Can Integrated Care Help in Meeting the Challenges Posed on Our Health Care Systems by COVID-19? Some Preliminary Lessons Learned from the European VIGOUR Project

The COVID-19 pandemic puts health and care systems under pressure globally. This current paper highlights challenges arising in the care for older and vulnerable populations in this context and reflects upon possible perspectives for different systems making use of nested integrated care approaches adapted during the work of the EU-funded project VIGOU

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Phenotypic characterization of SETD3 knockout Drosophila

<div><p>Lysine methylation is a reversible post-translational modification that affects protein function. Lysine methylation is involved in regulating the function of both histone and non-histone proteins, thereby influencing both cellular transcription and the activation of signaling pathways. To date, only a few lysine methyltransferases have been studied in depth. Here, we study the <i>Drosophila</i> homolog of the human lysine methyltransferase SETD3, CG32732/dSETD3. Since mammalian SETD3 is involved in cell proliferation, we tested the effect of dSETD3 on proliferation and growth of <i>Drosophila</i> S2 cells and whole flies. Knockdown of dSETD3 did not alter mTORC1 activity nor proliferation rate of S2 cells. Complete knock-out of dSETD3 in <i>Drosophila</i> flies did not affect their weight, growth rate or fertility. dSETD3 KO flies showed normal responses to starvation and hypoxia. In sum, we could not identify any clear phenotypes for SETD3 knockout animals, indicating that additional work will be required to elucidate the molecular and physiological function of this highly conserved enzyme.</p></div

Transcriptional effects of dSETD3 KO in adult virgin flies.

<p><b>(A)</b> Control and dSETD3 KO virgin female flies were lysed in TRIZOL, RNA extracted and subjected to microarray analysis. Volcano blot shows all genes that were statistically significantly up or downregulated (<i>t</i> test < 0.05). Genes that were up or downregulated at least 2-fold upon dSETD3 KO (<i>t</i> test < 0.05) are indicated in green (down) and red (up). Data are available under GEO accession number GSE113846. <b>(B)</b> Gene ontology analysis using DAVID of dSETD3-affected genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201609#pone.0201609.ref041" target="_blank">41</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201609#pone.0201609.ref042" target="_blank">42</a>].</p

Knockdown of dSETD3 in the wing disc reduces growth.

<p><b>(A-B)</b> Knockdown of dSETD3 in the posterior compartment of the wing decreases tissue size. (A) Size of the posterior compartment normalized to size of the control anterior compartment. (B) Cell size in the posterior compartment was determined by counting the number of cells via their trichomes in a defined area, and then calculating the inverse–ie area per cell. Animals were raised at 25°C, 10 wings per genotype were used for quantifications. Error bars: standard deviation (SD); *** <i>t</i> test < 0.001 <b>(C)</b> Knockdown of dSETD3 in Drosophila S2 cells does not change mTORC1 activity. dSETD3 was knocked-down by treating S2 cells with 2 independent dsRNAs for 5 days, and mTORC1 activity was assayed via immunoblotting for phosphorylation of the direct target S6K. The two lanes per sample represent biological duplicates. <b>(D)</b> S2 cells treated with 3 different dsRNAs against dSETD3 do not show a change in cell proliferation rates. S2 cells were treated with dsRNAs for 5 days and plated freshly at the same cell density. Cell number was then counted every 24 hours for 5 days.</p

Direct plantlet inoculation with soil or insect-associated fungi may control cabbage root fly maggots

A potential Delia radicum biological control strategy involving cauliflower plantlet inoculation with various fungi was investigated in a series of laboratory and glasshouse experiments. In addition to entomopathogenic fungi, fungi with a high rhizosphere competence and fungi with the ability to survive as saprotrophs in soil were tested. The following fungal species were evaluated in the experiments: Trichoderma atroviride, T. koningiopsis, T. gamsii, Beauveria bassiana, Metharhizium anisopliae, M. brunneum and Clonostachys solani. A commercial carbosulfan-based insecticide was used as a positive control. Additionally, two commercial products, one based on B. bassiana (Naturalis) and one on Bacillus thuringiensis (Delfin) were used as reference biocontrol agents. The aims were (i) to assess the pathogenicity of the selected fungal isolates to Delia radicum, (ii) to evaluate the fungal isolates’ rhizosphere competence, with the emphasis on the persistence of the original inoculum on the growing roots, (iii) to assess possible endophytic plant tissue colonization, and (iv) to evaluate potential plant growth stimulating effects of the added inoculi. Significant pathogenicity of tested fungi against Delia radicum was confirmed in in vitro and glasshouse experiments. All tested fungi persisted on cauliflower rhizoplane. More importantly, the added fungi were found on thoroughly washed roots outside the original point of inoculation. This provided us with evidence that our tested fungi could be transferred via or grow with the elongating roots. In addition to colonizing the rhizoplane, some fungi were found inside the plant root or stem tissue, thus exhibiting endophytic characteristics. The importance of fungal ecology as a criterion in appropriate biological control agent selection is discussed

dSETD3 does not play a role in hypoxia response in <i>Drosophila</i>.

<p><b>(A)</b> dSETD3 protein levels do not change upon hypoxia in S2 cells. S2 cells were treated for 5 days with control or dSETD3 dsRNA and then reseeded and subjected to control or hypoxic conditions (1% oxygen) for 36 hours. Cell lysates were analyzed by immunoblotting with indicated antibodies. Quantification of dSETD3 bands normalized to ERK bands was done with ImageJ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201609#pone.0201609.ref046" target="_blank">46</a>]. <b>(B)</b> Expression of hypoxia induced genes in S2 cells is not dependent on dSETD3. S2 cells were treated for 5 days with control or dSETD3 dsRNA and then reseeded and subjected to control or hypoxic conditions (1% oxygen) for 36 hours. RNA was isolated and analyzed by RT-qPCR. Error bars from technical triplicates: standard deviation (SD). <b>(C)</b> dSETD3 protein levels do not change upon hypoxia in flies. Control flies were subjected to hypoxia (5% oxygen) for 3 or 6 hours and lysates were analyzed by immunoblotting with indicated antibodies. Three samples (5 larvae each) per condition are shown. <b>(D)</b> Expression of hypoxia induced genes is not affected by dSETD3 KO in flies. Control and dSETD3 KO male flies were subjected to 1, 4 or 6 hours of hypoxia (1.8% oxygen). RNA was isolated and analyzed by RT-qPCR. Error bars from technical triplicates: standard deviation (SD). <b>(E-E’)</b> dSETD3 is not required for survival during hypoxic conditions. Control and dSETD3 KO flies were put into hypoxic conditions (1.8% oxygen (E, n = 4 x 10 flies) and 2% oxygen (E’, n = 6 x 10 flies)) for indicated amounts of times and surviving animals after the treatment were counted. Error bars: standard deviation (SD).</p

Phenotypic characterization of dSETD3 KO flies.

<p><b>(A)</b> Wing size of adult female control (n = 7) and dSETD3 KO (n = 8) flies. Error bars: standard deviation (SD). <b>(B)</b> Weight of control and dSETD3 KO flies (n = 4 x 8 flies for each condition). Error bars: standard deviation (SD). <b>(C)</b> Pupation timing of control and dSETD3 KO animals at 25°C (n = 8 x 30 flies for each condition). Error bars: standard deviation (SD). <b>(D)</b> Fat storage measured by TAG abundance in control and dSETD3 KO flies, normalized to total body weight (n = 4 x 8 flies for each condition). Error bars: standard deviation (SD). <b>(E)</b> Glycogen storage measured in control and dSETD3 KO flies, normalized to total body weight (n = 4 x 8 flies for each condition). Error bars: standard deviation (SD); * <i>t</i> test < 0.05. <b>(F-F’)</b> Full starvation of control and dSETD3 KO animals on PBS/agarose (0.7%) for male (F) or female (F’) adult flies (n = 3 x 20 flies for each condition). Error bars: standard deviation (SD). <b>(G)</b> Fertility of control and dSETD3 KO mated females over the course of three days (n = 4 x 8–10 flies). Error bars: standard deviation (SD); * <i>t</i> test < 0.05. (H) dSETD3 KO flies do not have impaired motility, assayed using a climbing assay with control and dSETD3 KO adult females. Flies were put into plastic tubes, tapped down, and observed climbing towards a light source at the top of the tube. The time was measured that was required for 50% of the flies in one tube to pass a set threshold (biological quadruplicates, each measured twice). Error bars: standard deviation (SD).</p

Generation of dSETD3 KO flies.

<p><b>(A)</b> Schematic of the dSETD3 genomic locus and the knockout region. Orange indicates coding exons. To avoid interfering with the splicing of CG14431 only the first exon of CG32732 (dSETD3) was removed and replaced with a dsRED expression cassette using CRISPR-induced homologous recombination. The location of the amplicon used for Q-RT-PCR in panel C is shown and labeled with ‘qPCR’. <b>(B)</b> Complete loss of dSETD3 protein in different dSETD3 KO lines. Knock-out stock 2 was used in this study. Homozygous flies were lysed and analyzed by immunoblotting with purified dSETD3 antibody. <b>(C)</b> dSETD3 mRNA is completely lost in dSETD3 KO flies. mRNA was isolated from control and dSETD3 KO homozygous flies and dSETD3 expression was analyzed by RT-qPCR against the region indicated in (A), and normalized to rp49. Error bars: standard deviation (SD).</p

dSETD3 is localized in the nucleus and the cytoplasm.

<p><b>(A)</b> Epitope tagged dSETD3 is present in the cytoplasm and the nucleus. S2 cells were transfected with constructs to express either N-terminally or C-terminally HA-tagged dSETD3, and then immunostained for HA-tag or myc-tag (green “FITC”), DAPI (blue) and phalloidin (red “phall”) to stain the actin skeleton. Scale bar: 10μm.</p