Histological assessment of cardiac amyloidosis in patients undergoing transcatheter aortic valve replacement.

AIMS: Studies have reported a strongly varying co-prevalence of aortic stenosis (AS) and cardiac amyloidosis (CA). We sought to histologically determine the co-prevalence of AS and CA in patients undergoing transcatheter aortic valve replacement (TAVR). Consequently, we aimed to derive an algorithm to identify cases in which to suspect the co-prevalence of AS and CA. METHODS AND RESULTS: In this prospective, monocentric study, endomyocardial biopsies of 162 patients undergoing TAVR between January 2017 and March 2021 at the University Medical Centre Göttingen were analysed by one pathologist blinded to clinical data using haematoxylin-eosin staining, Elastica van Gieson staining, and Congo red staining of endomyocardial biopsies. CA was identified in only eight patients (4.9%). CA patients had significantly higher N-terminal pro-brain natriuretic peptide (NT-proBNP) levels (4356.20 vs. 1938.00 ng/L, P = 0.034), a lower voltage-to-mass ratio (0.73 vs. 1.46 × 10-2 mVm2/g, P = 0.022), and lower transaortic gradients (Pmean 17.5 vs. 38.0 mmHg, P = 0.004) than AS patients. Concomitant CA was associated with a higher prevalence of post-procedural acute kidney injury (50.0% vs. 13.1%, P = 0.018) and sudden cardiac death [SCD; P (log-rank test) = 0.017]. Following propensity score matching, 184 proteins were analysed to identify serum biomarkers of concomitant CA. CA patients expressed lower levels of chymotrypsin (P = 0.018) and carboxypeptidase 1 (P = 0.027). We propose an algorithm using commonly documented parameters-stroke volume index, ejection fraction, NT-proBNP levels, posterior wall thickness, and QRS voltage-to-mass ratio-to screen for CA in AS patients, reaching a sensitivity of 66.6% with a specificity of 98.1%. CONCLUSIONS: The co-prevalence of AS and CA was lower than expected, at 4.9%. Despite excellent 1 year mortality, AS + CA patients died significantly more often from SCD. We propose a multimodal algorithm to facilitate more effective screening for CA containing parameters commonly documented during clinical routine. Proteomic biomarkers may yield additional information in the future.

Low-flow in aortic valve stenosis patients with reduced ejection fraction does not depend on left ventricular function.

BACKGROUND: Patients with severe aortic stenosis (AS) and reduced left ventricular ejection fraction (LVEF) can be distinguished into high- (HG) and low-gradient (LG) subgroups. However, less is known about their characteristics and underlying (pathophysiological) hemodynamic mechanisms. METHODS: 98 AS patients with reduced LVEF were included. Subgroup characteristics were analyzed by a multimodal approach using clinical and histological data, next-generation sequencing (NGS) and applying echocardiography as well as cardiovascular magnetic resonance (CMR) imaging. Biopsy samples were analyzed with respect to fibrosis and mRNA expression profiles. RESULTS: 40 patients were classified as HG-AS and 58 patients as LG-AS. Severity of AS was comparable between the subgroups. Comparison of both subgroups revealed no differences in LVEF (p = 0.1), LV mass (p = 0.6) or end-diastolic LV diameter (p = 0.12). Neither histological (HG: 23.2% vs. LG: 25.6%, p = 0.73) and circulating biomarker-based assessment (HG: 2.6 ± 2.2% vs. LG: 3.2 ± 3.1%; p = 0.46) of myocardial fibrosis nor global gene expression patterns differed between subgroups. Mitral regurgitation (MR), atrial fibrillation (AF) and impaired right ventricular function (MR: HG: 8% vs. LG: 24%; p < 0.001; AF: HG: 30% vs. LG: 51.7%; p = 0.03; RVSVi: HG 36.7 vs. LG 31.1 ml/m2, p = 0.045; TAPSE: HG 20.2 vs. LG 17.3 mm, p = 0.002) were more frequent in LG-AS patients compared to HG-AS. These pathologies could explain the higher mortality of LG vs. HG-AS patients. CONCLUSION: In patients with low-flow severe aortic stenosis, low transaortic gradient and cardiac output are not primarily due to LV dysfunction or global changes in gene expression, but may be attributed to other additional cardiac pathologies like mitral regurgitation, atrial fibrillation or right ventricular dysfunction. These factors should also be considered during planning of aortic valve replacement.

DNA Methylation Analysis Identifies Novel Epigenetic Loci in Dilated Murine Heart upon Exposure to Volume Overload.

Left ventricular (LV) dilatation, a prominent risk factor for heart failure (HF), precedes functional deterioration and is used to stratify patients at risk for arrhythmias and cardiac mortality. Aberrant DNA methylation contributes to maladaptive cardiac remodeling and HF progression following pressure overload and ischemic cardiac insults. However, no study has examined cardiac DNA methylation upon exposure to volume overload (VO) despite being relatively common among HF patients. We carried out global methylome analysis of LV harvested at a decompensated HF stage following exposure to VO induced by aortocaval shunt. VO resulted in pathological cardiac remodeling, characterized by massive LV dilatation and contractile dysfunction at 16 weeks after shunt. Although methylated DNA was not markedly altered globally, 25 differentially methylated promoter regions (DMRs) were identified in shunt vs. sham hearts (20 hypermethylated and 5 hypomethylated regions). The validated hypermethylated loci in Junctophilin-2 (Jph2), Signal peptidase complex subunit 3 (Spcs3), Vesicle-associated membrane protein-associated protein B (Vapb), and Inositol polyphosphate multikinase (Ipmk) were associated with the respective downregulated expression and were consistently observed in dilated LV early after shunt at 1 week after shunt, before functional deterioration starts to manifest. These hypermethylated loci were also detected peripherally in the blood of the shunt mice. Altogether, we have identified conserved DMRs that could be novel epigenetic biomarkers in dilated LV upon VO exposure.

Metabolomic Profiling in Patients with Different Hemodynamic Subtypes of Severe Aortic Valve Stenosis.

Severe aortic stenosis (AS) is a common pathological condition in an ageing population imposing significant morbidity and mortality. Based on distinct hemodynamic features, i.e., ejection fraction (EF), transvalvular gradient and stroke volume, four different AS subtypes can be distinguished: (i) normal EF and high gradient, (ii) reduced EF and high gradient, (iii) reduced EF and low gradient, and (iv) normal EF and low gradient. These subtypes differ with respect to pathophysiological mechanisms, cardiac remodeling, and prognosis. However, little is known about metabolic changes in these different hemodynamic conditions of AS. Thus, we carried out metabolomic analyses in serum samples of 40 AS patients (n = 10 per subtype) and 10 healthy blood donors (controls) using ultrahigh-performance liquid chromatography-tandem mass spectroscopy. A total of 1293 biochemicals could be identified. Principal component analysis revealed different metabolic profiles in all of the subgroups of AS (All-AS) vs. controls. Out of the determined biochemicals, 48% (n = 620) were altered in All-AS vs. controls (p < 0.05). In this regard, levels of various acylcarnitines (e.g., myristoylcarnitine, fold-change 1.85, p < 0.05), ketone bodies (e.g., 3-hydroxybutyrate, fold-change 11.14, p < 0.05) as well as sugar metabolites (e.g., glucose, fold-change 1.22, p < 0.05) were predominantly increased, whereas amino acids (e.g., leucine, fold-change 0.8, p < 0.05) were mainly reduced in All-AS. Interestingly, these changes appeared to be consistent amongst all AS subtypes. Distinct differences between AS subtypes were found for metabolites belonging to hemoglobin metabolism, diacylglycerols, and dihydrosphingomyelins. These findings indicate that relevant changes in substrate utilization appear to be consistent for different hemodynamic subtypes of AS and may therefore reflect common mechanisms during AS-induced heart failure. Additionally, distinct metabolites could be identified to significantly differ between certain AS subtypes. Future studies need to define their pathophysiological implications.

Hemodynamic stress-induced cardiac remodelling is not modulated by ablation of phosphodiesterase 4D interacting protein.

Adrenergic stimulation in the heart activates the protein kinase A (PKA), which phosphorylates key proteins involved in intracellular Ca2+ handling. PKA is held in proximity to its substrates by protein scaffolds, the A kinase anchoring proteins (AKAPs). We have previously identified the transcript of phosphodiesterase 4D interacting protein (Pde4dip; also known as myomegalin), one of the sarcomeric AKAPs, as being differentially expressed following hemodynamic overload, a condition inducing hyperadrenergic state in the heart. Here, we addressed whether PDE4DIP is involved in the adverse cardiac remodelling following hemodynamic stress. Homozygous Pde4dip knockout (KO) mice, generated by CRISPR-Cas9 technology, and wild-type (WT) littermates were exposed to aortocaval shunt (shunt) or transthoracic aortic constriction (TAC) to induce hemodynamic volume overload (VO) or pressure overload (PO), respectively. The mortality, cardiac structure, function and pathological cardiac remodelling were followed up after hemodynamic injuries. The PDE4DIP protein level was markedly downregulated in volume-overloaded- but upregulated in pressure-overloaded-WT hearts. Following shunt or TAC, mortality rates were comparably increased in both genotypes. Twelve weeks after shunt or TAC, Pde4dip-KO animals showed a similar degree of cardiac hypertrophy, dilatation and dysfunction as WT mice. Cardiomyocyte hypertrophy, myocardial fibrosis, reactivation of cardiac stress genes and downregulation of ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2 transcript did not differ between WT and Pde4dip-KO hearts following shunt or TAC. In summary, despite a differential expression of PDE4DIP protein in remodelled WT hearts, Pde4dip deficiency does not modulate adverse cardiac remodelling after hemodynamic VO or PO.

Heat shock protein A4 ablation leads to skeletal muscle myopathy associated with dysregulated autophagy and induced apoptosis.

BACKGROUND: Molecular chaperones assist protein folding, facilitate degradation of misfolded polypeptides, and thereby maintain protein homeostasis. Impaired chaperone activity leads to defective protein quality control that is implicated in multiple skeletal muscle diseases. The heat shock protein A4 (HSPA4) acts as a co-chaperone for HSP70. Previously, we showed that Hspa4 deletion causes impaired protein homeostasis in the heart. However, its functional role in skeletal muscle has not been explored. METHODS: We performed a comparative phenotypic and biochemical analyses of Hspa4 knockout (KO) mice with wild-type (WT) littermates. RESULTS: HSPA4 is markedly upregulated in regenerating WT muscle in vivo, and in differentiated myoblasts in vitro. Hspa4-KO mice are marked by growth retardation and increased variability in body weight, accompanied by 35% mortality rates during the peri-weaning period. The surviving Hspa4-KO mice experienced progressive skeletal muscle myopathy, characterized by increased number of muscle fibers with centralized nuclei, heterogeneous myofiber size distribution, inflammatory cell infiltrates and upregulation of embryonic and perinatal myosin heavy chain transcripts. Hspa4-KO muscles demonstrated an accumulation of autophagosome-associated proteins including microtubule associated protein1 light chain 3-II (LC3-II) and p62/sequestosome accompanied by increased number of TUNEL-positive nuclei. CONCLUSIONS: Our findings underscore the indispensable role of HSPA4 in maintenance of muscle integrity through contribution in skeletal muscle autophagy and apoptosis, which might provide a novel therapeutic strategy for skeletal muscle morbidities.

Murine Embryonic Stem Cell Culture, Self-Renewal, and Differentiation.

Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, can proliferate indefinitely in vitro (self-renewal) and can differentiate into cells of all three germ layers (pluripotency). These unique properties make them exceptionally valuable in basic science and clinical researches, including cell replacement therapies, drug discovery, and regenerative medicine. Mouse ESCs represent an important model system for studying gene function during development and disease.ESCs culture is time-consuming, laborious, and costly. Suboptimal ESCs culture conditions can alter their identity, pluripotency, and their compatibility with downstream differentiation protocols. In this chapter, we provide a general guideline for murine ESCs culture on murine fibroblast feeder layers. Moreover, we describe protocols for maintenance of ESCs pluripotency and induction of ESCs differentiation.

Perturbed differentiation of murine embryonic stem cells upon Pelota deletion due to dysregulated FOXO1/ß-catenin signaling.

Differentiation of the embryonic stem cells (ESCs) is regulated by a variety of different signaling pathways. Genetic depletion of murine Pelota gene (Pelo) leads to early embryonic lethality. Here, we aimed at determining the embryonic stage and deciphering the dysregulated signaling pathways affected upon Pelo deletion. We found that development of PELO-null embryos is perturbed between the embryonic days E4.5 and E5.5, at which first differentiation process of ESCs takes place. Molecular analysis revealed enhanced activity of phosphoinositide 3-kinase-protein kinase B/ AKT (PI3K-PKB/AKT) signaling, but nuclear accumulation of forkhead box O1 (FOXO1), and upregulation of the pluripotency-related gene, Oct4, in mutant ESCs cultured under differentiation condition. Despite increased levels of nuclear ß-catenin in PELO-null ESCs as a result of decreased activity of glycogen synthase kinase-3ß, the activity of the canonical wingless (Wnt)/ß-catenin/T-cell factor (TCF) was significantly attenuated as judged by the promoter reporter assay, downregulated Wnt/ß-catenin target genes, and impaired cell proliferation. Interestingly, we demonstrated an increased binding of ß-catenin to FOXO1 in PELO-mutant ESCs cultured under differentiation condition that could explain, on one side, the nuclear accumulation of FOXO1 protein and hence persistent pluripotency of PELO-mutant ESCs, and on the other side, the dysregulated transcriptional activity of ß-catenin/TCF and therefore attenuated PELO-null ESC self-renewal. Taken together, our results strongly suggest that PELO deletion averts ESC differentiation through promoting FOXO1/ß-catenin binding with subsequent dysregulation of FOXO1 and canonical ß-catenin/TCF signaling pathways.

In vivo [U-13C]glucose labeling to assess heart metabolism in murine models of pressure and volume overload.

Alterations in the metabolism of substrates such as glucose are integrally linked to the structural and functional changes that occur in the remodeling heart. Assessment of such metabolic changes under in vivo conditions would provide important insights into this interrelationship. We aimed to investigate glucose carbon metabolism in pressure-overload and volume-overload cardiac hypertrophy by using an in vivo [U-13C]glucose labeling strategy to enable analyses of the metabolic fates of glucose carbons in the mouse heart. Therefore, [U-13C]glucose was administered in anesthetized mice by tail vein infusion, and the optimal duration of infusion was established. Hearts were then excised for 13C metabolite isotopomer analysis by NMR spectroscopy. [U-13C]glucose infusions were performed in mice 2 wk following transverse aortic constriction (TAC) or aortocaval fistula (Shunt) surgery. At this time point, there were similar increases in left ventricular (LV) mass in both groups, but TAC resulted in concentric hypertrophy with impaired LV function, whereas Shunt caused eccentric hypertrophy with preserved LV function. TAC was accompanied by significant changes in glycolysis, mitochondrial oxidative metabolism, glucose metabolism to anaplerotic substrates, and de novo glutamine synthesis. In contrast to TAC, hardly any metabolic changes could be observed in the Shunt group. Taken together, in vivo [U-13C]glucose labeling is a valuable method to investigate the fate of nutrients such as glucose in the remodeling heart. We find that concentric and eccentric cardiac remodeling are accompanied by distinct differences in glucose carbon metabolism.NEW & NOTEWORTHY This study implemented a method for assessing the fate of glucose carbons in the heart in vivo and used this to demonstrate that pressure and volume overload are associated with distinct changes. In contrast to volume overload, pressure overload-induced changes affect the tricarboxylic acid cycle, glycolytic pathways, and glutamine synthesis. A better understanding of cardiac glucose metabolism under pathological conditions in vivo may provide new therapeutic strategies specific for different types of hemodynamic overload.

Assessing the role of extracellular signal-regulated kinases 1 and 2 in volume overload-induced cardiac remodelling.

AIMS: Volume overload (VO) and pressure overload (PO) induce differential cardiac remodelling responses including distinct signalling pathways. Extracellular signal-regulated kinases 1 and 2 (ERK1/2), key signalling components in the mitogen-activated protein kinase (MAPK) pathways, modulate cardiac remodelling during pressure overload (PO). This study aimed to assess their role in VO-induced cardiac remodelling as this was unknown. METHODS AND RESULTS: Aortocaval fistula (Shunt) surgery was performed in mice to induce cardiac VO. Two weeks of Shunt caused a significant reduction of cardiac ERK1/2 activation in wild type (WT) mice as indicated by decreased phosphorylation of the TEY (Thr-Glu-Tyr) motif (-28% as compared with Sham controls, P < 0.05). Phosphorylation of other MAPKs was unaffected. For further assessment, transgenic mice with cardiomyocyte-specific ERK2 overexpression (ERK2tg) were studied. At baseline, cardiac ERK1/2 phosphorylation in ERK2tg mice remained unchanged compared with WT littermates, and no overt cardiac phenotype was observed; however, cardiac expression of the atrial natriuretic peptide was increased on messenger RNA (3.6-fold, P < 0.05) and protein level (3.1-fold, P < 0.05). Following Shunt, left ventricular dilation and hypertrophy were similar in ERK2tg mice and WT littermates. Left ventricular function was maintained, and changes in gene expression indicated reactivation of the foetal gene program in both genotypes. No differences in cardiac fibrosis and kinase activation was found amongst all experimental groups, whereas apoptosis was similarly increased through Shunt in ERK2tg and WT mice. CONCLUSIONS: VO-induced eccentric hypertrophy is associated with reduced cardiac ERK1/2 activation in vivo. Cardiomyocyte-specific overexpression of ERK2, however, does not alter cardiac remodelling during VO. Future studies need to define the pathophysiological relevance of decreased ERK1/2 signalling during VO.

Genetic deletion of calcium/calmodulin-dependent protein kinase type II delta does not mitigate adverse myocardial remodeling in volume-overloaded hearts.

Calcium/calmodulin-dependent protein kinase type II delta (CaMKIIδ), the predominant CaMKII isoform expressed in the heart, has been implicated in the progression of myocardial infarction- and pressure overload-induced pathological remodeling. However, the role of CaMKIIδ in volume overload (VO) has not been explored. We have previously reported an activation of CaMKII during transition to HF in long-term VO. Here, we address whether CaMKIIδ is critically involved in the mortality, myocardial remodeling, and heart failure (HF) progression in response to VO. CaMKIIδ knockout (δ-KO) and wild-type (WT) littermates were exposed to aortocaval shunt-induced VO, and the progression of adverse myocardial remodeling was assessed by serial echocardiography, histological and molecular analyses. The mortality rates during 10 weeks of VO were similar in δ-KO and WT mice. Both genotypes displayed comparable eccentric myocardial hypertrophy, altered left ventricle geometry, perturbed systolic and diastolic functions after shunt. Additionally, cardiomyocytes hypertrophy, augmented myocyte apoptosis, and up-regulation of hypertrophic genes were also not significantly different in δ-KO versus WT hearts after shunt. Therefore, CaMKIIδ signaling seems to be dispensable for the progression of VO-induced maladaptive cardiac remodeling. Accordingly, we hypothesize that CaMKIIδ-inhibition as a therapeutic approach might not be helpful in the context of VO-triggered HF.

Pelota Regulates Epidermal Differentiation by Modulating BMP and PI3K/AKT Signaling Pathways.

The depletion of evolutionarily conserved pelota protein causes impaired differentiation of embryonic and spermatogonial stem cells. In this study, we show that temporal deletion of pelota protein before epidermal barrier acquisition leads to neonatal lethality due to perturbations in permeability barrier formation. Further analysis indicated that this phenotype is a result of failed processing of profilaggrin into filaggrin monomers, which promotes the formation of a protective epidermal layer. Molecular analyses showed that pelota protein negatively regulates the activities of bone morphogenetic protein and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in the epidermis. To address whether elevated activities of bone morphogenetic protein and PI3K/AKT signaling pathways were the cause for the perturbed epidermal barrier in Pelo-deficient mice, we made use of organotypic cultures of skin explants from control and mutant embryos at embryonic day 15.5. Inhibition of PI3K/AKT signaling did not significantly affect the bone morphogenetic protein activity. However, inhibition of bone morphogenetic protein signaling caused a significant attenuation of PI3K/AKT activity in mutant skin and, more interestingly, the restoration of profilaggrin processing and normal epidermal barrier function. Therefore, increased activity of the PI3K/AKT signaling pathway in Pelo-deficient skin might conflict with the dephosphorylation of profilaggrin and thereby affect its proper processing into filaggrin monomers and ultimately the epidermal differentiation.

Pelota mediates gonocyte maturation and maintenance of spermatogonial stem cells in mouse testes.

Pelota (Pelo) is an evolutionarily conserved gene, and its deficiency in Drosophila affects both male and female fertility. In mice, genetic ablation of Pelo leads to embryonic lethality at the early implantation stage as a result of the impaired development of extra-embryonic endoderm (ExEn). To define the consequences of Pelo deletion on male germ cells, we temporally induced deletion of the gene at both embryonic and postnatal stages. Deletion of Pelo in adult mice resulted in a complete loss of whole-germ cell lineages after 45 days of deletion. The absence of newly emerging spermatogenic cycles in mutants confirmed that spermatogonial stem cells (SSCs) were unable to maintain spermatogenesis in the absence of PELO protein. However, germ cells beyond the undifferentiated SSC stage were capable of completing spermatogenesis and producing spermatozoa, even in the absence of PELO. Following the deletion of Pelo during embryonic development, we found that although PELO is dispensable for maintaining gonocytes, it is necessary for the transition of gonocytes to SSCs. Immunohistological and protein analyses revealed the attenuation of FOXO1 transcriptional activity, which induces the expression of many SSC self-renewal genes. The decreased transcriptional activity of FOXO1 in mutant testes was due to enhanced activity of the PI3K/AKT signaling pathway, which led to phosphorylation and cytoplasmic sequestration of FOXO1. These results suggest that PELO negatively regulates the PI3K/AKT pathway and that the enhanced activity of PI3K/AKT and subsequent FOXO1 inhibition are responsible for the impaired development of SSCs in mutant testes.

Respiratory distress and early neonatal lethality in Hspa4l/Hspa4 double-mutant mice.

Heat shock proteins HSPA4L and HSPA4 are closely related members of the HSP110 family and act as cochaperones. We generated Hspa4l(-/-)Hspa4(-/-) mice to investigate a functional complementarity between HSPA4L and HSPA4 during embryonic development. Hspa4l(-/-)Hspa4(-/-) embryos exhibited marked pulmonary hypoplasia and neonatal death. Compared with lungs of wild-type, Hspa4l(-/-), and Hspa4(-/-) embryos, Hspa4l(-/-)Hspa4(-/-) lungs were characterized by diminished saccular spaces and increased mesenchymal septa. Mesenchymal hypercellularity was determined to be due to an increased cell proliferation index and decreased cell death. A significant increase in expression levels of prosurvival protein B cell leukemia/lymphoma 2 may be the cause for inhibition of apoptotic process in lungs of Hspa4(-/-)Hspa4l(-/-) embryos. Accumulation of glycogen and diminished expression of surfactant protein B, prosurfactant protein C, and aquaporin 5 in saccular epithelium suggested impaired maturation of type II and type I pneumocytes in the Hspa4l(-/-)Hspa4(-/-) lungs. Further experiments showed a significant accumulation of ubiquitinated proteins in the lungs of Hspa4l(-/-)Hspa4(-/-) embryos, indicating an impaired chaperone activity. Our study demonstrates that HSPA4L and HSPA4 collaborate in embryonic lung maturation, which is necessary for adaptation to air breathing at birth.

Zfp819, a novel KRAB-zinc finger protein, interacts with KAP1 and functions in genomic integrity maintenance of mouse embryonic stem cells.

Pluripotency is maintained by both known and unknown transcriptional regulatory networks. In the present study, we have identified Zfp819, a KRAB-zinc finger protein, as a novel pluripotency-related factor and characterized its role in pluripotent stem cells. We show that Zfp819 is expressed highly in various types of pluripotent stem cells but not in their differentiated counterparts. We identified the presence of non-canonical nuclear localization signals in particular zinc finger motifs and identified them as responsible for the nuclear localization of Zfp819. Analysis of the Zfp819 promoter region revealed the presence of a transcriptionally active chromatin signature. Moreover, we confirmed the binding of pluripotency-related factors, Oct4, Sox2, and Nanog to the distal promoter region of Zfp819, indicating that the expression of this gene is regulated by a pluripotency transcription factor network. We found that the expression of endogenous retroviral elements (ERVs) such as Intracisternal A Particle (IAP) retrotransposons, Long Interspersed Nuclear Elements (LINE1), and Short Interspersed Nuclear Elements (SINE B1) is significantly upregulated in Zfp819-knockdown (Zfp819_KD) cells. In line with the activation of ERVs, we observed the occurrence of spontaneous DNA damage in Zfp819_KD cells. Furthermore, we tested whether Zfp819 can interact with KAP1, a KRAB-associated protein with a transcriptional repression function, and found the interaction between these two proteins in both in vitro and in vivo experiments. The challenging of Zfp819_KD cells with DNA damaging agent revealed that these cells are inefficient in repairing the damaged DNA, as cells showed presence of γH2A.X foci for a prolonged time. Collectively, our study identified Zfp819 as a novel pluripotency-related factor and unveiled its function in genomic integrity maintenance mechanisms of mouse embryonic stem cells.

Spongiform encephalopathy in siblings with no evidence of protease-resistant prion protein or a mutation in the prion protein gene.

We discuss relevant aspects in two siblings with a neurodegenerative process of unclear aetiology who developed progressive dementia with global aphasia and hyperoral behaviour at the ages of 39 and 46 years and who died 6 and 5 years after disease onset. The cases were reported to the National Reference Center for TSE Surveillance in Göttingen, Germany. Detailed clinical examinations, CSF, blood samples, and copies of the important diagnostic tests (magnetic resonance imaging, electroencephalogram, laboratory tests) were obtained. Further neuropathological and genetic analyses were performed. Cerebral magnetic resonance imaging of both siblings showed prominent changes in signal intensity, especially in the left medial temporal cortex, but also the hippocampal formation. Neuropathological examination revealed spongiform changes, neuronal loss, and astrocytic gliosis, which are typical in Creutzfeldt-Jakob disease. However, no prion protein deposits were detectable by immunohistochemical analysis, Western blot, or PET blot, though abundant tau protein deposits were observed. A mutation in the coding region of the prion protein genes of both siblings was excluded. A detailed search of the literature revealed no other cases with a similar clinical and neuropathological appearance. While the disease aetiology remains unclear, the findings point to a neurodegenerative process and most likely a genetic disease.