Structural rearrangements of the central region of the morbillivirus attachment protein stalk domain trigger F protein refolding for membrane fusion

It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91-115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111-118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism.

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

Fusion toxins used for cancer-related therapy have demonstrated short circulation half-lives, which impairs tumor localization and, hence, efficacy. Here, we demonstrate that the pharmacokinetics of a fusion toxin composed of a designed ankyrin repeat protein (DARPin) and domain I–truncated Pseudomonas Exotoxin A (PE40/ETA″) can be significantly improved by facile bioorthogonal conjugation with a polyethylene glycol (PEG) polymer at a unique position. Fusion of the anti-EpCAM DARPin Ec1 to ETA″ and expression in methionine-auxotrophic E. coli enabled introduction of the nonnatural amino acid azidohomoalanine (Aha) at position 1 for strain-promoted click PEGylation. PEGylated Ec1-ETA″ was characterized by detailed biochemical analysis, and its potential for tumor targeting was assessed using carcinoma cell lines of various histotypes in vitro, and subcutaneous and orthotopic tumor xenografts in vivo. The mild click reaction resulted in a well-defined mono-PEGylated product, which could be readily purified to homogeneity. Despite an increased hydrodynamic radius resulting from the polymer, the fusion toxin demonstrated high EpCAM-binding activity and retained cytotoxicity in the femtomolar range. Pharmacologic analysis in mice unveiled an almost 6-fold increase in the elimination half-life (14 vs. 82 minutes) and a more than 7-fold increase in the area under the curve (AUC) compared with non-PEGylated Ec1-ETA″, which directly translated in increased and longer-lasting effects on established tumor xenografts. Our data underline the great potential of combining the inherent advantages of the DARPin format with bioorthogonal click chemistry to overcome the limitations of engineering fusion toxins with enhanced efficacy for cancer-related therapy.

Mechanism for active membrane fusion triggering by morbillivirus attachment protein.

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering.

β1 integrins regulate myoblast fusion and sarcomere assembly

The mechanisms that regulate the formation of multinucleated muscle fibers from mononucleated myoblasts are not well understood. We show here that extracellular matrix (ECM) receptors of the beta1 integrin family regulate myoblast fusion. beta1-deficient myoblasts adhere to each other, but plasma membrane breakdown is defective. The integrin-associated tetraspanin CD9 that regulates cell fusion is no longer expressed at the cell surface of beta1-deficient myoblasts, suggesting that beta1 integrins regulate the formation of a protein complex important for fusion. Subsequent to fusion, beta1 integrins are required for the assembly of sarcomeres. Other ECM receptors such as the dystrophin glycoprotein complex are still expressed but cannot compensate for the loss of beta1 integrins, providing evidence that different ECM receptors have nonredundant functions in skeletal muscle fibers.

An Early-Warning System for Hypo-/Hyperglycemic Events Based on Fusion of Adaptive Prediction Models

Introduction: Early warning of future hypoglycemic and hyperglycemic events can improve the safety of type 1 diabetes mellitus (T1DM) patients. The aim of this study is to design and evaluate a hypoglycemia / hyperglycemia early warning system (EWS) for T1DM patients under sensor-augmented pump (SAP) therapy. Methods: The EWS is based on the combination of data-driven online adaptive prediction models and a warning algorithm. Three modeling approaches have been investigated: (i) autoregressive (ARX) models, (ii) auto-regressive with an output correction module (cARX) models, and (iii) recurrent neural network (RNN) models. The warning algorithm performs postprocessing of the models′ outputs and issues alerts if upcoming hypoglycemic/hyperglycemic events are detected. Fusion of the cARX and RNN models, due to their complementary prediction performances, resulted in the hybrid autoregressive with an output correction module/recurrent neural network (cARN)-based EWS. Results: The EWS was evaluated on 23 T1DM patients under SAP therapy. The ARX-based system achieved hypoglycemic (hyperglycemic) event prediction with median values of accuracy of 100.0% (100.0%), detection time of 10.0 (8.0) min, and daily false alarms of 0.7 (0.5). The respective values for the cARX-based system were 100.0% (100.0%), 17.5 (14.8) min, and 1.5 (1.3) and, for the RNN-based system, were 100.0% (92.0%), 8.4 (7.0) min, and 0.1 (0.2). The hybrid cARN-based EWS presented outperforming results with 100.0% (100.0%) prediction accuracy, detection 16.7 (14.7) min in advance, and 0.8 (0.8) daily false alarms. Conclusion: Combined use of cARX and RNN models for the development of an EWS outperformed the single use of each model, achieving accurate and prompt event prediction with few false alarms, thus providing increased safety and comfort.

Improved Localization of Implanted Subdural Electrode Contacts on MRI Using an Elastic Image Fusion Algorithm in Invasive EEG Recording

BACKGROUND: Accurate projection of implanted subdural electrode contacts in presurgical evaluation of pharmacoresistant epilepsy cases by invasive EEG is highly relevant. Linear fusion of CT and MRI images may display the contacts in the wrong position due to brain shift effects. OBJECTIVE: A retrospective study in five patients with pharmacoresistant epilepsy was performed to evaluate whether an elastic image fusion algorithm can provide a more accurate projection of the electrode contacts on the pre-implantation MRI as compared to linear fusion. METHODS: An automated elastic image fusion algorithm (AEF), a guided elastic image fusion algorithm (GEF), and a standard linear fusion algorithm (LF) were used on preoperative MRI and post-implantation CT scans. Vertical correction of virtual contact positions, total virtual contact shift, corrections of midline shift and brain shifts due to pneumencephalus were measured. RESULTS: Both AEF and GEF worked well with all 5 cases. An average midline shift of 1.7mm (SD 1.25) was corrected to 0.4mm (SD 0.8) after AEF and to 0.0mm (SD 0) after GEF. Median virtual distances between contacts and cortical surface were corrected by a significant amount, from 2.3mm after LF to 0.0mm after AEF and GEF (p<.001). Mean total relative corrections of 3.1 mm (SD 1.85) after AEF and 3.0mm (SD 1.77) after GEF were achieved. The tested version of GEF did not achieve a satisfying virtual correction of pneumencephalus. CONCLUSION: The technique provided a clear improvement in fusion of pre- and post-implantation scans, although the accuracy is difficult to evaluate.

Landmark Detection for Fusion of Fundus and MRI Towards a Patient-Specific Multi-Modal Eye Model

Ophthalmologists typically acquire different image modalities to diagnose eye pathologies. They comprise e.g., Fundus photography, Optical Coherence Tomography (OCT), Computed Tomography (CT) and Magnetic Resonance Imaging (MRI). Yet, these images are often complementary and do express the same pathologies in a different way. Some pathologies are only visible in a particular modality. Thus, it is beneficial for the ophthalmologist to have these modalities fused into a single patient-specific model. The presented article’s goal is a fusion of Fundus photography with segmented MRI volumes. This adds information to MRI which was not visible before like vessels and the macula. This article’s contributions include automatic detection of the optic disc, the fovea, the optic axis and an automatic segmentation of the vitreous humor of the eye.