Quantitative analysis of mRNA-lipid nanoparticle stability in human plasma and serum by size-exclusion chromatography coupled with dual-angle light scattering.

Understanding the stability of mRNA loaded lipid nanoparticles (mRNA-LNPs) is imperative for their clinical development. Herein, we propose the use of size-exclusion chromatography coupled with dual-angle light scattering (SEC-MALS) as a new approach to assessing mRNA-LNP stability in pure human serum and plasma. By applying a dual-column configuration to attenuate interference from plasma components, SEC-MALS was able to elucidate the degradation kinetics and physical property changes of mRNA-LNPs, which have not been observed accurately by conventional dynamic light scattering techniques. Interestingly, both serum and plasma had significantly different impacts on the molecular weight and radius of gyration of mRNA-LNPs, suggesting the involvement of clotting factors in desorption of lipids from mRNA-LNPs. We also discovered that a trace impurity (~1 %) in ALC-0315, identified as its O-tert-butyloxycarbonyl-protected form, greatly diminished mRNA-LNP stability in serum. These results demonstrated the potential utility of SEC-MALS for optimization and quality control of LNP formulations.

Unveiling the Dynamic Self-Assembly of a Recombinant Dragline-Silk-Mimicking Protein.

Despite the considerable interest in the recombinant production of synthetic spider silk fibers that possess mechanical properties similar to those of native spider silks, such as the cost-effectiveness, tunability, and scalability realization, is still lacking. To address this long-standing challenge, we have constructed an artificial spider silk gene using Golden Gate assembly for the recombinant bacterial production of dragline-mimicking silk, incorporating all the essential components: the N-terminal domain, a 33-residue-long major-ampullate-spidroin-inspired segment repeated 16 times, and the C-terminal domain (N16C). This designed silk-like protein was successfully expressed in Escherichia coli, purified, and cast into films from formic acid. We produced uniformly 13C-15N-labeled N16C films and employed solid-state magic-angle spinning nuclear magnetic resonance (NMR) for characterization. Thus, we could demonstrate that our bioengineered silk-like protein self-assembles into a film where, when hydrated, the solvent-exposed layer of the rigid, ß-nanocrystalline polyalanine core undergoes a transition to an α-helical structure, gaining mobility to the extent that it fully dissolves in water and transforms into a highly dynamic random coil. This hydration-induced behavior induces chain dynamics in the glycine-rich amorphous soft segments on the microsecond time scale, contributing to the elasticity of the solid material. Our findings not only reveal the presence of structurally and dynamically distinct segments within the film's superstructure but also highlight the complexity of the self-organization responsible for the exceptional mechanical properties observed in proteins that mimic dragline silk.

Photoacoustic tomography versus cone-beam computed tomography versus micro-computed tomography: Accuracy of 3D reconstructions of human teeth.

OBJECTIVES: In this in-vitro study, teeth were imaged using photoacoustic tomography (PAT), cone-beam computed tomography (CBCT), and micro-computed tomography (µ-CT). The study had aim: to identify the best wavelength for PAT images to determine the accuracy of the three imaging methods, and to determine whether PAT images of teeth can achieve acceptable reconstruction quality. METHODS: Nineteen human mandibular single-rooted incisors were extracted from patients with trauma or periodontitis. To determine the best wavelength for acquiring photoacoustic images, all 19 teeth were scanned in vitro with PAT, using different laser wavelengths between 680 and 960 nm. The images were analyzed using image analysis software. To assess the accuracy of PAT and compare it with the accuracy of CBCT, each tooth was also scanned in vitro using CBCT and the reference standard technique of µ-CT. Subsequently, three different three-dimensional models, one for each imaging technique, were created for each tooth. Finally, the three different three-dimensional models acquired for the same tooth were matched and analyzed regarding volume and surface. RESULTS: The highest quality tooth images were achieved using the 680 nm wavelength, which showed the best contrast ratio. The full geometry of the dental root (µ-CT compared with PAT) could be visualized with relative standard deviations of 0.12 mm for the surface and -7.33 mm3 for the volume (n = 19). The full geometry of the dental root (µ-CT compared with CBCT) could be visualized with relative standard deviations of 0.06 mm for the surface and -14.56 mm3 for the volume (n = 19). The difference between the PAT-µ-CT group and CBCT-µ-CT group regarding the total average of the root surface area was not significant (p>0.06). CONCLUSION: Images, which were acquired using PAT at 680nm showed the best contrast ration, enabling the identification of dentin, cementum and the dental pulp. No significant differences were found between the PAT-µ-CT group and CBCT-µ-CT group regarding the total average of the RSA and the total volume. Thus, three-dimensional reconstructions based on in-vitro PAT are already of acceptable reconstruction quality.

3D-LC-MS with 2D Multimethod Option for Fully Automated Assessment of Multiple Attributes of Monoclonal Antibodies Directly from Cell Culture Supernatants.

Fully automated analysis of multiple structural attributes of monoclonal antibodies (mAbs) using three-dimensional liquid chromatography-mass spectrometry (3D-LC-MS) is described. The analyzer combines Protein A affinity chromatography in the first dimension (1D) with a multimethod option in the second dimension (2D) (choice between size exclusion (SEC), cation exchange (CEX), and hydrophobic interaction chromatography (HIC)) and desalting SEC-MS in the third dimension (3D). This innovative 3D-LC-MS setup allows simultaneous and sequential assessment of mAb titer, size/charge/hydrophobic variants, molecular weight (MW), amino acid (AA) sequence, and post-translational modifications (PTMs) directly from cell culture supernatants. The reported methodology that finds multiple uses throughout the biopharmaceutical development trajectory was successfully challenged by the analysis of different trastuzumab and tocilizumab samples originating from biosimilar development programs.

Pedestrian Crossing Decisions in Virtual Environments: Behavioral Validity in CAVEs and Head-Mounted Displays.

OBJECTIVE: To contribute to the validation of virtual reality (VR) as a tool for analyzing pedestrian behavior, we compared two types of high-fidelity pedestrian simulators to a test track. BACKGROUND: While VR has become a popular tool in pedestrian research, it is uncertain to what extent simulator studies evoke the same behavior as nonvirtual environments. METHOD: An identical experimental procedure was replicated in a CAVE automatic virtual environment (CAVE), a head-mounted display (HMD), and on a test track. In each group, 30 participants were instructed to step forward whenever they felt the gap between two approaching vehicles was adequate for crossing. RESULTS: Our analyses revealed distinct effects for the three environments. Overall acceptance was highest on the test track. In both simulators, crossings were initiated later, but a relationship between gap size and crossing initiation was apparent only in the CAVE. In contrast to the test track, vehicle speed significantly affected acceptance rates and safety margins in both simulators. CONCLUSION: For a common decision task, the results obtained in virtual environments deviate from those in a nonvirtual test bed. The consistency of differences indicates that restrictions apply when predicting real-world behavior based on VR studies. In particular, the higher susceptibility to speed effects warrants further investigation, since it implies that differences in perceptual processing alter experimental outcomes. APPLICATION: Our observations should inform the conclusions drawn from future research in pedestrian simulators, for example by accounting for a higher sensitivity to speed variations and a greater uncertainty associated with crossing decisions.

Complementing femur model validation with a variability-focused approach.

OBJECTIVE: This short communication presents an approach as an objective means to validate that population variability is potentially incorporated into human body models in an accurate way, complementing existing validation techniques based on individual experiment-simulation comparison. This shall provide a further option for the assessment of the quality of large-number statistical simulations with human body models regarding their biofidelic behavior. METHODS: This population-based approach uses mathematical clustering methods to group similar curves of a combined population of numerical simulation results and experimental curves together. The resulting clusters can be used to assess the biofidelic behavior of numerical simulations, also with characteristics substantially differing from the experimental objects. This developed population-based approach was tested on a reference load case, the dynamic 3-point bending of the femur (Forman et al. 2012). RESULTS: The clustering approach rendered a distinction into 4 groups of response curves. For this small number, the grouping can be manually assessed as plausible. All experimental, and most numerical responses were grouped into one cluster. Three result curves constitute a cluster of their own, with their meta-data ranking on the margins of the population in at least one of the crucial biomechanical parameters. Such a result can be considered in accordance with the included experimental and anthropometric data. CONCLUSIONS: The feasibility of using such a cluster analysis without individual comparisons is demonstrated on a small set of results. It is used to judge whether a finite element model including aspects of the variation in a population is in agreement with experimental and anthropometric data. For experiments as the femur bending addressed here, it is of high importance to firstly ensure a gross match of curve shapes between experiments and simulation, i.e., capturing the relevant biomechanical aspects.

Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: Evaluation of multi-systems reproducibility.

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

Online 2D-LC for Complex N-Glycan Analysis from Biopharmaceuticals.

EPO has a complex glycosylation pattern with differently branched and charged glycans. A combination of hydrophilic interaction chromatography (HILIC) with weak anion exchange chromatography (WAX) enables highly orthogonal separation. Comprehensive 2D-LC analysis with HILIC in the first and WAX in the second dimension provides high resolution 2D chromatography together with simultaneous charge profiling. Meanwhile, multiple heart-cutting 2D-LC analysis combining WAX and HILIC separation provides a flexible alternative whereby the user can select multiple peaks to be analyzed in the second dimension and, moreover, run longer gradients in the second dimension.

Analysis of Street-Crossing Behavior: Comparing a CAVE Simulator and a Head-Mounted Display among Younger and Older Adults.

Interactive pedestrian simulators have become a valuable research tool for investigating street-crossing behavior and developing solutions for improving pedestrian safety. There are two main kinds of pedestrian simulators: one uses a technology based on rear-projection screens (Cave Automatic Virtual Environment, or CAVE), the other a head-mounted display (HMD). These devices are used indiscriminately, regardless of the research objective, and it is not yet known whether they are equally effective for studying street crossing. The present study was aimed at comparing the street crossing behavior and subjective evaluations of younger and older adult pedestrians when they are using a CAVE-like or HMD-based (HTC Vive Pro) pedestrian simulator. Thirty younger adults and 25 older adults performed 36 street-crossing trials (combining different speeds, two-way traffic conditions, and gap sizes) on each of the two types of simulators. The results indicated that participants in the HMD condition crossed the street significantly more often (58.6 %) than in the CAVE condition (42.44%) and had shorter safety margins. The most striking difference pertained to crossing initiation, which occurred considerably earlier (1.78 s) in the HMD condition than in the CAVE condition. Synchronization of crossing initiation with oncoming traffic was not as good in the CAVE condition because visual information in front of the pedestrian was missing due to the absence of ground projection. In both simulators, older adults caused more collisions than did younger ones, had shorter safety margins, and a slower crossing speed. Hence, the HMD reproduced classical age-related differences in most street-crossing behaviors already found on the CAVE. Usually observed speed effects were also found for both simulators. Neither cybersickness nor any adverse effects on stereoacuity or postural balance were found for either simulator. The HMD produced a higher level of presence and preference than the CAVE did. These findings provide evidence that HMDs have a clear potential for studying pedestrian behaviour.

Measuring egocentric distance perception in virtual reality: Influence of methodologies, locomotion and translation gains.

Virtual reality has become a popular means to study human behavior in a wide range of settings, including the role of pedestrians in traffic research. To understand distance perception in virtual environments is thereby crucial to the interpretation of results, as reactions to complex and dynamic traffic scenarios depend on perceptual processes allowing for the correct anticipation of future events. A number of approaches have been suggested to quantify perceived distances. While previous studies imply that the selected method influences the estimates' accuracy, it is unclear how the respective estimates depend on depth information provided by different perceptual modalities. In the present study, six methodological approaches were compared in a virtual city scenery. The respective influence of visual and non-visual cues was investigated by manipulating the ratio between visually perceived and physically walked distances. In a repeated measures design with 30 participants, significant differences between methods were observed, with the smallest error occurring for visually guided walking and verbal estimates. A linear relation emerged between the visual-to-physical ratio and the extent of underestimation, indicating that non-visual cues during walking affected distance estimates. This relationship was mainly evident for methods building on actual or imagined walking movements and verbal estimates.

Does intraoperative bone density testing correlate with parameters of primary implant stability? A pilot study in minipigs.

Objectives: Bone density, surgical protocol, and implant design are the major determinants of primary stability. The goal of this animal trial was to investigate potential correlations of intraoperative bone density testing with clinical and histologic parameters of primary implant stability. Material and methods: Following extractions of all mandibular premolars and subsequent healing, four implants each were placed in a total of four minipigs. Bone density was determined by applying intraoperative compressive tests using a device named BoneProbe whereas measurements of implant insertion torque and resonance frequency analysis were used for evaluating implant stability. Bone mineral density (BMD) and bone to implant contact were quantified after harvesting mandibular block sections. Spearman rank correlation tests were performed for evaluating correlations (α = .05). Results: Due to variation in clinical measurements, only weak correlations could be identified. A positive correlation was found between the parameters bone to implant contact and BMD (Spearman's rho .53; p = .05) whereas an inverse correlation was observed between BMD and implant stability (Spearman's rho -.61; p = .03). Both BoneProbe measurements in the cortical and trabecular area positively correlated with implant insertion torque (Spearman's rho 0.60; p = .02). A slightly stronger correlation was observed between the average of both BoneProbe measurements and implant insertion torque (Spearman's rho.66; p = .01). Conclusions: While establishing exact relationships among parameters of implant stability and the measurement techniques applied would require greater sample size, intraoperative compressive testing of bone might, despite the weak correlations seen here, be a useful tool for predicting primary implant stability.

Recurrent malignant Triton tumour: first report on a long time survivor.

Malignant Triton tumour represents an extremely rare mesenchymal malignancy exhibiting histopathologic patterns of peripheral nerve sheath tumours and rhabdomyoblastic components, the latter usually determining the mostly fatal outcome. We report on a 26-year old patient diagnosed with malignant Triton tumour who developed multiple recurrences despite repeated aggressive surgery, chemo- and radiotherapy during an 8-year period. After Northern blotting analysis of an excised in-transit metastasis had revealed expression of retinoic receptors alpha and gamma, the patient received experimental treatment with isotretinoin and interferon-alpha for one year and remains without any evidence of disease for more than three years. This is the first report on a long-term survivor of multiple recurrences of malignant Triton tumour.

Heregulin and retinoids synergistically induce branching morphogenesis of breast cancer cells cultivated in 3D collagen gels.

C-erbB and retinoid receptor signaling control mammary epithelial cell proliferation, differentiation, and morphology. Here, we examined the morphogenetic activities of c-erbB specific ligands such as heregulin and of retinoids on non-malignant (primary, MTSV1-7) and malignant (T47D, SKBR-3) human mammary epithelial cells (HMEC) cultivated in 3D collagen type I gels. These cells are positive for both c-erbB and retinoid receptors. Non-malignant primary HMEC spontaneously formed branched structures in collagen, whereas SV40 large T antigen-immortalized non-tumorigenic MTSV1-7 spontaneously formed balls and required heregulin or retinoid X receptor alpha-selective retinoid Ro 25-7386 for branching, which was further stimulated by combination of both types of agents. In malignant cells, heregulin alone induced ball formation and cooperated either with Ro 25-7386 (T47D) or with retinoic acid receptor alpha-selective AM580 (SKBR-3) for branching morphogenesis, which was accompanied by changes in the subcellular distribution of alpha(2)beta(1)-integrin and E-cadherin, and by down-regulation of c-erbB-2, -3, or -4. Heregulin and/or retinoids correspondingly increased the integrin-dependent adhesion of malignant cells to type I collagen. Our data demonstrate cooperative signaling of c-erbB and retinoid receptor pathways at the levels of morphogenesis and immunophenotypic differentiation.