either myc-tagged beta(2)- or flag-tagged beta(1)-subunits to study the surface localization of these beta-subunit isoforms in polarized monolayers. We find that the beta(2)-isoform is targeted to the basolateral surface of the plasma membrane in a polarization pattern indistinguishable from the beta(1)-isoform. However, inclusion of butyrate in the growth medium leads to upregulation of overexpressed beta(1)- or beta(2)-subunits and to their appearance at the apical surface. The beta(2)-isoform expressed in MDCK cells does not assemble into alpha(1)beta(2) heterodimers with the endogenous alpha(1). Our findings demonstrate that expression of the beta(2)-isoform does not lead to apical localization of the Na-K-ATPase in MDCK cells and provides evidence for an unexpected effect of butyrate on the trafficking of Na pump subunits.