We explore properties of the class of Bekolle-Bonami weights B.sub.infinity introduced by the authors in a previous work. Although Bekolle-Bonami weights are known to be ill-behaved because they do ...not satisfy a reverse Holder property, we prove that when restricting to a class of weights that are "nearly constant on top halves", one recovers some of the classical properties of Muckenhoupt weights. We also provide an application of this result to the study of the spectra of certain integral operators.
•Overview of characterization techniques for molecularly imprinted polymers.•Several techniques are supported by own results.•Conventional techniques are covered such as EM, particle size analyzers, ...LC-MS/MS.•Advanced characterization techniques include NMR, TGA, DSC.•Specific characterization techniques exist for MIP on surfaces and sensors.
Molecularly imprinted polymers (MIP) represent synthetic recognition elements capable of interacting with a target analyte to obtain selective binding and/or detection and/or removal thereof. Various characterization techniques have been explored and evaluated. A first group concerns morphological characterization methods such as electron microscopy (EM), particle size analysis and pore size determination. Secondly, techniques have evolved for analysis of chemical structure characteristics (nuclear magnetic resonance, infrared investigation) and thermal features (thermogravimetric analysis, differential scanning calorimetry). Additionally, selective target binding is mostly studied by liquid chromatography coupled to different detectors. Finally, MIP structures on surfaces such as immobilized particles and films are frequently investigated by EM, atomic force microscopy, static contact angle measurements, X-ray photoelectron spectroscopy, quartz crystal microbalance and surface plasmon resonance.
The current review provides an overview of the above mentioned MIP characterization techniques. In most cases, the techniques covered are illustrated with data obtained for MIP against ergot alkaloids.
The goals of this review paper on deep learning (DL) in medical imaging and radiation therapy are to (a) summarize what has been achieved to date; (b) identify common and unique challenges, and ...strategies that researchers have taken to address these challenges; and (c) identify some of the promising avenues for the future both in terms of applications as well as technical innovations. We introduce the general principles of DL and convolutional neural networks, survey five major areas of application of DL in medical imaging and radiation therapy, identify common themes, discuss methods for dataset expansion, and conclude by summarizing lessons learned, remaining challenges, and future directions.
Robust and well-established orthogonal techniques for characterization of individual extracellular vesicles (EVs) are required to utilize them as therapeutic and diagnostic tools. However, to better ...utilize any analytical technology, we first need to determine the limitations of that technology.
We have established capabilities to reliably analyze EVs samples using three different orthogonal techniques; nanoparticle tracking analysis, flow cytometry and analytical HPLC. Using these three platforms, EV samples can be analyzed reliably and in agreement with MISEV and MIFlowCyt-EV guidelines.
Scattering and fluorescence mode of NTA suffer from masking effect and photobleaching respectively. We have optimized the labeling and measurement parameters to achieve reliable measurements. We will provide guidelines on how to characterize EVs accurately using any single particle analysis technique.
We tested EV standards from Sigma (tested at ISEVxTech 2022) and measured identical particle concentration on 3 orthogonal techniques sc-NTA, fl-NTA and flow cytometry.
We compared the size distribution using 5 techniques, sc-NTA (static), sc-NTA (flow), fl-NTA (flow), DLS and Cryo-EM. We found out that fl-NTA (flow) and Cryo-EM (as the gold standard) agreed the most with each other.
Different nanoparticle families such as liposomes, lipid nanoparticles, mammalian EVs, and bacterial EVs were successfully characterized using this analytical toolbox.
Single particle analysis techniques are less reliable when crude EV samples are tested. We used BSA as a model of non-EV associated protein contaminants. We observed that the free protein concentration must be below 50 µg/mL for scattering and 5 µg/mL for fluorescence NTA to characterize EVs accurately. Therefore, the relevant ratio of EVs compared to other non-EV components is a critical parameter in reliably analyzing crude EV samples.
MSC conditioned media can be successfully characterized throughout each step of the purification process including clarification, tangential flow filtration, and chromatography.
The optimization of labeling and measurement parameters is a critical step to characterize EVs reliably. Here, We provide guidelines on performing systematic and fundamental studies using standard samples and models for both EVs and protein contaminations in the background for any analytical technology.
Interest in gene therapy-based disease prevention and treatment has grown rapidly over the last decade. While lentivirus has been the viral vector of choice for gene therapy, recombinant ...adeno-associated viral (rAAV) vectors have seen recent widespread application due to the non-integrating ability. Baculoviruses are emerging as a highly adaptable vector with a broad tissue and host tropism. Regardless of vector, extensive quality control (QC) throughout the entire development and manufacturing process is essential. A robust QC process expedites safe, effective commercialization of final product. While Sanger sequencing can be ideal to verify and validate sequences pre-packaging, next generation sequencing (NGS) combined with post-viral production methodologies like CE analysis and transmission electron microscopy offer an effective high-throughput approach for monitoring quality, from initial construct assembly to analysis of the encapsulated product. Both short-read and long-read sequencing technologies offer distinct advantages including sequencing of the entire viral genome, with detection of potential mutations, truncations, and contaminants.
Here we describe our novel proprietary vector-agnostic workflows, for use in lentiviral, AAV or baculovirus settings. Starting with high quality synthesis of either vector plasmid or region of interest, full-length plasmid sequence is confirmed. Depending on these results, correction or new synthesis of plasmid options are applied. Following packaging within the viral vector system of choice, QC results using both short- and long-read NGS platforms are supported with a regulatory-compliant Sanger assay. State of the art synthesis reduces potential upstream errors. Sanger ITR sequencing and sequence correction upstream alleviates potential downstream issues in viral packaging. High-quality viral packaging ensures robust viral titers for downstream use. The combined NGS approach post-packaging alleviates current constraints for high throughput AAV sequencing and thereby enhance the overall QC process. Our Good Laboratory Practices (GLP) Sanger sequencing method extends read lengths through the entire ITR regions, allowing for rapid sequence confirmation of the final AAV product. The combination of these approaches enables a comprehensive solution, ideal for sequence confirmation of both transfer plasmid and final packaged product for improved viral vector gene therapy manufacturing in advance of FDA or EMA filings.
Neural stem cell-derived extracellular vesicles (NSC-EVs) are promising cell-free therapeutics but their application is limited by practical drawbacks and issues of inaccessibility. Like most ...therapeutic EV preparations, NSC-EVs are typically cryopreserved at -80°C. Cryopreservation is associated with freeze/thaw stress, as well as transportation challenges. Cryopreserved EV therapies are distributed to the point of care within the pharmaceutical cold chain which is among the most pressing barriers to widespread access of medicines globally. Lyophilization has been used to preserve various biological materials at room temperature, thereby circumventing the cold chain and enhancing long-term storage. Studies to date have shown that EVs can be lyophilized for long-term storage but have not evaluated the therapeutic ability of EVs post-lyophilization. The present study evaluates the character and therapeutic function of neural stem cell-derived EVs following lyophilization, toward the major aim of optimizing this storage technique for therapeutic EV products.
NSC-EVs were isolated by ultracentrifugation, then snap-frozen in liquid nitrogen, and stored 1) frozen at -80°C or 2) lyophilized at room temperature. Following 24 hours of storage, both groups of EVs were characterized by NanoFCM NanoAnalyzer, to determine the concentration and size of nanoparticles. Furthermore, nanoparticle population compositions were determined using Memglow, a lipid membrane probe, and CD63 antibody, an exosomal marker. Lyophilization did not have a destructive effect on EVs, based on their count and size, nor did it alter population composition relative to frozen samples.SLS1 To investigate their therapeutic capacity, the frozen and lyophilized NSC-EVs were analyzed in a microglia morphology assay using C20 immortalized human microglia cells. Trends indicate that both frozen and lyophilized NSC-EVs may reduce the inflammatory state of primed microglia cells in this assay. The successful lyophilization of therapeutic NSC-EVs significantly enhances their utility and accessibility by evading the otherwise necessary supply cold-chain, thus improving clinical translation and reaching more communities in need.
The use of plasmids is critical in the gene therapy viral vector workflow: plasmids are widely used for creation of the viral vector construct, along with helper and packaging plasmids for final ...product creation. While the construct insert is universally sequenced during initial design, sequencing of the backbone of the plasmid is not frequently assessed. Sequence errors within the plasmid backbone, helper and packaging plasmids can have major impacts in downstream packaging and viral vector production. Traditional Sanger approaches can be used for fast and frequent characterization, however limitations within the technology necessitate many sequencing reactions to span the full plasmid, with a known reference sequence. Traditional Next Generation Sequencing (NGS) approaches can overcome the need for multiple reactions and a reference sequence but are historically slower and cost-prohibitive.
Here we describe a fast and cost-effective sequencing approach for sequencing circular plasmid DNA to generate a highly accurate and annotated full-length consensus sequences without the need for primer design or a known reference sequence. This long-read sequencing method is comparable in cost and time to Sanger sequencing but unlike Sanger sequencing, overcomes common issues within hard to sequence regions like long nucleotide repeats or inverted terminal repeat (ITR) domains of adeno-associated viruses (AAVs). When used effectively, it can be harnessed to characterize gene therapy viral vector plasmid and associated packaging plasmids required throughout the gene therapy development life cycle without delay, ultimately accelerating the viral vector design and development process.
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