Multivalent dextran hybrids for efficient cytosolic delivery of biomolecular cargoes.

The development of novel biotherapeutics based on peptides and proteins is often limited to extracellular targets, because these molecules are not able to reach the cytosol. In recent years, several approaches were proposed to overcome this limitation. A plethora of cell-penetrating peptides (CPPs) was developed for cytoplasmic delivery of cell-impermeable cargo molecules. For many CPPs, multimerization or multicopy arrangement on a scaffold resulted in improved delivery but also higher cytotoxicity. Recently, we introduced dextran as multivalent, hydrophilic polysaccharide scaffold for multimerization of cell-targeting cargoes. Here, we investigated covalent conjugation of a CPP to dextran in multiple copies and assessed the ability of resulted molecular hybrid to enter the cytoplasm of mammalian cells without largely compromising cell viability. As a CPP, we used a novel, low-toxic cationic amphiphilic peptide L17E derived from M-lycotoxin. Here, we show that cell-penetrating properties of L17E are retained upon multivalent covalent linkage to dextran. Dextran-L17E efficiently mediated cytoplasmic translocation of an attached functional peptide and a peptide nucleic acid (PNA). Moreover, a synthetic route was established to mask the lysine side chains of L17E with a photolabile protecting group thus opening avenues for light-triggered activation of cellular uptake.

Significant others? Social groups, income expectations, and redistributive preferences.

While inequality between individuals is known to be an important determinant of redistributive preferences, research on inequality between groups has increased only recently. This paper argues that individuals infer income expectations from the economic standing of their social group, in particular groups based on characteristics determined at birth, such as sex, race, or parents class. High group incomes can lead individuals to oppose redistribution, even if they are currently poor. Analyses of US survey data from 1978 to 2014 support this argument. The uncovered effects on preferences exceed those of individual income by more than three times in magnitude.

Effect of Osocimab in Preventing Venous Thromboembolism Among Patients Undergoing Knee Arthroplasty: The FOXTROT Randomized Clinical Trial.

Importance: The efficacy of factor XIa inhibition for thromboprophylaxis is unknown. Osocimab is a long-acting, fully human monoclonal antibody that inhibits factor XIa. Objective: To compare different doses of osocimab with enoxaparin and apixaban for thromboprophylaxis in patients who have undergone knee arthroplasty. Design, Setting, and Participants: Randomized, open-label, adjudicator-blinded, phase 2 noninferiority trial with observer blinding for osocimab doses, conducted at 54 hospitals in 13 countries. Adult patients undergoing unilateral knee arthroplasty were randomized from October 2017 through August 2018 and followed up until January 2019. Interventions: Single intravenous osocimab postoperative doses of 0.3 mg/kg (n = 107), 0.6 mg/kg (n = 65), 1.2 mg/kg (n = 108), or 1.8 mg/kg (n = 106); preoperative doses of 0.3 mg/kg (n = 109) or 1.8 mg/kg (n = 108); or 40 mg of subcutaneous enoxaparin once daily (n = 105) or 2.5 mg of oral apixaban twice daily (n = 105) for at least 10 days or until venography. Main Outcomes and Measures: The primary outcome was venous thromboembolism incidence between 10 and 13 days postoperatively (assessed by mandatory bilateral venography performed 10 to 13 days after surgery or confirmed symptomatic deep vein thrombosis or pulmonary embolism). A 5% noninferiority margin compared with enoxaparin was chosen. The primary safety outcome of major or clinically relevant nonmajor bleeding was assessed until 10 to 13 days postoperatively. Results: Of 813 randomized participants (mean [SD] age, 66.5 years [8.2 years]; body mass index, 32.7 [5.7]; and 74.2% women), 600 were included in the per-protocol population used for the primary analysis. The primary outcome occurred in 18 patients (23.7%) receiving 0.3 mg/kg, 8 (15.7%) receiving 0.6 mg/kg, 13 (16.5%) receiving 1.2 mg/kg, and 14 (17.9%) receiving 1.8 mg/kg of osocimab postoperatively; 23 (29.9%) receiving 0.3 mg/kg and 9 (11.3%) receiving 1.8 mg/kg of osocimab preoperatively; 20 (26.3%) receiving enoxaparin; and 12 (14.5%) receiving apixaban. Osocimab given postoperatively met criteria for noninferiority compared with enoxaparin with risk differences (1-sided 95% CIs) of 10.6% (95% CI, -1.2% to ∞) at the 0.6-mg/kg dose; 9.9% (95% CI, -0.9% to ∞) at the 1.2-mg/kg dose, and 8.4% (95% CI, -2.6 to ∞) at the 1.8-mg/kg dose. The preoperative dose of 1.8 mg/kg of osocimab met criteria for superiority compared with enoxaparin with a risk difference of 15.1%; 2-sided 90% CI, 4.9% to 25.2%). Postoperative and preoperative doses of 0.3 mg/kg of osocimab did not meet the prespecified criteria for noninferiority, with risk differences (1-sided 95% CIs) of 2.6% (95% CI, -8.9% to ∞) and -3.6% (95% CI, -15.5% to ∞), respectively. Major or clinically relevant nonmajor bleeding was observed in up to 4.7% of those receiving osocimab, 5.9% receiving enoxaparin, and 2% receiving apixaban. Conclusions and Relevance: Among patients undergoing knee arthroplasty, postoperative osocimab 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg met criteria for noninferiority compared with enoxaparin, and the preoperative 1.8-mg/kg dose of osocimab met criteria for superiority compared with enoxaparin for the primary outcome of incidence of venous thromboembolism at 10 to 13 days postoperatively. Further studies are needed to establish efficacy and safety of osocimab relative to standard thromboprophylaxis. Trial Registration: ClinicalTrials.gov Identifier: NCT03276143.

TRAIL-Inspired Multivalent Dextran Conjugates Efficiently Induce Apoptosis upon DR5 Receptor Clustering.

Triggering apoptosis of tumor cells has been in focus of cancer-inspired research since decades. As clustering of death receptor 5 (DR5), which is overexpressed on various cancer cells, leads to formation of the death-inducing signaling cascade (DISC), DR5 has recently become a promising target for tumor treatment. Herein, we demonstrate that covalent multimerization of a death receptor targeting peptide (DR5TP) on a dextran scaffold generates potent apoptosis-inducing conjugates (EC50 =2-20 nm). A higher conformational flexibility compared to reported DR5TP multimerization approaches, introduced by the polysaccharide framework compensates the reported need for the defined ligand orientation that was considered as essential prerequisite for effective receptor clustering and apoptosis induction. Enzyme-catalyzed ligation of a hydrophilic dextran conjugate bearing multiple DR5-targeting sites to a human fragment crystallizable (Fc) receptor did not affect the potency (EC50 =2-7 nm), providing an option for improved in vivo half-life and prospective conjugation to an antibody of interest in view of bispecific tumor targeting.

BAY 81-8973 Efficacy and Safety in Previously Untreated and Minimally Treated Children with Severe Hemophilia A: The LEOPOLD Kids Trial.

INTRODUCTION: BAY 81-8973, a full-length recombinant factor VIII for hemophilia A treatment, has been extensively evaluated in previously treated patients in the LEOPOLD (Long-Term Efficacy Open-Label Program in Severe Hemophilia A Disease) clinical trials. AIM: To assess BAY 81-8973 efficacy and safety when used for bleed prophylaxis and treatment in previously untreated/minimally treated patients (PUPs/MTPs). METHODS: In this phase III, multicenter, open-label, uncontrolled study, PUPs/MTPs (<6 years old) with severe hemophilia A received BAY 81-8973 (15-50 IU/kg) at least once weekly as prophylaxis. Primary efficacy endpoint was the annualized bleeding rate (ABR) within 48 hours after prophylaxis infusion. Adverse events and immunogenicity were assessed. Patients who developed inhibitors were offered immune tolerance induction (ITI) treatment in an optional extension phase. RESULTS: Fifty-two patients were enrolled, with 43 patients (mean age: 13.6 months) treated. Median (interquartile range) ABR for all bleeds within 48 hours of prophylaxis infusion was 0.0 (0.0-1.8) among patients without inhibitors (n = 20) and 0.0 (0.0-2.2) among all patients. As expected, inhibitors were the most frequent treatment-related adverse event (high titer: 17 [39.5%] patients; low titer: 6 [13.9%] patients). Six of 12 patients who underwent ITI treatment in the extension phase (high titer [n = 5], low titer [n = 1]) achieved a negative inhibitor titer. CONCLUSION: BAY 81-8973 was effective for bleed prevention and treatment in PUPs/MTPs. The observed inhibitor rate was strongly influenced by a cluster of inhibitor cases, and consequently, slightly higher than in other PUP/MTP studies. Overall, the BAY 81-8973 benefit-risk profile remains unchanged and supported by ongoing safety surveillance. Immune tolerance can be achieved with BAY 81-8973.

The colonial struggle over polygamy: Consequences for educational expansion in sub-Saharan Africa.

Christian missions in colonial Africa have contributed significantly to the expansion of formal education and thereby shaped the continent's long-term economic and political development. This paper breaks new ground by showing that this process depended on local demand for education. It is argued that disagreements over norms, and in particular the struggle over polygamy, which resulted from missions' insistence on monogamy in traditionally polygamous areas, lowered African demand for education. Analyses of geocoded data from historical and contemporary sources, covering most of sub-Saharan Africa, show that the struggle is associated with worse educational outcomes today. Effects are not limited to formal attainments but carry over to informal outcomes, in particular literacy. The findings attest to considerable heterogeneity in missionary legacies and suggest that local conditions should be given greater consideration in future studies on the long-term consequences of colonial-era interventions.

"Caught in the Middle! Wealth Inequality and Conflict over Redistribution".

A vast literature documents that wealth inequality has risen throughout advanced democracies, especially the accumulation of wealth among the rich. Yet, instead of increasing wealth redistribution, governments have done the seemingly opposite. Key to understanding why democratic governments do not increase wealth redistribution in times of rising inequalities is to shed light on the public's preferences. In this paper, we map the public's redistributive preferences in fourteen countries based on new survey data. We show that traditional socioeconomic cleavages in preferences for wealth redistribution are undermined by diverging mobility expectations. People who expect to climb up the wealth distribution, mostly lower wealth groups, are less supportive of redistribution than people with high stakes of major wealth losses, mainly upper wealth groups. We show that future expectations among the rich and the poor have a highly moderating role for the class conflict over wealth redistribution. Moreover, the middle class, the decisive group in democracies, is highly unresponsive to future prospects. The findings suggest that the middle class does not have much to lose or to win, and therefore, wealth redistribution is of low salience among this group.

AdEPro: Animation of Adverse Event Profiles-Presentation of an Easy-to-Use App for Visually Exploring Individual Study Data.

MOTIVATION: Reviewing the adverse event data collected in clinical trials is a lengthy and tedious process when these data are presented in the form of tables, data listings, and static graphs. Thus, to enable anyone interested in exploring adverse event data efficiently and relatively independently, we developed AdEPro, a compact, powerful, and easy-to-use interactive app. DESCRIPTION AND USE OF THE APP: AdEPro is an app for (audio-)visualizing adverse event data from clinical trials. The app dynamically displays the onset, severity, and development of selected adverse events on the individual subject level and on the treatment group level. This paper illustrates that there are numerous questions related to adverse events that can be approached by means of AdEPro, e.g., questions about temporal aspects of adverse events, associations between adverse events, and the influence of subject characteristics. AdEPro provides quick first answers to such questions; however, it does not provide statistical proof. Essentially, it acts as a versatile "hypothesis generator," helping the user to decide whether further analyses are indicated. No programming knowledge is required for exploring data by means of AdEPro. However, the user needs some basic knowledge of the software R and of extracting data from a clinical data base. The software code is open source, allowing modifications and expansions of the app, if desired. AVAILABILITY AND IMPLEMENTATION: AdEPro can be freely obtained from https://cran.r-project.org/package=adepro . It runs on any computer on which R is installed. Patient data are stored and processed locally.

Simplifying the Detection of Surface Presentation Levels in Yeast Surface Display by Intracellular tGFP Expression.

Yeast surface display (YSD) is an ultra-high throughput method used in protein engineering. Protein-protein interactions as well as surface presentation on the yeast cell surface are verified through fluorophore-conjugated labeling agents.In this chapter we describe an improved setup for full-length surface presentation detection. To this end, we used a single open reading frame (ORF) encoding for the protein to be displayed and a 2A sequence and tGFP for an intracellular fluorescence signal. The 2A sequence allows the simultaneous generation of two separate proteins from the same ORF through ribosomal skipping. The entangled expression of the POI on the yeast surface and intracellular tGFP obviates the need for fluorescent staining steps.

Dextramabs: A Novel Format of Antibody-Drug Conjugates Featuring a Multivalent Polysaccharide Scaffold.

Antibody-drug conjugates (ADCs) are multicomponent biomolecules that have emerged as a powerful tool for targeted tumor therapy. Combining specific binding of an immunoglobulin with toxic properties of a payload, they however often suffer from poor hydrophilicity when loaded with a high amount of toxins. To address these issues simultaneously, we developed dextramabs, a novel class of hybrid antibody-drug conjugates. In these architectures, the therapeutic antibody trastuzumab is equipped with a multivalent dextran polysaccharide that enables efficient loading with a potent toxin in a controllable fashion. Our modular chemoenzymatic approach provides an access to synthetic dextramabs bearing monomethyl auristatin as releasable cytotoxic cargo. They possess high drug-to-antibody ratios, remarkable hydrophilicity, and high toxicity in vitro.

Structure of a glutamine donor mimicking inhibitory peptide shaped by the catalytic cleft of microbial transglutaminase.

The protein cross-linking enzyme transglutaminase from Streptomyces mobaraensis (MTG) is frequently used to modify therapeutic proteins. In order to reveal the binding mode of glutamine donor substrates, we have now crystallized MTG covalently linked to large inhibitory peptides. A series of peptide structures were examined but DIPIGSKMTG, which was chloroacetylated at serine, was the only inhibitory molecule that resulted in an interpretable density map. We found that, besides the warhead (modified Ser6), Ile4 and Gly5 of the inhibitory peptide occupy the tight but extended hydrophobic bottom of the MTG-binding cleft. Both termini of the peptide protrude along the cleft walls almost perpendicular to the bottom of the extended cleft. This peptide model suggests a zipper-like cross-linking mechanism of self-assembled substrate proteins by MTG.