Nitric Oxide Photorelease from Silicone Films Doped with N-Nitroso BODIPY.

Nitric oxide (NO) is a unique biochemical mediator involved in the regulation of vital processes. Light-controllable NO releasers show promise in the development of smart therapies. Here, we present a novel biocompatible material based on polydimethylsiloxane (PDMS) doped with BODIPY derivatives containing an N-nitroso moiety that is capable of the photoinduced generation of NO. We study the green-light-induced NO-release properties with the following three methods: electrochemical gas-phase sensor, liquid-phase sensor, and the Griess assay. Prolonged release of NO from the polymer films after short irradiation by narrow-band LED light sources and a laser beam is demonstrated. Importantly, this was accompanied by no or little release of the parent compound (BODIPY-based photodonor). Silicone films with the capability of controllable and clean NO release can potentially be used as a highly portable NO delivery system for different therapeutic applications.

Asymmetric nucleosome PARylation at DNA breaks mediates directional nucleosome sliding by ALC1.

The chromatin remodeler ALC1 is activated by DNA damage-induced poly(ADP-ribose) deposited by PARP1/PARP2 and their co-factor HPF1. ALC1 has emerged as a cancer drug target, but how it is recruited to ADP-ribosylated nucleosomes to affect their positioning near DNA breaks is unknown. Here we find that PARP1/HPF1 preferentially initiates ADP-ribosylation on the histone H2B tail closest to the DNA break. To dissect the consequences of such asymmetry, we generate nucleosomes with a defined ADP-ribosylated H2B tail on one side only. The cryo-electron microscopy structure of ALC1 bound to such an asymmetric nucleosome indicates preferential engagement on one side. Using single-molecule FRET, we demonstrate that this asymmetric recruitment gives rise to directed sliding away from the DNA linker closest to the ADP-ribosylation site. Our data suggest a mechanism by which ALC1 slides nucleosomes away from a DNA break to render it more accessible to repair factors.

Single-molecule studies reveal the off-pathway elemental pause state as a target of streptolydigin inhibition of RNA polymerase and its dramatic enhancement by Gre factors.

Antibiotic streptolydigin (Stl) inhibits bacterial transcription by blocking the trigger loop folding in the active center of RNA polymerase (RNAP), which is essential for catalysis. We use acoustic force spectroscopy to characterize the dynamics of transcription elongation in ternary elongation complexes of RNAP (ECs) in the presence of Stl at a single-molecule level. We found that Stl induces long-lived stochastic pauses while the instantaneous velocity of transcription between the pauses is unaffected. Stl enhances the short-lived pauses associated with an off-pathway elemental paused state of the RNAP nucleotide addition cycle. Unexpectedly, we found that transcript cleavage factors GreA and GreB, which were thought to be Stl competitors, do not alleviate the streptolydigin-induced pausing; instead, they synergistically increase transcription inhibition by Stl. This is the first known instance of a transcriptional factor enhancing antibiotic activity. We propose a structural model of the EC-Gre-Stl complex that explains the observed Stl activities and provides insight into possible cooperative action of secondary channel factors and other antibiotics binding at the Stl-pocket. These results offer a new strategy for high-throughput screening for prospective antibacterial agents.

Spatiotemporally controlled generation of NTPs for single-molecule studies.

Many essential processes in the cell depend on proteins that use nucleoside triphosphates (NTPs). Methods that directly monitor the often-complex dynamics of these proteins at the single-molecule level have helped to uncover their mechanisms of action. However, the measurement throughput is typically limited for NTP-utilizing reactions, and the quantitative dissection of complex dynamics over multiple sequential turnovers remains challenging. Here we present a method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations. We demonstrate the effectiveness of LAGOON in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding and RNA polymerase elongation. LAGOON can be readily integrated with many single-molecule techniques, and we anticipate that it will facilitate studies of a wide range of crucial NTP-driven processes.

Design and Synthesis of New Acridone-Based Nitric Oxide Fluorescent Probe.

Nitric oxide (NO) is an important signaling molecule involved in a wide range of physiological and pathological processes. Fluorescent imaging is a useful tool for monitoring NO concentration, which could be essential in various biological and biochemical studies. Here, we report the design of a novel small-molecule fluorescent probe based on 9(10H)acridone moiety for nitric oxide sensing. 7,8-Diamino-4-carboxy-10-methyl-9(10H)acridone reacts with NO in aqueous media in the presence of O2, yielding a corresponding triazole derivative with fivefold increased fluorescence intensity. The probe was shown to be capable of nitric oxide sensing in living Jurkat cells.

Generalized nonequilibrium capillary relations for two-phase flow through heterogeneous media.

For two-phase flow in porous media, the natural medium heterogeneity necessarily gives rise to capillary nonequilibrium effects. The relaxation to the equilibrium is a slow process which should be introduced in macroscopic flow models. Many nonequilibrium models are based on a phenomenological approach. At the same time there exists a rigorous mathematical way to develop the nonequilibrium equations. Its formalism, developed by Bourgeat and Panfilov [Computational Geosciences 2, 191 (1998)], is based on the homogenization of the microscale flow equations over medium heterogeneities. In contrast with the mentioned paper, in which the case of a sufficiently fast relaxation was analyzed, we consider the case of long relaxation, which leads to the appearance of long-term memory on the macroscale. Due to coupling between the nonlinearity and nonlocality in time, the macroscopic model remains, however, incompletely homogenized, in the general case. At the same time, frequently only the relationship for the nonequilibrium capillary pressure is of interest for applications. In the present paper, we obtain such an exact relationship in two different independent forms for the case of long-term memory. This relationship is more general than that obtained by Bourgeat and Panfilov. In addition, we prove the comparison theorem which determines the upper and lower bounds for the macroscopic model. These bounds represent linear flow models, which are completely homogenized. The results obtained are illustrated by numerical simulations.