Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Dynamic defense workshop : from research to practice.

On September 5th and 6th, 2012, the Dynamic Defense Workshop: From Research to Practice brought together researchers from academia, industry, and Sandia with the goals of increasing collaboration between Sandia National Laboratories and external organizations, de ning and un- derstanding dynamic, or moving target, defense concepts and directions, and gaining a greater understanding of the state of the art for dynamic defense. Through the workshop, we broadened and re ned our de nition and understanding, identi ed new approaches to inherent challenges, and de ned principles of dynamic defense. Half of the workshop was devoted to presentations of current state-of-the-art work. Presentation topics included areas such as the failure of current defenses, threats, techniques, goals of dynamic defense, theory, foundations of dynamic defense, future directions and open research questions related to dynamic defense. The remainder of the workshop was discussion, which was broken down into sessions on de ning challenges, applications to host or mobile environments, applications to enterprise network environments, exploring research and operational taxonomies, and determining how to apply scienti c rigor to and investigating the eld of dynamic defense

Identifying continence options after stroke (ICONS): a cluster randomised controlled feasibility trial.

Background: Urinary incontinence (UI) affects half of patients hospitalised after stroke and is often poorly managed. Cochrane systematic reviews have shown some positive impact of conservative interventions (such as bladder training) in reducing UI, but their effectiveness has not been demonstrated with stroke patients. Methods: We conducted a cluster randomised controlled feasibility trial of a systematic voiding programme (SVP) for the management of UI after stroke. Stroke services were randomised to receive SVP (n = 4), SVP plus supported implementation (SVP+, n = 4), or usual care (UC, n = 4). Feasibility outcomes were participant recruitment and retention. The main effectiveness outcome was presence or absence of UI at six and 12 weeks post-stroke. Additional effectiveness outcomes included were the effect of the intervention on different types of UI, continence status at discharge, UI severity, functional ability, quality of life, and death. Results: It was possible to recruit patients (413; 164 SVP, 125 SVP+, and 124 UC) and participant retention was acceptable (85% and 88% at six and 12 weeks, respectively). There was no suggestion of a beneficial effect on the main outcome at six (SVP versus UC: odds ratio (OR) 0.94, 95% CI: 0.46 to 1.94; SVP+ versus UC: OR: 0.62, 95% CI: 0.28 to 1.37) or 12 weeks (SVP versus UC: OR: 1.02, 95% CI: 0.54 to 1.93; SVP+ versus UC: OR: 1.06, 95% CI: 0.54 to 2.09). No secondary outcomes showed a strong suggestion of clinically meaningful improvement in SVP and/or SVP+ arms relative to UC at six or 12 weeks. However, at 12 weeks both intervention arms had higher estimated odds of continence than UC for patients with urge incontinence. Conclusions: The trial has met feasibility outcomes of participant recruitment and retention. It was not powered to demonstrate effectiveness, but there is some evidence of a potential reduction in the odds of specific types of incontinence. A full trial should now be considered

The timing of Rapid-Response Team activations: a multicentre international study

Background:Most studies of Rapid-Response Teams (RRTs) assess their effect on outcomes of all hospitalised patients. Little information exists on RRT activation patterns or why RRT calls are needed. Triage error may necessitate RRT review of ward patients shortly after hospital admission. RRT diurnal activation rates may reflect the likely frequency of caregiver visits.Objectives:To study the timing of RRT calls in relation to time of day and day of week, and their frequency and outcomes in relation to days after hospital admission.Methods:We prospectively studied RRT calls over 1 month in seven hospitals during 2009, collecting data on patient age, sex, admitting unit, admission source, limitations of medical therapy (LOMTs), and admission and discharge dates. We assessed the timing of RRT calls in relation to hospital admission and circadian variation; and differences in characteristics and outcomes of calls occurring early (Days 0 and 1) versus late (after Day 7) after hospital admission.Results:There were 652 RRT calls for 518 patients. Calls were more likely on Mondays (P=0.018) and during work hours (P&lt;0.0001) but less likely on weekends (P=0.003) or overnight (P&lt;0.001). There were 177 early calls (27.1%) and 198 late calls (30.4%). Early calls involved younger patients (median ages, 67.5 years [early calls] v 73 years [late calls]; P= 0.01), fewer LOMTs (P=0.029), and lower in hospital mortality (12.8% [early calls] v 32.3% [late calls]; P&lt;0.0001). The mortality difference remained in patients without LOMTs (5.6% [early calls] v 19.6% [late calls]; P=0.003).Conclusions:About one-quarter of RRT calls occurred shortly after hospital admission, and were more common when caregivers were around. Early calls may partially reflect suboptimal triage, though the associated mortality appeared low. Late calls may reflect suboptimal end-of-life care planning, and the associated mortality was high. There is a need to further assess the epidemiology of RRT calls at different phases of the hospital stay

Evaluating a systematic voiding programme for patients with urinary incontinence after stroke in secondary care using soft systems analysis and Normalisation Process Theory: findings from the ICONS case study phase.International journal of nursing studies

Background: Urinary incontinence (UI) affects between 40 and 60% of people in hospital after stroke, but is often poorly managed in stroke units. Objectives: To inform an exploratory trial by three methods: identifying the organisational context for embedding the SVP; exploring health professionals' views around embedding the SVP and measuring presence/absence of UI and frequency of UI episodes at baseline and six weeks post-stroke. Design: A mixed methods single case study included analysis of organisational context using interviews with clinical leaders analysed with soft systems methodology, a process evaluation using interviews with staff delivering the intervention and analysed with Normalisation Process Theory, and outcome evaluation using data from patients receiving the SVP and analysed using descriptive statistics. Setting: An 18 bed acute stroke unit in a large Foundation Trust (a 'not for profit' privately controlled entity not accountable to the UK Department of Health) serving a population of 370,000. Participants: Health professionals and clinical leaders with a role in either delivering the SVP or linking with it in any capacity were recruited following informed consent. Patients were recruited meeting the following inclusion criteria: aged 18 or over with a diagnosis of stroke; urinary incontinence (UI) as defined by the International Continence Society; conscious; medically stable as judged by the clinical team and with incontinence classified as stress, urge, mixed or 'functional'. All patients admitted to the unit during the intervention period were screened for eligibility; informed consent to collect baseline and outcome data was sought from all eligible patients. Results: Organisational context: 18 health professionals took part in four group interviews. Findings suggest an environment not conducive to therapeutic continence management and a focus on containment of UI. Embedding the SVP into practice: 21 nursing staff took part in six group interviews. Initial confusion gave way to embedding of processes facilitated by new routines and procedures. Patient outcome: 43 patients were recruited; 28 of these commenced the SVP. Of these, 6/28 (21%) were continent at six weeks post-stroke or discharge. Conclusion: It was possible to embed the SVP into practice despite an organisational context not conducive to therapeutic continence care. Recommendations are made for introducing the SVP in a trial context

When Should the Customer Really Be King? On the Optimum Level of Salesperson Customer Orientation in Sales Encounters

In today’s age of relational selling, it is a key challenge for salespeople to determine to what degree customer-oriented behaviors also drive sales performance. There-fore, this study analyzes whether a salesperson’s customer orientation in sales encounters has an optimum level with regard to sales performance and customer attitudes. Using triadic data from a cross-industry survey of 56 sales managers, 195 sales representatives, and 538 customers, the authors provide strong empirical support for a curvilinear, inverted U-shaped effect of a salesperson’s customer orientation on sales performance, while the effect of customer orientation on customer attitudes is continuously positive. Moreover, the findings reveal that the optimum level of customer orientation with regard to sales performance is higher for salespeople selling individualized products, in firms pursuing a premium price strategy and in markets with a high degree of competitive intensity