Investigating the photophysical properties of rhodamines using a spectroscopic single-molecule fluorescence method.

The photophysical properties of rhodamine molecules play a critical role in their performance across various applications. The spectroscopic single-molecule fluorescence (sSMF) technique overcomes the limitations of conventional SMF by distinguishing individual fluorophores based on their emission spectra. This enables precise measurement and direct comparison of photophysical properties among distinct molecules under identical conditions, without requiring separation of molecules.

An oxygen-scavenging system without impact on DNA mechanical properties in single-molecule fluorescence experiments.

Oxygen scavenging systems (OSSs) are critical for dye stability in single-molecule fluorescence (SMF) experiments. However, the commonly used protocatechuic acid (PCA)/protocatechuate-3,4-dioxygenase (PCD) OSS alters DNA mechanical properties, limiting its applicability. To address this limitation, we examine the bilirubin oxidase (BOD) OSS, which had not been previously used in single-molecule experiments, alongside the pyranose oxidase and catalase (POC) OSS.

Application of Multi-Department Cooperation, Intelligent Prevention, and Supervision to Reduce the Incidence of Central Line-Associated Bloodstream Infections.

Unlabelled: Central line-associated bloodstream infections (CLABSIs) can result in worse outcomes and high hospitalization cost for patients. This study aimed to assess the effectiveness of multi-department cooperation, intelligent prevention, and supervision (MDCIPS) in reducing the incidence of CLABSIs and improving the clinical outcomes of the patients. Key issues were identified through a literature review and survey on the status quo.

Chromatin Buffers Torsional Stress During Transcription.

Transcription through chromatin under torsion represents a fundamental problem in biology. Pol II must overcome nucleosome obstacles and, because of the DNA helical structure, must also rotate relative to the DNA, generating torsional stress. However, there is a limited understanding of how Pol II transcribes through nucleosomes while supercoiling DNA.

A versatile and high-throughput flow-cell system combined with fluorescence imaging for simultaneous single-molecule force measurement and visualization.

A flow-cell offers many advantages for single-molecule studies. But, its merit as a quantitative single-molecule tool has long been underestimated. In this work, we developed a gas-pumped fully calibrated flow-cell system combined with fluorescence imaging for simultaneous single-molecule force measurement and visualization.

Design and simulation for seeding performance of high-speed inclined corn metering device based on discrete element method (DEM).

Mechanical precision corn seed-metering planter has a compact structure, missed and repeated seeding advantages during high-speed operation. In this regard, the current research study focuses on the development of a corn seed planter that features an inclined seed-metering device. The spatial layout of the seed-metering device is optimized to change the seed-filling mode to meet the needs of high-speed operation.

Transcription factor regulation of RNA polymerase's torque generation capacity.

During transcription, RNA polymerase (RNAP) supercoils DNA as it translocates. The resulting torsional stress in DNA can accumulate and, in the absence of regulatory mechanisms, becomes a barrier to RNAP elongation, causing RNAP stalling, backtracking, and transcriptional arrest. Here we investigate whether and how a transcription factor may regulate both torque-induced RNAP stalling and the torque generation capacity of RNAP.

Single-Molecule Angular Optical Trapping for Studying Transcription Under Torsion.

Optical tweezers are flexible and powerful single-molecule tools that have been extensively utilized in biophysical studies. With their ability to stretch and twist DNA, and measure its force and torque simultaneously, they provide excellent opportunities to gain novel insights into the function of protein motors and protein-DNA interactions. Recently, a novel DNA supercoiling assay using an angular optical tweezers (AOT) has been developed to investigate torque generation during transcription.

Mfd Dynamically Regulates Transcription via a Release and Catch-Up Mechanism.

The bacterial Mfd ATPase is increasingly recognized as a general transcription factor that participates in the resolution of transcription conflicts with other processes/roadblocks. This function stems from Mfd's ability to preferentially act on stalled RNA polymerases (RNAPs). However, the mechanism underlying this preference and the subsequent coordination between Mfd and RNAP have remained elusive.

The molecular origin of high DNA-repair efficiency by photolyase.

The primary dynamics in photomachinery such as charge separation in photosynthesis and bond isomerization in sensory photoreceptor are typically ultrafast to accelerate functional dynamics and avoid energy dissipation. The same is also true for the DNA repair enzyme, photolyase. However, it is not known how the photoinduced step is optimized in photolyase to attain maximum efficiency.

Direct determination of resonance energy transfer in photolyase: structural alignment for the functional state.

Photoantenna is essential to energy transduction in photoinduced biological machinery. A photoenzyme, photolyase, has a light-harvesting pigment of methenyltetrahydrofolate (MTHF) that transfers its excitation energy to the catalytic flavin cofactor FADH¯ to enhance DNA-repair efficiency. Here we report our systematic characterization and direct determination of the ultrafast dynamics of resonance energy transfer from excited MTHF to three flavin redox states in E.

Dynamic Determination of Active-Site Reactivity in Semiquinone Photolyase by the Cofactor Photoreduction.

Photolyase contains a flavin cofactor in a fully reduced form as its functional state to repair ultraviolet-damaged DNA upon blue light absorption. However, after purification, the cofactor exists in its oxidized or neutral semiquinone state. Such oxidization eliminates the repair function, but it can be reverted by photoreduction, a photoinduced process with a series of electron-transfer (ET) reactions.

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase.

The flavin cofactor in photoenzyme photolyase and photoreceptor cryptochrome may exist in an oxidized state and should be converted into reduced state(s) for biological functions. Such redox changes can be efficiently achieved by photoinduced electron transfer (ET) through a series of aromatic residues in the enzyme. Here, we report our complete characterization of photoreduction dynamics of photolyase with femtosecond resolution.

Dynamic determination of the functional state in photolyase and the implication for cryptochrome.

The flavin adenine dinucleotide cofactor has an unusual bent configuration in photolyase and cryptochrome, and such a folded structure may have a functional role in initial photochemistry. Using femtosecond spectroscopy, we report here our systematic characterization of cyclic intramolecular electron transfer (ET) dynamics between the flavin and adenine moieties of flavin adenine dinucleotide in four redox forms of the oxidized, neutral, and anionic semiquinone, and anionic hydroquinone states. By comparing wild-type and mutant enzymes, we have determined that the excited neutral oxidized and semiquinone states absorb an electron from the adenine moiety in 19 and 135 ps, whereas the excited anionic semiquinone and hydroquinone states donate an electron to the adenine moiety in 12 ps and 2 ns, respectively.

Electron tunneling pathways and role of adenine in repair of cyclobutane pyrimidine dimer by DNA photolyase.

Electron tunneling pathways in enzymes are critical to their catalytic efficiency. Through electron tunneling, photolyase, a photoenzyme, splits UV-induced cyclobutane pyrimidine dimer into two normal bases. Here, we report our systematic characterization and analyses of photoinitiated three electron transfer processes and cyclobutane ring splitting by following the entire dynamical evolution during enzymatic repair with femtosecond resolution.

Arabidopsis cryptochrome 2 (CRY2) functions by the photoactivation mechanism distinct from the tryptophan (trp) triad-dependent photoreduction.

Cryptochromes are blue-light receptors mediating various light responses in plants and animals. The photochemical mechanism of cryptochromes is not well understood. It has been proposed that photoactivation of cryptochromes involves the blue-light-dependent photoreduction of flavin adenine dinucleotide via the electron transport chain composed of three evolutionarily conserved tryptophan residues known as the "trp triad.

Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase.

Photolyase uses blue light to restore the major ultraviolet (UV)-induced DNA damage, the cyclobutane pyrimidine dimer (CPD), to two normal bases by splitting the cyclobutane ring. Our earlier studies showed that the overall repair is completed in 700 ps through a cyclic electron-transfer radical mechanism. However, the two fundamental processes, electron-tunneling pathways and cyclobutane ring splitting, were not resolved.

Dynamics and mechanism of repair of ultraviolet-induced (6-4) photoproduct by photolyase.

One of the detrimental effects of ultraviolet radiation on DNA is the formation of the (6-4) photoproduct, 6-4PP, between two adjacent pyrimidine rings. This lesion interferes with replication and transcription, and may result in mutation and cell death. In many organisms, a flavoenzyme called photolyase uses blue light energy to repair the 6-4PP (ref.

Ultrafast solvation dynamics at binding and active sites of photolyases.

Dynamic solvation at binding and active sites is critical to protein recognition and enzyme catalysis. We report here the complete characterization of ultrafast solvation dynamics at the recognition site of photoantenna molecule and at the active site of cofactor/substrate in enzyme photolyase by examining femtosecond-resolved fluorescence dynamics and the entire emission spectra. With direct use of intrinsic antenna and cofactor chromophores, we observed the local environment relaxation on the time scales from a few picoseconds to nearly a nanosecond.

Comparative photochemistry of animal type 1 and type 4 cryptochromes.

Cryptochromes (CRYs) are blue-light photoreceptors with known or presumed functions in light-dependent and light-independent gene regulation in plants and animals. Although the photochemistry of plant CRYs has been studied in some detail, the photochemical behavior of animal cryptochromes remains poorly defined in part because it has been difficult to purify animal CRYs with their flavin cofactors. Here we describe the purification of type 4 CRYs of zebrafish and chicken as recombinant proteins with full flavin complement and compare the spectroscopic properties of type 4 and type 1 CRYs.

Ultrafast dynamics and anionic active states of the flavin cofactor in cryptochrome and photolyase.

We report here our systematic studies of the dynamics of four redox states of the flavin cofactor in both photolyases and insect type 1 cryptochromes. With femtosecond resolution, we observed ultrafast photoreduction of oxidized state flavin adenine dinucleotide (FAD) in subpicosecond and of neutral radical semiquinone (FADH(*)) in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the photolyase/cryptochrome family.