Functional specialization of the yeast Rho1 GTP exchange factors

Rho GTPases are regulated in complex spatiotemporal patterns that may be dependent, in part at least, on the multiplicity of their GTP exchange factors (GEFs). Here, we examine the extent of and basis for functional specialization of the Rom2 and Tus1 GEFs that activate the yeast Rho1 GTPase, the ortholog of mammalian RhoA. First, we find that these GEFs selectively activate different Rho1-effector branches. Second, the synthetic genetic networks around ROM2 and TUS1 confirm very different global in vivo roles for these GEFs. Third, the GEFs are not functionally interchangeable: Tus1 cannot replace the essential role of Rom2, even when overexpressed. Fourth, we find that Rom2 and Tus1 localize differently: Rom2 to the growing bud surface and to the bud neck at cytokinesis; Tus1 only to the bud neck but in a distinct pattern. Finally, we find that these GEFs are dependent on different protein co-factors: Rom2 function and localization is largely dependent on Ack1, a SEL1 domain containing protein; Tus1 function and localization is largely dependent on the Tus1-interacting protein Ypl066w (which we name Rgl1). We have revealed a surprising level of diversity among the Rho1 GEFs that contributes another level of complexity to the spatiotemporal control of Rho1

Living well while providing support: validation of LTCQ-Carer for assessing informal carers’ quality of life

Purpose Despite international policies to support the health and wellbeing of informal (family) caregivers, there is no consensus on how to evaluate the effectiveness of carer support. We aimed to develop and validate a new quality-of-life measure for carers (LTCQ-Carer) and to assess its potential for use within a clinical pathway. Methods Psychometric properties of LTCQ-Carer were tested through cognitive interviews (qualitative phase) and a pilot survey (quantitative phase). Participants were family caregivers of people recently diagnosed with mild cognitive impairment (MCI) or dementia, recruited through one of 14 memory clinics in south-east England. They self-completed the new measure and comparative existing measures (EQ-5D, ASCOT-Carer). Ongoing feedback from memory clinic staff on potential use of LTCQ-Carer was collected. Results Interview participants (n = 10) found all draft items of LTCQ-Carer relevant and prompted inclusion of a new item on ‘time to yourself’. Responses from survey participants (n = 107) indicated acceptability (low missing data), high internal reliability (Cronbach’s α = 0.95), and a general construct (single factor loadings 0.43–0.86 for all items). Observation of predicted associations with EQ-5D and ASCOT-Carer supported construct validity. Responsiveness requires further testing as evidence was inconclusive. Clinical staff feedback on potential use was positive. Conclusion LTCQ-Carer is a valid new measure for assessing family caregivers’ quality of life across broad health and social care domains, expanding the range of high-quality tools for evaluating carer support. When used concurrently with patient assessment, it could highlight carer needs and prompt appropriate family support at the earliest point in the clinical pathway

Differential activation of inflammatory pathways in A549 type II pneumocytes by Streptococcus pneumoniae strains with different adherence properties

BACKGROUND: Adherence of Streptococcus pneumoniae bacteria to lung cells is a first step in the progression from asymptomatic carriage to pneumonia. Adherence abilities vary widely among S. pneumoniae patient isolates. In this study, the binding properties of S. pneumoniae isolates and the effects of binding on activation of the Nuclear Factor-Kappa-B (NFκB) pathway and cytokine secretion by type II pneumocytes were measured. METHODS: Mechanisms of high- and low-binding S. pneumoniae adherence to A549 cells were investigated by blocking putative receptors on bacteria and host cells with antibody and by eluting choline-binding proteins off of bacterial surfaces. NFκB activation was measured by western blot and immunocytochemistry and cytokine secretion was detected by a protein array. RESULTS: This study shows that S. pneumoniae isolates from pneumonia patients (n = 298) can vary by as much as 1000-fold in their ability to bind to human lung epithelial cells. This difference resulted in differential activation of the NFκB pathway. High-, but not low-binding S. pneumoniae used Choline-binding protein A (CbpA) to bind to complement component C3 on epithelial cell surfaces. Interleukin-8 (IL-8) was the only cytokine secreted by cells treated with either low- or high-binding S. pneumoniae. CONCLUSION: These results indicate that S. pneumoniae clinical isolates are not homogeneous in their interaction with host epithelial cells. The differential activation of host cells by high- and low-binding S. pneumoniae strains could have implications for the treatment of pneumococcal pneumonia and for vaccine development

Monocytes regulate the mechanism of T-cell death by inducing Fas-mediated apoptosis during bacterial infection.

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed 'classic' features of apoptosis following exposure to pneumococci. Conversely, purified CD3(+) T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3(+) T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3(+) T-cells in PBMC cultures required 'classical' CD14(+) monocytes, which enhanced T-cell activation. CD3(+) T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3(+) T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease

Evaluating Detection and Diagnostic Decision Support Systems for Bioterrorism Response

We evaluated the usefulness of detection systems and diagnostic decision support systems for bioterrorism response. We performed a systematic review by searching relevant databases (e.g., MEDLINE) and Web sites for reports of detection systems and diagnostic decision support systems that could be used during bioterrorism responses. We reviewed over 24,000 citations and identified 55 detection systems and 23 diagnostic decision support systems. Only 35 systems have been evaluated: 4 reported both sensitivity and specificity, 13 were compared to a reference standard, and 31 were evaluated for their timeliness. Most evaluations of detection systems and some evaluations of diagnostic systems for bioterrorism responses are critically deficient. Because false-positive and false-negative rates are unknown for most systems, decision making on the basis of these systems is seriously compromised. We describe a framework for the design of future evaluations of such systems

Streptococcus pneumoniae in Biofilms Are Unable to Cause Invasive Disease Due to Altered Virulence Determinant Production

It is unclear whether Streptococcus pneumoniae in biofilms are virulent and contribute to development of invasive pneumococcal disease (IPD). Using electron microscopy we confirmed the development of mature pneumococcal biofilms in a continuous-flow-through line model and determined that biofilm formation occurred in discrete stages with mature biofilms composed primarily of dead pneumococci. Challenge of mice with equal colony forming units of biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Biofilm pneumococci of numerous serotypes were hyper-adhesive and bound to A549 type II pneumocytes and Detroit 562 pharyngeal epithelial cells at levels 2 to 11-fold greater than planktonic counterparts. Using genomic microarrays we examined the pneumococcal transcriptome and determined that during biofilm formation S. pneumoniae down-regulated genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide (CPS) production, and virulence. We confirmed these changes by measuring CPS by ELISA and immunoblotting for the toxin pneumolysin and the bacterial adhesins phosphorylcholine (ChoP), choline-binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP). We conclude that biofilm pneumococci were avirulent due to reduced CPS and pneumolysin production along with increased ChoP, which is known to bind C-reactive protein and is opsonizing. Likewise, biofilm pneumococci were hyper-adhesive due to selection for the transparent phase variant, reduced CPS, and enhanced production of PsrP, CbpA, and ChoP. These studies suggest that biofilms do not directly contribute to development of IPD and may instead confer a quiescent mode of growth during colonization

Pneumococcal Capsular Polysaccharide Structure Predicts Serotype Prevalence

There are 91 known capsular serotypes of Streptococcus pneumoniae. The nasopharyngeal carriage prevalence of particular serotypes is relatively stable worldwide, but the host and bacterial factors that maintain these patterns are poorly understood. Given the possibility of serotype replacement following vaccination against seven clinically important serotypes, it is increasingly important to understand these factors. We hypothesized that the biochemical structure of the capsular polysaccharides could influence the degree of encapsulation of different serotypes, their susceptibility to killing by neutrophils, and ultimately their success during nasopharyngeal carriage. We sought to measure biological differences among capsular serotypes that may account for epidemiological patterns. Using an in vitro assay with both isogenic capsule-switch variants and clinical carriage isolates, we found an association between increased carriage prevalence and resistance to non-opsonic neutrophil-mediated killing, and serotypes that were resistant to neutrophil-mediated killing tended to be more heavily encapsulated, as determined by FITC-dextran exclusion. Next, we identified a link between polysaccharide structure and carriage prevalence. Significantly, non-vaccine serotypes that have become common in vaccinated populations tend to be those with fewer carbons per repeat unit and low energy expended per repeat unit, suggesting a novel biological principle to explain patterns of serotype replacement. More prevalent serotypes are more heavily encapsulated and more resistant to neutrophil-mediated killing, and these phenotypes are associated with the structure of the capsular polysaccharide, suggesting a direct relationship between polysaccharide biochemistry and the success of a serotype during nasopharyngeal carriage and potentially providing a method for predicting serotype replacement

Estimating EQ-5D utilities based on the Short-Form Long Term Conditions Questionnaire (LTCQ-8)

Purpose: The aim of this work was to develop a mapping algorithm for estimating EuroQoL 5 Dimension (EQ-5D) utilities from responses to the Long-Term Conditions Questionnaire (LTCQ), thus increasing LTCQ’s potential as a comprehensive outcome measure for evaluating integrated care initiatives. Methods: We combined data from three studies to give a total sample of 1334 responses. In each of the three datasets, we randomly selected 75% of the sample and combined the selected random samples to generate the estimation dataset, which consisted of 1001 patients. The unselected 25% observations from each dataset were combined to generate an internal validation dataset of 333 patients. We used direct mapping models by regressing responses to the LTCQ-8 directly onto EQ-5D-5L and EQ-5D-3L utilities as well as response (or indirect) mapping to predict the response level that patients selected for each of the five EQ-5D-5L domains. Several models were proposed and compared on mean squared error and mean absolute error. Results: A two-part model with OLS was the best performing based on the mean squared error (0.038) and mean absolute error (0.147) when estimating the EQ-5D-5L utilities. A multinomial response mapping model using LTCQ-8 responses was used to predict EQ-5D-5L responses levels. Conclusions: This study provides a mapping algorithm for estimating EQ-5D utilities from LTCQ responses. The results from this study can help broaden the applicability of the LTCQ by producing utility values for use in economic analyses

Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II

In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes

Identifying Host Genetic Risk Factors in the Context of Public Health Surveillance for Invasive Pneumococcal Disease

Host genetic factors that modify risk of pneumococcal disease may help target future public health interventions to individuals at highest risk of disease. We linked data from population-based surveillance for invasive pneumococcal disease (IPD) with state-based newborn dried bloodspot repositories to identify biological samples from individuals who developed invasive pneumococcal disease. Genomic DNA was extracted from 366 case and 732 anonymous control samples. TagSNPs were selected in 34 candidate genes thought to be associated with host response to invasive pneumococcal disease, and a total of 326 variants were successfully genotyped. Among 543 European Americans (EA) (182 cases and 361 controls), and 166 African Americans (AA) (53 cases and 113 controls), common variants in surfactant protein D (SFTPD) are consistently underrepresented in IPD. SFTPD variants with the strongest association for IPD are intronic rs17886286 (allelic OR 0.45, 95% confidence interval (CI) [0.25, 0.82], with p = 0.007) in EA and 5′ flanking rs12219080 (allelic OR 0.32, 95%CI [0.13, 0.78], with p = 0.009) in AA. Variants in CD46 and IL1R1 are also associated with IPD in both EA and AA, but with effects in different directions; FAS, IL1B, IL4, IL10, IL12B, SFTPA1, SFTPB, and PTAFR variants are associated (p≤0.05) with IPD in EA or AA. We conclude that variants in SFTPD may protect against IPD in EA and AA and genetic variation in other host response pathways may also contribute to risk of IPD. While our associations are not corrected for multiple comparisons and therefore must be replicated in additional cohorts, this pilot study underscores the feasibility of integrating public health surveillance with existing, prospectively collected, newborn dried blood spot repositories to identify host genetic factors associated with infectious diseases