Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes

<p>Abstract</p> <p>Background</p> <p>Hypoxic culturing of chondrocytes is gaining increasing interest in cartilage research. Culturing of chondrocytes under low oxygen tension has shown several advantages, among them increased synthesis of extracellular matrix and increased redifferentiation of dedifferentiated chondrocytes. Quantitative gene expression analyses such as quantitative real-time PCR (qRT-PCR) are powerful tools in the investigation of underlying mechanisms of cell behavior and are used routinely for differentiation and phenotype assays. However, the genes used for normalization in normoxic cell-cultures might not be suitable in the hypoxic environment. The objective of this study was to determine hypoxia-stable housekeeping genes (HKG) for quantitative real-time PCR (qRT-PCR) in human chondrocytes cultured in 21%, 5% and 1% oxygen by geNorm and NormFinder analyses.</p> <p>Results</p> <p>The chondrocytic response to the hypoxic challange was validated by a significant increase in expression of the hypoxia-inducible gene ankyrin repeat 37 as well as SOX9 in hypoxia. When cultured on the 3-dimentional (3D) scaffold TATA-binding protein (TBP) exhibited the highest expression stability with NormFinder while Ribosomal protein L13a (RPL13A) and beta2-microglobulin (B2M) were the most stable using geNorm analysis. In monolayer RPL13A were the most stable gene using NormFinder, while geNorm assessed RPL13A and human RNA polymerase II (RPII) as most stable. When examining the combination of (3D) culturing and monolayer RPL13A and B2M showed the highest expression stability from geNorm analysis while RPL13A also showed the highest expression stability using NormFinder. Often used HKG such as beta actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were the most unstable genes investigated in all comparisons. The pairwise variations for the two most stable HKG in each group were all below the cut-off value of 0.15, suggesting that the two most stable HKG from geNorm analysis would be sufficient for qRT-PCR.</p> <p>Conclusion</p> <p>All data combined we recommend RPL13A, B2M and RPII as the best choice for qRT-PCR analyses when comparing normoxic and hypoxic cultured human chondrocytes although other genes might also be suitable. However, the matching of HKG to target genes by means of a thorough investigation of the stability in each study would always be preferable.</p

Mortality risk of long-term amiodarone therapy for atrial fibrillation patients without structural heart disease

Background: Amiodarone is often prescribed in the management of atrial fibrillation (AF) but is known to cause significant end-organ toxicities. In this study, we examined the impact of amiodarone on all-cause mortality in AF patients with structurally normal hearts. Methods: We performed a retrospective cohort analysis of all AF patients with structurally normal hearts who were prescribed antiarrhythmic drugs (AAD) for rhythm control of AF at our institution from 2006 to 2013 (n = 2,077). Baseline differences between the amiodarone (AMIO: n = 403) and other AADs (NON-AMIO: n = 1,674) groups were corrected for using propensity score matching. Results: Amiodarone use as first-line therapy decreased significantly with a higher degree of prescriber specialization in arrhythmia management (31%, 22%, and 9% for primary care physicians, general cardiologists and cardiac electrophysiologists, respectively, p &lt; 0.001). After propensity score matching, baseline comorbidities were balanced between the AMIO and NON-AMIO groups. Over a median follow-up of 28.2 months (range 6.0–100.9 months), amiodarone was associated with increased all-cause (HR 2.41, p = 0.012) and non-cardiac (HR 3.55, p = 0.008) mortality, but not cardiac mortality. AF recurrence and cardiac hospi­talizations were similar between the two study groups. Conclusions: Amiodarone treatment of AF is associated with increased mortality in patients without structural heart disease and therefore should be avoided or only used as a second-line therapy, when other AF therapies fail. Adherence to guideline recommendations in the management of AF patients impacts clinical outcome

Effect of Oxygen Consumption of Thylakoid Membranes (Chloroplasts) From Spinach after Inhibition Using JNN

Abstract: In this research, an Artificial Neural Network (ANN) model was developed and tested to predict effect of oxygen consumption of thylakoid membranes (chloroplasts) from spinach after inhibition. A number of factors were identified that may affect of oxygen consumption of thylakoid membranes from spinach. Factors such as curve, herbicide, dose, among others, as input variables for the ANN model. A model based on multi-layer concept topology was developed and trained using the data from some inhibition of photosynthesis in farms. The evaluation of testing the dataset shows that the ANN model is capable of correctly predicting the effect of oxygen consumption of thylakoid membranes (chloroplasts) from spinach after inhibition with 100% accuracy

Public views of different sources of health advice : pharmacists, social media and mobile health applications

Abstract Objectives Investigating public perceptions of community pharmacists (CP) in public health and their use of social media (SM) and mobile health applications (MH apps) in that regard. Methods Two surveys were created. One sought public perceptions of SM and the other of MH apps for health advice. Both included a section on perceptions of the role of CPs in public health. A convenience sampling strategy, based on proximity, was used.The study population was the public (n = 8 500 000) living in Greater London. The general public were recruited face-to-face in public spaces. A minimum sample (95% confidence interval/5% margin of error) of 385 was needed. Ethical approval was obtained from the university ethics committee. Responses were analysed in SPSS. Key findings About 820/1800 (45.6%) completed one/both surveys. Respondents seek health advice primarily from GPs, followed by digital mediums and then CPs. Under 35s use digital mediums more frequently (P = 0.039). Those who had used SM (41.7%) or MH apps (61.8%) for health information did not confirm its accuracy with a healthcare professional (HCP). Of those that did (MH apps = 39.2%; SM = 58.3%), the HCP disagreed with the information on MH apps and SM on 19.6% and 36.7% of occasions, respectively. Nevertheless, 64.5% stated that if a SM page was maintained by an HCP they would use it. Conclusions The public are using digital mediums for health advice instead of speaking to an HCP. If CPs want to have an impact on public health they must start imbedding digital mediums into their services. </jats:sec

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10&nbsp;years; 78.2% included were male with a median age of 37&nbsp;years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p&lt;0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p&lt;0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised