Prevalence of Sarcocystis calchasi in free-ranging host species: Accipiter hawks and Common Woodpigeon in Germany

The apicomplexan parasite Sarcocystis calchasi (S. calchasi) triggers pigeon protozoal encephalitis, a neurologic disease in columbids. Accipiter hawks have been identified as the final host, and Columbidae and Psittaciformes as intermediate hosts. In this study, 368 free-ranging Accipiter hawks and 647 free-ranging common woodpigeons were sampled in a country-wide study in order to identify the prevalence of S. calchasi in these populations. A semi-nested PCR specific for S. calchasi tested positive in 7.3% (4.9-10.5) of submitted samples from Accipiter hawks. Juvenile Accipiter hawks (13.7%; 7.7-22.0) had a significantly higher infection rate with S. calchasi than adult Accipiter hawks (5.8%; 2.7-9.3). The prevalence of S. calchasi in common woodpigeons was 3.3% (5.4-9.7). Positive pigeons were identified in 14/16 federal states, and a region-dependency was detected, with higher rates of infection in the eastern parts of Germany. The results of this study suggest that the common woodpigeon is a natural reservoir for S. calchasi. In a study of one region for four consecutive years, an increase in prevalence was not detected. Findings indicate that the parasite is not newly introduced to Germany, but rather long established. The prevalence suggests that there is a substantial risk of S. calchasi infections in other free-ranging as well as captive host species

Associations with age and glomerular filtration rate in a referred population with chronic kidney disease: Methods and baseline data from a UK multicentre cohort study (NURTuRE-CKD)

BACKGROUND: Chronic kidney disease (CKD) is common but heterogenous and is associated with multiple adverse outcomes. The National Unified Renal Translational Research Enterprise (NURTuRE)-CKD cohort was established to investigate risk factors for clinically important outcomes in persons with CKD referred to secondary care. METHODS: Eligible participants with CKD stages G3-4 or stages G1-2 plus albuminuria > 30 mg/mmol were enrolled from 16 nephrology centres in England, Scotland and Wales from 2017 to 2019. Baseline assessment included demographic data, routine laboratory data and research samples. Clinical outcomes are being collected over 15 years by the UK Renal Registry using established data linkage. Baseline data are presented with subgroup analysis by age, sex and estimated GFR (eGFR). RESULTS: 2996 participants were enrolled. Median (interquartile range) age was 66 (54 to 74) years, 58.5% were male, eGFR 33.8 (24.0 to 46.6) ml/min/1.73m2 and UACR 209 (33 to 926) mg/g. 1883 participants (69.1%) were in high-risk CKD categories. Primary renal diagnosis was CKD of unknown cause in 32.3%, glomerular disease in 23.4% and diabetic kidney disease in 11.5%. Older participants and those with lower eGFR had higher systolic blood pressure and were less likely to be treated with renin-angiotensin system inhibitors (RASi) but were more likely to receive a statin. Female participants were less likely to receive a RASi or statin. CONCLUSIONS: NURTuRE-CKD is a prospective cohort of persons who are at relatively high risk of adverse outcomes. Long-term follow-up and a large biorepository create opportunities for research to improve risk prediction and investigate underlying mechanisms to inform new treatment development

Glycoproteomic Analysis of the Aortic Extracellular Matrix in Marfan Patients.

OBJECTIVE: Marfan syndrome (MFS) is caused by mutations in FBN1 (fibrillin-1), an extracellular matrix (ECM) component, which is modified post-translationally by glycosylation. This study aimed to characterize the glycoproteome of the aortic ECM from patients with MFS and relate it to aortopathy. Approach and Results: ECM extracts of aneurysmal ascending aortic tissue from patients with and without MFS were enriched for glycopeptides. Direct N-glycopeptide analysis by mass spectrometry identified 141 glycoforms from 47 glycosites within 35 glycoproteins in the human aortic ECM. Notably, MFAP4 (microfibril-associated glycoprotein 4) showed increased and more diverse N-glycosylation in patients with MFS compared with control patients. MFAP4 mRNA levels were markedly higher in MFS aortic tissue. MFAP4 protein levels were also increased at the predilection (convexity) site for ascending aorta aneurysm in bicuspid aortic valve patients, preceding aortic dilatation. In human aortic smooth muscle cells, MFAP4 mRNA expression was induced by TGF (transforming growth factor)-β1 whereas siRNA knockdown of MFAP4 decreased FBN1 but increased elastin expression. These ECM changes were accompanied by differential gene expression and protein abundance of proteases from ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family and their proteoglycan substrates, respectively. Finally, high plasma MFAP4 concentrations in patients with MFS were associated with a lower thoracic descending aorta distensibility and greater incidence of type B aortic dissection during 68 months follow-up. CONCLUSIONS: Our glycoproteomics analysis revealed that MFAP4 glycosylation is enhanced, as well as its expression during the advanced, aneurysmal stages of MFS compared with control aneurysms from patients without MFS

National Unified Renal Translational Research Enterprise: Idiopathic Nephrotic Syndrome (NURTuRE-INS) study

BackgroundIdiopathic Nephrotic Syndrome (INS) is a heterogenous disease and current classification is based on observational responses to therapies or kidney histology. The National Unified Renal Translational Research Enterprise (NURTuRE)-INS cohort aims to facilitate novel ways of stratifying INS patients to improve disease understanding, therapeutics, and design of clinical trials.MethodsNURTuRE-INS is a prospective cohort study of children and adults with INS with a linked biorepository. All recruits had at least one sampling visit collecting serum, plasma, urine and blood for RNA and DNA extraction, frozen within 2 hours of collection. Clinical histology slides and biopsy tissue blocks were also collected.ResultsIn total, 739 participants were recruited from 23 centres to NURTuRE-INS, half of whom were diagnosed in childhood (n = 365, 49%). The majority were white (n = 525, 71%) and the median age at recruitment was 32 (interquartile range 12-54). Steroid-sensitive nephrotic syndrome (SSNS) was the most common clinical diagnosis (n = 518, 70%). Of patients diagnosed in childhood who underwent a kidney biopsy - for SSNS (n=103), 76 demonstrated minimal change disease (MCD); whereas for steroid-resistant nephrotic syndrome (n=80), 21 had MCD. Almost all patients diagnosed in adulthood had a kidney biopsy (n = 352, 94%); 187 MCD and 162 focal segmental glomerulosclerosis.ConclusionsNURTuRE-INS is a prospective cohort study with high-quality biosamples and longitudinal data that will assist research into the mechanistic stratification of INS. Samples and data will be available through a Strategic Access and Oversight Committee

GSKIP – ein neues A-Kinase-Ankerprotein, das PKA- und GSK3β-Signalwege integriert

A-kinase anchoring proteins (AKAPs) localise the cAMP-dependent protein kinase A (PKA) to distinct cellular compartments and thereby facilitate access to PKA substrates in close vicinity. In addition, AKAPs bind other signalling proteins, allowing for the integration of cAMP signalling with other signalling pathways in a temporally and spatially controlled manner. GSK3β interaction protein (GSKIP) had been identified in a database screen as a novel AKAP and had been shown to bind regulatory RII subunits of PKA in vitro. In this thesis, the AKAP function of GSKIP was demonstrated in live cells by cAMP agarose pull-down experiments and by fluorescence cross-correlation spectroscopy (FCCS). In addition, it was shown that GSKIP is a ubiquitously expressed, cytosolic AKAP that forms a complex with PKA RIIα and GSK3β in vitro as determined by enzyme-linked immunosorbent assays (ELISA). Moreover, GSKIP was found to increase the PKA-dependent phosphorylation of GSK3β serine-9 and thus inhibition of GSK3β. GSKIP is highly conserved among fungi, invertebrates and vertebrates. In a peptide spot array-based approach, the potential RII- and GSK3β-interaction domains of GSKIP orthologues were tested for RIIα and GSK3β binding, respectively. The interaction with RIIα and thus an AKAP function was only observed in vertebrate orthologues of GSKIP. In contrast, the binding to GSK3β was found for most of the peptides derived from fungal, invertebrate and vertebrate GSKIP proteins. In order to develop a basis for the elucidation of the physiological function of GSKIP, a mouse model for the conditional knockout of GSKIP was developed during this thesis. Genomic mouse DNA containing the GSKIP coding sequence was used to generate a targeting vector in which exon 2 of the GSKIP gene was flanked by loxP sites (floxed). This allowed a Cre-catalysed removal of exon 2 and thereby a disruption of the gene. The targeting vector was site-specifically integrated into the genome of embryonic stem (ES) cells. Recombinant ES cells were injected into mouse embryos which developed into mice carrying a floxed GSKIP allele. The genetic modification was inherited to filial generations and by breeding to a Cre-expressing mouse strain, the floxed sequence containing exon 2 was removed. The resulting GSKIP+/- mice were viable and fertile. Male and female GSKIP+/- mice were crossed but did not yield GSKIP / offspring, implying that a homozygous deletion of GSKIP is embryonically lethal. In future experiments, a detailed phenotypic analysis of GSKIP knockout mice will be performed with the prospect of gaining insight into the cause of embryonic lethality and into the physiological function of GSKIP.A-Kinase-Ankerproteine (AKAP) binden die cAMP-abhängige Proteinkinase A (PKA) durch direkte Proteininteraktion in spezifischen zellulären Kompartimenten. Dadurch wird die Phosphorylierung nahe gelegener Substrate ermöglicht. Außerdem interagieren AKAP direkt mit weiteren Signalproteinen und können so räumlich und zeitlich verschiedene Signalwege integrieren. In einer in unserer Arbeitsgruppe durchgeführten Datenbanksuche wurde das GSK3β interaction protein (GSKIP) als neues AKAP identifiziert. Seine AKAP-Funktion (Interaktion mit regulatorischen RII-Untereinheiten der PKA) wurde in vitro nachgewiesen. In der vorliegenden Arbeit wurde die AKAP-Funktion von GSKIP durch cAMP- Agarose-Präzipitationen und durch Fluoreszenz-Kreuzkorrelations-Spektroskopie erstmals auch in lebenden Zellen nachgewiesen. Es wurde gezeigt, dass GSKIP ein ubiquitär exprimiertes, zytosolisches AKAP ist, das in vitro einen Komplex mit der RIIα-Untereinheit der PKA und der GSK3β bildet. Weiterhin wurde festgestellt, dass GSKIP in Zellen zu einer Erhöhung der PKA-abhängigen inhibitorischen Phosphorylierung der GSK3β führt. GSKIP ist evolutionär konserviert. Die den RII- und GSK3β-Bindedomänen entsprechenden Regionen verschiedener Orthologe von GSKIP wurden als Peptid-Spots synthetisiert und hinsichtlich einer RIIα- bzw. GSK3β-Bindung untersucht. Dabei wurde festgestellt, dass die Interaktion mit GSK3β in GSKIP-Orthologen aus Pilzen, Invertebraten und Vertebraten konserviert ist. Die RIIα-Bindung hingegen wurde nur für Vertebraten gezeigt. Als Basis für die Aufklärung der physiologischen Funktion von GSKIP wurde in dieser Arbeit ein konditionelles GSKIP-knockout- Mausmodell generiert. Genomische DNA aus der Maus, welche die GSKIP-kodierende Sequenz enthält, wurde für die Klonierung eines Targeting-Vektors verwendet, in dem das Exon 2 des GSKIP-Gens von loxP-Sequenzen flankiert (gefloxt) wurde. Diese ermöglichten eine von der Cre-Rekombinase katalysierte Inaktivierung des Gens. Der Targeting-Vektor wurde sequenzspezifisch in das Genom embryonaler Stamm- (ES)-Zellen integriert. Rekombinante ES-Zellen wurden in Mausembryonen injiziert, aus denen sich Mäuse mit einem gefloxten GSKIP-Allel entwickelten. Diese genetische Modifikation wurde an Folgegenerationen vererbt. Durch das Verpaaren mit einem Cre-Rekombinase exprimierenden Mausstamm wurde die gefloxte Sequenz entfernt. Die resultierenden GSKIP+/--Mäuse waren lebens- und fortpflanzungsfähig. Allerdings brachten GSKIP+/- × GSKIP+/--Verpaarungen keine GSKIP-/--Nachkommen hervor, was auf eine embryonale Lethalität der homozygoten Gendeletion hindeutet. Zukünftig soll eine detaillierte Analyse des Phänotyps der GSKIP-knockout-Maus erfolgen, um die Ursache der embryonalen Lethalität und die physiologische Funktion von GSKIP aufzuklären

A-kinase anchoring proteins as potential drug targets

A-kinase anchoring proteins (AKAPs) crucially contribute to the spatial and temporal control of cellular signalling. They directly interact with a variety of protein binding partners and cellular constituents, thereby directing pools of signalling components to defined locales. In particular, AKAPs mediate compartmentalization of cAMP signalling. Alterations in AKAP expression and their interactions are associated with or cause diseases including chronic heart failure, various cancers and disorders of the immune system such as HIV. A number of cellular dysfunctions result from mutations of specific AKAPs. The link between malfunctions of single AKAP complexes and a disease makes AKAPs and their interactions interesting targets for the development of novel drugs

MicroRNA Biomarkers and Platelet Reactivity:The Clot Thickens

Over the last few years, several groups have evaluated the potential of microRNAs (miRNAs) as biomarkers for cardiometabolic disease. In this review, we discuss the emerging literature on the role of miRNAs and other small noncoding RNAs in platelets and in the circulation, and the potential use of miRNAs as biomarkers for platelet activation. Platelets are a major source of miRNAs, YRNAs, and circular RNAs. By harnessing multiomics approaches, we may gain valuable insights into their potential function. Because not all miRNAs are detectable in the circulation, we also created a gene ontology annotation for circulating miRNAs using the gene ontology term extracellular space as part of blood plasma. Finally, we share key insights for measuring circulating miRNAs. We propose ways to standardize miRNA measurements, in particular by using platelet-poor plasma to avoid confounding caused by residual platelets in plasma or by adding RNase inhibitors to serum to reduce degradation. This should enhance comparability of miRNA measurements across different cohorts. We provide recommendations for future miRNA biomarker studies, emphasizing the need for accurate interpretation within a biological and methodological context

"Young at heart":Regenerative potential linked to immature cardiac phenotypes

AbstractThe adult human myocardium is incapable of regeneration; yet, the zebrafish (Danio rerio) can regenerate damaged myocardium. Similar to the zebrafish heart, hearts of neonatal, but not adult mice are capable of myocardial regeneration. We performed a proteomics analysis of adult zebrafish hearts and compared their protein expression profile to hearts from neonatal and adult mice. Using difference in-gel electrophoresis (DIGE), there was little overlap between the proteome from adult mouse (>8weeks old) and adult zebrafish (18months old) hearts. Similarly, there was a significant degree of mismatch between the protein expression in neonatal and adult mouse hearts. Enrichment analysis of the selected proteins revealed over-expression of DNA synthesis-related proteins in the cardiac proteome of the adult zebrafish heart similar to neonatal and 4days old mice, whereas in hearts of adult mice there was a mitochondria-related predominance in protein expression. Importantly, we noted pronounced differences in the myofilament composition: the adult zebrafish heart lacks many of the myofilament proteins of differentiated adult cardiomyocytes such as the ventricular isoforms of myosin light chains and nebulette. Instead, troponin I and myozenin 1 were expressed as skeletal isoforms rather than cardiac isoforms. The relative immaturity of the adult zebrafish heart was further supported by cardiac microRNA data. Our assessment of zebrafish and mammalian hearts challenges the assertions on the translational potential of cardiac regeneration in the zebrafish model. The immature myofilament composition of the fish heart may explain why adult mouse and human cardiomyocytes lack this endogenous repair mechanism