Hemin (iron protoporphyrin IX) is a crucial component of many physiological processes acting either as a prosthetic group or as an intracellular messenger. Some unnatural, synthetic porphyrins have potent anti-scrapie activity and can interact with normal prion protein (PrP C ). These observations raised the possibility that hemin, as a natural porphyrin, is a physiological ligand for PrP C . Accordingly, we evaluated PrP C interactions with hemin. When hemin (3-10 M) was added to the medium of cultured cells, clusters of PrP C formed on the cell surface, and the detergent solubility of PrP C decreased. The addition of hemin also induced PrP C internalization and turnover. The ability of hemin to bind directly to PrP C was demonstrated by hemin-agarose affinity chromatography and UV-visible spectroscopy. Multiple hemin molecules bound primarily to the N-terminal third of PrP C , with reduced binding to PrP C lacking residues 34 -94. These hemin-PrP C interactions suggest that PrP C may participate in hemin homeostasis, sensing, and/or uptake and that hemin might affect PrP C functions.

To date most mid-infrared spectroscopic studies have been limited, due to lack of sensitivity, to the structural characterization of a single oligonucleotide probe immobilized over the entire surface of a gold-coated slide or other infrared substrate. By contrast, widely used and commercially available glass slides and a microarray spotter that prints approximately 120-μm-diameter DNA spots were employed in the present work. To our knowledge, mid-infrared chemical imaging (IRCI) in the external reflection mode has been applied in the present study for the first time to the detection of nanostructure-based DNA microarrays spotted on glass slides. Alkyl amine-modified oligonucleotide probes were immobilized on glass slides that had been prefunctionalized with succinimidyl ester groups. This molecular fluorophore-free method entailed the binding of gold-nanoparticle-streptavidin conjugates to biotinylated DNA targets. Hybridization was visualized by the silver enhancement of gold nanoparticles. The adlayer of silver, selectively bound only to hybridized spots in a microarray, formed the external reflective infrared substrate that was necessary for the detection of DNA hybridization by IRCI in the present proof-of-concept study. IRCI made it possible to discriminate between diffuse and specular external reflection modes. The promising qualitative results are presented herein, and the implications for quantitative determination of DNA microarrays are discussed.

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We report on the optimization of a recently proposed mid-infrared chemical imaging (IRCI) detection method for the analysis of DNA microarrays. The improved protocol allowed for a ten-fold reduction in the time needed to generate a mosaic image of an entire microarray and the production of IR images with high contrast that would facilitate data analysis and interpretation. Advantages of using this protocol were evaluated by applying it to the analysis of four virulence genes in the genomes of 19 strains of the food bacterial pathogen Yersinia enterocolitica.

Clostridium botulinum is a pathogen of concern for low-acid canned foods. Here we report draft genomes of a neurotoxin-producing C. botulinum strain isolated from water samples used for cooling low-acid canned foods at a canning facility. The genome sequence confirmed that this strain belonged to C. botulinum serotype B1, albeit with major differences, including thousands of unique single nucleotide polymorphisms (SNPs) compared to other genomes of the same serotype.

Background: Inhibitors of the PD-1/PD-L1 checkpoint pathway were among the first FDA approved immunotherapies and are now among the standard of care for cancer patients. In vivo animal models that recreate crucial aspects of human tumor biology and anti-tumor immunity are needed for evaluating new therapy developments across cancer types. In 2019, we reported an animal model using non-adult (cord blood derived) hCD34+ hematopoietic stem cells, considered the standard cell source, for humanizing the immune system of NCG mice. In this study we assessed a mouse model using adult patient mobilized hCD34+ stem cells as an alternative source and evaluated engraftment and tumor modulation using checkpoint inhibitors. Study Details: We evaluated the anti-tumor effects of an anti-human PD-1 checkpoint inhibitor on basal lung cell adenocarcinoma (A549) in NCGs humanized with adult patient mobilized hCD34+ stem cells. Humanized mice were implanted subcutaneously with A549 cells, and tumor bearing mice (TBM) were randomized into treatment groups when the average tumor size reached a volume of ~80-120 mm3. Vehicle control mice were treated with isotype control IgG antibody, whereas A549 TBM were dosed with anti-hPD-1 antibody biweekly (10 mg/kg) for three weeks. Clinical observations, body weights and tumor growth kinetics were recorded throughout the study. At study termination, whole blood, spleen and tumor tissue were collected and processed for immune profiling by multi-color flow cytometry. Results: Anti-hPD-1 monotherapy significantly inhibited A549 tumor growth. NCG mice injected with adult patient mobilized hCD34+ stem cells sustained engraftment. At study termination common human leukocytes were distributed in peripheral blood, spleen and tumors of surviving mice. Phenotyping of the cancer microenvironment revealed the presence of lymphoid (T-lymphocytes and subsets including Tregs) and myeloid immune cells (DC, TAM) with the latter lineage less detectable overall. PD-1 checkpoint blockade on average increased CD8+ T-cell infiltration and decreased Treg frequencies among the TIL population. Generally, human cytokines (IFN-γ, IL-2 and TNF-α) were secreted by viable tumor-infiltrating total T-cells (CD3+) and subsets (CD4+ and CD8+) following ex vivo PMA/Ionomycin stimulation in the presence of Brefeldin A, demonstrating the capability to produce polyfunctional responses. Conclusions: This study demonstrates that adult patient mobilized hCD34+ cells can successfully reconstitute NCG mice. An immune modulating anti-tumor response was elicited with tissue infiltration patterns of human leukocytes overall comparable to those found in cord blood derived hCD34+ humanized NCG mice. These findings make this model utilizing NCG mice engrafted with adult patient mobilized hCD34+ stem cells a viable approach for cell transfer studies when assessing targeted immuno-oncology therapies. Citation Format: Jenny Rowe, Christoph Eberle, Ann Fiore, Robert Mihalek, Brianna Schoen, Stephen Festin. Assessment of a novel tumor model using adult patient mobilized human CD34+ hematopoietic stem cells in NCG mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1647.

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