FindFoci: a focus detection algorithm with automated parameter training that closely matches human assignments, reduces human inconsistencies and increases speed of analysis

Accurate and reproducible quantification of the accumulation of proteins into foci in cells is essential for data interpretation and for biological inferences. To improve reproducibility, much emphasis has been placed on the preparation of samples, but less attention has been given to reporting and standardizing the quantification of foci. The current standard to quantitate foci in open-source software is to manually determine a range of parameters based on the outcome of one or a few representative images and then apply the parameter combination to the analysis of a larger dataset. Here, we demonstrate the power and utility of using machine learning to train a new algorithm (FindFoci) to determine optimal parameters. FindFoci closely matches human assignments and allows rapid automated exploration of parameter space. Thus, individuals can train the algorithm to mirror their own assignments and then automate focus counting using the same parameters across a large number of images. Using the training algorithm to match human assignments of foci, we demonstrate that applying an optimal parameter combination from a single image is not broadly applicable to analysis of other images scored by the same experimenter or by other experimenters. Our analysis thus reveals wide variation in human assignment of foci and their quantification. To overcome this, we developed training on multiple images, which reduces the inconsistency of using a single or a few images to set parameters for focus detection. FindFoci is provided as an open-source plugin for ImageJ

Caloric Restriction Alters the Metabolic Response to a Mixed-Meal: Results from a Randomized, Controlled Trial

OBJECTIVES: To determine if caloric restriction (CR) would cause changes in plasma metabolic intermediates in response to a mixed meal, suggestive of changes in the capacity to adapt fuel oxidation to fuel availability or metabolic flexibility, and to determine how any such changes relate to insulin sensitivity (S(I)). METHODS: Forty-six volunteers were randomized to a weight maintenance diet (Control), 25% CR, or 12.5% CR plus 12.5% energy deficit from structured aerobic exercise (CR+EX), or a liquid calorie diet (890 kcal/d until 15% reduction in body weight)for six months. Fasting and postprandial plasma samples were obtained at baseline, three, and six months. A targeted mass spectrometry-based platform was used to measure concentrations of individual free fatty acids (FFA), amino acids (AA), and acylcarnitines (AC). S(I) was measured with an intravenous glucose tolerance test. RESULTS: Over three and six months, there were significantly larger differences in fasting-to-postprandial (FPP) concentrations of medium and long chain AC (byproducts of FA oxidation) in the CR relative to Control and a tendency for the same in CR+EX (CR-3 month P = 0.02; CR-6 month P = 0.002; CR+EX-3 month P = 0.09; CR+EX-6 month P = 0.08). After three months of CR, there was a trend towards a larger difference in FPP FFA concentrations (P = 0.07; CR-3 month P = 0.08). Time-varying differences in FPP concentrations of AC and AA were independently related to time-varying S(I) (P<0.05 for both). CONCLUSIONS: Based on changes in intermediates of FA oxidation following a food challenge, CR imparted improvements in metabolic flexibility that correlated with improvements in S(I). TRIAL REGISTRATION: ClinicalTrials.gov NCT00099151

Knee complaints vary with age and gender in the adult population. Population-based reference data for the Knee injury and Osteoarthritis Outcome Score (KOOS)

BACKGROUND: Self-reported knee complaints may vary with age and gender. Reference data from the adult population would help to better interpret the outcome of interventions due to knee complaints. The objectives of the present study were to describe the variation of self-reported knee pain, function and quality of life with age and gender in the adult population and to establish population-based reference data for the Knee injury and Osteoarthritis Outcome Score (KOOS). METHODS: Population-based cohort retrieved from the national population register. The knee-specific Knee injury and Osteoarthritis Outcome Score (KOOS) was mailed to 840 subjects aged 18–84 yrs. RESULTS: 68% response rate. Women in the age group 55–74 reported more knee-related complaints in all the KOOS subscales than age-matched men. The differences were significant for the subscales Pain (p = 0.027), Symptoms (p = 0.003) and ADL function (p = 0.046). In men, worse ADL and Sport and Recreation function was seen in the oldest age group 75–84 years compared to the younger age groups (p < 0.030). In women, worse Pain (p < 0.007), ADL (p < 0.030), Sport and Recreation (p < 0.001) and QOL (p < 0.002) were seen already in the age group 55–74 compared to the younger age groups. CONCLUSION: We found pain and other symptoms, physical function, and knee-related quality of life to vary with age and gender implying the use of age- and gender matched reference values for improved understanding of the outcome after interventions due to knee injury and knee OA

Factors promoting health-related quality of life in people with rheumatic diseases: a 12 month longitudinal study

<p>Abstract</p> <p>Background</p> <p>Rheumatic diseases have a significant adverse impact on the individual from physical, mental and social aspects, resulting in a low health-related quality of life (HRQL). There is a lack of longitudinal studies on HRQL in people with rheumatic diseases that focus on factors promoting HRQL instead of risk factors. The aim of this study was to investigate the associations between suggested health promoting factors at baseline and outcome in HRQL at a 12 month follow-up in people with rheumatic diseases.</p> <p>Methods</p> <p>A longitudinal cohort study was conducted in 185 individuals with rheumatic diseases with questionnaires one week and 12 months after rehabilitation in a Swedish rheumatology clinic. HRQL was assessed by SF-36 together with suggested health factors. The associations between SF-36 subscales and the health factors were analysed by multivariable logistic regressions.</p> <p>Results</p> <p>Factors predicting better outcome in HRQL in one or several SF-36 subscales were being younger or middle-aged, feeling painless, having good sleep structure, feeling rested after sleep, performing low effort of exercise more than twice per week, having strong sense of coherence (SOC), emotional support and practical assistance, higher educational level and work capacity. The most important factors were having strong SOC, feeling rested after sleep, having work capacity, being younger or middle-aged, and having good sleep structure.</p> <p>Conclusions</p> <p>This study identified several factors that promoted a good outcome in HRQL to people with rheumatic diseases. These health factors could be important to address in clinical work with rheumatic diseases in order to optimise treatment strategies.</p

Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H⁺ secretion

The cystic fibrosis (CF) airway surface liquid (ASL) provides a nutrient rich environment for bacterial growth including elevated glucose, which together with defective bacterial killing due to aberrant HCO3− transport and acidic ASL, make the CF airways susceptible to colonisation by respiratory pathogens such as Pseudomonas aeruginosa. Approximately half of adults with CF have CF related diabetes (CFRD) and this is associated with increased respiratory decline. CF ASL contains elevated lactate concentrations and hyperglycaemia can also increase ASL lactate. We show that primary human bronchial epithelial (HBE) cells secrete lactate into ASL, which is elevated in hyperglycaemia. This leads to ASL acidification in CFHBE, which could only be mimicked in non-CF HBE following HCO3− removal. Hyperglycaemia-induced changes in ASL lactate and pH were exacerbated by the presence of P. aeruginosa and were attenuated by inhibition of monocarboxylate lactate-H+ co-transporters (MCTs) with AR-C155858. We conclude that hyperglycaemia and P. aeruginosa induce a metabolic shift which increases lactate generation and efflux into ASL via epithelial MCT2 transporters. Normal airways compensate for MCT-driven H+ secretion by secreting HCO3−, a process which is dysfunctional in CF airway epithelium leading to ASL acidification and that these processes may contribute to worsening respiratory disease in CFRD

Local and systemic effect of transfection-reagent formulated DNA vectors on equine melanoma

Background Equine melanoma has a high incidence in grey horses. Xenogenic DNA vaccination may represent a promising therapeutic approach against equine melanoma as it successfully induced an immunological response in other species suffering from melanoma and in healthy horses. In a clinical study, twenty- seven, grey, melanoma-bearing, horses were assigned to three groups (n = 9) and vaccinated on days 1, 22, and 78 with DNA vectors encoding for equine (eq) IL-12 and IL-18 alone or in combination with either human glycoprotein (hgp) 100 or human tyrosinase (htyr). Horses were vaccinated intramuscularly, and one selected melanoma was locally treated by intradermal peritumoral injection. Prior to each injection and on day 120, the sizes of up to nine melanoma lesions per horse were measured by caliper and ultrasound. Specific serum antibodies against hgp100 and htyr were measured using cell based flow- cytometric assays. An Analysis of Variance (ANOVA) for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. For post-hoc testing a Tukey-Kramer Multiple-Comparison Test was performed to compare the relative volumes on the different examination days. An ANOVA for repeated measurements was performed to analyse changes in body temperature over time. A one-way ANOVA was used to evaluate differences in body temperature between the groups. A p–value < 0.05 was considered significant for all statistical tests applied. Results In all groups, the relative tumor volume decreased significantly to 79.1 ± 26.91% by day 120 (p < 0.0001, Tukey-Kramer Multiple-Comparison Test). Affiliation to treatment group, local treatment and examination modality had no significant influence on the results (ANOVA for repeated measurements). Neither a cellular nor a humoral immune response directed against htyr or hgp100 was detected. Horses had an increased body temperature on the day after vaccination. Conclusions This is the first clinical report on a systemic effect against equine melanoma following treatment with DNA vectors encoding eqIL12 and eqIL18 and formulated with a transfection reagent. Addition of DNA vectors encoding hgp100 respectively htyr did not potentiate this effect

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