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

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

Evidence-Based Medicine in Daily Surgical Decision Making: A Survey-Based Comparison between the UK and Germany

Background: Evidence-based medicine (EbM) is a vital part of reasonable and conclusive decision making for clinicians in daily clinical work. To analyze the knowledge and the attitude of surgeons towards EbM, a survey was performed in the UK and Germany. Methods: A web-based questionnaire was distributed via mailing lists from the Royal College of Surgeons of England (RCSE) and the Berufsverband Deutscher Chirurgen (BDC). Our primary aim was to get information about knowledge of EbM amongst German and British surgeons. Results: A total of 549 individuals opened the questionnaire, but only 198 questionnaires were complete and valid for analysis. In total, 40,000 recipients were approached via the mailing lists of the BDC and RCSE. The response rate was equally low in both countries. On a scale from 1 (unimportant) to 10 (very important), all participants rated EbM as very important for daily clinical decision making (7.3 ± 1.9) as well as for patients (7.8 ± 1.9) and the national health system (7.8 ± 1.9). On a scale from 1 (unimportant) to 5 (very important), systematic reviews (4.6 ± 0.6) and randomized controlled trials (4.6 ± 0.6) were identified as the highest levels of study designs to enhance evidence in medicine. British surgeons considered EbM to be more important in daily clinical work when compared to data from German surgeons (7.9 ± 1.6 vs. 6.7 ± 2.1, p < 0.001). Subgroup analysis showed different results in some categories; however, a pattern to explain the differences was not evident. Personal requirements expressed in a free text field emphasized the results and reflected concerns such as broad unwillingness and lack of interdisciplinary approaches for patients (n = 59: 25 in the UK and 34 in Germany). Conclusion: The overall results show that EbM is believed to be important by surgeons in the UK and Germany. However, perception of EbM in the respective health system (UK vs. Germany) may be different. Nonetheless, EbM is an important tool to navigate through daily clinical problems although a discrepancy between the knowledge of theoretical abstract terms and difficulties in implementing EbM in daily clinical work has been detected. The provision of infrastructure, courses and structured education as a permanent instrument will advance the knowledge, application and improvement of EbM in the future

Complex multicomponent patterns rendered on a 3D DNA-barrel pegboard

DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of similar to 90nm diameter and similar to 250nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of similar to 8nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology

HCC recurrence in HCV‐infected patients after liver transplantation: SiLVER Study reveals benefits of sirolimus in combination with CNIs – a post‐hoc analysis

Factors affecting outcomes in liver transplant (LTx) recipients with hepatocellular carcinoma (HCC) and hepatitis C viral (HCV) infection include the choice of immunosuppression. Here, we analyzed the HCV+ subgroup of patients from the randomized controlled, international SiLVER Study. We performed a post hoc analysis of 166 HCV+ SiLVER Study patients regarding HCC outcome after LTx. Control patients (group A: n = 88) received mTOR inhibitor (mTORi)-free, calcineurin inhibitor (CNI)-based versus sirolimus-based immunosuppression (group B: n = 78). We found no significant difference regarding HCV-RNA titers between group A and B. Since no effect in group B could be due to variable sirolimus dosing, we split group B into patients receiving sirolimus-based immunosuppression + CNIs for >50% (B1; n = 44) or <50% (B2; n = 34) of the time. While there remained no difference in HCV-RNA titer between groups, HCC recurrence-free survival in group B1 (81.8%) was markedly better versus both group A (62.7%; P = 0.0136) and group B2 (64.7%; P = 0.0326); Interestingly, further subgroup analysis revealed an increase (P = 0.0012) in liver enzyme values in group B2. Taken together, in HCV-infected patients with HCC and LTx, mTORi immunosuppression + CNIs yields excellent outcomes. Unexpectedly, higher levels of liver inflammation and poorer outcomes occur with mTORi monotherapy in the HCV+ subgroup

Higher Postoperative Mortality and Inferior Survival After Right-Sided Liver Resection for Perihilar Cholangiocarcinoma:Left-Sided Resection is Preferred When Possible

BACKGROUND: A right- or left-sided liver resection can be considered in about half of patients with perihilar cholangiocarcinoma (pCCA), depending on tumor location and vascular involvement. This study compared postoperative mortality and long-term survival of right- versus left-sided liver resections for pCCA.METHODS: Patients who underwent major liver resection for pCCA at 25 Western centers were stratified according to the type of hepatectomy-left, extended left, right, and extended right. The primary outcomes were 90-day mortality and overall survival (OS).RESULTS: Between 2000 and 2022, 1701 patients underwent major liver resection for pCCA. The 90-day mortality was 9% after left-sided and 18% after right-sided liver resection (p &lt; 0.001). The 90-day mortality rates were 8% (44/540) after left, 11% (29/276) after extended left, 17% (51/309) after right, and 19% (108/576) after extended right hepatectomy (p &lt; 0.001). Median OS was 30 months (95% confidence interval [CI] 27-34) after left and 23 months (95% CI 20-25) after right liver resection (p &lt; 0.001), and 33 months (95% CI 28-38), 27 months (95% CI 23-32), 25 months (95% CI 21-30), and 21 months (95% CI 18-24) after left, extended left, right, and extended right hepatectomy, respectively (p &lt; 0.001). A left-sided resection was an independent favorable prognostic factor for both 90-day mortality and OS compared with right-sided resection, with similar results after excluding 90-day fatalities.CONCLUSIONS: A left or extended left hepatectomy is associated with a lower 90-day mortality and superior OS compared with an (extended) right hepatectomy for pCCA. When both a left and right liver resection are feasible, a left-sided liver resection is preferred.</p

Study Protocol: A Pilot Study to Determine the Safety and Efficacy of Induction-Therapy, De Novo MPA and Delayed mTOR-Inhibition in Liver Transplant Recipients with Impaired Renal Function. PATRON-Study

<p>Abstract</p> <p>Background</p> <p>Patients undergoing liver transplantation with preexisting renal dysfunction are prone to further renal impairment with the early postoperative use of Calcineurin-inhibitors. However, there is only little scientific evidence for the safety and efficacy of de novo CNI free "bottom-up" regimens in patients with impaired renal function undergoing liver transplantation. This is a single-center study pilot-study (<b>PATRON07</b>) investigating safety and efficacy of CNI-free, "bottom-up" immunosuppressive (IS) strategy in patients undergoing liver transplantation (LT) with renal impairment prior to LT.</p> <p>Methods/Design</p> <p>Patients older than 18 years with renal impairment at the time of liver transplantation eGFR < 50 ml/min and/or serum creatinine levels > 1.5 mg/dL will be included. Patients in will receive a CNI-free combination therapy (basiliximab, MMF, steroids and delayed Sirolimus). Primary endpoint is the incidence of steroid resistant acute rejection within the first 30 days after LT. The study is designed as prospective two-step trial requiring a maximum of 29 patients. In the first step, 9 patients will be included. If 8 or more patients show no signs of biopsy proven steroid resistant rejection, additional 20 patients will be included. If in the second step a total of 27 or more patients reach the primary endpoint the regimen is regarded to be safe and efficient.</p> <p>Discussion</p> <p>If a CNI-free-"bottom-up" IS strategy is safe and effective, this may be an innovative concept in contrast to classic top-down strategies that could improve the patient short and long-time renal function as well as overall complications and survival after LT. The results of <b>PATRON07 </b>may be the basis for a large multicenter RCT investigating the new "bottom-up" immunosuppressive strategy in patients with poor renal function prior to LT.</p> <p><url>http://www.clinicaltrials.gov</url>-identifier: NCT00604357</p

Liver transplantation for patients with acute-on-chronic liver failure (ACLF) in Europe: Results of the ELITA/EF-CLIF collaborative study (ECLIS)

BACKGROUND AND AIMS: Liver transplantation (LT) has been proposed to be an effective salvage therapy even for the sickest patients with acute-on-chronic liver failure (ACLF). This large collaborative study was designed to address the current clinical practice and outcomes of ACLF patients wait listed (WL) for LT in Europe. METHODS: Retrospective study including 308 consecutive ACLF patients, listed in 20 centres across 8 European countries, from January 2018 to June 2019. RESULTS: 2677 patients received a LT, 1216 (45.4%) for decompensated cirrhosis (DC). Of these, 234 (19.2%) had ACLF at LT: ACLF-1, 58 (4.8%); ACLF-2, 78 (6.4%); and ACLF-3, 98 (8.1%). Wide variations were observed amongst countries: France and Germany had high rates of ACLF-2/3 (27-41%); Italy, Switzerland, Poland and Netherlands had medium rates (9-15%); and United Kingdom and Spain had low rates (3-5%) (p 4 mmol/L (HR 3.14, 95% CI 1.37-7.19), recent infection from multi-drug resistant organisms (HR 3.67, 95% CI 1.63-8.28), and renal replacement therapy (HR 2.74, 95% CI 1.37-5.51) were independent predictors of post-LT mortality. During the same period, 74 patients with ACLF died on the WL. In an intention-to-treat analysis, one-year survival of ACLF patients on the LT WL was 73% for ACLF-1 or -2 and 50% for ACLF-3. CONCLUSION: The results reveal wide variations in listing patients with ACLF in Europe despite favorable post-LT survival. Risk factors for mortality were identified, allowing a more precise prognostic assessment of ACLF patients for potential LT. LAY SUMMARY: Acute on chronic liver failure (ACLF) is a severe clinical condition for which liver transplantation is an effective therapeutic option. This study has demonstrated that in Europe, referral and access to liver transplantation (LT) for patients with ACLF needs to be harmonized to avoid inequities. Post-LT survival for patients with ACLF was >80% after 1 year and some factors have been identified for selecting patients with favorable outcomes

HCC recurrence in HCV-infected patients after liver transplantation: SiLVER Study reveals benefits of sirolimus in combination with CNIs - a post-hoc analysis

Factors affecting outcomes in liver transplant (LTx) recipients with hepatocellular carcinoma (HCC) and hepatitis C viral (HCV) infection include the choice of immunosuppression. Here, we analyzed the HCV+ subgroup of patients from the randomized controlled, international SiLVER Study. We performed a post hoc analysis of 166 HCV+ SiLVER Study patients regarding HCC outcome after LTx. Control patients (group A: n&nbsp;=&nbsp;88) received mTOR inhibitor (mTORi)-free, calcineurin inhibitor (CNI)-based versus sirolimus-based immunosuppression (group B: n&nbsp;=&nbsp;78). We found no significant difference regarding HCV-RNA titers between group A and B. Since no effect in group B could be due to variable sirolimus dosing, we split group B into patients receiving sirolimus-based immunosuppression&nbsp;+&nbsp;CNIs for &gt;50% (B1; n&nbsp;=&nbsp;44) or &lt;50% (B2; n&nbsp;=&nbsp;34) of the time. While there remained no difference in HCV-RNA titer between groups, HCC recurrence-free survival in group B1 (81.8%) was markedly better versus both group A (62.7%; P&nbsp;=&nbsp;0.0136) and group B2 (64.7%; P&nbsp;=&nbsp;0.0326); Interestingly, further subgroup analysis revealed an increase (P&nbsp;=&nbsp;0.0012) in liver enzyme values in group B2. Taken together, in HCV-infected patients with HCC and LTx, mTORi immunosuppression&nbsp;+&nbsp;CNIs yields excellent outcomes. Unexpectedly, higher levels of liver inflammation and poorer outcomes occur with mTORi monotherapy in the HCV+ subgroup