Genome-wide association study identifies genetic variants underlying footrot in Portuguese Merino sheep.

BACKGROUND: Ovine footrot caused by Dichelobacter nodosus (D. nodosus) is a contagious disease with serious economic and welfare impacts in sheep production systems worldwide. A better understanding of the host genetic architecture regarding footrot resistance/susceptibility is crucial to develop disease control strategies that efficiently reduce infection and its severity. A genome-wide association study was performed using a customized SNP array (47,779 SNPs in total) to identify genetic variants associated to footrot resistance/susceptibility in two Portuguese native breeds, i.e. Merino Branco and Merino Preto, and a population of crossbred animals. A cohort of 1375 sheep sampled across 17 flocks, located in the Alentejo region (southern Portugal), was included in the analyses. RESULTS: Phenotypes were scored from 0 (healthy) to 5 (severe footrot) based on visual inspection of feet lesions, following the Modified Egerton System. Using a linear mixed model approach, three SNPs located on chromosome 24 reached genome-wide significance after a Bonferroni correction (p < 0.05). Additionally, six genome-wide suggestive SNPs were identified each on chromosomes 2, 4, 7, 8, 9 and 15. The annotation and KEGG pathway analyses showed that these SNPs are located within regions of candidate genes such as the nonsense mediated mRNA decay associated PI3K related kinase (SMG1) (chromosome 24) and the RALY RNA binding protein like (RALYL) (chromosome 9), both involved in immunity, and the heparan sulfate proteoglycan 2 (HSPG2) (chromosome 2) and the Thrombospodin 1 (THBS1) (chromosome 7) implicated in tissue repair and wound healing processes. CONCLUSION: This is the first attempt to identify molecular markers associated with footrot in Portuguese Merino sheep. These findings provide relevant information on a likely genetic association underlying footrot resistance/susceptibility and the potential candidate genes affecting this trait. Genetic selection strategies assisted on the information obtained from this study could enhance Merino sheep-breeding programs, in combination with farm management strategies, for a more effective and sustainable long-term solution for footrot control.

Effect of Carbodiimide (EDC) on the Bond Strength Longevity of Epoxy Resin-based Endodontic Sealer to Root Dentin: An In-Vitro Study.

PURPOSE: EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) can increase dentin bonding longevity. This study aimed to evaluate the effect of final irrigation of the root canal with EDC on the bond strength (BS) longevity of an epoxy resin-based root-canal sealer. MATERIALS AND METHODS: Twenty maxillary canines were sectioned and standardized for root length at 17 mm. Roots were instrumented and distributed into 2 groups according to the final irrigation protocol: EDTA 17%+NaOCl 2.5% (C) and EDTA 17%+NaOCl 2.5%+EDC 0.5M (EDC). The canals were dried and filled with AH Plus (Dentsply Sirona). Three slices were obtained per third, and the first slice from each third was used for the immediate push-out test (i) followed by analysis of the failure pattern (n = 10); the second slice from each third was used for the push-out test after 6-month aging (A) followed by analysis of the failure pattern (n = 10); the third slice from each third was used to examine the adhesive interface under confocal laser scanning microscopy (CLSM) (n = 10). Data were analyzed with ANOVA, Fisher's exact and Kruskal-Wallis tests. RESULTS: Higher BSs were found for EDC-A (5.6 ± 1.9) than for EDC-I (3.3 ± 0.7), C-i (2.5 ± 1.0) and C-i (2.6 ± 1.0) (p = 0.0001), while C-A values were in some cases similar to C-i and in others similar to EDC-i. No statistically significant difference was observed between the thirds (p > 0.05), except for EDC-i, which showed lower BS for the cervical (2.79 ± 0.46) compared to the apical third (3.8 ± 0.5), while the middle third in some cases had values similar to those of the apical and in others to the cervical third (3.2 ± 0.7) (p = 0.032). More mixed adhesive failures were found in the cervical third, and more adhesive failures to the sealer occurred in the middle and apical thirds (p = 0.014). A significant difference was observed between treatments in terms of adaptation of the adhesive interface, with a higher percentage of good adaptation using EDC (66.7%) than using C (40%), and a lower percentage of poor adaptation with EDC (10%) compared to C (20%) (p < 0.05). CONCLUSION: Root canal irrigation with EDC increased the longevity of the adhesive interface of an epoxy resin-based root-canal sealer.

A metagenomics approach to characterize the footrot microbiome in Merino sheep.

In the Portuguese Alentejo region, Merino sheep breed is the most common breed, reared for the production of meat, dairy, and wool. Footrot is responsible for lameness, decreased animal welfare, and higher production losses, generating a negative economic impact. The disease is caused by Dichelobacter nodosus that interacts with the sheep foot microbiome, to date largely uncharacterized. In fact, Dichelobacter nodosus is not able to induce footrot by itself being required the presence of a second pathogen known as Fusobacterium necrophorum. To understand and characterize the footrot microbiome dynamics of different footrot lesion scores, a whole metagenome sequencing (WMGS) approach was used. Foot tissue samples were collected from 212 animals with different degrees of footrot lesion scores, ranging from 0 to 5. Distinct bacterial communities were associated with feet with different footrot scores identifying a total of 63 phyla and 504 families. As the severity of footrot infection increases the microorganisms' diversity decreases triggering a shift in the composition of the microbiome from a dominant gram-positive in mild stages to a dominant gram-negative in the severe stages. Several species previously associated with footrot and other polymicrobial diseases affecting the epidermis and provoking inflammatory responses such as Treponema spp., Staphylococcus spp., Streptococcus spp. and Campylobacter spp. were identified proliferating along with the lesions' severity. Although these bacteria are not able to initiate footrot, several evidences have been described supporting their association with the severity and incidence increase of footrot lesions caused by Dichelobacter nodosus and Fusobacterium necrophorum. Further investigation is required to establish the roles of particular taxa and identify which of them play a role in the disease process and which are opportunistic pathogens.

Physical Rehabilitation Programs for Bedridden Patients with Prolonged Immobility: A Scoping Review.

Bedridden patients usually stay in bed for long periods, presenting several problems caused by immobility, leading to a long recovery process. Thus, identifying physical rehabilitation programs for bedridden patients with prolonged immobility requires urgent research. Therefore, this scoping review aimed to map existing physical rehabilitation programs for bedridden patients with prolonged immobility, the rehabilitation domains, the devices used, the parameters accessed, and the context in which these programs were performed. This scoping review, guided by the Joanna Briggs Institute's (JBI) methodology and conducted in different databases (including grey literature), identified 475 articles, of which 27 were included in this review. The observed contexts included research institutes, hospitals, rehabilitation units, nursing homes, long-term units, and palliative care units. Most of the programs were directed to the musculoskeletal domain, predominantly toward the lower limbs. The devices used included lower limb mobilization, electrical stimulation, inclined planes, and cycle ergometers. Most of the evaluated parameters were musculoskeletal, cardiorespiratory, or vital signs. The variability of the programs, domains, devices and parameters found in this scoping review revealed no uniformity, a consequence of the personalization and individualization of care, which makes the development of a standard intervention program challenging.

Ovine footrot in Southern Portugal: Detection of Dichelobacter nodosus and Fusobacterium necrophorum in sheep with different lesion scores.

The Mediterranean climate region of Alentejo in the Southern of Portugal is an important sheep production centre but little is known about the presence and characteristics of Dichelobacter nodosus in association with Fusobacterium necrophorum in the different footrot lesion scores. DNA from 261 interdigital biopsy samples, taken from 14 footrot affected flocks and from three non-affected flocks, were analysed for the presence of D. nodosus and F. necrophorum by real-time PCR. Both virulence and serogroup were determined for 132 and 53 D. nodosus positive biopsy samples, respectively. The co-infection with both bacteria was the commonest epidemiological finding associated with a greater disease severity. There was a statistically significant association (p = 0.002) between footrot-affected flocks and the presence of D. nodosus. Most D. nodosus positive samples were virulent (96.2 %) and belonged to serogroup B (90 %).

Metabolic and proliferation evaluation of human adipose-derived mesenchymal stromal cells (ASC) in different culture medium volumes: standardization of static culture.

Adipose-derived mesenchymal stromal/stem cells (ASC) have acquired a prominent role in tissue engineering and regenerative medicine. However, the standardization of basic culture procedures in this cellular type is still not well established according to the main qualitative cellular attributes. We evaluate the cell growth profile of human ASC in a different culture medium volumes and their nutritional composition utilizing static cultivation. Culture medium volumes (5, 10 and 15 mL/25 cm2) in T-flasks were evaluated by kinetic parameters and the metabolic composition was determined by biochemical analysis and Fourier transform infrared (FT-IR) absorption spectroscopy. 50% renewal of culture medium volume every 48 h was adopted. Immunophenotypic characterization and cell differentiation were performed. There was no difference (p > 0.05) in the kinetic parameters of cell proliferation between the culture medium volumes or in FT-IR composition. However, the concentrations of glucose, glutamine, lactate, and glutamate varied significantly during the cultivation process as a function of the medium volume. ASC presented specific antigens and differentiation potential of mesenchymal stromal/stem cells. It was concluded that the minimal culture medium volume (5 mL/25 cm2 in static culture) was sufficient to maintain the stability, potency, and growth of ASC, representing an economic and safe standardization for this cell culture process.

Effectiveness of educational outreach visits compared with usual guideline dissemination to improve family physician prescribing-an 18-month open cluster-randomized trial.

BACKGROUND: Educational outreach visits are meant to improve the practice of health professionals by promoting face-to-face visits to deliver educational contents. They have been shown to change prescription behavior, but long-term effects are still uncertain. This trial aimed to determine if they improve family physician prescribing compared with passive guideline dissemination. METHODS: Parallel, open, superiority, and cluster-randomized trial. National Health Service primary care practices (clusters) were recruited in the Lisbon region-Portugal between March 2013 and January 2014. They could enter if they had at least four family physicians willing to participate and not planning to retire in the follow-up period. Three national guidelines were chosen for dissemination: acid secretion modifiers, non-steroidal anti-inflammatory drugs, and antiplatelets. Physicians in the intervention group received one 15 to 20 min educational outreach visit at their workplace for each guideline. Physicians in the control group had access to guidelines through the Directorate-General for Health's website (passive dissemination). Primary outcomes were the proportion of COX-2 inhibitors prescribed within the NSAID class and the proportion of omeprazole within the PPI class at 18 months after the intervention. A cost-benefit analysis was performed. Practices were randomized by minimization. Data analyses were done at individual physician level using generalized mixed-effects regression models. Participants could not be blinded. RESULTS: Thirty-eight practices with 239 physicians were randomized (120 to intervention and 119 to control). Of 360 planned visits, 322 were delivered. No differences were found between physicians in the intervention and control groups regarding the proportion of omeprazole prescribed among PPIs 18 months after the visit (46.28 vs 47.15%, p = 0.971) or the proportion of COX-2 inhibitors among NSAIDs (12.07 vs 13.08%, p = 0.085). All secondary outcome comparisons showed no effect. There was no difference in cumulative drug costs at 18 months (3223.50€/1000 patients in the intervention group and 3143.92€/1000 patients in the control group, p = 0.848). CONCLUSIONS: Educational outreach visits were unsuccessful in improving compliance with guideline recommendations among Portuguese family physicians. No effects were observed at 1, 6, and 18 months after the intervention, and there were no associated cost savings. TRIAL REGISTRATION: ClinicalTrials.gov NCT01984034 . Registered 7 November 2013.

Effect of European Medicines Agency's restrictions on trimetazidine utilization in Portugal.

PURPOSE: Following safety concerns regarding trimetazidine, the European Medicines Agency (EMA) recommended restrictions on its use. Our objective was to determine the impact of regulatory actions on trimetazidine utilization in Portugal. METHODS: Retrospective interrupted time-series analysis of monthly ambulatory pharmacy reimbursement records for the Portuguese National Health Service between January 2006 and December 2015. Regulatory actions were identified by searching the EMA, Portuguese Medicines Authority, and European Commission's websites. Concurrent factors in the same period were also identified. The main outcome was the dispensing of trimetazidine-containing products per month in Portugal. RESULTS: Two interruption periods were defined in the series: May 2011, when EMA announced it would review trimetazidine safety, and June 2012 to January 2013, when EMA announced it had reached a final opinion recommending restrictions; the European Commission approved EMA's recommendation; the Portuguese Medicines Authority issued safety alerts, changed the summary of product characteristics, and approved a direct health-care professional letter; and a regional bulletin was issued. Interruption 1 had no effect on trimetazidine use, but interruption 2 resulted in decreases in level and trend-from 8.3 million defined daily doses in 2010 to 2.8 million in 2015. After interruption 2, trimetazidine use tended towards a lower steady state. CONCLUSIONS: There was a significant decrease in trimetazidine use in Portugal following a complex intervention that included safety alerts, changes to the summary of product characteristics, a direct health-care professional letter, and a regional drug bulletin. No effect was seen when EMA announced its review of trimetazidine safety.

An open cluster-randomized, 18-month trial to compare the effectiveness of educational outreach visits with usual guideline dissemination to improve family physician prescribing.

BACKGROUND: The Portuguese National Health Directorate has issued clinical practice guidelines on prescription of anti-inflammatory drugs, acid suppressive therapy, and antiplatelets. However, their effectiveness in changing actual practice is unknown. METHODS: The study will compare the effectiveness of educational outreach visits regarding the improvement of compliance with clinical guidelines in primary care against usual dissemination strategies. A cost-benefit analysis will also be conducted. We will carry out a parallel, open, superiority, randomized trial directed to primary care physicians. Physicians will be recruited and allocated at a cluster-level (primary care unit) by minimization. Data will be analyzed at the physician level. Primary care units will be eligible if they use electronic prescribing and have at least four physicians willing to participate. Physicians in intervention units will be offered individual educational outreach visits (one for each guideline) at their workplace during a six-month period. Physicians in the control group will be offered a single unrelated group training session. Primary outcomes will be the proportion of cyclooxygenase-2 inhibitors prescribed in the anti-inflammatory class, and the proportion of omeprazole in the proton pump inhibitors class at 18 months post-intervention. Prescription data will be collected from the regional pharmacy claims database. We estimated a sample size of 110 physicians in each group, corresponding to 19 clusters with a mean size of 6 physicians. Outcome collection and data analysis will be blinded to allocation, but due to the nature of the intervention, physicians and detailers cannot be blinded. DISCUSSION: This trial will attempt to address unresolved issues in the literature, namely, long term persistence of effect, the importance of sequential visits in an outreach program, and cost issues. If successful, this trial may be the cornerstone for deploying large scale educational outreach programs within the Portuguese National Health Service. TRIAL REGISTRATION: ClinicalTrials.gov number NCT01984034.

Cancer stem cells markers CD44, CD24 and ALDH1 in breast cancer special histological types.

AIMS: CD44, CD24 and ALDH1 are the most consistently used biomarkers to identify and characterise the breast cancer stem cell (CSC) phenotype. However, most studies performed until now analysed samples of invasive ductal carcinomas of no special type (IDC-NST). Therefore, prevalence and clinical significance of these CSC markers in breast carcinomas of special histological types (SHT) is largely unknown. For that reason, this study aims to determine the distribution of the breast CD44, CD24 and ALDH1 CSC markers among a series of invasive breast carcinomas of SHT, in comparison with a series of IDC-NST. METHODS: 117 invasive SHT breast carcinomas were analysed for the expression of CD44, CD24 and ALDH1, by immuhohistochemistry. The distribution of these CSC markers was evaluated among the distinct histological special types, and the results were compared with a series of 466 IDC-NST. RESULTS: The expression prevalence of the breast CSC markers differed between special types and IDC-NST. Medullary, papillary and tubular carcinomas were enriched in the CSC phenotype CD44(+)/CD24(-/low) (80.0%, 100.0% and 100.0%, respectively, vs 45.3% in IDC-NST). Considering the ALDH1 cytoplasmic tumour expression, only medullary and metaplastic carcinomas displayed significant increase in CD44(+)/CD24(-/low)/ALDH1(+) CSC phenotype frequency (36.4% and 28.6%, respectively, vs 4.8% in IDC-NST). CONCLUSIONS: The expression distribution of breast CSC markers is largely dependent on histological type. Interestingly, within the distinct SHT, medullary and metaplastic carcinomas are the two types highly associated with high-grade carcinomas, basal-like and claudin-low molecular subtypes, and to the CSC phenotype CD44(+)/CD24(-/low)/ALDH1(+).