Variability of RNA quality extracted from biofilms of foodborne pathogens using different kits impacts mRNA quantification by qPCR

The biofilm formation by foodborne pathogens is known to increase the problem related with surface disinfection procedure in the food processing environment and consequent transmission of these pathogens into the population. Messenger RNA has been increasingly used to understand the action and the consequences of disinfectants in the virulence on such biofilms. RNA quality is an important requirement for any RNA-based analysis since the quality can impair the mRNA quantification. Therefore, we evaluated five different RNA extraction kits using biofilms of the foodborne pathogens Listeria monocytogenes, Escherichia coli, and Salmonella enterica. The five kits yielded RNA with different quantities and qualities. While for E. coli the variability of RNA quality did not affect the quantification of mRNA, the same was not true for L. monocytogenes or S. enterica. Therefore, our results indicate that not all kits are suitable for RNA extraction from bacterial biofilms, and thus, the selection of RNA extraction kit is crucial to obtain accurate and meaningful mRNA quantification.AF and JCB acknowledge the financial support of individual grants SFRH/BD/62359/2009 and SFRH/BD/66250/2009, respectively. The authors acknowledge the gift of bacterial strains to Joana Azeredo and Maria Olivia Pereira.

Stage at presentation of breast cancer in Luanda, Angola - a retrospective study

Background: It is expected that, by 2020, 15 million new cases of cancer will occur every year in the world, one million of them in Africa. Knowledge of cancer trends in African countries is far from adequate, and improvements in cancer prevention efforts are urgently needed. The aim of this study was to characterize breast cancer clinically and pathologically at presentation in Luanda, Angola; we additionally provide quality information that will be useful for breast cancer care planning in the country. Methods: Data on breast cancer cases were retrieved from the Angolan Institute of Cancer Control, from 2006 to 2014. For women diagnosed in 2009 (5-years of follow-up), demographic, clinical and pathological information, at presentation, was collected, namely age at diagnosis, parity, methods used for pathological diagnoses, tumor pathological characteristics, stage of disease and treatment. Descriptive statistics were performed. Results: The median age of women diagnosed with breast cancer in 2009 was 47 years old (range 25–89). The most frequent clinical presentation was breast swelling with axillary lymph nodes metastasis (44.9 %), followed by a mass larger than 5 cm (14.2 %) and lump (12.9 %). Invasive ductal carcinoma was the main histologic type (81.8 %). Only 10.1 % of cancer cases had a well differentiated histological grade. Cancers were diagnosed mostly at advanced stages (66.7 % in stage III and 11.1 % in stage IV). Discussion: In this study, breast cancer was diagnosed at a very advanced stage. Although it reports data from a single cancer center in Luanda, Angola it reinforces the need for early diagnosis and increasing awareness. According to the main challenges related to breast cancer diagnosis and treatment herein presented, we propose a realistic framework that would allow for the implementation of a breast cancer care program, built under a strong network based on cooperation, teaching, audit, good practices and the organization of health services. Conclusion: Angola needs urgently a program for early diagnosis of breast cancer.We thank Susana Santos for correction of the article in English language, and a Cancer Registry Staff from IACC, particularly Pedro Luis Hernandez Gonzalez, Paulo Ernesto Alves, Xacu Parica and Alberto Sivi Lutumba for their support in data acquisition. We also thank SEMED -Portugal in support for publication

The role of cognitive dysfunction in the symptoms and remission from depression.

The disability and burden associated with major depression comes only in part from its affective symptoms; cognitive dysfunctions associated with depression also play a crucial role. Furthermore, these cognitive impairments during depression are manifold and multilevel affecting elementary and more complex cognitive processes equally. Several models from different directions tried to evaluate, conceptualize and understand the depth and magnitude of cognitive dysfunctions in depression and their bidirectional interactions with other types of depressive symptomatology including mood symptoms. In the current review, we briefly overview different types of cognitive symptoms and deficits related to major depression including hot and cold as well as trait- and state-like cognitive alterations and we also describe current knowledge related to the impact of cognitive impairments on the course and outcomes of depression including remission, residual symptoms, function, and response to treatment. We also emphasize shortcomings of currently available treatments for depression in sufficiently improving cognitive dysfunctions and point out the need for newer pharmacological approaches especially in cooperation with psychotherapeutic interventions

Optimizing a qPCR Gene Expression Quantification Assay for S. epidermidis Biofilms: A Comparison between Commercial Kits and a Customized Protocol

Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psmβ1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used

Fit between humanitarian professionals and project requirements: hybrid group decision procedure to reduce uncertainty in decision-making

Choosing the right professional that has to meet indeterminate requirements is a critical aspect in humanitarian development and implementation projects. This paper proposes a hybrid evaluation methodology for some non-governmental organizations enabling them to select the most competent expert who can properly and adequately develop and implement humanitarian projects. This methodology accommodates various stakeholders’ perspectives in satisfying the unique requirements of humanitarian projects that are capable of handling a range of uncertain issues from both stakeholders and project requirements. The criteria weights are calculated using a two-step multi-criteria decision-making method: (1) Fuzzy Analytical Hierarchy Process for the evaluation of the decision maker weights coupled with (2) Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) to rank the alternatives which provide the ability to take into account both quantitative and qualitative evaluations. Sensitivity analysis have been developed and discussed by means of a real case of expert selection problem for a non-profit organisation. The results show that the approach allows a decrease in the uncertainty associated with decision-making, which proves that the approach provides robust solutions in terms of sensitivity analysis

The use of global rating scales for OSCEs in veterinary medicine

OSCEs (Objective Structured Clinical Examinations) are widely used in health professions to assess clinical skills competence. Raters use standardized binary checklists (CL) or multi-dimensional global rating scales (GRS) to score candidates performing specific tasks. This study assessed the reliability of CL and GRS scores in the assessment of veterinary students, and is the first study to demonstrate the reliability of GRS within veterinary medical education. Twelve raters from two different schools (6 from University of Calgary [UCVM] and 6 from Royal (Dick) School of Veterinary Studies [R(D)SVS] were asked to score 12 students (6 from each school). All raters assessed all students (video recordings) during 4 OSCE stations (bovine haltering, gowning and gloving, equine bandaging and skin suturing). Raters scored students using a CL, followed by the GRS. Novice raters (6 R(D)SVS) were assessed independently of expert raters (6 UCVM). Generalizability theory (G theory), analysis of variance (ANOVA) and t-tests were used to determine the reliability of rater scores, assess any between school differences (by student, by rater), and determine if there were differences between CL and GRS scores. There was no significant difference in rater performance with use of the CL or the GRS. Scores from the CL were significantly higher than scores from the GRS. The reliability of checklist scores were .42 and .76 for novice and expert raters respectively. The reliability of the global rating scale scores were .7 and .86 for novice and expert raters respectively. A decision study (D-study) showed that once trained using CL, GRS could be utilized to reliably score clinical skills in veterinary medicine with both novice and experienced raters