Flow cytometric quantification of apoptotic and proliferating cells applying an improved method for dissociation of spheroids

Spheroids are a promising tool for many cell culture applications, but their microscopic analysis is limited. Flow cytometry on a single cell basis, which requires a gentle but also efficient dissociation of spheroids, could be an alternative analysis. Mono-culture and coculture spheroids consisting of human fibroblasts and human endothelial cells were generated by the liquid overlay technique and were dissociated using AccuMax as a dissociation agent combined with gentle mechanical forces. This study aimed to quantify the number of apoptotic and proliferative cells. We were able to dissociate spheroids of differing size, age, and cellular composition in a single-step dissociation protocol within 10 min. The number of single cells was higher than 95% and in most cases, the viability of the cells after dissociation was higher than 85%. Coculture spheroids exhibited a higher sensitivity as shown by lower viability, higher amount of cellular debris, and a higher amount of apoptotic cells. Considerable expression of the proliferation marker Ki67 could only be seen in 1-day-old spheroids but was already downregulated on Day 3. In summary, our dissociation protocol enabled a fast and gentle dissociation of spheroids for the subsequent flow cytometric analysis. The chosen cell type had a strong influence on cell viability and apoptosis. Initially high rates of proliferative cells decreased rapidly and reached values of healthy tissue 3 days after generation of the spheroids. In conclusion, the flow cytometry of dissociated spheroids could be a promising analytical tool, which could be ideally combined with microscopic techniques

Vorwort zum Themenheft "Hochschuldidaktik im Spiegel der Fachkulturen"

Anlass, sich im vorliegenden Themenheft intensiv mit Verbindungen zwischen Hochschuldidaktik und Fachkulturen auseinanderzusetzen, war die fünfte Konferenz zur Qualität in der Lehre an der Fachhochschule Kiel, die am 9./10. Mai 2019 unter dem Titel Hochschuldidaktik im Spiegel der Fachkulturen stattfand. Die Herausgeberinnen freuen sich sehr, dass die Ergebnisse und darüber hinausgehende Überlegungen in diesem Heft als Publikation realisiert werden konnten. Das Vorwort gibt Einblick in die Anlässe für die Entstehung und fasst Beiträge wie Zusammenhänge des Themenheftes "Hochschuldidaktik im Spiegel der Fachkulturen" zusammen

Cholecystokinin effects on feeding, glucose, and pancreatic hormones in rhesus monkeys

The effects of cholecystokinin on feeding, and on plasma glucose and pancreatic hormone responses to a mixed-meal were determined in lean rhesus monkeys. Following an overnight fast the octapeptide of cholecystokinin (CCK-8) was administered intravenously over a 6 minute period coincident with the initiation of free feeding or an intragastric infusion of a mixed liquid diet. CCK-8 inhibited feeding and delayed the plasma glucose and insulin response to a mixed-meal. The threshold for the feeding effect ranged from 30 to 120 ng/kg/min across monkeys and did not extend beyond 15 minutes of the start of the CCK infusion. The delays in plasma glucose and insulin were not dependent on rate or amount of food entering the stomach. Further, there were no alterations from basal levels in plasma glucose or insulin prior to the onset of CCK-induced feeding inhibition. There was no evidence that CCK-8 stimulated insulin release, nor was the usual close relationship between plasma glucose and insulin levels in response to a mixed-meal changed by CCK-8.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25259/1/0000702.pd

Predictive performance and clinical application of COV50, a urinary proteomic biomarker in early COVID-19 infection: a prospective multicentre cohort study

Background: The SARS-CoV-2 pandemic is a worldwide challenge. The CRIT-CoV-U pilot study generated a urinary proteomic biomarker consisting of 50 peptides (COV50), which predicted death and disease progression from SARS-CoV-2. After the interim analysis presented for the German Government, here, we aimed to analyse the full dataset to consolidate the findings and propose potential clinical applications of this biomarker. Methods: CRIT-CoV-U was a prospective multicentre cohort study. In eight European countries (Austria, France, Germany, Greece, North Macedonia, Poland, Spain, and Sweden), 1012 adults with PCR-confirmed COVID-19 were followed up for death and progression along the 8-point WHO scale. Capillary electrophoresis coupled with mass spectrometry was used for urinary proteomic profiling. Statistical methods included logistic regression and receiver operating characteristic curve analysis with a comparison of the area under curve (AUC) between nested models. Hospitalisation costs were derived from the care facility corresponding with the Markov chain probability of reaching WHO scores ranging from 3 to 8 and flat-rate hospitalisation costs adjusted for the gross per capita domestic product of each country. Findings: From June 30 to Nov 19, 2020, 228 participants were recruited, and from April 30, 2020, to April 14, 2021, 784 participants were recruited, resulting in a total of 1012 participants. The entry WHO scores were 1-3 in 445 (44%) participants, 4-5 in 529 (52%) participants, and 6 in 38 (4%) participants; and of all participants, 119 died and 271 had disease progression. The odds ratio (OR) associated with COV50 in all 1012 participants for death was 2·44 (95% CI 2·05-2·92) unadjusted and 1·67 (1·34-2·07) when adjusted for sex, age, BMI, comorbidities, and baseline WHO score; and for disease progression, the OR was 1·79 (1·60-2·01) when unadjusted and 1·63 (1·41-1·91) when adjusted (p Interpretation: The urinary proteomic COV50 marker might be predictive of adverse COVID-19 outcomes. Even in people with mild-to-moderate PCR-confirmed infections (WHO scores 1-4), the 0·04 COV50 threshold justifies earlier drug treatment, thereby potentially reducing the number of days in hospital and associated costs. Funding: German Federal Ministry of Health

Money Minute: Using short informational videos during COVID-19

The COVID-19 pandemic has created a money crunch for some families. To help families struggling financially while capitalizing on at-home time, The University of Tennessee (UT) Extension consumer economics leadership team developed a series of money management videos called Money Minute. The primary purpose of the videos was to provide research-based financial education during this time of financial hardships. Filmed using Zoom, each video offers a piece of research-based information, additional resources, and a call to action. The video series proved to be effective in reaching clientele with financial information in the midst of a pandemic

T-Cell Protein Tyrosine Phosphatase Attenuates STAT3 and Insulin Signaling in the Liver to Regulate Gluconeogenesis

OBJECTIVE: Insulin-induced phosphatidylinositol 3-kinase (PI3K)/Akt signaling and interleukin-6 (IL-6)-instigated JAK/STAT3-signaling pathways in the liver inhibit the expression of gluconeogenic genes to decrease hepatic glucose output. The insulin receptor (IR) and JAK1 tyrosine kinases and STAT3 can serve as direct substrates for the T-cell protein tyrosine phosphatase (TCPTP). Homozygous TCPTP-deficiency results in perinatal lethality prohibiting any informative assessment of TCPTP's role in glucose homeostasis. Here we have used Ptpn2+/- mice to investigate TCPTP's function in glucose homeostasis. RESEARCH DESIGN AND METHODS: We analyzed insulin sensitivity and gluconeogenesis in chow versus high-fat-fed (HFF) Ptpn2+/- and Ptpn2+/+ mice and insulin and IL-6 signaling and gluconeogenic gene expression in Ptpn2+/- and Ptpn2+/+ hepatocytes. RESULTS: HFF Ptpn2+/- mice exhibited lower fasted blood glucose and decreased hepatic glucose output as determined in hyperinsulinemic euglycemic clamps and by the decreased blood glucose levels in pyruvate tolerance tests. The reduced hepatic glucose output coincided with decreased expression of the gluconeogenic genes G6pc and Pck1 and enhanced hepatic STAT3 phosphorylation and PI3K/Akt signaling in the fasted state. Insulin-induced IR-beta-subunit Y1162/Y1163 phosphorylation and PI3K/Akt signaling and IL-6-induced STAT3 phosphorylation were also enhanced in isolated Ptpn2+/- hepatocytes. The increased insulin and IL-6 signaling resulted in enhanced suppression of G6pc and Pck1 mRNA. CONCLUSIONS: Liver TCPTP antagonises both insulin and STAT3 signaling pathways to regulate gluconeogenic gene expression and hepatic glucose output

Outcomes and Complications With Off-Label Use of Drug-Eluting Stents Results From the STENT (Strategic Transcatheter Evaluation of New Therapies) Group

ObjectivesThis study evaluates outcomes and complications in patients treated with drug-eluting stents (DES) for “off-label” indications.BackgroundDrug-eluting stents have been effective in randomized trials, but their safety and efficacy for off-label indications has not been well studied.MethodsThe STENT (Strategic Transcatheter Evaluation of New Therapies) Registry is the largest multicenter U.S. registry evaluating outcomes of DES. Off-label indications included ostial, left main, long, bifurcation, and in-stent restenotic lesions, saphenous vein grafts, chronic total occlusions, small or large vessels, multilesion or multivessel percutaneous coronary interventions, and ST-segment elevation myocardial infarction. Outcomes were adjusted using Cox proportional hazards regression and propensity analyses.ResultsDrug-eluting stents were used in an off-label manner in 59% of patients. The patients who received off-label treatment were more often male, had a higher incidence of prior infarction and bypass surgery, and lower ejection fractions. Off-label versus “on-label” use of DES was associated with higher rates of death, myocardial infarction, target vessel revascularization, major adverse cardiac events, and stent thrombosis at 9 months and 2 years. Off-label use of DES compared with off-label use of bare-metal stents (BMS) had lower rates of death, myocardial infarction, target vessel revascularization, and major adverse cardiac events at 9 months and 2 years and lower rates of stent thrombosis at 9 months.ConclusionsOff-label use of DES is associated with higher event rates compared with on-label use of DES, which is consistent with a higher risk clinical and lesion profile. However, event rates with off-label use of DES are lower compared with off-label use of BMS. Pending results from randomized trials, our data support the use of DES for off-label indications in selected patients

Phenotype Selection Reveals Coevolution of Muscle Glycogen and Protein and PTEN as a Gate Keeper for the Accretion of Muscle Mass in Adult Female Mice

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice

Audio computer-assisted self-interviewing (ACASI) may avert socially desirable responses about infant feeding in the context of HIV

BACKGROUND: Understanding infant feeding practices in the context of HIV and factors that put mothers at risk of HIV infection is an important step towards prevention of mother to child transmission of HIV (PMTCT). Face-to-face (FTF) interviewing may not be a suitable way of ascertaining this information because respondents may report what is socially desirable. Audio computer-assisted self-interviewing (ACASI) is thought to increase privacy, reporting of sensitive issues and to eliminate socially desirable responses. We compared ACASI with FTF interviewing and explored its feasibility, usability, and acceptability in a PMTCT program in Kenya. METHODS: A graphic user interface (GUI) was developed using Macromedia Authorware(® )and questions and instructions recorded in local languages Kikuyu and Kiswahili. Eighty mothers enrolled in the PMTCT program were interviewed with each of the interviewing mode (ACASI and FTF) and responses obtained in FTF interviews and ACASI compared using McNemar's χ(2 )for paired proportions. A paired Student's t-test was used to compare means of age, marital-time and parity when measuring interview mode effect and two-sample Student's t-test to compare means for samples stratified by education level – determined during the exit interview. A Chi-Square (χ(2)test) was used to compare ability to use ACASI by education level. RESULTS: Mean ages for intended time for breastfeeding as reported by ACASI were 11 months by ACASI and 19 months by FTF interviewing (p < 0.001). Introduction of complementary foods at ≤3 months was reported more frequently by respondents in ACASI compared to FTF interviews for 7 of 13 complementary food items commonly utilized in the study area (p < 0.05). More respondents reported use of unsuitable utensils for infant feeding in ACASI than in FTF interviewing (p = 0.001). In other sensitive questions, 7% more respondents reported unstable relationships with ACASI than when interviewed FTF (p = 0.039). Regardless of education level, respondents used ACASI similarly and majority (65%) preferred it to FTF interviewing mainly due to enhanced usability and privacy. Most respondents (79%) preferred ACASI to FTF for future interviewing. CONCLUSION: ACASI seems to improve quality of information by increasing response to sensitive questions, decreasing socially desirable responses, and by preventing null responses and was suitable for collecting data in a setting where formal education is low