A patient's admission to an intensive care unit (ICU) has a significant impact on family members and other relatives. In order for them to be able to cope with such a stressful situation, the availability of appropriate understandable and accessible information is crucial. The information asymmetry between relatives and medical professionals may adversely affect satisfaction of relatives and their risk of subsequent anxiety, depression and stress symptoms. The aim of this study was therefore to understand which topics are most important to the relatives of ICU patients and to quantify the perceptions of medical professionals regarding the information needs of relatives. A cross-sectional survey was conducted in 2015. The survey had 42 questions, such as 'diagnosis', 'treatment', 'comfort', 'family' and 'end of life'. In total, the survey was handed out to four different groups. A total of 336 persons answered the survey (26 relatives, 28 ICU physicians, 202 ICU nurses and 80 ICU medical professionals in a closed Facebook© group [Facebook, Menlo Park, California, USA]). Relatives ranked the five most important topics as follows: 'recent events (crisis)', 'my participation', 'contamination in hospital', 'physical pain', and 'probability'. Several significant differences (p<0.001) were detected, for example for the topics fever, medication, recent events (crisis), appointments, relapse, and investigations. Even the topic with the lowest ranking (religion) had a score of 3.15 (min. 1.00, max. 5.00) among relatives. The ICU professionals appear to have divergent opinions regarding the most important topics for ICU relatives as compared to relatives themselves.

Blood-brain barrier (BBB) impairment in systemic inflammation leads to neuroinflammation. Several factors including cytokines, chemokines and signal transduction molecules are implicated in BBB dysfunction in response to systemic inflammation. Here, we have adopted a novel in vivo technique; namely, cerebral open flow microperfusion (cOFM), to perform time-dependent cytokine analysis (TNF-alpha, IL-6 and IL-10) in the frontal cortex of the rat brain in response to a single peripheral administration of lipopolysaccharide (LPS). In parallel, we monitored BBB function using sodium fluorescein as low molecular weight reporter in the cOFM sample. In response to the systemic LPS administration, we observed a rapid increase of TNF-alpha in the serum and brain, which coincides with the BBB disruption. Brain IL-6 and IL-10 synthesis was delayed by approximately 1 h. Our data demonstrate that cOFM can be used to monitor changes in brain cytokine levels and BBB disruption in a rat sepsis model.

No abstract available

The age-associated deterioration in cellular and organismal functions associates with dysregulation of nutrient-sensing pathways and disabled autophagy. The reactivation of autophagic flux may prevent or ameliorate age-related metabolic dysfunctions. Non-toxic compounds endowed with the capacity to reduce the overall levels of protein acetylation and to induce autophagy have been categorized as caloric restriction mimetics (CRMs). Here, we show that aspirin or its active metabolite salicylate induce autophagy by virtue of their capacity to inhibit the acetyltransferase activity of EP300. While salicylate readily stimulates autophagic flux in control cells, it fails to further increase autophagy levels in EP300-deficient cells, as well as in cells in which endogenous EP300 has been replaced by salicylate-resistant EP300 mutants. Accordingly, the pro-autophagic activity of aspirin and salicylate on the nematode Caenorhabditis elegans is lost when the expression of the EP300 ortholog cpb-1 is reduced. Altogether, these findings identify aspirin as an evolutionary conserved CRM.

Virus genomics provides novel approaches for virus identification and classification. Based on the comparative analyses of sequenced lepidopteran-specific baculovirus genomes, degenerate oligonucleotides were developed that allow the specific amplification of several regions of the genome using polymerase chain reaction (PCR) followed by DNA sequencing. The DNA sequences within the coding regions of three highly conserved genes, namely polyhedrin/granulin (polh/gran), late expression factor 8 (lef-8), and late expression factor 9 (lef-9), were targeted for amplification. The oligonucleotides were tested on viral DNAs isolated from historical field samples, and amplification products were generated from 12 isolated nucleopolyhedrovirus (NPV) and 8 granulovirus (GV) DNAs. The PCR products were cloned or directly sequenced, and phylogenetic trees were inferred from individual and combined data sets of these three genes and compared to a phylogeny, which includes 22 baculoviruses using a combined data set of 30 core genes. This method allows a fast and reliable detection and identification of lepidopteran-specific NPVs and GVs. Furthermore, a strong correlation of the base composition of these three genome areas with that of the complete virus genome was observed and used to predict the base composition of uncharacterized baculovirus genomes. These analyses suggested that GVs have a significantly higher AT content than NPVs.

We analyzed the role of the peroxisomal peroxiredoxin Pmp20 of the yeast Hansenula polymorpha. Cells of a PMP20 disruption strain (pmp20) grew normally on substrates that are not metabolized by peroxisomal enzymes, but showed a severe growth defect on methanol, the metabolism of which involves a hydrogen peroxide producing peroxisomal oxidase. This growth defect was paralleled by leakage of peroxisomal matrix proteins into the cytosol. Methanol-induced pmp20 cells accumulated enhanced levels of reactive oxygen species and lipid peroxidation products. Moreover, the fatty acid composition of methanol-induced pmp20 cells differed relative to WT controls, suggesting an effect on fatty acid homeostasis. Plating assays and FACS-based analysis of cell death markers revealed that pmp20 cells show loss of clonogenic efficiency and membrane integrity, when cultured on methanol. We conclude that the absence of the peroxisomal peroxiredoxin leads to loss of peroxisome membrane integrity and necrotic cell death.

The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. We present a systems biology study integrating mRNA profiles with DNA-binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf), activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3), and microRNA profiles obtained in wild-type and RNAi-mediated knockdown. Finally, we confirmed conclusions primarily obtained in cardiomyocyte cell culture in a time-course of cardiac maturation in mouse around birth. We provide insights into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, we found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, we found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, we present an Srf-centered transcription network providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, we show the combinatorial contribution of four DNA-binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders.

The double-blind Trial Comparing Cardiovascular Safety of Insulin Degludec vs Insulin Glargine in Patients with Type 2 Diabetes at High Risk of Cardiovascular Events (DEVOTE) assessed the cardiovascular safety of insulin degludec. The incidence and rates of adjudicated severe hypoglycaemia, and all-cause mortality were also determined. This paper reports a secondary analysis investigating associations of severe hypoglycaemia with cardiovascular outcomes and mortality.

Worldwide, ∼1 million people manage their type 1 diabetes with an insulin pump and a continuous subcutaneous insulin infusion (CSII) catheter. Patients routinely insert a new catheter every 2-3 days due to increasing variability of insulin absorption over time. Catheter insertion and maintenance damage capillaries, lymphatics, cells, and connective tissue leading to an acute inflammatory response.

Adult hematopoietic stem cells (HSCs) are rare multipotent cells in bone marrow that are responsible for generating all blood cell types. HSCs are a heterogeneous group of cells with high plasticity, in part, conferred by epigenetic mechanisms. PHF19, a subunit of the Polycomb repressive complex 2 (PRC2), is preferentially expressed in mouse hematopoietic precursors. Here, we now show that, in stark contrast to results published for other PRC2 subunits, genetic depletion of Phf19 increases HSC identity and quiescence. While proliferation of HSCs is normally triggered by forced mobilization, defects in differentiation impede long-term correct blood production, eventually leading to aberrant hematopoiesis. At molecular level, PHF19 deletion triggers a redistribution of the histone repressive mark H3K27me3, which notably accumulates at blood lineage-specific genes. Our results provide novel insights into how epigenetic mechanisms determine HSC identity, control differentiation, and are key for proper hematopoiesis.

Background: Besides anemia, iron deficiency may cause more subtle symptoms, including the restless legs syndrome (RLS), the chronic fatigue syndrome (CFS) or sleeping disorders. Objective: The aim of this pre-planned secondary analysis of the IronWoMan randomized controlled trial (RCT) was to compare the frequency and severity of symptoms associated with iron deficiency before and after (intravenous or oral) iron supplementation in iron deficient blood donors.

Aging is associated with cardiac hypertrophy and progressive decline in heart function. One of the hallmarks of cellular aging is the dysfunction of mitochondria. These organelles occupy around 1/4 to 1/3 of the cardiomyocyte volume. During cardiac aging, the removal of defective or dysfunctional mitochondria by mitophagy as well as the dynamic equilibrium between mitochondrial fusion and fission is distorted. Here, we hypothesized that these changes affect the number of mitochondria and alter their three-dimensional (3D) characteristics in aged mouse hearts. The polyamine spermidine stimulates both mitophagy and mitochondrial biogenesis, and these are associated with improved cardiac function and prolonged lifespan. Therefore, we speculated that oral spermidine administration normalizes the number of mitochondria and their 3D morphology in aged myocardium. Young (4-months old) and old (24-months old) mice, treated or not treated with spermidine, were used in this study (n = 10 each). The number of mitochondria in the left ventricles was estimated by design-based stereology using the Euler-Poincaré characteristic based on a disector at the transmission electron microscopic level. The 3D morphology of mitochondria was investigated by 3D reconstruction (using manual contour drawing) from electron microscopic z-stacks obtained by focused ion beam scanning electron microscopy. The volume of the left ventricle and cardiomyocytes were significantly increased in aged mice with or without spermidine treatment. Although the number of mitochondria was similar in young and old control mice, it was significantly increased in aged mice treated with spermidine. The interfibrillar mitochondria from old mice exhibited a lower degree of organization and a greater variation in shape and size compared to young animals. The mitochondrial alignment along the myofibrils in the spermidine-treated mice appeared more regular than in control aged mice, however, old mitochondria from animals fed spermidine also showed a greater diversity of shape and size than young mitochondria. In conclusion, mitochondria of the aged mouse left ventricle exhibited changes in number and 3D ultrastructure that is likely the structural correlate of dysfunctional mitochondrial dynamics. Spermidine treatment reduced, at least in part, these morphological changes, indicating a beneficial effect on cardiac mitochondrial alterations associated with aging.

Electron tomography allows one to obtain 3D reconstructions visualizing a tissue's ultrastructure from a series of 2D projection images. An inherent problem with this imaging technique is that its projection images contain unwanted shifts, which must be corrected for to achieve reliable reconstructions. Commonly, the projection images are aligned with each other by means of fiducial markers prior to the reconstruction procedure. In this work, we propose a joint alignment and reconstruction algorithm that iteratively solves for both the unknown reconstruction and the unintentional shift and does not require any fiducial markers. We evaluate the approach first on synthetic phantom data where the focus is not only on the reconstruction quality but more importantly on the shift correction. Subsequently, we apply the algorithm to healthy C57BL/6J mice and then compare it with non-obese diabetic (NOD) mice, with the aim of visualizing the attack of immune cells on pancreatic beta cells within type 1 diabetic mice at a more profound level through 3D analysis. We empirically demonstrate that the proposed algorithm is able to compute the shift with a remaining error at only the sub-pixel level and yields high-quality reconstructions for the limited-angle inverse problem. By decreasing labour and material costs, the algorithm facilitates further research directed towards investigating the immune system's attacks in pancreata of NOD mice for numerous samples at different stages of type 1 diabetes.

Autophagy defects accelerate aging, while stimulation of autophagy decelerates aging. Acyl-coenzyme A binding protein (ACBP), which is encoded by a diazepam-binding inhibitor (DBI), acts as an extracellular feedback regulator of autophagy. As shown here, knockout of the gene coding for the yeast orthologue of ACBP/DBI (ACB1) improves chronological aging, and this effect is reversed by knockout of essential autophagy genes (ATG5, ATG7) but less so by knockout of an essential mitophagy gene (ATG32). In humans, ACBP/DBI levels independently correlate with body mass index (BMI) as well as with chronological age. In still-healthy individuals, we find that high ACBP/DBI levels correlate with future cardiovascular events (such as heart surgery, myocardial infarction, and stroke), an association that is independent of BMI and chronological age, suggesting that ACBP/DBI is indeed a biomarker of "biological" aging. Concurringly, ACBP/DBI plasma concentrations correlate with established cardiovascular risk factors (fasting glucose levels, systolic blood pressure, total free cholesterol, triglycerides), but are inversely correlated with atheroprotective high-density lipoprotein (HDL). In mice, neutralization of ACBP/DBI through a monoclonal antibody attenuates anthracycline-induced cardiotoxicity, which is a model of accelerated heart aging. In conclusion, plasma elevation of ACBP/DBI constitutes a novel biomarker of chronological aging and facets of biological aging with a prognostic value in cardiovascular disease.

Mitochondrial function declines during brain aging and is suspected to play a key role in age-induced cognitive decline and neurodegeneration. Supplementing levels of spermidine, a body-endogenous metabolite, has been shown to promote mitochondrial respiration and delay aspects of brain aging. Spermidine serves as the amino-butyl group donor for the synthesis of hypusine (Nε-[4-amino-2-hydroxybutyl]-lysine) at a specific lysine residue of the eukaryotic translation initiation factor 5A (eIF5A). Here, we show that in the Drosophila brain, hypusinated eIF5A levels decline with age but can be boosted by dietary spermidine. Several genetic regimes of attenuating eIF5A hypusination all similarly affect brain mitochondrial respiration resembling age-typical mitochondrial decay and also provoke a premature aging of locomotion and memory formation in adult Drosophilae. eIF5A hypusination, conserved through all eukaryotes as an obviously critical effector of spermidine, might thus be an important diagnostic and therapeutic avenue in aspects of brain aging provoked by mitochondrial decline.

The hyperinsulinaemic-hypoglycaemic glucose clamp technique has been developed and applied to assess effects of and responses to hypoglycaemia under standardised conditions. However, the degree to which the methodology of clamp studies is standardised is unclear. This systematic review examines how hyperinsulinaemic-hypoglycaemic clamps have been performed and elucidates potential important differences.

The naked mole-rat (Heterocephalus glaber) is characterized by a more than tenfold higher life expectancy compared to another rodent species of the same size, namely, the laboratory mouse (Mus musculus). We used mass spectrometric metabolomics to analyze circulating plasma metabolites in both species at different ages. Interspecies differences were much more pronounced than age-associated alterations in the metabolome. Such interspecies divergences affected multiple metabolic pathways involving amino, bile and fatty acids as well as monosaccharides and nucleotides. The most intriguing metabolites were those that had previously been linked to pro-health and antiaging effects in mice and that were significantly increased in the long-lived rodent compared to its short-lived counterpart. This pattern applies to α-tocopherol (also known as vitamin E) and polyamines (in particular cadaverine, N8-acetylspermidine and N1,N8-diacetylspermidine), all of which were more abundant in naked mole-rats than in mice. Moreover, the age-associated decline in spermidine and N1-acetylspermidine levels observed in mice did not occur, or is even reversed (in the case of N1-acetylspermidine) in naked mole-rats. In short, the present metabolomics analysis provides a series of testable hypotheses to explain the exceptional longevity of naked mole-rats.

To investigate the pharmacokinetic and pharmacodynamic properties and safety of a novel formulation of insulin aspart (AT247) versus two currently marketed insulin aspart formulations (NovoRapid [IAsp] and Fiasp [faster IAsp]).

Spermidine is a natural polyamine which was shown to prolong lifespan of organisms and to improve cardiac and cognitive function. Spermidine was also reported to reduce inflammation and modulate T-cells. Autophagy is one of the mechanisms that spermidine exerts its effect. Autophagy is vital for β-cell homeostasis and autophagy deficiency was reported to lead to exacerbated diabetes in mice. The effect of spermidine in type 1 diabetes pathogenesis remains to be elucidated. Therefore, we examined the effect of spermidine treatment in non-obese diabetic (NOD) mice, a mouse model for type 1 diabetes. NOD mice were given untreated or spermidine-treated water ad libitum from 4 weeks of age until diabetes onset or 35 weeks of age. We found that treatment with 10 mM spermidine led to higher diabetes incidence in NOD mice despite unchanged pancreatic insulitis. Spermidine modulated tissue polyamine levels and elevated signs of autophagy in pancreas. Spermidine led to increased proportion of pro-inflammatory T-cells in pancreatic lymph nodes (pLN) in diabetic mice. Spermidine elevated the proportion of regulatory T-cells in early onset mice, whereas it reduced the proportion of regulatory T-cells in late onset mice. In summary spermidine treatment led to higher diabetes incidence and elevated proportion of T-cells in pLN.

The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.