Engineering Escherichia coli for Conversion of Dietary Isoflavones in the Gut.

Introducing metabolic pathways to the gut is important to tailor the biochemical components ultimately absorbed by the host. Given identical diets, hosts possessing different consortia of gut bacteria can exhibit distinct health outcomes regulated by metabolic capabilities of the gut microbiota. The disparate competency of the population to metabolize isoflavones, such as dietary daidzein, has shown health benefits for those individuals possessing gut bacteria capable of producing equol from daidzein-rich diets. To begin addressing health inequalities due to gut metabolic pathway deficiencies, we developed a probiotic that allows metabolism of isoflavones to provide a gut phenotype paralleling that of natural equol producers. Toward this goal, we engineered Escherichia coli to produce the enzymes necessary for conversion of daidzein to equol, and as demonstrated in a murine model, these bacteria enabled elevated serum equol levels to dietary daidzein, thus serving as a starting point for more sophisticated systems.

Introduction of Plasmid to the Murine Gut via Consumption of an Escherichia coli Carrier and Examining the Impact of Bacterial Dosing and Antibiotics on Persistence.

Purpose: We examine the impacts of dosing strategies of plasmids on bacterial communities in the murine gut by measuring the quantity of plasmids in mouse feces. Methods: We fed mice carrier bacteria, E. coli, that contain plasmids with both a reporter gene and an antibiotic resistant gene. We varied the quantity of the plasmid-carrying bacteria and the length of time the mice consumed the bacteria. We also pretreated the gut with broad-spectrum antibiotics and used continuous antibiotic treatment to investigate selection pressure. We collected bacteria from fecal pellets to quantify the number of plasmid-carrying bacteria via plate assay. Results: Dosing regimens with plasmid-carrying bacteria resulted in a significantly increased duration of persistence of the plasmid within the gut when supplemented continuously with kanamycin during as well as after completion of bacterial dosing. The carrier bacteria concentration influenced the short-term abundance of carrier bacteria. Conclusion: We evaluated the persistence of plasmid-carrying bacteria in the murine gut over time using varying dosage strategies. In future work, we will study how bacterial diversity in the gut impacts the degree of plasmid transfer and the prevalence of plasmid-carrying bacteria over time. Lay Summary: Observing how plasmids persist within the gut can help us understand how newly introduced genes, including antibiotic resistance, are transmitted within the gut microbiome. In our experiments, mice were given bacteria containing a genetically engineered plasmid and were examined for the persistence of the plasmid in the gut. We found long-term persistence of the plasmid in the gut when administering antibiotics during and following dosing of the mice with bacteria carrying the plasmid. The use of higher concentrations of carrier bacteria influenced the short-term abundance of the plasmid-carrying bacteria in the gut. Description of Future Works: Building on evidence from these initial studies that persistence of plasmids within the gut can be regulated by the dosage strategy, we will explore future studies and models of gene uptake in the context of spatial and taxonomic control and further determine if dosing strategies alter the compositional diversity of the gut microbiome.

Corrigendum: Notch Intracellular Domain Plasmid Delivery via Poly(Lactic-Co-Glycolic Acid) Nanoparticles to Upregulate Notch Pathway Molecules.

[This corrects the article DOI: 10.3389/fcvm.2021.707897.].

Notch Intracellular Domain Plasmid Delivery via Poly(Lactic-Co-Glycolic Acid) Nanoparticles to Upregulate Notch Pathway Molecules.

Notch signaling is a highly conserved signaling system that is required for embryonic development and regeneration of organs. When the signal is lost, maldevelopment occurs and leads to a lethal state. Delivering exogenous genetic materials encoding Notch into cells can reestablish downstream signaling and rescue cellular functions. In this study, we utilized the negatively charged and FDA approved polymer poly(lactic-co-glycolic acid) to encapsulate Notch Intracellular Domain-containing plasmid in nanoparticles. We show that primary human umbilical vein endothelial cells (HUVECs) readily uptake the nanoparticles with and without specific antibody targets. We demonstrated that our nanoparticles are non-toxic, stable over time, and compatible with blood. We further demonstrated that HUVECs could be successfully transfected with these nanoparticles in static and dynamic environments. Lastly, we elucidated that these nanoparticles could upregulate the downstream genes of Notch signaling, indicating that the payload was viable and successfully altered the genetic downstream effects.

Engineering a reporter cell line to mimic the high oligomannose presenting surface immunoglobulin of follicular lymphoma B cells.

Subtypes of B cell non-Hodgkin's lymphomas, including follicular lymphomas, have shown a unique high oligomannose presentation on their immunoglobulins that will interact with natural receptors of the innate immunity, reportedly causing stimulation and proliferation. From deep sequencing of the variable heavy and light chain sequences of follicular lymphoma involved tissue sections, we identified the consensus variable sequences possessing glycosylation sites at the complementarity determining region. Using this information, we developed a cell line, referred to here as BZ, which displays the consensus variable segments as part of a surface antibody (IgM) and confirmed its presentation of high oligomannose on the heavy chain both in vitro and in vivo. An mCherry expressing variant provided a reporter cell line displaying the high oligomannose surface biomarker while affording clear fluorescent signals for FACS screening as well as for fluorescent in vivo imaging of ectopic xenograft tumors. In developing this reporter cell line that displays the biomarker glycan of follicular lymphoma, we provide a tool that may be used for future screening and validation of receptive moieties for selectively binding high oligomannose for development of targeted diagnostics or therapeutics to such B cell malignancies that display this unique glycan.

A Peptide-Lectin Fusion Strategy for Developing a Glycan Probe for Use in Various Assay Formats.

While nucleic acid and protein analysis approaches continue to see significant breakthroughs, analytical strategies for glycan determination have by comparison seen slower technological advances. Here we provide a strategy for glycan probe development using an engineered lectin fusion that can be incorporated into various common pathology lab assay formats including Western blot and agglutination assays. In this proof of concept, we use the natural lectin, Pseudomonas fluorescens agglutinin (PFA), capable of binding core Man alpha(1-3)-Man alpha(1-6)-Man units, where this lectin has previously been shown to bind to the glycans presented by the gp120 coat protein of (HIV) Human Immunodeficiency Virus. In our strategy, we engineered the lectin to possess a fusion of the biotin mimetic tag equence of amino acids V-S-H-P-Q-A-P-F. With the glycan receptive PFA directly linked to the biotin mimic, we could facilitate a probe for various standard clinical assay formats by virtue of coupling to streptavidin-HRP (horseradish peroxidase) or streptavidin beads for Western blot and agglutination assays respectively. We found the PFA fusion retained low nanomolar affinity for gp120 by ELISA (Enzyme Linked Immunosorbent Assay) and microscale thermophoresis. This probe engineering strategy proved effective in the relevant assay formats that may now allow detection for the presence of glycans containing the core Man alpha(1-3)-Man alpha(1-6)-Man units recognized by PFA.

IR-783 Labeling of a Peptide Receptor for 'Turn-On' Fluorescence Based Sensing.

In this study, we examine a means for developing near-IR fluorescent sensors through streamlined, site-specific coupling with peptide-based receptors. As the penultimate step of solid-phase synthesis of a peptide-based receptor, we show a simple means of labeling the N' terminus with the near IR fluorophore IR-783 to afford a viable fluorescent sensor after cleavage from the resin. The proof-of-concept probe utilized a biotin mimetic peptide sequence as the receptive moiety. Here we revealed a "turn-on" fluorescence enhancement upon binding of the biotin mimetic probe to its intended streptavidin target. Not all peptide-receptive moieties tested were able to generate such an enhancement upon target binding, and as such, the rationale for the observed fluorescence response properties is discussed.

Luminescent Probe Based Techniques for Hypoxia Imaging.

Hypoxia is a condition of tissue environments wherein a lower than normal level of oxygen is available, and it serves as the root cause and indicator of various diseases. Detection of hypoxia in tumors is imperative for furthering our understanding of the pathological effects and the development of proper treatments, as it is well established that hypoxic tumors are able to impede the cancer treatment process by being resistant to many therapies. It is important therefore to be able to detect hypoxia in tissues and tumors through in vivo imaging methods. A growing area for detection of hypoxia in vivo is the use of fluorescent/luminescent probes which has accelerated in recent years. The continued quest for improvements in selectivity and sensitivity has inspired researchers to pursue new strategies for fluorescence/luminescent probe design. This review will discuss various luminescent probes based on small molecules, dyes, macromolecules, and nanoparticles for sensitive and specific detection of oxygen levels directly or by indirect mechanisms such as the presence of enzymes or related factors that arise in a hypoxic environment. Following the particular mechanism of detection, each probe has specific structural and photophysical properties which permit its selectivity and sensitivity. These probes show promise in terms of low toxicity and high specificity among other merits discussed, and in providing new dimensions for hypoxia detection, these works contribute to future potential methods for clinical diagnosis of hypoxic tissues and tumors.