Fluoromycobacteriophages for rapid, specific, and sensitive antibiotic susceptibility testing of Mycobacterium tuberculosis

Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively- drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections. © 2009 Piuri et al

Sexual function in Britain: Findings from the third National Survey of Sexual Attitudes and Lifestyles (Natsal-3)

Background Despite its importance to sexual health and wellbeing, sexual function is given little attention in sexual health policy. Population-based studies are needed to understand sexual function across the life course. Methods We undertook a probability sample survey (the third National Survey of Sexual Attitudes and Lifestyles [Natsal-3]) of 15 162 individuals aged 16-74 years who lived in Britain (England, Scotland, and Wales). Interviews were done between Sept 6, 2010, and Aug 31, 2012. We assessed the distribution of sexual function by use of a novel validated measure (the Natsal-SF), which assessed problems with individual sexual response, sexual function in a relationship context, and self-appraisal of sex life (17 items; 16 items per gender). We assess factors associated with low sexual function (defi ned as the lowest quintile of distribution of Natsal-SF scores) and the distribution of components of the measure. Participants reporting one or more sexual partner in the past year were given a score on the Natsal-SF (11 690 participants). 4122 of these participants were not in a relationship for all of the past year and we employed the full information maximum likelihood method to handle missing data on four relationship items. Findings We obtained data for 4913 men and 6777 women for the Natsal-SF. For men and women, low sexual function was associated with increased age, and, after age-adjustment, with depression (adjusted odds ratio 3.70 [95% CI 2.90-4.72] for men and 4.11 [3.36-5.04] for women) and self-reported poor health status (2.63 [1.73-3.98] and 2.41 [1.72-3.39]). Low sexual function was also associated with experiencing the end of a relationship (1.52 [1.18-1.95] and 1.77 [1.44-2.17]), inability to talk easily about sex with a partner (2.36 [1.94-2.88] and 2.82 [2.28-3.48]), and not being happy in the relationship (2.89 [2.32-3.61] and 4.10 [3.39-4.97]). Associations were also noted with engaging in fewer than four sex acts in the past 4 weeks (3.13 [2.58-3.79] and 3.38 [2.80-4.09]), having had same sex partners (2.28 [1.56-3.35] and 1.60 [1.16-2.20]), paying for sex (in men only; 2.62 [1.46-4.71]), and higher numbers of lifetime sexual partners (in women only; 2.12 [1.68-2.67] for ten or more partners). Low sexual function was also associated with negative sexual health outcomes such as experience of non-volitional sex (1.98 [1.14-3.43] and 2.18 [1.79-2.66]) and STI diagnosis (1.50 [1.06-2.11] and 1.83 [1.35-2.47]). Among individuals reporting sex in the past year, problems with sexual response were common (41.6% of men and 51.2% of women reported one or more problem) but selfreported distress about sex lives was much less common (9.9% and 10.9%). For individuals in a sexual relationship for the past year, 23.4% of men and 27.4% of women reported an imbalance in level of interest in sex between partners, and 18.0% of men and 17.1% of women said that their partner had had sexual diffi culties. Most participants who did not have sex in the past year were not dissatisfi ed, distressed, or avoiding sex because of sexual diffi culties. Interpretation Wide variability exists in the distribution of sexual function scores. Low sexual function is associated with negative sexual health outcomes, supporting calls for a greater emphasis on sexual function in sexual health policy and interventions. Funding Grants from the UK Medical Research Council and the Wellcome Trust, with support from the Economic and Social Research Council and the Department of Health

Spirituality and end-of-life care in disadvantaged men dying of prostate cancer

Despite the positive influence of spiritual coping on the acceptance of a cancer diagnosis, higher spirituality is associated with receipt of more high intensity care at the end of life. The purpose of our study was to assess the association between spirituality and type of end-of-life care received by disadvantaged men with prostate cancer. We studied low-income, uninsured men in IMPACT, a state-funded public assistance program, who had died since its inception in 2001. Of the 60 men who died, we included the 35 who completed a spirituality questionnaire at program enrollment. We abstracted sociodemographic and clinical information as well as treatment within IMPACT, including zolendroic acid, chemotherapy, hospice use, and palliative radiation therapy. We measured spirituality with the Functional Assessment of Chronic Illness Therapy—Spiritual Well-Being questionnaire (FACIT-Sp) and compared end-of-life care received between subjects with low and high FACIT-Sp scores using chi-squared analyses. A higher proportion of men with high (33%) versus low (13%) spirituality scores enrolled in hospice, although our analysis was not adequately powered to demonstrate statistical significance. Likewise, we saw a trend toward increased receipt of palliative radiation among those with higher spirituality (37% vs. 25%, P = 0.69). The differences in end-of-life care received among those with low and high spirituality varied little by the FACIT-Sp peace and faith subscales. End-of-life care was similar between men with lower and higher spirituality. Men with higher spirituality trended toward greater hospice use, suggesting that they redirected the focus of their care from curative to palliative goals

Serum Response Factor Regulates Immediate Early Host Gene Expression in Toxoplasma gondii-Infected Host Cells

Toxoplasma gondii is a wide spread pathogen that can cause severe and even fatal disease in fetuses and immune-compromised hosts. As an obligate intracellular parasite, Toxoplasma must alter the environment of its host cell in order to establish its replicative niche. This is accomplished, in part, by secretion of factors into the host cell that act to modulate processes such as transcription. Previous studies demonstrated that genes encoding transcription factors such as c-jun, junB, EGR1, and EGR2 were amongst the host genes that were the most rapidly upregulated following infection. In cells stimulated with growth factors, these genes are regulated by a transcription factor named Serum Response Factor. Serum Response Factor is a ubiquitously expressed DNA binding protein that regulates growth and actin cytoskeleton genes via MAP kinase or actin cytoskeletal signaling, respectively. Here, we report that Toxoplasma infection leads to the rapid activation of Serum Response Factor. Serum Response Factor activation is a Toxoplasma-specific event since the transcription factor is not activated by the closely related protozoan parasite, Neospora caninum. We further demonstrate that Serum Response Factor activation requires a parasite-derived secreted factor that signals via host MAP kinases but independently of the host actin cytoskeleton. Together, these data define Serum Response Factor as a host cell transcription factor that regulates immediate early gene expression in Toxoplasma-infected cells

Brain Circuitries Involved in Semantic Interference by Demands of Emotional and Non-Emotional Distractors

BACKGROUND: Previous studies have indicated that the processes leading to the resolution of emotional and non-emotional interference conflicts are unrelated, involving separate networks. It is also known that conflict resolution itself suggests a considerable overlap of the networks. Our study is an attempt to examine how these findings may be related. METHODOLOGY/PRINCIPAL FINDINGS: We used functional magnetic resonance imaging (fMRI) to study neural responses of 24 healthy subjects to emotional and non-emotional conflict paradigms involving the presentation of congruent and incongruent word-face pairs based on semantic incompatibility between targets and distractors. In the emotional task, the behavioral interference conflict was greater (compared to the non-emotional task) and was paralleled by involvement of the extrastriate visual and posterodorsal medial frontal cortices. In both tasks, we also observed a common network including the dorsal anterior cingulate, the supplemental motor area, the anterior insula and the inferior prefrontal cortex, indicating that these brain structures are markers of experienced conflict. However, the emotional task involved conflict-triggered networks to a considerably higher degree. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that responses to emotional and non-emotional distractors involve the same systems, which are capable of flexible adjustments based on conflict demands. The function of systems related to conflict resolution is likely to be adjusted on the basis of an evaluation process that primarily involves the extrastriate visual cortex, with target playing a significant role

Capturing judgement strategies in risk assessments with improved quality of clinical information: How nurses' strategies differ from the ecological model

Background: Nurses' risk assessments of patients at risk of deterioration are sometimes suboptimal. Advances in clinical simulation mean higher quality information can be used as an alternative to traditional paper-based approaches as a means of improving judgement. This paper tests the hypothesis that nurses' judgement strategies and policies change as the quality of information used by nurses in simulation changes. Methods: Sixty-three student nurses and 34 experienced viewed 25 paper-case based and 25 clinically simulated scenarios, derived from real cases, and judged whether the (simulated) patient was at 'risk' of acute deterioration. Criteria of judgement "correctness" came from the same real cases. Information relative weights were calculated to examine judgement policies of individual nurses. Group comparisons of nurses and students under both paper and clinical simulation conditions were undertaken using non parametric statistical tests. Judgment policies were also compared to the ecological statistical model. Cumulative relative weights were calculated to assess how much information nurses used when making judgements. Receiver operating characteristic (ROC) curves were generated to examine predictive accuracy amongst the nurses. Results: There were significant variations between nurses' judgement policies and those optimal policies determined by the ecological model. Nurses significantly underused the cues of consciousness level, respiration rate, and systolic blood pressure than the ecological model requires. However, in clinical simulations, they tended to make appropriate use of heart rate, with non-significant difference in the relative weights of heart rate between clinical simulations and the ecological model. Experienced nurses paid substantially more attention to respiration rate in the simulated setting compared to paper cases, while students maintained a similar attentive level to this cue. This led to a non-significant difference in relative weights of respiration rate between experienced nurses and students. Conclusions: Improving the quality of information by clinical simulations significantly impacted on nurses' judgement policies of risk assessments. Nurses' judgement strategies also varied with the increased years of experience. Such variations in processing clinical information may contribute to nurses' suboptimal judgements in clinical practice. Constructing predictive models of common judgement situations, and increasing nurses' awareness of information weightings in such models may help improve judgements made by nurses

Symbiotic Legume Nodules Employ Both Rhizobial Exo- and Endo-Hydrogenases to Recycle Hydrogen Produced by Nitrogen Fixation

BACKGROUND: In symbiotic legume nodules, endosymbiotic rhizobia (bacteroids) fix atmospheric N(2), an ATP-dependent catalytic process yielding stoichiometric ammonium and hydrogen gas (H(2)). While in most legume nodules this H(2) is quantitatively evolved, which loss drains metabolic energy, certain bacteroid strains employ uptake hydrogenase activity and thus evolve little or no H(2). Rather, endogenous H(2) is efficiently respired at the expense of O(2), driving oxidative phosphorylation, recouping ATP used for H(2) production, and increasing the efficiency of symbiotic nodule N(2) fixation. In many ensuing investigations since its discovery as a physiological process, bacteroid uptake hydrogenase activity has been presumed a single entity. METHODOLOGY/PRINCIPAL FINDINGS: Azorhizobium caulinodans, the nodule endosymbiont of Sesbania rostrata stems and roots, possesses both orthodox respiratory (exo-)hydrogenase and novel (endo-)hydrogenase activities. These two respiratory hydrogenases are structurally quite distinct and encoded by disparate, unlinked gene-sets. As shown here, in S. rostrata symbiotic nodules, haploid A. caulinodans bacteroids carrying single knockout alleles in either exo- or-endo-hydrogenase structural genes, like the wild-type parent, evolve no detectable H(2) and thus are fully competent for endogenous H(2) recycling. Whereas, nodules formed with A. caulinodans exo-, endo-hydrogenase double-mutants evolve endogenous H(2) quantitatively and thus suffer complete loss of H(2) recycling capability. More generally, from bioinformatic analyses, diazotrophic microaerophiles, including rhizobia, which respire H(2) may carry both exo- and endo-hydrogenase gene-sets. CONCLUSIONS/SIGNIFICANCE: In symbiotic S. rostrata nodules, A. caulinodans bacteroids can use either respiratory hydrogenase to recycle endogenous H(2) produced by N(2) fixation. Thus, H(2) recycling by symbiotic legume nodules may involve multiple respiratory hydrogenases

HUNK phosphorylates EGFR to regulate breast cancer metastasis

Epidermal growth factor receptor (EGFR) is commonly over-expressed in metastatic breast cancer yet metastatic breast cancer is generally resistant to anti-EGFR therapies, and the mechanism for resistance to EGFR inhibitors in this setting is not fully understood. Hormonally up-regulated neu-associated kinase (HUNK) kinase is up-regulated in aggressive breast cancers and is thought to play a role in breast cancer metastasis. However, no studies have been conducted to examine a relationship between EGFR and HUNK in breast cancer metastasis. We performed a kinase substrate screen and identified that EGFR is phosphorylated by HUNK. Our studies show that HUNK phosphorylates EGFR at T654, enhancing receptor stability and downstream signaling. We found that increased phosphorylation of T654 EGFR correlates with increased epithelial to mesenchymal, migration and invasion, and metastasis. In addition, we found that HUNK expression correlates with overall survival and distant metastasis free survival. This study shows that HUNK directly phosphorylates EGFR at T654 to promote metastasis and is the first study to show that the phosphorylation of this site in EGFR regulates metastasis

Inter-individual variations of human mercury exposure biomarkers: a cross-sectional assessment

BACKGROUND: Biomarkers for mercury (Hg) exposure have frequently been used to assess exposure and risk in various groups of the general population. We have evaluated the most frequently used biomarkers and the physiology on which they are based, to explore the inter-individual variations and their suitability for exposure assessment. METHODS: Concentrations of total Hg (THg), inorganic Hg (IHg) and organic Hg (OHg, assumed to be methylmercury; MeHg) were determined in whole blood, red blood cells, plasma, hair and urine from Swedish men and women. An automated multiple injection cold vapour atomic fluorescence spectrophotometry analytical system for Hg analysis was developed, which provided high sensitivity, accuracy, and precision. The distribution of the various mercury forms in the different biological media was explored. RESULTS: About 90% of the mercury found in the red blood cells was in the form of MeHg with small inter-individual variations, and part of the IHg found in the red blood cells could be attributed to demethylated MeHg. THg in plasma was associated with both IHg and MeHg, with large inter-individual variations in the distribution between red blood cells and plasma. THg in hair reflects MeHg exposure at all exposure levels, and not IHg exposure. The small fraction of IHg in hair is most probably emanating from demethylated MeHg. The inter-individual variation in the blood to hair ratio was very large. The variability seemed to decrease with increasing OHg in blood, most probably due to more frequent fish consumption and thereby blood concentrations approaching steady state. THg in urine reflected IHg exposure, also at very low IHg exposure levels. CONCLUSION: The use of THg concentration in whole blood as a proxy for MeHg exposure will give rise to an overestimation of the MeHg exposure depending on the degree of IHg exposure, why speciation of mercury forms is needed. THg in RBC and hair are suitable proxies for MeHg exposure. Using THg concentration in plasma as a measure of IHg exposure can lead to significant exposure misclassification. THg in urine is a suitable proxy for IHg exposure

Blood Flow and Glucose Metabolism in Stage IV Breast Cancer: Heterogeneity of Response During Chemotherapy

Objective: The purpose of the study was to compare early changes in blood flow (BF) and glucose metabolism (MRglu) in metastatic breast cancer lesions of patients treated with chemotherapy. Methods: Eleven women with stage IV cancer and lesions in breast, lymph nodes, liver, and bone were scanned before treatment and after the first course of chemotherapy. BF, distribution volume of water (Vd), MRglu/BF ratio, MRgluand its corresponding rate constants K1and k3were compared per tumor lesion before and during therapy. Results: At baseline, mean BF and MRgluvaried among different tumor lesions, but mean Vdwas comparable in all lesions. After one course of chemotherapy, mean MRgludecreased in all lesions. Mean BF decreased in breast and node lesions and increased in bone lesions. Vddecreased in breast and nodes, but did not change in bone lesions. The MRglu/BF ratio decreased in breast and bone lesions and increased in node lesions. In patients with multiple tumor lesions BF and MRgluresponse could be very heterogeneous, even within similar types of metastases. BF and MRgluincreased in lesions of patients who experienced early disease progression or showed no response during clinical follow-up. Conclusion: BF and MRgluchanges separately give unique information on different aspects of tumor response to chemotherapy. Changes in BF and MRgluparameters can be remarkably heterogeneous in patients with multiple lesions