Structural basis for dual-mode inhibition of the ABC transporter MsbA.

The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.

Unsafe by design: Infusion task reallocation and safety perceptions in U.S. hospitals.

BACKGROUND: Research suggests that changes in nurse roles can compromise perceived organizational safety. However, over the past 15 years, many infusion tasks have been reallocated from specialty nurse infusion teams to individual generalist nurses-a process we call infusion task reallocation . These changes purportedly benefit employees by allowing care providers to practice at the "top of their license." However, job demands-resources theory suggests that changing core task arrangements can either enrich or merely enlarge jobs depending on their effects on demands and resources, with corresponding consequences for performance (e.g., safety). There is relatively little research directly exploring these effects and their mechanisms. PURPOSE: This study examines the relationship between infusion task reallocation and perceptions of organizational safety. We also explore the extent to which this relationship may be mediated by infusion-related resources and psychological safety. METHODOLOGY: Data were collected through a survey of 623 nurses from 580 U.S. hospitals. The relationship between infusion task reallocation and perceptions of organizational safety, as well as the potential mediating roles of infusion-related resources and psychological safety, was examined using structural equation modeling. RESULTS: Infusion task reallocation was negatively associated with respondents' perceptions of organizational safety, with nurses working in organizations without an infusion team indicating lower perceptions of organizational safety than nurses working in organizations with an infusion team. This relationship was mediated by nurse perceptions of psychological safety within the organization, but not by infusion-related resources, suggesting that task reallocation is associated with lower perceived organizational safety because nurses feel less psychologically safe rather than because of perceived technical constraints. PRACTICE IMPLICATIONS: The results indicate that, although infusion task reallocation may be a cost-reducing approach to managing clinical responsibilities, it enlarges rather than enriches the job through higher demands and fewer resources for nurses and, in turn, lower perceived organizational safety.

A Novel Inhibitor of the LolCDE ABC Transporter Essential for Lipoprotein Trafficking in Gram-Negative Bacteria.

The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral ß-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors of Escherichia coli growth followed by selection of compounds that induced the extracytoplasmic σE stress response. Mutations in lolC, lolD, and lolE conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of E. coli cells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolCQ258K), which confers high-level resistance to G0507 in vivo but no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.

Disrupting Gram-Negative Bacterial Outer Membrane Biosynthesis through Inhibition of the Lipopolysaccharide Transporter MsbA.

There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill Escherichia coli through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.

Identifying potential therapeutic targets of methicillin-resistant Staphylococcus aureus through in vivo proteomic analysis.

BACKGROUND: Detailed knowledge on protein repertoire of a pathogen during host infection is needed for both developing a better understanding of the pathogenesis and defining potential therapeutic targets. Such data, however, have been missing for Staphylococcus aureus, a major human pathogen. METHODS: We determined the surface proteome of methicillin-resistant S. aureus (MRSA) clone usa300 derived directly from murine systemic infectiON. RESULTS: The majority of the in vivo-expressed surface-associated proteins were lipoproteins involved in nutrient acquisition, especially uptake of metal ions. Enzyme-linked immunosorbent assay (ELISA) of convalescent human serum samples revealed that proteins that were highly produced during murine experimental infection were also produced during natural human infection. We found that among the 7 highly abundant lipoproteins only MntC, which is the manganese-binding protein of the MntABC system, was essential for MRSA virulence during murine systemic infection. Moreover, we show that MntA and MntB are equally important for MRSA virulence. CONCLUSIONS: Besides providing experimental evidence that MntABC might be a potential therapeutic target for the development of antibiotics, our in vivo proteomics data will serve as a valuable basis for defining potential antigen combinations for multicomponent vaccines.

The inactivation of tolC sensitizes Escherichia coli to perturbations in lipopolysaccharide transport.

The Escherichia coli outer membrane channel TolC complexes with several inner membrane efflux pumps to export compounds across the cell envelope. All components of these complexes are essential for robust efflux activity, yet E. coli is more sensitive to antimicrobial compounds when tolC is inactivated compared to the inactivation of genes encoding the inner membrane drug efflux pumps. While investigating these susceptibility differences, we identified a distinct class of inhibitors targeting the core-lipopolysaccharide translocase, MsbA. We show that tolC null mutants are sensitized to structurally unrelated MsbA inhibitors and msbA knockdown, highlighting a synthetic-sick interaction. Phenotypic profiling revealed that tolC inactivation induced cell envelope softening and increased outer membrane permeability. Overall, this work identified a chemical probe of MsbA, revealed that tolC is associated with cell envelope mechanics and integrity, and highlighted that these findings should be considered when using tolC null mutants to study efflux deficiency.

Visceral adiposity index, fitness and clustered cardiovascular disease risk in adolescents.

BACKGROUND:  Clustering of cardiovascular disease (CVD) risk factors have been observed in children and adolescents, but its association with visceral adiposity index (VAI) and cardiorespiratory fitness (CRF) in adolescents has rarely been studied. AIM:  This study determines the independent associations of VAI and CRF with the clustering of cardiovascular disease risk (CVDr) among Nigerian adolescents. SETTING:  Adolescents from specific secondary schools in Kogi East, North Central Nigeria participated in the study. METHODS:  A cross-sectional sample of 403 adolescents (202 boys and 201 girls) aged 11 years - 19 years were evaluated for VAI, CRF and CVDr. Using identified risk factors, a clustered CVDr score was generated. The association between VAI, CRF and clustered CVDr was evaluated using regression models that controlled for age, gender and maturity status. RESULTS:  Fitness was negatively associated with CVDr (ß = -0.268, p  0.001), while VAI was positively correlated with CVDr (ß = 0.379, p  0.001). After CRF or VAI adjustment, the independent association with the dependent variable remained significant. The odds of an adolescent with elevated VAI being at risk of CVD was 4.7 times higher than his peers. Unfit adolescents were 2.1 times more likely to develop CVDr. CONCLUSION:  Both VAI and CRF were independently associated with the clustering of CVDr in Nigerian adolescents. The findings suggest that health promotion efforts focusing on healthy diet and aerobic-type physical activity programmes should be encouraged among the youth to reduce the risk of CVD.Contribution: This study shows that improving visceral adipose tissue and fitness may lower CVD risk factors in adolescents, which is significant for public health.

Motherhood in women with serious mental illness.

This study aimed to determine the prevalence of motherhood among inpatient females at a large state psychiatric hospital in suburban New York, as well as develop an understanding of the characteristics and needs of this unique population. Data on motherhood status was gathered from October 2010 through April 2011 via medical records. Data on custody status, frequency of contacts with children, and effect of mental illness on parenting was assessed through patient surveys and focus groups. 38.5 % of female inpatients were found to be mothers, almost half of whom reported at least weekly contact with children despite their inpatient status. The majority of identified mothers reported having maintained custody of their minor children and expressed great pride at being primary caretakers for their children, yet also emphasized the challenging effects of stigma associated with mental illness and parenting. A significant proportion of women at this psychiatric hospital were found to be mothers. Although acknowledged by some clinicians at the individual level, motherhood appears to remain a forgotten role systemically. Determining motherhood status and recognizing the varied roles our patients have is one more way mental health providers can model and promote recovery-oriented care.

Differences between homologous alleles of olfactory receptor genes require the Polycomb Group protein Eed.

A number of mammalian genes are expressed from only one of the two homologous chromosomes, selected at random in each cell. These include genes subject to X-inactivation, olfactory receptor (OR) genes, and several classes of immune system genes. The means by which monoallelic expression is established are only beginning to be understood. Using a cytological assay, we show that the two homologous alleles of autosomal random monoallelic loci differ from each other in embryonic stem (ES) cells, before establishment of monoallelic expression. The Polycomb Group gene Eed is required to establish this distinctive behavior. In addition, we found that when Eed mutant ES cells are differentiated, they fail to establish asynchronous replication timing at OR loci. These results suggest a common mechanism for random monoallelic expression on autosomes and the X chromosome, and implicate Eed in establishing differences between homologous OR loci before and after differentiation.

Discovery of Inhibitors of the Lipopolysaccharide Transporter MsbA: From a Screening Hit to Potent Wild-Type Gram-Negative Activity.

The dramatic increase in the prevalence of multi-drug resistant Gram-negative bacterial infections and the simultaneous lack of new classes of antibiotics is projected to result in approximately 10 million deaths per year by 2050. We report on efforts to target the Gram-negative ATP-binding cassette (ABC) transporter MsbA, an essential inner membrane protein that transports lipopolysaccharide from the inner leaflet to the periplasmic face of the inner membrane. We demonstrate the improvement of a high throughput screening hit into compounds with on-target single digit micromolar (µM) minimum inhibitory concentrations against wild-type uropathogenic Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. A 2.98 Å resolution X-ray crystal structure of MsbA complexed with an inhibitor revealed a novel mechanism for inhibition of an ABC transporter. The identification of a fully encapsulated membrane binding site in Gram-negative bacteria led to unique physicochemical property requirements for wild-type activity.

Relationship between endometriosis, endometrioid adenocarcinoma, gliomatosis peritonei, and carcinoid tumor in a patient with recurrent ovarian teratoma.

Gliomatosis peritonei (GP) describes the implantation of mature neuroglial tissue in the peritoneum and is usually associated with mature ovarian teratoma but is also found in cases of immature teratoma. We report the case of a patient with recurrent mature ovarian teratoma, GP, endometriosis (with malignant transformation), and carcinoid tumor, found at the time of hysterectomy for a primary endometrial adenocarcinoma. This unusual combination of tumor types has not been reported earlier. Metaplasia of the totipotential subcoelomic or submesothelial stem cells is a recognized pathway for the development of endometriosis. Evidence from molecular genetic studies suggests that a similar process of stem cell differentiation may explain at least some cases of GP. The coexistence (and colocalization) of endometriosis, GP, and carcinoid tumor in this case raises the possibility that peritoneal stem cells may occasionally show an even wider spectrum of aberrant differentiation. This has relevance for the assessment and management of patients with synchronous gynecologic tumors or presumed metastatic disease.

Incidence of cleft pathology in Greater New Orleans before and after Hurricane Katrina.

BACKGROUND: Reports after the 2005 Hurricane Katrina have documented an increase in stress reactions and environmental teratogens (arsenic, mold, alcohol). OBJECTIVE: To assess the incidence of cleft pathology before and after the hurricane, and the distribution of cleft cases by gender and race. METHODS: Retrospective chart review of cleft lip with or without cleft palate (CL/P) and cleft palate (CP) cases registered with the Cleft and Craniofacial Team at Children's Hospital of New Orleans, the surgical center that treated cleft cases in Greater New Orleans between 2004 and 2007. Live birth data were obtained from the Louisiana State Center for Health Statistics. RESULTS: The incidence of cleft cases, beginning 9 months after the hurricane (i.e., June 1, 2006) was significantly higher compared with the period before the hurricane (0.80 versus 1.42; p = .008). Within racial group comparisons showed a higher incidence among African Americans versus whites (0.42 versus 1.22; p = .01). The distribution of CL/P and CP cases by gender was significant (p = .05). CONCLUSION: The increase in the incidence of cleft cases after the hurricane may be attributable to increased stress and teratogenic factors associated with the hurricane. The increase among African Americans may have been due to comparatively higher exposure to environmental risk factors. These findings warrant further investigation to replicate the results elsewhere in the Gulf to determine whether there is a causal relationship between environmental risk factors and increased cleft pathology.

Natural disaster and crisis: lessons learned about cleft and craniofacial care from Hurricane Katrina and the West Bank.

Cleft care is generally characterized by staged, carefully timed surgeries and long-term, team-centered follow-up. Acute and chronic crises can wreak havoc on the comprehensive team care required by children with craniofacial anomalies. In addition, there is evidence that crises, including natural disasters and chronic disruptions, such as political turmoil and poverty, can lead to an increased incidence of craniofacial anomalies. The purpose of this article is to delineate the impact of acute and chronic crises on cleft care. Hurricane Katrina in New Orleans, Louisiana, in 2005, resulted in an acute crisis that temporarily disrupted the infrastructure necessary to deliver cleft care; chronic turmoil in the West Bank/Palestine has resulted in an absence of infrastructure to deliver cleft care. Through these central examples, this article will illustrate-through the prism of cleft care-the need for (1) disaster preparedness for acute crises, (2) changing needs following acute crises that may lead to persistent chronic disruption, and (3) baseline and long-term monitoring of population changes after a disaster has disrupted a health care delivery system.

Introducing spirituality into psychiatric care.

Spirituality is important to many psychiatric patients, and these patients may be moved toward recovery more effectively if their spiritual needs are addressed in treatment. This, however, is rarely given expression in the psychiatric services of teaching hospitals. In order to develop this potential area of improved care, we (1) evaluated the differential attitudes of patients and psychiatric trainees toward the value of spirituality in the recovery process, (2) established a program of group meetings conducted by psychiatric residents and staff where patients can discuss how to draw on their spirituality in coping with their problems, and (3) established related training experiences for psychiatric residents. The results and implications of these three initiatives are presented.

Reaching consensus on a home infusion central line-associated bloodstream infection surveillance definition via a modified Delphi approach.

BACKGROUND: A consensus on a central line-associated bloodstream infection (CLABSI) surveillance definition in home infusion is needed to standardize measurement and benchmark CLABSI to provide data to drive improvement initiatives METHODS: Experts across fields including home infusion therapy, infectious diseases, and healthcare epidemiology convened to perform a 3-step modified Delphi approach to obtain input and achieve consensus on a candidate home infusion CLABSI definition. RESULTS: The numerator criterion was identified by participants as involving one of the 2 following: (1) recognized pathogen isolated from blood culture and pathogen is not related to infection at another site, or (2) one of the following signs or symptoms: fever of 38°C (100.4°F), chills, or hypotension (systolic blood pressure ≤90 mm Hg), and one of the 2 following: (A) common skin contaminant isolated from 2 blood cultures drawn on separate occasions and organism is not related to infection at another site, or (B) common skin contaminant isolated from blood culture from patient with intravascular access device and provider institutes appropriate antimicrobial therapy. The criteria for a denominator included days from the day of admission with a central venous catheter to day of removal of central venous catheter. In addition, 11 inclusion criteria and 4 exclusion criteria were included. DISCUSSION: Home infusion therapy and healthcare epidemiology experts developed candidate criteria for a home infusion CLABSI surveillance definition. CONCLUSIONS: Home care and home infusion agencies can use this definition to monitor their own CLABSI rates and implement preventative strategies.

X chromosomes alternate between two states prior to random X-inactivation.

Early in the development of female mammals, one of the two X chromosomes is silenced in half of cells and the other X chromosome is silenced in the remaining half. The basis of this apparent randomness is not understood. We show that before X-inactivation, the two X chromosomes appear to exist in distinct states that correspond to their fates as the active and inactive X chromosomes. Xist and Tsix, noncoding RNAs that control X chromosome fates upon X-inactivation, also determine the states of the X chromosomes prior to X-inactivation. In wild-type ES cells, X chromosomes switch between states; among the progeny of a single cell, a given X chromosome exhibits each state with equal frequency. We propose a model in which the concerted switching of homologous X chromosomes between mutually exclusive future active and future inactive states provides the basis for the apparently random silencing of one X chromosome in female cells.

Trends in Infusion Administrative Practices in US Health Care Organizations: An Exploratory Analysis.

While specialized infusion clinical services remain the standard of care, widespread curtailing and disbanding of infusion teams as a cost-cutting measure has been documented in health care organizations for nearly 2 decades. Owing to this trend, as well as recent government interventions in medical error control, the authors engaged in an exploratory study of infusion administration practices in the US health care industry. This article presents the authors' exploratory findings, as well as their potential implications.

Counting chromosomes: not as easy as 1, 2, 3.

Mammalian cells must count their X chromosomes to determine whether to initiate X chromosome inactivation. A region that may be important for X chromosome counting has been identified, but the puzzle pieces still do not quite fit.