We demonstrated that SPAAC-NAD sequencing (SPAAC-NAD-seq), when combined with immunodepletion of m7G-RNAs, enables NAD-RNA identification with accuracy and sensitivity, leading to the discovery of new NAD-RNA profiles in Arabidopsis Furthermore, SPAAC-NAD-seq retained full-length sequence information. Therefore, SPAAC-NAD-seq would enable specific and efficient discovery of NAD-RNAs in prokaryotes and, when combined with m7G-RNA depletion, in eukaryotes.Hyperpolarized fumarate is a promising biosensor for carbon-13 magnetic resonance metabolic imaging. Such molecular imaging applications require nuclear hyperpolarization to attain sufficient signal strength. Dissolution dynamic nuclear polarization is the current state-of-the-art methodology for hyperpolarizing fumarate, but this is expensive and relatively slow. Alternatively, this important biomolecule can be hyperpolarized in a cheap and convenient manner using parahydrogen-induced polarization. However, this process requires a chemical reaction, and the resulting solutions are contaminated with the catalyst, unreacted reagents, and reaction side-product molecules, and are hence unsuitable for use in vivo. In this work we show that the hyperpolarized fumarate can be purified from these contaminants by acid precipitation as a pure solid, and later redissolved to a desired concentration in a clean aqueous solvent. Significant advances in the reaction conditions and reactor equipment allow for formation of hyperpolarized fumarate at 13C polarization levels of 30-45%.Foxp3+CD4+ regulatory T cells (Tregs) regulate most types of immune response as well as several processes important for tissue homeostasis, for example, metabolism and repair. Dedicated Treg compartments-with distinct transcriptomes, T cell receptor repertoires, and growth/survival factor dependencies-have been identified in several nonlymphoid tissues. These Tregs are specifically adapted to function and operate in their home tissue-When, where, and how do they take on their specialized characteristics? We recently reported that a splenic Treg population expressing low levels of the transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) contains precursors of Tregs residing in visceral adipose tissue. This finding made sense given that PPARγ, the "master regulator" of adipocyte differentiation, is required for the accumulation and function of Tregs in visceral adipose tissue but not in lymphoid tissues. Here we use single-cell RNA sequencing, single-cell Tcra and Tcrb sequencing, and adoptive-transfer experiments to show that, unexpectedly, the splenic PPARγlo Treg population is transcriptionally heterogeneous and engenders Tregs in multiple nonlymphoid tissues beyond visceral adipose tissue, such as skin and liver. The existence of a general pool of splenic precursors for nonlymphoid-tissue Tregs opens possibilities for regulating their emergence experimentally or therapeutically.As the core component of the adherens junction in cell-cell adhesion, the cadherin-catenin complex transduces mechanical tension between neighboring cells. Structural studies have shown that the cadherin-catenin complex exists as an ensemble of flexible conformations, with the actin-binding domain (ABD) of α-catenin adopting a variety of configurations. Here, we have determined the nanoscale protein domain dynamics of the cadherin-catenin complex using neutron spin echo spectroscopy (NSE), selective deuteration, and theoretical physics analyses. NSE reveals that, in the cadherin-catenin complex, the motion of the entire ABD becomes activated on nanosecond to submicrosecond timescales. By contrast, in the α-catenin homodimer, only the smaller disordered C-terminal tail of ABD is moving. Molecular dynamics (MD) simulations also show increased mobility of ABD in the cadherin-catenin complex, compared to the α-catenin homodimer. Biased MD simulations further reveal that the applied external forces promote the transition of ABD in the cadherin-catenin complex from an ensemble of diverse conformational states to specific states that resemble the actin-bound structure. The activated motion and an ensemble of flexible configurations of the mechanosensory ABD suggest the formation of an entropic trap in the cadherin-catenin complex, serving as negative allosteric regulation that impedes the complex from binding to actin under zero force. Mechanical tension facilitates the reduction in dynamics and narrows the conformational ensemble of ABD to specific configurations that are well suited to bind F-actin. Our results provide a protein dynamics and entropic explanation for the observed force-sensitive binding behavior of a mechanosensitive protein complex.Asymmetric cell division generates two daughter cells with distinct characteristics and fates. Positioning different regulatory and signaling proteins at the opposing ends of the predivisional cell produces molecularly distinct daughter cells.  https://www.selleckchem.com/products/mpi-0479605.html  Here, we report a strategy deployed by the asymmetrically dividing bacterium Caulobacter crescentus where a regulatory protein is programmed to perform distinct functions at the opposing cell poles. We find that the CtrA proteolysis adaptor protein PopA assumes distinct oligomeric states at the two cell poles through asymmetrically distributed c-di-GMP dimeric at the stalked pole and monomeric at the swarmer pole. Different polar organizing proteins at each cell pole recruit PopA where it interacts with and mediates the function of two molecular machines the ClpXP degradation machinery at the stalked pole and the flagellar basal body at the swarmer pole. We discovered a binding partner of PopA at the swarmer cell pole that together with PopA regulates the length of the flagella filament. Our work demonstrates how a second messenger provides spatiotemporal cues to change the physical behavior of an effector protein, thereby facilitating asymmetry.Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes.