Characterization of Drosophila GDNF Receptor-Like and Evidence for Its Evolutionarily Conserved Interaction with Neural Cell Adhesion Molecule (NCAM)/FasII

Peer reviewe

Influence of age on the clinical outcomes of coronary revascularisation for the treatment of patients with multivessel de novo coronary artery lesions: sirolimus-eluting stent vs. coronary artery bypass surgery and bare metal stent, insight from the multicentre randomised Arterial Revascularisation Therapy Study Part I (ARTS-I) and Part II (ARTS-II)

peer reviewedAims: We sought to evaluate the prognostic impact of age on the procedural results and subsequent clinical outcomes in patients with multivessel disease (MVD) treated either by coronary artery bypass surgery (CABG) or by percutaneous coronary intervention (PCI) with or without drug eluting stents, based on data of the Arterial Revascularisation Therapies Study (ARTS) part I and part II. The potential influence of age in determining the most appropriate revascularisation strategy for patients with MVD is largely unknown. Methods and results: Three year clinical outcome of ARTS I patients randomised to PCI with bare metal stent (BMS) (n= 600) or CABG (n= 605), and matched patients treated by PCI with sirolimus-eluting stents (SES) in ARTS II (n= 607) were reviewed according to four age quartiles. Endpoints were measured in terms of major adverse cardiac and cerebrovascular events MACCE) during hospital stay and up to three years. The frequency of female, diabetes, hypertension, peripheral vascular disease, pulmonary disease, as well as lesion complexity increased with age. At three years, MACCE free survival was comparable between patients treated by CABG or SES PCI, regardless of age quartile. The incidence of MACCE was higher among ARTS I BMS treated patients in all but the second age quartile. This was primarily related to a higher need for repeat revascularisation among BMS treated patients. However, age, which emerged as a strong independent predictor of MACCE following CABG (p<0.005), was not predictive of adverse events following PCI. Conversely, diabetes was the strongest independent predictor of MACCE among PCI treated patients (p<0.02), but didn’t affect three-year outcomes following CABG. Conclusions: Age seems to influence the CABG outcome in-hospital but not PCI. PCI-SES could offer lower immediate risk in patients with MVD and comparable long-term outcome as CABG especially in older patients. The worst outcome of PCI-BMS group is primarily related to the need for repeat revascularisation. Diabetes is the most important predictor of MACCE following PCI

Kohti avointa kehittämistä - matkaoppaana Innokylä!

Innokylä on avoin kansallinen verkkopalvelu, joka tarjoaa välineitä ja toimintamalleja sosiaali- ja terveysalan kehittäjille ja päättäjille. Innokylässä ei kuitenkaan ole kyse vain työkaluista, vaan uudenlaisesta kehittämisen kulttuurista, joka arvoja ovat avoimuus ja moninäkökulmaisuus. Innokylässä kehittäminen lähtee tunnistetuista tarpeista, joihin etsitään ratkaisuja yhdessä kaikkien osapuolten asiantuntemusta hyödyntäen. Kehittämistä on tehty Suomessa tyypillisesti hankkeissa, joiden tulosten juurruttaminen käytäntöön on usein jäänyt kesken. Innokylässä painotetaan pelkkien hankkeiden sijaan kehittämistä osana perustyötä. Tulosten jakaminen ja arvioiminen ovat myös tärkeä osa toimintaa. Oppaassa esitellään Innokylän työvälineet ja kuvataan miten niitä voidaan hyödyntää kehittämisen eri vaiheissa. Innokyläläiset jakavat myös omat kokemuksensa yhteiskehittämisestä, sen vaikeuksista ja innostavuudesta. Parasta ja pahinta on yhteistyö! Opas sopii kehittäjille, omaa työtään kehittäville käytännön työntekijöille sekä heidän esimiehilleen

Experimental Infection of Mink with SARS-COV-2 Omicron Variant and Subsequent Clinical Disease

We report an experimental infection of American mink with SARS-CoV-2 Omicron variant and show that mink remain positive for viral RNA for days, experience clinical signs and histopathologic changes, and transmit the virus to uninfected recipients. Preparedness is crucial to avoid spread among mink and spillover to human populations.Peer reviewe

<i>In situ</i> hybridization analysis of <i>DmGfrl</i> expression during embryogenesis.

<p>Cellular blastoderm (B) and early stage embryos up to stage 10 (C) do not express <i>DmGfrl</i>. Expression (blue staining) first appeared in the seven abdominal segments in the ventral nerve cord (D, vnc) and in unidentified ganglia in the head region at about stage 13. The expression became more widespread in the central and peripheral nervous system through the later stages of embryogenesis (E, F). <i>DmGfrl</i> was expressed in single cells or clusters of a few cells in the head sensory ganglia (E, sg), ventral nerve chord (D–F) and lateral sensory ganglia (E, arrowheads). At stage 15, dorsal vessel (dv) also expressed <i>DmGfrl</i> (E). Control hybridization with a sense probe did not show specific staining (A). (G–L) Following whole mount in situ hybridization, the embryos were subjected to immunoperoxidase staining (brown color) with neuronal and glial marker antibodies. Co-staining for the neuronal marker FasII showed that <i>DmGfrl</i>-expressing cells are localized within the VNC, along the longitudinal axon bundles (G). A lateral view on the VNC shows that the <i>DmGfrl</i> signal did not colocalize with the nuclear staining for REPO, a glial cell marker (H, arrowheads show <i>DmGfrl</i>+ cells). A ventral view of the VNC also shows the paired <i>DmGfrl</i>+ cells in each segment do not co-localize with REPO (I, large arrows show <i>DmGfrl</i>+ cells). However, in the more lateral and dorsal cells there may be some overlap in the signals for DmGfrl and REPO (I, small arrows). The <i>DmGfrl</i>+ cells localized posteriorly from the dMP2 interneurons (J, brown staining) in late-stage embryos, indicating that they are not dMP2 neurons and likely not vMP2 neurons either. Futsch/22C10 staining visualizes that the lateral axonal projections along which the <i>DmGfrl</i>+ cells were located (K, arrows). Staining for cut, a sensory neuron marker showed that the <i>DmGfrl</i>-expressing cells were within the external sensory organ cell clusters and likely all positive for cut (L, arrows). Original magnification was 400X in h, i and l, and 200X in all other images.</p

Genomic and transcript structure of the <i>DmGfrl</i> locus.

<p>A 105-kb region of chromosome 3R is shown. The annotation is according to Flybase version FB2009_07, released August 10, 2009. On the basis of our cDNA cloning and sequencing results, the currently annotated <i>mun</i>/<i>DmGfrl</i> locus is extended into the 5′ direction by approximately 85 kb, and the genes previously annotated as CG34118 and CG17208 are fused with <i>DmGfrl</i>. CG4335 and CG10881, predicted intronless genes encoding a trimethyllysine dioxygenase and a translation initiation factor, respectively, are embedded on opposite strands in the 7.5-kb intron between exons 6 and 7. The Flybase entries previously annotated as CG17208 and CG34118 were identified as being the 5′ termini of the <i>DmGfrl</i> transcripts A and B, respectively. <i>PBac{SAstopDsRed}LL00378</i> is a hypomorphic insertion utilized in the biochemical detection of endogenous DmGfrl protein (see Fig. 4). <i>Df(3R)Exel6185</i> (<i>Df1</i>) and <i>Df(3R)BSC519</i> (<i>Df2</i>) are the genomic deficiencies used in the genetic experiments. <i>delDmGfrl</i> denotes the deletion allele generated by means excision between the FRT-carrying PBac elements <i>PBac{WH}mun[f00705]</i> and <i>PBac{WH}f03437</i>. Exon-intron structures of the six <i>DmGfrl</i> transcripts (A, Ab, B, Bb, C, D) assembled from experimental and <i>in silico</i> data are presented below the genomic locus drawing. Transcripts C and D were not detected on Northern blots, which suggest that they are present at very low levels compared to the other transcripts, and thus, their physiological significance remains to be investigated.</p

Quantitation of <i>DmGfrl</i> null female fertility and oogenesis phenotype.

<p>(A) <i>delDmGfrl</i> females displayed markedly reduced fertility. The absolute fertility of <i>delDmGfrl/Df1</i> transheterozygous females was reduced by ∼60% as compared to heterozygous control females. Their fecundity (measured as the average number of progeny produced by individual females in a given time) was reduced to ∼10% of the fecundity of control females. (B) Morphology of eggs laid by control, <i>delDmGfrl</i> and rescue females. Uppermost row shows the morphology of heterozygous (<i>delDemGfl/+</i>) control eggs. Second row exemplifies the morphology of eggs laid by <i>DmGfrl</i> null females (<i>delDmGfrl/Df1</i>). The eggs were small and often translucent (not visible in this image), and ∼60% of them display lack of or abnormal dorsal appendages (arrows). <i>LacZ</i> transgene under the <i>daughterless</i> (<i>da)</i> driver (3^rd row) did not rescue the egg morphology, whereas eggs laid by females expressing the <i>DmGfrl</i> transgene under the <i>da</i> driver in <i>DmGfrl</i> null background (4^th row) were almost fully wild-type by appearance. (C) Quantitation of the size of eggs laid by control, <i>delDmGfrl</i> and rescue females. Average egg length was reduced from 0.528 mm in heterozygous control eggs (female genotype <i>delDmGfrl/+)</i> to 0.466 mm in eggs laid by homozygous <i>delDmGfrl</i>/<i>delDmGfrl</i> females and to 0.450 mm in eggs laid by <i>delDmGfrl/Df1</i> females (1^st to 3^rd bars). <i>DmGfrl</i> transgene (UAS-Tg), but not <i>LacZ</i> transgene (UAS-LacZ), partially rescued the dumpless-like phenotype (4^th and 5^th bars). Statistical significance from Tukey’s post hoc test after one-way ANOVA are shown with asterisk (*) with respect to the <i>del/+</i> genotype and hash (#) with respect to the transgene rescue genotype (<i>del da-G4/del UAS-DmGfrl</i>). (D) Quantitation and rescue of the malformed egg phenotype. The percentage of malformed eggs laid by <i>DmGfrl</i> null females, displaying either dumpless-like phenotype or malformed dorsal appendages or both, was ∼60–70% depending on the genetic background (2^nd, 4^th and 5^th bars). Expression of DmGfrl under the <i>da-GAL4</i> driver diminished the percentage of malformed eggs from 63% (driver only, 4^th bar) to ∼3% (driver and transgene, 6^th bar). Expression of <i>LacZ</i> transgene in the same background did not rescue the egg phenotype (59%, 5^th bar). Asterisk (*) represent statistical significance obtained from Dunn’s post hoc test after non-parametric Kruskal-Wallis ANOVA with respect to the <i>del/+</i> genotype. The error bars represent standard deviation in graphs A, C, and D. Two asterisks correspond to p-values of <0.01 and three asterisk to p-values of <0.001.</p

Northern blot and RT-PCR analysis of <i>DmGfrl</i> and <i>DmRet</i> mRNA expression.

<p>(A) Two transcripts of approximately 7000 and 7500 nucleotides were detected in embryonic (E), pupal (P), adult female (F) and adult male (M) stages, but not in 3rd instar larvae (L). An essentially identical transcript pattern was detected with a probe corresponding to a region coding for the first two GFRα-like domains (exons 5–12) and with a probe corresponding to a region coding for the latter two GFRα-like domains (exons 11–17). With a <i>DmRet</i> probe, a major band of ∼5000 nt and a weaker band of ∼6500 nt were detected at all developmental stages. Hybridization for the rp49 gene showed roughly equal poly-A RNA loading. (B) Hybridization with 5′ untranslated region (UTR) probes revealed that the two major <i>DmGfrl</i> mRNA bands (∼7000-nt and ∼7500-nt) arise from differential transcription start site usage, with the ∼7000-nt band corresponding to transcript A and the ∼7500-nt band correcponding to transcripts B. The calculated size difference between the two 5′UTRs is ∼590 nt. Note that the 5′UTR probes give a markedly weaker signal in adults (A) than in pupae (P), which may be due to alternative splicing of the 5′ non-coding exons in adult tissues. Equally spaced arrows on the right illustrate the size difference of the two transcripts. (C) In RT-PCR with primers designed to amplify a 1085-bp fragment from exons 15–23 of <i>DmGfrl</i> one major major DNA band was detected in embryonic (E), larval (L), pupal (P) and adult (A) tissues (left image). With primers designed to amplify the entire coding region of <i>DmGfrl</i> transcript B, two bands of approximately 2.3 kb and 2.6 kb were detected in adult tissues (right image). The 2.3-kb band was sequenced and found to correspond to an alternative transcript (named Bb) that lacks exon 12. The lower band was weaker in embryonic tissues, suggesting developmentally regulated splicing. In pupal tissues a lower band barely separated from the 2.6-kb band is seen (right panel). This band may represent additional alternative splicing in this tissue. The faint bands above the major <i>DmGfrl</i> band in the left panel likely represent partially spliced mRNAs.</p

Amino acid sequence of DmGfrlA and schematic structures of the predicted DmGfrl protein isoforms identified in this study.

<p>(A) In the amino acid sequence of DmGfrl protein isoform A the signal sequence is marked with solid double underlining. The amino acid sequence of isoform B is identical to that of isoform A except for 26 N-terminal amino acids preceding the D0 domain, which reads MLKPFAVIIGIFYLGSTIKGVVAILN in DmGfrlB. V5 after the signal sequence denotes the site (between Q31 and G32) where a sequence encoding the V5 tag (GKPIPNPLLGLDST) was inserted in some of the expression constructs. The GFRα-like domains 0 to 3 (D0 to D3) are marked with solid underlining and the sequence (MKKCDRI) similar to mammalian heparin binding sites with bold underlining. The three predicted N-glycosylation sites are marked with N above the amino acid sequence and the mucin-type glycosylation sites with asterisks (*). A predicted low-score (big-PI Predictor) GPI anchoring site (G1008, underlined) is marked with ‘GPI’. It precedes a C-terminal hydrophobic region typical of GPI anchored proteins (dash underlining). (B) Schematic structures of four predicted DmGfrl isoforms and comparison to human GFRα1 and predicted <i>C. elegans</i> Gfr-like protein. Several insect genomes encode Gfr-like proteins that are approximately double the size of vertebrate Gfrα proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051997#pone.0051997-Airaksinen2" target="_blank">[30]</a>. Most of this size difference is due to the long sequence between the fourth GFRα-like domain and the plasma membrane anchor in the insect proteins.</p