Loss of Furin in ß-Cells Induces an mTORC1-ATF4 Anabolic Pathway That Leads to ß-Cell Dysfunction.

FURIN is a proprotein convertase (PC) responsible for proteolytic activation of a wide array of precursor proteins within the secretory pathway. It maps to the PRC1 locus, a type 2 diabetes susceptibility locus, but its specific role in pancreatic ß-cells is largely unknown. The aim of this study was to determine the role of FURIN in glucose homeostasis. We show that FURIN is highly expressed in human islets, whereas PCs that potentially could provide redundancy are expressed at considerably lower levels. ß-cell-specific Furin knockout (ßFurKO) mice are glucose intolerant as a result of smaller islets with lower insulin content and abnormal dense-core secretory granule morphology. mRNA expression analysis and differential proteomics on ßFurKO islets revealed activation of activating transcription factor 4 (ATF4), which was mediated by mammalian target of rapamycin C1 (mTORC1). ßFurKO cells show impaired cleavage or shedding of vacuolar-type ATPase (V-ATPase) subunits Ac45 and prorenin receptor, respectively, and impaired lysosomal acidification. Blocking V-ATPase pharmacologically in ß-cells increased mTORC1 activity, suggesting involvement of the V-ATPase proton pump in the phenotype. Taken together, these results suggest a model of mTORC1-ATF4 hyperactivation and impaired lysosomal acidification in ß-cells lacking Furin, causing ß-cell dysfunction.

Differentiation of Human Cardiac Atrial Appendage Stem Cells into Adult Cardiomyocytes: A Role for the Wnt Pathway?

Human cardiac stem cells isolated from atrial appendages based on aldehyde dehydrogenase activity (CASCs) can be expanded in vitro and differentiate into mature cardiomyocytes. In this study, we assess whether Wnt activation stimulates human CASC proliferation, whereas Wnt inhibition induces cardiac maturation. CASCs were cultured as described before. Conventional PCR confirmed the presence of the Frizzled receptors. Small-molecule inhibitors (IWP2, C59, XAV939, and IWR1-endo) and activator (CHIR99021) of the Wnt/ß -catenin signaling pathway were applied, and the effect on ß-catenin and target genes for proliferation and differentiation was assessed by Western blot and RT-qPCR. CASCs express multiple early cardiac differentiation markers and are committed toward myocardial differentiation. They express several Frizzled receptors, suggesting a role for Wnt signaling in clonogenicity, proliferation, and differentiation. Wnt activation increases total and active ß-catenin levels. However, this does not affect CASC proliferation or clonogenicity. Wnt inhibition upregulated early cardiac markers but could not induce mature myocardial differentiation. When CASCs are committed toward myocardial differentiation, the Wnt pathway is active and can be modulated. However, despite its role in cardiogenesis and myocardial differentiation of pluripotent stem-cell populations, our data indicate that Wnt signaling has limited effects on CASC clonogenicity, proliferation, and differentiation.

Equine Allogeneic Chondrogenic Induced Mesenchymal Stem Cells Are an Effective Treatment for Degenerative Joint Disease in Horses.

Degenerative joint disease is one of the main causes of equine early retirement from pleasure riding or a performance career. The disease is initially triggered by an abnormal loading of normal cartilage or a normal loading of abnormal cartilage. This primary insult is accompanied with joint inflammation, which leads to further progressive degeneration of the articular cartilage and changes in the surrounding tissues. Therefore, in search for an effective treatment, 75 adult horses with early signs of degenerative fetlock joint disease were enrolled in a randomized, multicenter, double-blinded, and placebo-controlled study. Fifty animals were injected intra-articularly with the investigational veterinary product (IVP) consisting of allogeneic chondrogenic induced mesenchymal stem cells (ciMSCs) with equine allogeneic plasma, and 25 horses were injected with 0.9% NaCl (saline) control product. From week 3 to 18 after treatment, lameness scores (P < 0.001), flexion test responses (P < 0.034), and joint effusion scores (P < 0.001) were remarkably superior in IVP-treated horses. Besides nasal discharge in both treatment groups, no adverse events were observed during the entire study period. On long-term follow-up (1 year), significantly more investigational product-treated horses were working at training level or were returned to their previous level of work (P < 0.001).

Archival May-Grünwald-Giemsa-Stained Bone Marrow Smears Are an Eligible Source for Molecular DNA Research.

Biobanking is increasingly important in studying complex heterogeneous diseases. Therefore, it is essential to ensure the sample quality after long-term storage for reliable downstream analyses. The Clinical Biobank of the Jessa Hospital and the University Biobank Limburg (UBiLim) hold a continuously growing collection of hematological samples, including May-Grünwald-Giemsa (MGG)- and Perls' Prussian Blue (PPB)-stained bone marrow (BM) smears, stored at room temperature (RT) for up to 20 years. In this study, we investigated the effect of short- and long-term storage on the quality of DNA and RNA extracted from these BM smears to assess their fitness-for-purpose in downstream molecular applications, including agarose gel electrophoresis, bio-analyzer analysis, quantitative polymerase chain reaction (qPCR), and targeted next-generation sequencing (NGS). The RNA quality was very low for all samples, independent of storage time or staining method. The DNA from PPB-stained BM smears was already degraded after 1 year of storage and correspondingly could not be used for reliable downstream molecular analysis. In contrast, DNA extracted from MGG-stained BM smears stored for up to 10 years was able to generate high-quality data in qPCR and targeted NGS analyses. Longer storage periods (>15 years) of these samples revealed a high degree of degradation and a significant amount of DNA transitions and transversions. In conclusion, the DNA extracted from archival MGG-stained BM smears with a storage time up to at least 10 years was qualitatively good and fit for downstream analysis, including targeted NGS. This indicates that these samples are an eligible source for molecular DNA research and for studying complex diseases.

Prognostic Biomarkers in the Progression From MGUS to Multiple Myeloma: A Systematic Review.

Multiple myeloma (MM), characterized by malignant plasma cells in the bone marrow, is consistently preceded by asymptomatic premalignant stage monoclonal gammopathy of undetermined significance (MGUS). These MGUS patients have an annual risk of 1% to progress to MM. Clinical, imaging, and genomic (genetic and epigenetic) factors were identified, whose presence increased the risk of progression from MGUS to MM. In this systematic review we summarize the currently identified clinical, imaging, and genomic biomarkers suggested to increase the progression risk or shown to be differentially expressed/present between both cohorts of patients. Despite the wide range of proposed markers, there are still no reliable biomarkers to individually predict which MGUS patient will progress to MM and which will not. Research on biomarkers in the progression from MGUS to MM will give more insight in the unknown pathogenesis of this hematological malignancy. This would improve research by elucidating new pathways and potential therapeutic targets as well as clinical management by closer follow-up and earlier treatment of high-risk MGUS patients.

Tenogenically Induced Allogeneic Peripheral Blood Mesenchymal Stem Cells in Allogeneic Platelet-Rich Plasma: 2-Year Follow-up after Tendon or Ligament Treatment in Horses.

Poor healing of tendon and ligament lesions often results in early retirement of sport horses. Therefore, regenerative therapies are being explored as potentially promising treatment for these injuries. In this study, an intralesional injection was performed with allogeneic tenogenically induced mesenchymal stem cells and platelet-rich plasma 5-6 days after diagnosis of suspensory ligament (SL) (n = 68) or superficial digital flexor tendon (SDFT) (n = 36) lesion. Clinical, lameness and ultrasonographic evaluation was performed at 6 and 12 weeks. Moreover, a survey was performed 12 and 24 months after treatment to determine how many horses were competing at original level and how many were re-injured. At 6 weeks, 88.2% of SL (n = 68) and 97.3% of SDFT lesions (n = 36) demonstrated moderate ultrasonographic improvement. At 12 weeks, 93.1% of SL (n = 29) and 95.5% of SDFT lesions (n = 22) improved convincingly. Moreover, lameness was abolished in 78.6% of SL (n = 28) and 85.7% (n = 7) of SDFT horses at 12 weeks. After 12 months (n = 92), 11.8% of SL and 12.5% of SDFT horses were re-injured, whereas 83.8 of SL and 79.2% of SDFT returned to previous performance level. At 24 months (n = 89) after treatment, 82.4 (SL) and 85.7% (SDFT) of the horses returned to previous level of performance. A meta-analysis was performed on relevant published evidence evaluating re-injury 24 months after stem cell-based [17.6% of the SL and 14.3% of the SDFT group (n = 89)] versus conventional therapies. Cell therapies resulted in a significantly lower re-injury rate of 18% [95% confidence interval (CI), 0.11-0.25] 2 years after treatment compared to the 44% re-injury rate with conventional treatments (95% CI, 0.37-0.51) based on literature data (P < 0.0001).

Endosome to trans-Golgi network transport of Proprotein Convertase 7 is mediated by a cluster of basic amino acids and palmitoylated cysteines.

Proprotein Convertase 7 (PC7) is a Furin-like endoprotease that cleaves precursor proteins at basic amino acids. PC7 is concentrated in the trans-Golgi network (TGN) but it shuttles between the plasma membrane and the TGN depending on sequences in the cytoplasmic tail. A short region containing a three amino acids motif, P724-L725-C726, is essential and sufficient for internalization of PC7 but not for TGN localization, which requires the additional presence of the juxtamembrane region. In this study we have investigated the contribution of a cluster of basic amino acids and two reversibly palmitoylated cysteine residues to endocytic trafficking. Stable cell lines overexpressing chimeric proteins (CD25 and CD46) containing the cytoplasmic domain of PC7 in which the basic cluster alone or together with both palmitoylated cysteines are mutated showed enhanced surface expression as demonstrated by immunofluorescence experiments and surface biotinylation. The mutant proteins no longer recycled to the TGN in antibody uptake experiments and accumulated in an endosomal compartment. Recycling of wild type PC7 to the TGN is blocked by nocodazole, suggesting that PC7 shuttles to the TGN via late endosomes, similar to Furin. Unlike furin, however, PC7 was found to recycle to a region within the TGN, which is deficient in sialyltransferase, as shown by resialylation experiments. In conclusion, a novel motif, composed of a basic amino acid cluster and two palmitoylated cysteines are essential for TGN localization and endocytic trafficking.

Cardiac atrial appendage stem cells promote angiogenesis in vitro and in vivo.

Cardiac atrial appendage stem cells (CASCs) show extraordinary myocardial differentiation properties, making them ideal candidates for myocardial regeneration. However, since the myocardium is a highly vascularized tissue, revascularization of the ischemic infarct area is essential for functional repair. Therefore, this study assessed if CASCs contribute to cardiac angiogenesis via paracrine mechanisms. First, it was demonstrated that CASCs produce and secrete high levels of numerous angiogenic growth factors, including vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) and insulin-like growth factor binding protein 3 (IGFBP-3). Functional in vitro assays with a human microvascular endothelial cell line (HMEC-1) and CASC CM showed that CASCs promote endothelial cell proliferation, migration and tube formation, the most important steps of the angiogenesis process. Addition of inhibitory antibodies against identified growth factors could significantly reduce these effects, indicating their importance in CASC-induced neovascularization. The angiogenic potential of CASCs and CASC CM was also confirmed in a chorioallantoic membrane assay, demonstrating that CASCs promote blood vessel formation in vivo. In conclusion, this study shows that CASCs not only induce myocardial repair by cardiomyogenic differentiation, but also stimulate blood vessel formation by paracrine mechanisms. The angiogenic properties of CASCs further strengthen their therapeutic potential and make them an optimal stem cell source for the treatment of ischemic heart disease.

From Bone Marrow to Cardiac Atrial Appendage Stem Cells for Cardiac Repair: A Review.

Traditionally the heart is considered a terminally differentiated organ. However, at the beginning of this century increased mitotic activity was reported in ischemic and idiopathic dilated cardiomyopathy hearts, compared to healthy controls, underscoring the potential of regeneration after injury. Due to the presence of adult stem cells in bone marrow and their purported ability to differentiate into other cell lineages, this cell population was soon estimated to be the most suited candidate for cardiac regeneration. Clinical trials with autologous bone marrow-derived mononuclear cells, using either an intracoronary or direct intramyocardial injection approach consistently showed only minor improvement in global left ventricular ejection fraction. This was explained by their limited cardiomyogenic differentiation potential. To obtain more convincing improvement in cardiac function, based on true myocardial regeneration, the focus of research has shifted towards resident cardiac progenitor cells. Several isolation procedures have been described: the c-kit surface marker was the first to be used, however experimental research has clearly shown that c-kit+ cells only marginally contribute to regeneration post myocardial infarction. Sphere formation was used to isolate the so-called cardiosphere derived cells (CDC), and also in this cell population cardiomyogenic differentiation is a rare event. Recently a new type of stem cells derived from atrial tissue (cardiac atrial stem cells - CASCs) was identified, based on the presence of the enzyme aldehyde dehydrogenase (ALDH). Those cells significantly improve both regional and global LV ejection fraction, based on substantial engraftment and consistent differentiation into mature cardiomyocytes (98%).

Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription.

BACKGROUND: Neurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed. METHODS: Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression. RESULTS: Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated. CONCLUSION: Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for neural development.

Cardiac atrial appendage stem cells engraft and differentiate into cardiomyocytes in vivo: A new tool for cardiac repair after MI.

BACKGROUND: This study assessed whether autologous transplantation of cardiac atrial appendage stem cells (CASCs) preserves cardiac function after myocardial infarction (MI) in a minipig model. METHODS AND RESULTS: CASCs were isolated from right atrial appendages of Göttingen minipigs based on high aldehyde dehydrogenase activity and expanded. MI was induced by a 2h snare ligation of the left anterior descending coronary artery. Upon reperfusion, CASCs were intramyocardially injected under NOGA guidance (MI-CASC, n=10). Non-transplanted pigs (MI, n=8) received sham treatment. 3D electromechanical mapping (EMM) and cardiac MRI were performed to assess left ventricular (LV) function. MI pigs developed LV dilatation at 2 months (2M), while in the MI-CASC group volumes remained stable. Global LV ejection fraction decreased by 16 ± 8% in MI animals vs 3 ± 10% in MI-CASC animals and regional wall thickening in border areas was better preserved in the MI-CASC group. EMM showed decreased viability and wall motion in the LV for both groups POST-MI, whereas at 2M these parameters only improved in the MI-CASC. Substantial cell retention was accompanied by cardiomyogenic differentiation in 98±1% of the transplanted CASCs, which functionally integrated. Second harmonic generation microscopy confirmed the formation of mature sarcomeres in transplanted CASCs. Absence of cardiac arrhythmias indicated the safety of CASC transplantation. CONCLUSION: CASCs preserve cardiac function by extensive engraftment and cardiomyogenic differentiation. Our data indicate the enormous potential of CASCs in myocardial repair.

Metabolic and Behavioural Phenotypes in Nestin-Cre Mice Are Caused by Hypothalamic Expression of Human Growth Hormone.

The Nestin-Cre driver mouse line has mild hypopituitarism, reduced body weight, a metabolic phenotype and reduced anxiety. Although several causes have been suggested, a comprehensive explanation is still lacking. In this study we examined the molecular mechanisms leading to this compound phenotype. Upon generation of the Nestin-Cre mice, the human growth hormone (hGH) minigene was inserted downstream of the Cre recombinase to ensure efficient transgene expression. As a result, hGH is expressed in the hypothalamus. This results in the auto/paracrine activation of the GH receptor as demonstrated by the increased phosphorylation of signal transducer and activator of transcription 5 (STAT5) and reduced expression of growth hormone releasing hormone (Ghrh). Low Ghrh levels cause hypopituitarism consistent with the observed mouse growth hormone (mGH) deficiency. mGH deficiency caused reduced activation of the GH receptor and hence reduced phosphorylation of STAT5 in the liver. This led to decreased levels of hepatic Igf-1 mRNA and consequently postnatal growth retardation. Furthermore, genes involved in lipid uptake and synthesis, such as CD36 and very low-density lipoprotein receptor were upregulated, resulting in liver steatosis. In conclusion, this study demonstrates the unexpected expression of hGH in the hypothalamus of Nestin-Cre mice which is able to activate both the GH receptor and the prolactin receptor. Increased hypothalamic GH receptor signaling explains the observed hypopituitarism, reduced growth and metabolic phenotype of Nestin-Cre mice. Activation of either receptor is consistent with reduced anxiety.

Single Nucleotide Polymorphism (rs4932178) in the P1 Promoter of FURIN Is Not Prognostic to Colon Cancer.

High expression of the proprotein processing enzyme FURIN has been associated with tumor progression and metastasis. A SNP (rs4932178) in the promoter of FURIN has been reported to affect expression in liver, with the T allele resulting in higher expression than the C allele. In this study we have investigated the association of this SNP with prognostic and biological subgroups of colorectal cancer (CRC). In a panel of 1382 patients with CRC, this SNP had no impact on overall survival or on postoperative risk of relapse. This SNP also could not be linked with FURIN expression levels in CRC samples from the patients. Furthermore, we demonstrate in luciferase reporter experiments in the colon cancer cell lines Caco-2 and SW480 and in the hepatocellular carcinoma cell line Huh 7 that expression is not affected by the SNP. Since, FURIN inhibition in human colon cancer cell lines has previously been shown to repress tumor metastases, association between FURIN gene expression levels and postoperative relapse-free survival was also investigated. However, no association could be found. Altogether, we could not confirm an effect of the SNP on FURIN expression in vitro and no correlations could be found in vivo with FURIN expression or outcome.

Liver-Specific Inactivation of the Proprotein Convertase FURIN Leads to Increased Hepatocellular Carcinoma Growth.

Proprotein convertases are subtilisin-like serine endoproteases that cleave and hence activate a variety of proproteins, including growth factors, receptors, metalloproteases, and extracellular matrix proteins. Therefore, it has been suggested that inhibition of the ubiquitously expressed proprotein convertase FURIN might be a good therapeutic strategy for several tumor types. Whether this is also the case for hepatocellular carcinoma (HCC) is currently not clear. In a mouse model for HCC expression of Furin was not altered in the tumors, while those of PC7, PC5/6, and PACE4 significantly decreased, at least at some time points. To investigate the impact of Furin inhibition on the development and progression of HCC in this model, Furin was genetically ablated in the liver. Furin inactivation resulted in an increased tumor mass after 5 weeks. This was not caused by decreased apoptosis, since no differences in the apoptosis index could be observed. However, it could at least partially be explained by increased hepatocyte proliferation at 5 weeks. The tumors of the Furin knockout mice were histologically similar to those in wild type mice. In conclusion, liver-specific Furin inhibition in HCC enhances the tumor formation and will not be a good therapeutic strategy for this tumor type.

Impaired islet function in commonly used transgenic mouse lines due to human growth hormone minigene expression.

The human growth hormone (hGH) minigene is frequently used in the derivation of transgenic mouse lines to enhance transgene expression. Although this minigene is present in the transgenes as a secondcistron, and thus not thought to be expressed, we found that three commonly used lines, Pdx1-Cre(Late), RIP-Cre, and MIP-GFP, each expressed significant amounts of hGH in pancreatic islets. Locally secreted hGH binds to prolactin receptors on ß cells, activates STAT5 signaling, and induces pregnancy-like changes in gene expression, thereby augmenting pancreatic ß cell mass and insulin content. In addition, islets of Pdx1-Cre(Late) mice have lower GLUT2 expression and reduced glucose-induced insulin release and are protected against the ß cell toxin streptozotocin. These findings may be important when interpreting results obtained when these and other hGH minigene-containing transgenic mice are used.

Phlorizin pretreatment reduces acute renal toxicity in a mouse model for diabetic nephropathy.

Streptozotocin (STZ) is widely used as diabetogenic agent in animal models for diabetic nephropathy (DN). However, it is also directly cytotoxic to kidneys, making it difficult to distinguish between DN-related and STZ-induced nephropathy. Therefore, an improved protocol to generate mice for DN studies, with a quick and robust achievement of the diabetic state, without direct kidney toxicity is required. To investigate the mechanism leading to STZ-induced nephropathy, kidney damage was induced with a high dose of STZ. This resulted in delayed gastric emptying, at least partially caused by impaired desacyl ghrelin clearance. STZ uptake in the kidneys is to a large extent mediated by the sodium/glucose cotransporters (Sglts) because the Sglt inhibitor phlorizin could reduce STZ uptake in the kidneys. Consequently, the direct toxic effects in the kidney and the gastric dilatation were resolved without interfering with the ß-cell toxicity. Furthermore, pancreatic STZ uptake was increased, hereby decreasing the threshold for ß-cell toxicity, allowing for single low non-nephrotoxic STZ doses (70 mg/kg). In conclusion, this study provides novel insights into the mechanism of STZ toxicity in kidneys and suggests a more efficient regime to induce DN with little or no toxic side effects.

Zic3 enhances the generation of mouse induced pluripotent stem cells.

Zinc finger protein of the cerebellum (Zic)3, a member of Gli family of transcription factors (TFs), is essential for maintaining pluripotency of embryonic stem cells (ESCs) and has been reported to activate TF Nanog in an Oct4/Sox2-independent manner. Previously, we showed that Zic3 (Z), in combination with the Yamanka factors OCT4, SOX2, and KLF4 (OSK), induces neural progenitor-like cells from human fibroblasts. However, a similar combination of TFs (OSKZ) transduced in mouse embryonic fibroblasts resulted in enhanced induced pluripotent stem cells (iPSCs) formation compared with OSK alone, but not neuroprogenitors. OSKZ-derived iPSCs are indistinguishable from mESCs in colony morphology, expression of alkaline phosphatase and pluripotency genes, and embryoid body and teratoma formation. Zic3 activates the transcription of Nanog, a key pluripotency regulator, as evidenced by a luciferase promoter assay. During the course of iPSC derivation, Zic3-mediated enhanced expression of Nanog and Tbx3, gene known to enhance iPSCs derivation, is observed. Not only does Zic3 enhance the reprogramming efficiency, but also reactivation of the endogenous Zic3 protein is essential for the generation of iPSCs, as knockdown of Zic3 during the iPSC generation with OSKM significantly reduced the number of colonies. Together, our result uncovers an important role of Zic3 in generating mouse iPSCs.

Generation and characterization of non-competitive furin-inhibiting nanobodies.

The PC (proprotein convertase) furin cleaves a large variety of proproteins and hence plays a major role in many pathologies. Therefore furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy-chain-derived nanobodies against catalytically active furin were developed as specific furin inhibitors. The nanobodies bound only to furin but not to other PCs. Upon overexpression in cell lines, they inhibited the cleavage of two different furin substrates, TGFß (transforming growth factor ß) and GPC3 (glypican 3). Purified nanobodies could inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but did not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode-of-action based on steric hindrance. The dissociation constant of purified nanobody 14 is in the nanomolar range. The nanobodies were non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies could protect HEK (human embryonic kidney)-293T cells from diphtheria-toxin-induced cytotoxicity as efficiently as the PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-domain nanobodies represent the first generation of highly specific non-competitive furin inhibitors.