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The use of coumarin caged molecules has been well documented in numerous photocaging applications including for the spatiotemporal control of Cre-estrogen receptor (Cre-ERT2) recombinase activity. In this article, we report that 4-hydroxytamoxifen (4OHT) caged with coumarin via a conventional ether linkage led to an unexpected photo-Claisen rearrangement which significantly competed with the release of free 4OHT. The basis for this unwanted reaction appears to be related to the coumarin structure and its radical-based mechanism of uncaging, as it did not occur in ortho-nitrobenzyl (ONB) caged 4OHT that was otherwise linked in the same manner. In an effort to perform design optimization, we introduced a self-immolative linker longer than the ether linkage and identified an optimal linker which allowed rapid 4OHT release by both single-photon and two-photon absorption mechanisms. The ability of this construct to actively control Cre-ERT2 mediated gene modifications was investigated in mouse embryonic fibroblasts (MEFs) in which the expression of a green fluorescent protein (GFP) reporter dependent gene recombination was controlled by 4OHT release and measured by confocal fluorescence microscopy and flow cytometry. In summary, we report the implications of this photo-Claisen rearrangement in coumarin caged compounds and demonstrate a rational linker strategy for addressing this unwanted side reaction.

The tamoxifen inducible Cre-ER/loxP system provides tissue specific temporal control of gene recombination events, and can be used to induce expression of reporter genes (e.g. GFP, LacZ) for lineage tracing studies. Cre enzyme fused with estrogen receptor (Cre-ER) is released upon tamoxifen binding, resulting in permanent activation of reporter genes within cells and their progeny. Tamoxifen and its active metabolite, hydroxytamoxifen (4OHT) diffuses rapidly in vivo, making it difficult to restrict labeling to specific locations. In this study, we developed a photocaged 4OHT molecule by covalently attaching 4OHT to an ortho-nitrobenzyl (ONB1) group, rendering 4OHT inactive. Exposure to UV radiation cleaves the bond between ONB1 and 4OHT, freeing the 4OHT to bind Cre-ER to result in downstream genetic recombination and reporter activation. We show that caged ONB1-4OHT crosses the cell membrane and uncages after short UV exposure, resulting in Cre-driven genetic recombination that can be localized to specific regions or tissues. ONB1-4OHT can provide spatial control of reporter activation and be adapted with any existing Cre-ER/loxP based system.

Fluorescently labeled human -[beta]40, combined with membrane conductivity measurements are used to demonstrate the application of single-molecule optical detection to monitor the interaction of single-oligomeric peptide structures with model planar black lipid membranes (BLMs). Larger extended and dynamic structures which are found with large conductances at higher concentrations indicated a major disruption of the membrane.

We report on the practicality of a heteromultivalent design strategy for a nanoplatform that targets lipopolysaccharide molecules (LPS) present on the surface of Gram-negative bacteria. This design is based on the conjugation of a poly(amido amine) (PAMAM) dendrimer with two types of ligands, each having distinct affinities: (i) polymyxin B (PMB) as a primary high affinity ligand; (ii) a PMB-mimicking dendritic branch as an auxiliary low affinity ligand. Co-conjugation of these two ligands maximizes the efficiency of the primary ligand even when the primary ligand is present at a low valency on the nanoplatform (mean nPMB≈ 1). By performing surface plasmon resonance studies using a LPS-immobilized cell wall model, we identified an ethanolamine (EA)-terminated branch as the auxiliary ligand that promotes binding avidity via heteromultivalent association. PMB conjugation of the dendrimer with excess EA branches led to LPS avidity two orders of magnitude greater than unconjugated PMB. Such tight binding observed by SPR corresponded well with adsorption to E. coli cells and with potent bactericidal activity in vitro. [ABSTRACT FROM AUTHOR]

The rational design of a nanoplatform in drug delivery plays a crucial role in determining its targeting specificity and efficacy in vivo. A conventional approach relies on the surface conjugation of a nanometer-sized particle with two functionally distinct types of molecules, one as a targeting ligand, and the other as a therapeutic agent to be delivered to the diseased cell. However, an alternative simplified approach can be used, in which a single type of molecule displaying dual function as both a targeting ligand and therapeutic agent is conjugated to the nanoparticle. In this review, we evaluate the validity of this new strategy by using methotrexate, which displays multifunctional mechanisms of action. Methotrexate binds to the folate receptor, a surface biomarker frequently overexpressed in tumor cells, and also inhibits dihydrofolate reductase, an enzyme critical for cell survival and division. Thus we describe a series of fifth generation poly(amido amine) dendrimers conjugated with methotrexate, and discuss several lines of evidence supporting the efficacy of this new platform strategy based on surface plasmon resonance spectroscopy, enzyme activity assays, and cell-based studies with folate receptor (+) KB cancer cells. [ABSTRACT FROM AUTHOR]

Poly(amidoamine) (PAMAM) dendrimers constitute an important classof nonviral, cationic vectors in gene delivery. Here we report ona new concept for dendrimer vector design based on the incorporationof dual binding motifs: DNA intercalation, and receptor recognitionfor targeted delivery. We prepared a series of dendrimer conjugatesderived from a fifth generation (G5) PAMAM dendrimer, each conjugatedwith multiple folate (FA) or riboflavin (RF) ligands for cell receptortargeting, and with 3,8-diamino-6-phenylphenanthridinium (“DAPP”)-derivedligands for anchoring a DNA payload. Polyplexes of each dendrimerwith calf thymus dsDNA were made and characterized by surface plasmonresonance (SPR) spectroscopy, dynamic light scattering (DLS) and zetapotential measurement. These studies provided evidence supportingpolyplex formation based on the observation of tight DNA-dendrimeradhesion, and changes in particle size and surface charge upon coincubation.Further SPR studies to investigate the adhesion of the polyplex toa model surface immobilized with folate binding protein (FBP), demonstratedthat the DNA payload has only a minimal effect on the receptor bindingactivity of the polyplex: KD= 0.22 nMfor G5(FA)(DAPP) versus 0.98 nM for its polyplex. Finally, we performedin vitro transfection assays to determine the efficiency of conjugatemediated delivery of a luciferase-encoding plasmid into the KB cancercell line and showed that RF-conjugated dendrimers were 1 to 2 ordersof magnitude more effective in enhancing luciferase gene transfectionthan a plasmid only control. In summary, this study serves as a proofof concept for DNA-ligand intercalation as a motif in the design ofmultivalent dendrimer vectors for targeted gene delivery. [ABSTRACT FROM AUTHOR]

Pyridinium aldoxime (PAM) molecules constitute a group of small molecule antidotes essential for the treatment of reactive organophosphate (OP) poisoning. Their therapeutic efficacy stems from a combination of two activities: (i) reactivation of acetylcholine esterase inhibited by OP; (ii) scavenging of free OP. Here, we report the design, synthesis and in vitro functional characterization of fifth generation poly(amidoamine)dendrimer conjugates, each tethered with PAM or hydroxamate, as OP scavenging macromolecules. We chose paraoxon (POX) as the model OP, and performed extensive reaction kinetic studies in aqueous media to determine the activities and mechanisms of these dendrimer conjugates in POX hydrolysis with ¹H NMR, UV-vis and LCMS/MS methods. Quantitative kinetic analysis suggests that the conjugate hydrolyzed POX through a catalytic mechanism as effective as that of the unconjugated molecules. In summary, we report the first class of dendrimer conjugates which are stable in the plasma and functioning as OP-responsive catalytic scavengers. [ABSTRACT FROM AUTHOR]

Many pathogens enter the host through mucosal sites. Thus, interfering with pathogen entry through local neutralization at mucosal sites therefore is an effective strategy for preventing disease. Mucosally administered vaccines have the potential to induce protective immune responses at mucosal sites. This manuscript delves into some of the latest developments in mucosal vaccination, particularly focusing on advancements in adjuvant technologies and the role of these adjuvants in enhancing vaccine efficacy against respiratory pathogens. It highlights the anatomical and immunological complexities of the respiratory mucosal immune system, emphasizing the significance of mucosal secretory IgA and tissue-resident memory T cells in local immune responses. We further discuss the differences between immune responses induced through traditional parenteral vaccination approaches vs. mucosal administration strategies, and explore the protective advantages offered by immunization through mucosal routes.

Importance: The degree of immune protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants provided by infection versus vaccination with wild-type virus remains unresolved, which could influence future vaccine strategies. The gold-standard for assessing immune protection is viral neutralization; however, few studies involve a large-scale analysis of viral neutralization against the Omicron variant by sera from individuals infected with wild-type virus., Objectives: 1) To define the degree to which infection versus vaccination with wild-type SARS-CoV-2 induced neutralizing antibodies against Delta and Omicron variants.2) To determine whether clinically available data, such as infection/vaccination timing or antibody status, can predict variant neutralization., Methods: We examined a longitudinal cohort of 653 subjects with sera collected three times at 3-to-6-month intervals from April 2020 to June 2021. Individuals were categorized according to SARS-CoV-2 infection and vaccination status. Spike and nucleocapsid antibodies were detected via ADVIA Centaur ® (Siemens) and Elecsys ® (Roche) assays, respectively. The Healgen Scientific ® lateral flow assay was used to detect IgG and IgM spike antibody responses. Pseudoviral neutralization assays were performed on all samples using human ACE2 receptor-expressing HEK-293T cells infected with SARS-CoV-2 spike protein pseudotyped lentiviral particles for wild-type (WT), B.1.617.2 (Delta), and B.1.1.529 (Omicron) variants., Results: Vaccination after infection led to the highest neutralization titers at all timepoints for all variants. Neutralization was also more durable in the setting of prior infection versus vaccination alone. Spike antibody clinical testing effectively predicted neutralization for wild-type and Delta. However, nucleocapsid antibody presence was the best independent predictor of Omicron neutralization. Neutralization of Omicron was lower than neutralization of either wild-type or Delta virus across all groups and timepoints, with significant activity only present in patients that were first infected and later immunized., Conclusions: Participants having both infection and vaccination with wild-type virus had the highest neutralizing antibody levels against all variants and had persistence of activity. Neutralization of WT and Delta virus correlated with spike antibody levels against wild-type and Delta variants, but Omicron neutralization was better correlated with evidence of prior infection. These data help explain why 'breakthrough' Omicron infections occurred in previously vaccinated individuals and suggest better protection is observed in those with both vaccination and previous infection. This study also supports the concept of future SARS-CoV-2 Omicron-specific vaccine boosters., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 O’Shea, Schuler, Chen, Troost, Wong, Chen, O’Shea, Peng, Gherasim, Manthei, Valdez, Baldwin and Baker.)