Simultaneous Evalulation of Contrast Pulse Sequences for Super-Resolution Ultrasound Imaging – Preliminary In Vitro and In Vivo Results

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

10.26434/chemrxiv.5844393 ◽

2018 ◽

Author(s):

Noor H. Dashti ◽

Rufika S. Abidin ◽

Frank Sainsbury

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Super Resolution ◽

Cell Engineering ◽

Particle Analysis ◽

Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both in vitro and in vivo cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for in vivo self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by in vitro self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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Comparison of pulse sequences used for super-resolution ultrasound imaging with deep learning

2020 IEEE International Ultrasonics Symposium (IUS) ◽

10.1109/ius46767.2020.9251473 ◽

2020 ◽

Author(s):

Katherine Brown ◽

Kenneth Hoyt

Keyword(s):

Deep Learning ◽

Ultrasound Imaging ◽

Pulse Sequences ◽

Super Resolution

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A Tumbling Magnetic Microrobot System for Biomedical Applications

Micromachines ◽

10.3390/mi11090861 ◽

2020 ◽

Vol 11 (9) ◽

pp. 861

Author(s):

Elizabeth E. Niedert ◽

Chenghao Bi ◽

Georges Adam ◽

Elly Lambert ◽

Luis Solorio ◽

...

Keyword(s):

Ultrasound Imaging ◽

Biomedical Applications ◽

Imaging System ◽

Ex Vivo ◽

Negative Control ◽

Circular Path ◽

Rotational Axis ◽

Significant Difference

A microrobot system comprising an untethered tumbling magnetic microrobot, a two-degree-of-freedom rotating permanent magnet, and an ultrasound imaging system has been developed for in vitro and in vivo biomedical applications. The microrobot tumbles end-over-end in a net forward motion due to applied magnetic torque from the rotating magnet. By turning the rotational axis of the magnet, two-dimensional directional control is possible and the microrobot was steered along various trajectories, including a circular path and P-shaped path. The microrobot is capable of moving over the unstructured terrain within a murine colon in in vitro, in situ, and in vivo conditions, as well as a porcine colon in ex vivo conditions. High-frequency ultrasound imaging allows for real-time determination of the microrobot’s position while it is optically occluded by animal tissue. When coated with a fluorescein payload, the microrobot was shown to release the majority of the payload over a 1-h time period in phosphate-buffered saline. Cytotoxicity tests demonstrated that the microrobot’s constituent materials, SU-8 and polydimethylsiloxane (PDMS), did not show a statistically significant difference in toxicity to murine fibroblasts from the negative control, even when the materials were doped with magnetic neodymium microparticles. The microrobot system’s capabilities make it promising for targeted drug delivery and other in vivo biomedical applications.

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Differential FSH Glycosylation Modulates FSHR Oligomerization and Subsequent cAMP Signaling

Frontiers in Endocrinology ◽

10.3389/fendo.2021.765727 ◽

2021 ◽

Vol 12 ◽

Author(s):

Uchechukwu T. Agwuegbo ◽

Emily Colley ◽

Anthony P. Albert ◽

Viktor Y. Butnev ◽

George R. Bousfield ◽

...

Keyword(s):

Super Resolution ◽

Hek293 Cells ◽

Camp Signaling ◽

Camp Production ◽

Functional Roles ◽

Menopausal Women ◽

Resolution Imaging ◽

Post Menopausal

Follicle-stimulating hormone (FSH) and its target G protein-coupled receptor (FSHR) are essential for reproduction. Recent studies have established that the hypo-glycosylated pituitary FSH glycoform (FSH21/18), is more bioactive in vitro and in vivo than the fully-glycosylated variant (FSH24). FSH21/18 predominates in women of reproductive prime and FSH24 in peri-post-menopausal women, suggesting distinct functional roles of these FSH glycoforms. The aim of this study was to determine if differential FSH glycosylation modulated FSHR oligomerization and resulting impact on cAMP signaling. Using a modified super-resolution imaging technique (PD-PALM) to assess FSHR complexes in HEK293 cells expressing FSHR, we observed time and concentration-dependent modulation of FSHR oligomerization by FSH glycoforms. High eFSH and FSH21/18 concentrations rapidly dissociated FSHR oligomers into monomers, whereas FSH24 displayed slower kinetics. The FSHR β-arrestin biased agonist, truncated eLHβ (Δ121-149) combined with asparagine56-deglycosylated eLHα (dg-eLHt), increased FSHR homomerization. In contrast, low FSH21/18 and FSH24 concentrations promoted FSHR association into oligomers. Dissociation of FSHR oligomers correlated with time points where higher cAMP production was observed. Taken together, these data suggest that FSH glycosylation may modulate the kinetics and amplitude of cAMP production, in part, by forming distinct FSHR complexes, highlighting potential avenues for novel therapeutic targeting of the FSHR to improve IVF outcomes.

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Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling

10.1101/2021.10.27.466054 ◽

2021 ◽

Author(s):

Jitske Jansen ◽

Bartholomeus T van den Berge ◽

Martijn van den Broek ◽

Rutger J Maas ◽

Brigith Willemsen ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Stem Cell ◽

Pluripotent Stem Cell ◽

Idiopathic Nephrotic Syndrome ◽

Super Resolution ◽

Induced Pluripotent Stem Cell ◽

Glomerular Injury ◽

Kidney Organoids

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. Here, we report human induced pluripotent stem cell derived kidney organoids containing a podocyte population that heads towards adult podocytes and were superior compared to 2D counterparts, based on scRNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

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Intravascular ultrasound imaging of human coronary arteries in vivo. Analysis of tissue characterizations with comparison to in vitro histological specimens.

Circulation ◽

10.1161/01.cir.83.3.913 ◽

1991 ◽

Vol 83 (3) ◽

pp. 913-926 ◽

Cited By ~ 410

Author(s):

J M Tobis ◽

J Mallery ◽

D Mahon ◽

K Lehmann ◽

P Zalesky ◽

...

Keyword(s):

Intravascular Ultrasound ◽

Ultrasound Imaging ◽

Coronary Arteries ◽

Intravascular Ultrasound Imaging ◽

In Vivo Analysis

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An Innovative Ex Vivo Vascular Bioreactor as Comprehensive Tool to Study the Behavior of Native Blood Vessels Under Physiologically Relevant Conditions

Journal of Engineering and Science in Medical Diagnostics and Therapy ◽

10.1115/1.4044472 ◽

2019 ◽

Vol 2 (4) ◽

Author(s):

Noemi Vanerio ◽

Marco Stijnen ◽

Bas A. J. M. de Mol ◽

Linda M. Kock

Keyword(s):

Shear Stress ◽

Blood Vessels ◽

Ultrasound Imaging ◽

Ex Vivo ◽

Biological Response ◽

Vessel Diameter ◽

Tissue Growth ◽

In Vivo Models

Abstract Ex vivo systems represent important models to study vascular biology and to test medical devices, combining the advantages of in vitro and in vivo models such as controllability of parameters and the presence of biological response, respectively. The aim of this study was to develop a comprehensive ex vivo vascular bioreactor to long-term culture and study the behavior of native blood vessels under physiologically relevant conditions. The system was designed to allow for physiological mechanical loading in terms of pulsatile hemodynamics, shear stress, and longitudinal prestretch and ultrasound imaging for vessel diameter and morphology evaluation. In this first experience, porcine carotid arteries (n = 4) from slaughterhouse animals were cultured in the platform for 10 days at physiological temperature, CO2 and humidity using medium with blood-mimicking viscosity, components, and stability of composition. As expected, a significant increase in vessel diameter was observed during culture. Flow rate was adjusted according to diameter values to reproduce and maintain physiological shear stress, while pressure was kept physiological. Ultrasound imaging showed that the morphology and structure of cultured arteries were comparable to in vivo. Histological analyses showed preserved endothelium and extracellular matrix and neointimal tissue growth over 10 days of culture. In conclusion, we have developed a comprehensive pulsatile system in which a native blood vessel can be cultured under physiological conditions. The present model represents a significant step toward ex vivo testing of vascular therapies, devices, drug interaction, and as basis for further model developments.

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