Thymidine phosphorylase mediates SARS-CoV-2 spike protein enhanced thrombosis in K18-hACE2 mice.

Introduction: Thymidine phosphorylase (TYMP), which facilitates platelet activation and thrombosis, is significantly increased in COVID-19 patients. We hypothesize that TYMP mediates SARS-CoV-2 spike protein (SP)-induced thrombosis.

Materials And Methods: Plasmids encoding wildtype SP or empty vector (p3.

Damped Oscillating Phosphoryl Transfer Reaction in the Cyanobacterial Circadian Clock.

Most organisms have circadian clocks to ensure the metabolic cycle to resonate with the rhythmic environmental changes without "damping," or losing robustness. Cyanobacteria is the oldest and simplest form of life that is known to harbor this biological intricacy. Its KaiABC-based central oscillator proteins can be reconstituted inside a test tube, and the post-translational modification cycle occurs with 24 h periodicity.

Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry.

Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and time-consuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation.

Shift in Conformational Equilibrium Underlies the Oscillatory Phosphoryl Transfer Reaction in the Circadian Clock.

Oscillatory phosphorylation/dephosphorylation can be commonly found in a biological system as a means of signal transduction though its pivotal presence in the workings of circadian clocks has drawn significant interest: for example in a significant portion of the physiology of PCC 7942. The biological oscillatory reaction in the cyanobacterial circadian clock can be visualized through its reconstitution in a test tube by mixing three proteins-KaiA, KaiB and KaiC-with adenosine triphosphate and magnesium ions. Surprisingly, the oscillatory phosphorylation/dephosphorylation of the hexameric KaiC takes place spontaneously and almost indefinitely in a test tube as long as ATP is present.

The Circadian Clock-A Molecular Tool for Survival in Cyanobacteria.

Cyanobacteria are photosynthetic organisms that are known to be responsible for oxygenating Earth's early atmosphere. Having evolved to ensure optimal survival in the periodic light/dark cycle on this planet, their genetic codes are packed with various tools, including a sophisticated biological timekeeping system. Among the cyanobacteria is PCC 7942, the simplest clock-harboring organism with a powerful genetic tool that enabled the identification of its intricate timekeeping mechanism.

Absolute Quantitation of Proteins by Coulometric Mass Spectrometry.

Accurate quantification is essential in the fields of proteomics, clinical assay, and biomarker discovery. Popular methods for absolute protein quantitation by mass spectrometry (MS) involve the digestion of target protein and employ isotope-labeled peptide internal standards to quantify chosen surrogate peptides. Although these methods have gained success, syntheses of isotope-labeled peptides are time-consuming and costly.

CikA, an Input Pathway Component, Senses the Oxidized Quinone Signal to Generate Phase Delays in the Cyanobacterial Circadian Clock.

The circadian clock is a timekeeping system in most organisms that keeps track of the time of day. The rhythm generated by the circadian oscillator must be constantly synchronized with the environmental day/night cycle to make the timekeeping system truly advantageous. In the cyanobacterial circadian clock, quinone is a biological signaling molecule used for entraining and fine-tuning the oscillator, a process in which the external signals are transduced into biological metabolites that adjust the phase of the circadian oscillation.

Magnesium Regulates the Circadian Oscillator in Cyanobacteria.

The circadian clock controls 24-h biological rhythms in our body, influencing many time-related activities such as sleep and wake. The simplest circadian clock is found in cyanobacteria, with the proteins KaiA, KaiB, and KaiC generating a self-sustained circadian oscillation of KaiC phosphorylation and dephosphorylation. KaiA activates KaiC phosphorylation by binding the A-loop of KaiC, while KaiB attenuates the phosphorylation by sequestering KaiA from the A-loop.

CikA Modulates the Effect of KaiA on the Period of the Circadian Oscillation in KaiC Phosphorylation.

Cyanobacteria contain a circadian oscillator that can be reconstituted in vitro. In the reconstituted circadian oscillator, the phosphorylation state of KaiC oscillates with a circadian period, spending about 12 h in the phosphorylation phase and another 12 h in the dephosphorylation phase. Although some entrainment studies have been performed using the reconstituted oscillator, they were insufficient to fully explain entrainment mechanisms of the cyanobacterial circadian clock due to the lack of input pathway components in the in vitro oscillator reaction mixture.

Circadian rhythms. A protein fold switch joins the circadian oscillator to clock output in cyanobacteria.

Organisms are adapted to the relentless cycles of day and night, because they evolved timekeeping systems called circadian clocks, which regulate biological activities with ~24-hour rhythms. The clock of cyanobacteria is driven by a three-protein oscillator composed of KaiA, KaiB, and KaiC, which together generate a circadian rhythm of KaiC phosphorylation. We show that KaiB flips between two distinct three-dimensional folds, and its rare transition to an active state provides a time delay that is required to match the timing of the oscillator to that of Earth's rotation.

Detecting KaiC phosphorylation rhythms of the cyanobacterial circadian oscillator in vitro and in vivo.

The central oscillator of the cyanobacterial circadian clock is unique in the biochemical simplicity of its components and the robustness of the oscillation. The oscillator is composed of three cyanobacterial proteins: KaiA, KaiB, and KaiC. If very pure preparations of these three proteins are mixed in a test tube in the right proportions and with ATP and MgCl2, the phosphorylation states of KaiC will oscillate with a circadian period, and these states can be analyzed simply by SDS-PAGE.

Oxidized quinones signal onset of darkness directly to the cyanobacterial circadian oscillator.

Synchronization of the circadian clock in cyanobacteria with the day/night cycle proceeds without an obvious photoreceptor, leaving open the question of its specific mechanism. The circadian oscillator can be reconstituted in vitro, where the activities of two of its proteins, KaiA and KaiC, are affected by metabolites that reflect photosynthetic activity: KaiC phosphorylation is directly influenced by the ATP/ADP ratio, and KaiA stimulation of KaiC phosphorylation is blocked by oxidized, but not reduced, quinones. Manipulation of the ATP/ADP ratio can reset the timing of KaiC phosphorylation peaks in the reconstituted in vitro oscillator.

Simplicity and complexity in the cyanobacterial circadian clock mechanism.

The circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942 is built on a three-protein central oscillator that can be reconstituted in vitro, a redox-sensitive input for synchronization with the environment, and a bacterial two-component signal transduction pathway for global transcriptional regulation. This review covers the most recent progress in our understanding of the biological and biochemical mechanism of this bacterial clock, such as the discovery of a quinone-binding activity of the oscillator protein KaiA, the molecular mechanism of circadian control of cell division, and the global control of gene expression via modulation of DNA topology.

The KaiA protein of the cyanobacterial circadian oscillator is modulated by a redox-active cofactor.

The circadian rhythms exhibited in the cyanobacterium Synechococcus elongatus are generated by an oscillator comprised of the proteins KaiA, KaiB, and KaiC. An external signal that commonly affects the circadian clock is light. Previously, we reported that the bacteriophytochrome-like protein CikA passes environmental signals to the oscillator by directly binding a quinone and using cellular redox state as a measure of light in this photosynthetic organism.

Elevated ATPase activity of KaiC applies a circadian checkpoint on cell division in Synechococcus elongatus.

A circadian clock coordinates physiology and behavior in diverse groups of living organisms. Another major cyclic cellular event, the cell cycle, is regulated by the circadian clock in the few cases where linkage of these cycles has been studied. In the cyanobacterium Synechococcus elongatus, the circadian clock gates cell division by an unknown mechanism.

A novel allele of kaiA shortens the circadian period and strengthens interaction of oscillator components in the cyanobacterium Synechococcus elongatus PCC 7942.

The basic circadian oscillator of the unicellular fresh water cyanobacterium Synechococcus elongatus PCC 7942, the model organism for cyanobacterial circadian clocks, consists of only three protein components: KaiA, KaiB, and KaiC. These proteins, all of which are homomultimers, periodically interact to form large protein complexes with stoichiometries that depend on the phosphorylation state of KaiC. KaiA stimulates KaiC autophosphorylation through direct physical interactions.

The day/night switch in KaiC, a central oscillator component of the circadian clock of cyanobacteria.

The circadian oscillator of the cyanobacterium Synechococcus elongatus is composed of only three proteins, KaiA, KaiB, and KaiC, which, together with ATP, can generate a self-sustained approximately 24 h oscillation of KaiC phosphorylation for several days. KaiA induces KaiC to autophosphorylate, whereas KaiB blocks the stimulation of KaiC by KaiA, which allows KaiC to autodephosphorylate. We propose and support a model in which the C-terminal loops of KaiC, the "A-loops", are the master switch that determines overall KaiC activity.

Computational and empirical trans-hydrogen bond deuterium isotope shifts suggest that N1-N3 A:U hydrogen bonds of RNA are shorter than those of A:T hydrogen bonds of DNA.

Density functional theory calculations of isolated Watson-Crick A:U and A:T base pairs predict that adenine 13C2 trans-hydrogen bond deuterium isotope shifts due to isotopic substitution at the pyrimidine H3, (2h)Delta13C2, are sensitive to the hydrogen-bond distance between the N1 of adenine and the N3 of uracil or thymine, which supports the notion that (2h)Delta13C2 is sensitive to hydrogen-bond strength. Calculated (2h)Delta13C2 values at a given N1-N3 distance are the same for isolated A:U and A:T base pairs. Replacing uridine residues in RNA with 5-methyl uridine and substituting deoxythymidines in DNA with deoxyuridines do not statistically shift empirical (2h)Delta13C2 values.