Protein-adaptive differential scanning fluorimetry using conformationally responsive dyes.

Differential scanning fluorimetry (DSF) is a technique that reports protein thermal stability via the selective recognition of unfolded states by fluorogenic dyes. However, DSF applications remain limited by protein incompatibilities with existing DSF dyes. Here we overcome this obstacle with the development of a protein-adaptive DSF platform (paDSF) that combines a dye library 'Aurora' with a streamlined procedure to identify protein-dye pairs on demand. paDSF was successfully applied to 94% (66 of 70) of proteins, tripling the previous compatibility and delivering assays for 66 functionally and biochemically diverse proteins, including 10 from severe acute respiratory syndrome coronavirus 2. We find that paDSF can be used to monitor biological processes that were previously inaccessible, demonstrated for the interdomain allostery of O-GlcNAc transferase. The chemical diversity and varied selectivities of Aurora dyes suggest that paDSF functionality may be readily extended. paDSF is a generalizable tool to interrogate protein stability, dynamics and ligand binding.

State of the Art of Silica Nanoparticles: An Overview on Biodistribution and Preclinical Toxicity Studies.

Over the past few years, nanoparticles have drawn particular attention in designing and developing drug delivery systems due to their distinctive advantages like improved pharmacokinetics, reduced toxicity, and specificity. Along with other successful nanosystems, silica nanoparticles (SNPs) have shown promising effects for therapeutic and diagnostic purposes. These nanoparticles are of great significance owing to their modifiable surface with various ligands, tunable particle size, and large surface area. The rate and extent of degradation and clearance of SNPs depend on factors such as size, shape, porosity, and surface modification, which directly lead to varying toxic mechanisms. Despite SNPs' enormous potential for clinical and pharmaceutical applications, safety concerns have hindered their translation into the clinic. This review discusses the biodistribution, toxicity, and clearance of SNPs and the formulation-related factors that ultimately influence clinical efficacy and safety for treatment. A holistic view of SNP safety will be beneficial for developing an enabling SNP-based drug product.

Protocol for performing and optimizing differential scanning fluorimetry experiments.

Differential scanning fluorimetry (DSF) is a widely used technique for determining the apparent melting temperature (Tma) of a purified protein. Here, we present a protocol for performing and optimizing DSF experiments. We describe steps for designing and performing the experiment, analyzing data, and optimization. We provide benchmarks for typical Tmas and ΔTmas, standard assay conditions, and upper and lower limits of commonly altered experimental variables. We also detail common pitfalls of DSF and ways to avoid, identify, and overcome them.

Genotoxicity assessment of eight nitrosamines using 2D and 3D HepaRG cell models.

N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates. There are only limited data on the genotoxicity of nitrosamines in human cell systems. In this study, we used metabolically competent human HepaRG cells, whose metabolic capability is comparable to that of primary human hepatocytes, to evaluate the genotoxicity of eight nitrosamines [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. Under the conditions we used to culture HepaRG cells, three-dimensional (3D) spheroids possessed higher levels of CYP activity compared to 2D monolayer cells; thus the genotoxicity of the eight nitrosamines was investigated using 3D HepaRG spheroids in addition to more conventional 2D cultures. Genotoxicity was assessed as DNA damage using the high-throughput CometChip assay and as aneugenicity/clastogenicity in the flow-cytometry-based micronucleus (MN) assay. Following a 24-h treatment, all the nitrosamines induced DNA damage in 3D spheroids, while only three nitrosamines, NDBA, NDEA, and NDMA, produced positive responses in 2D HepaRG cells. In addition, these three nitrosamines also caused significant increases in MN frequency in both 2D and 3D HepaRG models, while NMBA and NMPA were positive only in the 3D HepaRG MN assay. Overall, our results indicate that HepaRG spheroids may provide a sensitive, human-based cell system for evaluating the genotoxicity of nitrosamines.

Conformationally responsive dyes enable protein-adaptive differential scanning fluorimetry.

Flexible in vitro methods alter the course of biological discoveries. Differential Scanning Fluorimetry (DSF) is a particularly versatile technique which reports protein thermal unfolding via fluorogenic dye. However, applications of DSF are limited by widespread protein incompatibilities with the available DSF dyes. Here, we enable DSF applications for 66 of 70 tested proteins (94%) including 10 from the SARS-CoV2 virus using a chemically diverse dye library, Aurora, to identify compatible dye-protein pairs in high throughput. We find that this protein-adaptive DSF platform (paDSF) not only triples the previous protein compatibility, but also fundamentally extends the processes observable by DSF, including interdomain allostery in O-GlcNAc Transferase (OGT). paDSF enables routine measurement of protein stability, dynamics, and ligand binding.

Systemic Therapy for Atopic Dermatitis in Older Adults and Adults With Comorbidities: A Scoping Review and International Eczema Council Survey.

BACKGROUND: Clinical trials of systemic therapies for atopic dermatitis (AD) often exclude patients based on age and comorbidities. OBJECTIVES: We conducted a scoping review of observational studies and survey of International Eczema Council (IEC) members on the treatment of AD in patients with liver disease, renal disease, viral hepatitis, HIV, or history of malignancy. METHODS: We searched MEDLINE via Ovid, Embase via Ovid, and Web of Science from inception to September 14, 2020. We mapped the available evidence on the use of cyclosporine, methotrexate, azathioprine, mycophenolate, systemic corticosteroids, and dupilumab for AD in older adults (≥65 years) and adults with the previously mentioned comorbidities. We surveyed IEC members on their preferred systemic medications for each patient population. RESULTS: We identified 25 studies on the use of systemic medications in special populations of adults with AD. Although IEC members preferred dupilumab as the first-line systemic agent across all special populations, many could not identify viable third-line systemic therapy options for some populations. CONCLUSIONS: Data on systemic therapy for AD for older adults and adults with comorbidities are limited. Although IEC members' access to systemic therapies differs geographically, expert opinion suggests that dupilumab is preferred for those patients.

Assessment of Axillary Lymph Nodes for Metastasis on Ultrasound Using Artificial Intelligence.

The purpose of this study was to evaluate an artificial intelligence (AI) system for the classification of axillary lymph nodes on ultrasound compared to radiologists. Ultrasound images of 317 axillary lymph nodes from patients referred for ultrasound guided fine needle aspiration or core needle biopsy and corresponding pathology findings were collected. Lymph nodes were classified into benign and malignant groups with histopathological result serving as the reference. Google Cloud AutoML Vision (Mountain View, CA) was used for AI image classification. Three experienced radiologists also classified the images and gave a level of suspicion score (1-5). To test the accuracy of AI, an external testing dataset of 64 images from 64 independent patients was evaluated by three AI models and the three readers. The diagnostic performance of AI and the humans were then quantified using receiver operating characteristics curves. In the complete set of 317 images, AutoML achieved a sensitivity of 77.1%, positive predictive value (PPV) of 77.1%, and an area under the precision recall curve of 0.78, while the three radiologists showed a sensitivity of 87.8% ± 8.5%, specificity of 50.3% ± 16.4%, PPV of 61.1% ± 5.4%, negative predictive value (NPV) of 84.1% ± 6.6%, and accuracy of 67.7% ± 5.7%. In the three external independent test sets, AI and human readers achieved sensitivity of 74.0% ± 0.14% versus 89.9% ± 0.06% (p = .25), specificity of 64.4% ± 0.11% versus 50.1 ± 0.20% (p = .22), PPV of 68.3% ± 0.04% versus 65.4 ± 0.07% (p = .50), NPV of 72.6% ± 0.11% versus 82.1% ± 0.08% (p = .33), and accuracy of 69.5% ± 0.06% versus 70.1% ± 0.07% (p = .90), respectively. These preliminary results indicate AI has comparable performance to trained radiologists and could be used to predict the presence of metastasis in ultrasound images of axillary lymph nodes.

Roundup negatively impacts the behavior and nerve function of the Madagascar hissing cockroach (Gromphadorhina portentosa).

Glyphosate is the active ingredient in Roundup formulations. Glyphosate-based herbicides are used globally in agriculture, forestry, horticulture, and in urban settings. Glyphosate can persist for years in our soil, potentially impacting the soil-dwelling arthropods that are primary drivers of a suite of ecosystem services. Furthermore, although glyphosate is not generally classified as neurotoxic to insects, evidence suggests that it may cause nerve damage in other organisms. In a series of experiments, we used food to deliver environmentally realistic amounts of Roundup ready-to-use III, a common 2% glyphosate-based herbicide formulation that lists isopropylamine salt as its active ingredient, to Madagascar hissing cockroaches. We then assessed the impact of contamination on body mass, nerve health, and behavior. Contaminated food contained both 30.6 mg glyphosate and so-called inert ingredients. Food was refreshed weekly for 26-60 days, depending on the experiment. We found that consumption of contaminated food did not impact adult and juvenile survivorship or body weight. However, consumption of contaminated food decreased ventral nerve cord action-potential velocity by 32%, caused a 29% increase in respiration rate, and caused a 74.4% decrease in time spent on a motorized exercise wheel. Such changes in behavior may make cockroaches less capable of fulfilling their ecological service, such as pollinating or decomposing litter. Furthermore, their lack of coordination may make them more susceptible to predation, putting their population at risk. Given the decline of terrestrial insect abundance, understanding common risks to terrestrial insect populations has never been more critical. Results from our experiments add to the growing body of literature suggesting that this popular herbicide can act as a neurotoxin.

Design and Generation of Self-Assembling Peptide Virus-like Particles with Intrinsic GPCR Inhibitory Activity.

Synthetic analogs of the second transmembrane domain (TM) containing a portion of the extracellular loop 1 of G-protein-coupled receptors (GPCR) can serve as biased antagonists of the corresponding receptor. Analogs with negative charges added to the extracellular end self-assemble into round structures. Addition of polyethylene glycol chains of defined length to the C-terminus of the peptides prevents super aggregation and results in highly uniform particles that can fuse with cell membranes spontaneously. Added PEG chains slow down cell fusion, while attachment of receptor ligands to the surface of particles results in receptor-mediated membrane fusion and cell-selective delivery. Critical assembly concentration of TM peptide particles is in the nanomolar range and thus requires nontraditional methods of determination. In this chapter, we outline sequence selection and design of self-assembling GPCR antagonists, methods of the preparation of the nanoparticles, and biophysical methods of particle characterization. The protocols allow for straightforward rational design, generation, and characterization of self-assembling GPCR antagonists for a variety of applications.

Development of plasma and whole blood taurine reference ranges and identification of dietary features associated with taurine deficiency and dilated cardiomyopathy in golden retrievers: A prospective, observational study.

INTRODUCTION: A surge in Food and Drug Administration (FDA) consumer complaints identified concerns that legume-rich, grain-free diets were associated with nutritionally-mediated dilated cardiomyopathy (DCM). Golden retrievers represent the most reported breed affected by this condition and previous studies documented the disease is responsive to dietary change and taurine supplementation. Although dietary findings across cases are compelling, prospective studies with control groups are lacking. The role of diet in developing taurine deficiency and echocardiographic changes consistent with DCM in healthy dogs is unknown. OBJECTIVES: We hypothesized that golden retrievers eating non-traditional diets are at a higher risk of having taurine deficiency and nutritionally-mediated DCM compared with those eating traditional commercial diets. We aimed to compare taurine concentrations and echocardiographic indices of systolic function between golden retrievers in each diet group and elucidate associations between diet and these variables. Additionally, we aimed to generate breed-specific reference intervals for whole blood and plasma taurine concentrations. ANIMALS: 86 golden retrievers. METHODS: Golden retrievers eating traditional or non-traditional diets were evaluated and diet history, taurine concentrations and echocardiographic data were collected. Dietary features, taurine concentrations and echocardiographic findings were compared between diet groups. Relative risks were calculated for the likelihood of echocardiographic abnormalities and taurine deficiency in each diet group. Breed-specific reference intervals were constructed for taurine concentrations in dogs from the traditional diet group. RESULTS: Golden retrievers eating non-traditional diets had significantly lower taurine concentrations and more frequent systolic dysfunction. Breed specific reference intervals are higher than previously reported across breeds. CONCLUSIONS: Non-traditional diets, which were typically grain-free and contained legumes in this study, were significantly associated with and have increased relative risk for the identification of taurine deficiency and echocardiographic abnormalities consistent with nutritionally-mediated DCM. These findings were identifiable in the absence of clinical signs and support the findings of multiple previous studies and the ongoing FDA investigation.

Demonstration of a Tritiated Nitroxide Nuclear Battery.

Unattended, compact, terrestrial and space sensors require sources that have high energy and power densities to continuously operate for 3 to 99 years depending on application. Currently, chemical sources cannot fully satisfy these applications, especially in solid state form. Betavoltaic (ßV) nuclear batteries using ß--emitting radioisotopes possess energy densities 1000 times greater than conventional chemical sources. Their power density is a function of ß- flux saturation point relative to the planar (2D) configuration, ß- emission range, and the semiconductor converter, the betavoltaic (ßV) cell, properties. The figure of merit is the beta (ß-)-flux surface power density ( [Formula: see text] in µWn per cm2 footprint), where an optimal portion of incident beta particles penetrates the surrounding semiconductor depletion region. Tritiated nitroxides are favorable radioisotope sources with the potential to have the highest specific activity (Am in Ci/g) and [Formula: see text] for an organic compound in solid form. The goal of this research is to demonstrate a tritiated nitroxide nuclear battery using the planar (2D) coupling configuration. The reproducible tritiation procedure produced stable product with a Am of approximately 635 Ci/g, which was 70% of the theoretical Am. For the nuclear battery demonstration, the tritiated nitroxide, dissolved in methanol, was deposited on a 4H-SiC ßV and InGaP photovoltaic (PV) cell using a dispensing apparatus and micropipette. Both devices' characteristics were measured beforehand using a controlled electron beam source to approximate the surface radioactivity from the deposited radioisotope. The maximum power point (MPP) of the 4H-SiC and InGaP were 7.77 nW/cm2 and 1.63 nW/cm2 with 100 mCi and 67 mCi, respectively. The power and total efficiency were lower than expected due to partial solvent evaporation and droplet thickness. Numerical models using MCNP6 Monte Carlo code were used to simulate an optimal nuclear battery prototype. The models' accuracy was confirmed with the device calibration curves and a previous metal tritide model based on empirical results. Based on optimal model results, the tritiated nitroxide saturation layer thickness (D0.99) and [Formula: see text] (D0.99) were 10 µm and 558 nW/cm2, respectively, using a 4H-SiC.

Cell-targeting fusion constructs containing recombinant gelonin.

Therapeutic agents capable of targeting tumor cells present as established tumors and micrometastases have already demonstrated their potential in clinical trials. Immunotoxins targeting hematological malignancies and solid tumors have additionally demonstrated excellent clinical activity. This review focuses on our design and characterization studies of constructs composed of recombinant gelonin toxin fused to either growth factors or single-chain antibodies targeting solid tumor cells, tumor vasculature or hematological malignancies. These agents demonstrate cytotoxicity at nanomolar or sub-nanomolar levels. All of these constructs display impressive selectivity and specificity for antigen-bearing target cells in vitro and in vivo and are excellent clinical trial candidates.