The [4Fe4S] Cluster of Yeast DNA Polymerase ε Is Redox Active and Can Undergo DNA-Mediated Signaling.

Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase ε (Pol ε) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2CORE, are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2COREexo-, was used to limit DNA degradation. While no redox signal is apparent for Pol2COREexo- on DNA-modified electrodes, a large cathodic signal centered at -140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2COREexo-, which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S]2+, to oxidized Pol2COREexo- bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIIIY82A, a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2COREexo-. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2COREexo- via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

Therapeutic Potential of Two Visible Light Responsive Luminescent photoCORMs: Enhanced Cellular Internalization Driven by Lipophilicity.

Herein we report the synthesis, characterization, and cellular internalization properties of two visible-light active luminescent Mn-based photoCORMs. The enhanced membrane permeability of the photoactive Mn carbonyl complex (photoCORM) derived from a designed lipophilic ligand namely, [Mn(CO)3(Imdansyl)(L1)](CF3SO3) (1) (where L1 = a diazabutadiene-based ligand containing two highly lipophilic adamantyl motifs, Imdansyl = dansylimidazole) promoted rapid internalization within human colorectal adenocarcinoma (HT-29) cells compared to [Mn(CO)3(Imdansyl)(L2)](CF3SO3) (2) (where L2 = a diazabutadiene ligand bearing two hydrophilic 1,3,5-triazaadamantyl group). Colocalization experiments using membrane stain indicate different extents of localization of the two CO complexes within the cellular matrix. Visible-light triggered CO release from the lipophilic photoCORM induced caspase-3/7 activation on HT-29 cells, which was detected using confocal microscopy. The rapid accumulation of the lipophilic photoCORM 1 in the cellular membrane resulted in more efficient CO-induced cell death compared to the hydrophilic analogue 2.

Incorporation of a Theranostic "Two-Tone" Luminescent Silver Complex into Biocompatible Agar Hydrogel Composite for the Eradication of ESKAPE Pathogens in a Skin and Soft Tissue Infection Model.

Microbial invasion and colonization of the skin and underlying soft tissues are among the most common types of infections, becoming increasingly prevalent in hospital settings. Systemic antibiotic chemotherapies are now extremely limited due to emergence of drug-resistant Gram-positive and multidrug-resistant Gram-negative bacterial strains. Topical administration of antimicrobials provides an effective route for the treatment of skin and soft tissue infections (SSTIs). Therefore, the development of new and effective materials for the delivery of these agents is of paramount importance. Silver is a broad-spectrum antibiotic used for the treatment and prevention of infections since ancient times. However, the high reactivity of silver cation (Ag+) makes its incorporation into delivery materials quite challenging. Herein we report a novel soft agar hydrogel composite for the delivery of Ag+ into infected wound sites. This material incorporates a Ag(I) complex [Ag2(DSX)2(NO3)2] (1; DSX = 5-(dimethylamino)- N, N-bis(pyridin-2-ylmethyl) naphthalene-1-sulfonamide) that exhibits a change in fluorescence upon Ag+ release and qualitatively indicates the end point of silver delivery. The antibacterial efficacy of the material was tested against several bacterial strains in an SSTI model. The complex 1-agar composite proved effective at eradicating the pathogens responsible for the majority of SSTIs. The theranostic (therapeutic/diagnostic) properties coupled with its stability, softness, ease of application, and removal make this material an attractive silver-delivery vehicle for the treatment and prevention of SSTIs.

Eradication of HT-29 colorectal adenocarcinoma cells by controlled photorelease of CO from a CO-releasing polymer (photoCORP-1) triggered by visible light through an optical fiber-based device.

The gaseous signaling molecule carbon monoxide (CO) has recently been recognized for its wide range of physiological activity as well as its antineoplastic properties. However, site-specific delivery of this noxious gas presents a major challenge in hospital settings. In this work, a visible light-sensitive CO-releasing molecule (photoCORM) derived from manganese(I) and 2-(quinolyl)benzothiazole (qbt) namely, [Mn(CO)3(qbt)(4-vpy)](CF3SO3) (1), has been co-polymerized within a gas-permeable HEMA/EGDMA hydrogel. The resulting photoactive CO-releasing polymer (photoCORP-1) incorporates 1 such that neither the carbonyl complex nor its photoproduct(s) exits the polymer at any time. The material can be triggered to photorelease CO remotely by low-power broadband visible light (<1mWcm-2) with the aid of fiber optics technology. The CO photorelease rates of photoCORP-1 (determined by spectrophotometry) can be modulated by both the concentration of 1 in the hydrogel and the intensity of the light. A CO-delivery device has been assembled to deliver CO to a suspension of human colorectal adenocarcinoma cells (HT-29) under the control of visible light and the extent of CO-induced apoptotic death of the cancer cells has been determined via Annexin V/Propidium iodide stain and flow cytometry. This photoactive CO-releasing polymer could find use in delivering controlled doses of CO to cellular targets such as malignant tissues in remote parts of the body.

Tracking silver delivery to bacteria using turn-on fluorescence.

Herein we present a novel silver complex [Ag(qBODIPY)(CF3SO3)] (1) that exhibits dramatic increase in fluorescence upon silver release. Complex 1 has a minimum inhibitory concentration comparable to that of silver nitrate. Confocal microscopy was used to track the delivery of silver to bacterial targets.