124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics

Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.We thank Martin Spitaler and the imaging facility of the MPI of Biochemistry for confocal imaging support

Follow-up Computertomographie (CT) nach endovaskulärer Aneurysmareparatur (EVAR) der abdominellen Aorta: Diagnostische Genauigkeit von Diametermessungen zum Nachweis von Aneurysmasackvergrösserungen

Zielsetzung: Evaluation der diagnostischen Genauigkeit von Durchmessermessungen zur Detektion einer Aneurysmavolumenzunahme in der Nachsorge nach endovaskulärer Aneurysmareparatur (EVAR) bei infrarenalen Bauchaortenaneurysmen (BAA). Material und Methodik: In dieser retrospektiven Studie wurden 100 Paare von Nachsorge-CT-Untersuchungen, die zufällig aus unserer EVAR-Datenbank ausgewählt wurden, untersucht (Verhältnis männlich/weiblich, 91/9; mittleres Alter, 71 J; biiliakale und uniiliakale Gefäßprothesen, 96% und 4%; mittleres Intervall, 359 T). Fünf maximale Durchmesser (Dmax) wurden gemessen (anteroposterior, transversal, axial, coronar, und perpendicular). Das Aneruysmasackvolumen wurde durch manuelle Segmentation gemessen und als Referenzstandard verwendet. Insgesamt hatten 37% der Patienten ein persistierendes Typ II Endoleak. Ergebnisse: Der anteroposteriore, transversale, axiale, coronare, und perpendiculare Dmax nahm in 39 Patienten (Mittelwert, 4,3 mm), 30 Patienten (Mittelwert, 4,0), 35 Patienten (Mittelwert, 3,9 mm), 43 Patienten (Mittelwert, 3,9 mm), und 41 Patienten (Mittelwert, 4,3 mm) zu. Das Aneurysmasackvolumen nahm bei 39 Patienten (Mittelwert, 25,7 cm3) zu. Die Cutoff-Werte für den Reporting Standard für eine Zunahme des Aneurysmasackvolumens (Diameter ≥ 5,0 mm, Volumen ≥ 5,0%) hatten eine Sensitivität/Spezifität von 29%/95%, 33%/97%, 29%/99%, 33%/93%, und 38%/95% für fünf Dmax-Werte. Der Referenzstandard konnte die Zunahme des Aneurysmasackvolumens nicht detektieren bei 72%, 67%, 72%, 61% und 67% der Patienten mit einem persistierenden Typ II Endoleak. Schlussfolgerungen: Abhängig vom gewählten Cutoff-Wert, zeigten Duchmessermessungen eine niedrige bis mäßige Sensitivität zur Detektion einer Aneurysmavolumenzunahme. Die Durchmessermessungen konnten in einer hohen Zahl von Patienten mit pesistierendem Typ II Endoleak nach EVAR von BAA eine Aneurysmasackzunahme nicht detektieren

Adult Living Donor Liver Transplantation with ABO-Incompatible Grafts: A German Single Center Experience

Adult living donor liver transplantations (ALDLTs) across the ABO blood group barrier have been reported in Asia, North Americas, and Europe, but not yet in Germany. Several strategies have been established to overcome the detrimental effects that are attached with such a disparity between donor and host, but no gold standard has yet emerged. Here, we present the first experiences with three ABO-incompatible adult living donor liver transplantations in Germany applying different immunosuppressive strategies. Four patient-donor couples were considered for ABO-incompatible ALDLT. In these patients, resident ABO blood group antibodies (isoagglutinins) were depleted by plasmapheresis or immunoadsorption and replenishment was inhibited by splenectomy and/or B-cell-targeted immunosuppression. Despite different treatments ALDLT could safely be performed in three patients and all patients had good initial graft function without signs for antibody-mediated rejection (AMR). Two patients had long-term graft survival with stable graft function. We thus propose the feasibility of ABO-incompatible ALDLT with these protocols and advocate further expansion of ABO incompatible ALDLT in multicenter trials to improve efficacy and safety

Absence of claudin-3 does not alter intestinal absorption of phosphate in mice

Intestinal absorption of phosphate is bimodal, consisting of a transcellular pathway and a poorly characterized paracellular mode, even though the latter one contributes to the bulk of absorption under normal dietary conditions. Claudin-3 (Cldn3), a tight junction protein present along the whole intestine in mice, has been proposed to tighten the paracellular pathway for phosphate. The aim of this work was to characterize the phosphate-related phenotype of Cldn3-deficient mice. Cldn3-deficient mice and wildtype littermates were fed standard diet or challenged for 3 days with high dietary phosphate. Feces, urine, blood, intestinal segments and kidneys were collected. Measurements included fecal, urinary, and plasma concentrations of phosphate and calcium, plasma levels of phosphate-regulating hormones, evaluation of trans- and paracellular phosphate transport across jejunum and ileum, and analysis of intestinal phosphate and calcium permeabilities. Fecal and urinary excretion of phosphate as well as its plasma concentration was similar in both genotypes, under standard and high-phosphate diet. However, Cldn3-deficient mice challenged with high dietary phosphate had a reduced urinary calcium excretion and increased plasma levels of calcitriol. Intact FGF23 concentration was also similar in both groups, regardless of the dietary conditions. We found no differences either in intestinal phosphate transport (trans- or paracellular) and phosphate and calcium permeabilities between genotypes. The intestinal expression of claudin-7 remained unaltered in Cldn3-deficient mice. Our data do not provide evidence for a decisive role of Cldn3 for intestinal phosphate absorption and phosphate homeostasis. In addition, our data suggest a novel role of Cldn3 in regulating calcitriol levels

Erythropoietin stimulates fibroblast growth factor 23 (FGF23) in mice and men

Fibroblast growth factor 23 (FGF23) is a major endocrine regulator of phosphate and 1,25 (OH)2 vitamin D3 metabolism and is mainly produced by osteocytes. Its production is upregulated by a variety of factors including 1,25 (OH)2 vitamin D3, high dietary phosphate intake, and parathyroid hormone (PTH). Recently, iron deficiency and hypoxia have been suggested as additional regulators of FGF23 and a role of erythropoietin (EPO) was shown. However, the regulation of FGF23 by EPO and the impact on phosphate and 1,25(OH)2 vitamin D3 are not completely understood. Here, we demonstrate that acute administration of recombinant human EPO (rhEPO) to healthy humans increases the C-terminal fragment of FGF23 (C-terminal FGF23) but not intact FGF23 (iFGF23). In mice, rhEPO stimulates acutely (24 h) C-terminal FGF23 but iFGF23 only after 4 days without effects on PTH and plasma phosphate. 1,25 (OH)2 D3 levels and αklotho expression in the kidney decrease after 4 days. rhEPO induced FGF23 mRNA in bone marrow but not in bone, with increased staining of FGF23 in CD71+ erythroid precursors in bone marrow. Chronic elevation of EPO in transgenic mice increases iFGF23. Finally, acute injections of recombinant FGF23 reduced renal EPO mRNA expression. Our data demonstrate stimulation of FGF23 levels in mice which impacts mostly on 1,25 (OH)2 vitamin D3 levels and metabolism. In humans, EPO is mostly associated with the C-terminal fragment of FGF23; in mice, EPO has a time-dependent effect on both FGF23 forms. EPO and FGF23 may form a feedback loop controlling and linking erythropoiesis and mineral metabolism

Phosphate Restriction Prevents Metabolic Acidosis and Curbs Rise in FGF23 and Mortality in Murine Folic Acid–Induced AKI

Background: In AKI, plasma FGF23 and P$_{i}$ rise rapidly and are independently associated with disease severity and outcome. Methods: The effects of normal (NP) and low (LP) dietary P$_{i}$ were investigated in mice with FA-AKI after 3, 24, and 48 hours and 14 days. Results: After 24 hours of AKI, the LP diet curbed the rise in plasma FGF23 and prevented that of parathyroid hormone and calcitriol as well as of osseous but not splenic or thymic Fgf23 mRNA expression. The absence of Pth prevented the rise in calcitriol and reduced the elevation of FGF23 in FA-AKI with the NP diet. Furthermore, the LP diet attenuated the rise in renal and plasma IL-6 and mitigated the decline in renal α-Klotho. After 48 hours, the LP diet further dampened renal IL-6 expression and resulted in lower urinary neutrophil gelatinase-associated lipocalin. In addition, the LP diet prevented the increased formation of CPPs. Fourteen days after AKI induction, the LP diet group maintained less elevated plasma FGF23 levels and had greater survival than the NP diet group. This was associated with prevention of metabolic acidosis, hypocalcemia, hyperkalemia, and cardiac electrical disturbances. Conclusions: This study reveals P$_{i}$-sensitive FGF23 expression in the bone but not in the thymus or spleen in FA-AKI and demonstrates that P$_{i}$ restriction mitigates CPP formation, inflammation, acidosis, and mortality in this model. These results suggest that dietary P$_{i}$ restriction could have prophylactic potential in patients at risk for AKI

Benefits and barriers to accreditation of HPB center and fellowship programs in Europe:a strength-weakness-opportunity-and-threats (SWOT) analysis by an E-AHPBA-ESSO-UEMS ad hoc working committee

Background: Training in HPB surgery lacks uniformity across regions covered by the E-AHPBA. Accreditation has been in place for centers and fellowship programs, but with low uptake. The decision whether to continue, change or cease such accreditation is being discussed. Thus, a strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted. Methods: A mixed-methods, cross-sectional study among stakeholders in E-AHPBA, ESSO and UEMS under the E-AHPBA executive council was founded, ensuring representation by gender and geographic distribution. Results: Responses were collected from across E-AHPBA regions, with response from 15 of 24 subchapters. The most frequent and recurring themes are presented in a SWOT matrix which allows for paired evaluations of factors deemed to be helpful (Strengths and Opportunities), those that are harmful (Weaknesses and Threats). Conclusion: This study identified both helpful and harmful effects to an accreditation process of HPB centers or HPB fellowship training across the E-AHPBA membership region. Formal accreditation of centers is not within the scope, nor jurisdiction nor financial capacity for E-AHPBA in the current situation. A strong interest in formal HPB training should be capitalized into E-AHPBA strategic planning towards a structured accreditation system for HPB fellowship programs or HPB training tracks.</p

Hepatocellular carcinoma progression during bridging before liver transplantation

Background Recipient selection for liver transplantation in hepatocellular carcinoma (HCC) is based primarily on criteria affecting the chance of long-term success. Here, the relationship between pretransplant bridging therapy and long-term survival was investigated in a subgroup analysis of the SiLVER Study. Methods Response to bridging, as defined by comparison of imaging at the time of listing and post-transplant pathology report, was categorized into controlled versus progressive disease (more than 20 per cent tumour growth or development of new lesions). Results Of 525 patients with HCC who had liver transplantation, 350 recipients underwent pretransplant bridging therapy. Tumour progression despite bridging was an independent risk factor affecting overall survival (hazard ratio 1.80; P = 0.005). For patients within the Milan criteria (MC) at listing, mean overall survival was longer for those with controlled versus progressive disease (6.8 versus 5.8 years; P < 0.001). Importantly, patients with HCCs outside the MC that were downsized to within the MC before liver transplantation had poor outcomes compared with patients who never exceeded the MC (mean overall survival 6.2 versus 6.6 years respectively; P = 0.030). Conclusion Patients with HCCs within the MC that did not show tumour progression under locoregional therapy had the best outcomes after liver transplantation. Downstaging into the limits of the MC did not improve the probability of survival. Prognostic factors determining the long-term success of liver transplantation in patients with hepatocellular carcinoma are still under discussion. A subgroup analysis of the SiLVER trial showed that disease control under bridging therapy is strongly associated with improved prognosis in terms of overall survival. However, in tumours exceeding the limits of the Milan criteria, downstaging did not restore the probability of survival compared with that of patients within the Milan criteria

Safety and feasibility of third-party multipotent adult progenitor cells for immunomodulation therapy after liver transplantation--a phase I study (MISOT-I)

BACKGROUND: Liver transplantation is the definitive treatment for many end-stage liver diseases. However, the life-long immunosuppression needed to prevent graft rejection causes clinically significant side effects. Cellular immunomodulatory therapies may allow the dose of immunosuppressive drugs to be reduced. In the current protocol, we propose to complement immunosuppressive pharmacotherapy with third-party multipotent adult progenitor cells (MAPCs), a culture-selected population of adult adherent stem cells derived from bone marrow that has been shown to display potent immunomodulatory and regenerative properties. In animal models, MAPCs reduce the need for pharmacological immunosuppression after experimental solid organ transplantation and regenerate damaged organs. METHODS: Patients enrolled in this phase I, single-arm, single-center safety and feasibility study (n=3-24) will receive 2 doses of third-party MAPCs after liver transplantation, on days 1 and 3, in addition to a calcineurin-inhibitor-free "bottom-up" immunosuppressive regimen with Basiliximab, mycophenolic acid, and steroids. The study objective is to evaluate the safety and clinical feasibility of MAPC administration in this patient cohort. The primary endpoint of the study is safety, assessed by standardized dose-limiting toxicity events. One secondary endpoint is the time until first biopsy-proven acute rejection, in order to collect first evidence of efficacy. Dose escalation (150, 300, 450, and 600 million MAPCs) will be done according to a 3 + 3 classical escalation design (4 groups of 3-6 patients each). DISCUSSION: If MAPCs are safe for patients undergoing liver transplantation in this study, a phase II/III trial will be conducted to assess their clinical efficacy