Differentiated embryonic chondrocyte expressed gene-1 is a central signaling component in the development of collagen-induced rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases and affects almost 1% of the population. Differentiated embryo-chondrocyte expressed gene-1 (DEC1) has been associated with both osteogenesis and osteoclastogenesis. RA condition is marked by inflammatory hyperplasia, and DEC1 is known to support inflammatory reactions and implicated in antiapoptosis and cell invasion. Here, our goal was to test the hypothesis that DEC1 enhances RA development induced by collagen-induced arthritis (CIA), a well-recognized protocol for developing RA animal models. DEC1+/+ and DEC1-/- mice were subjected to CIA protocol, and the development of RA condition was monitored. We found that CIA robustly induced RA phenotypes (e.g., synovial hyperplasia) and greatly increased the expression of proinflammatory cytokines such as TNF-α. However, these changes were detected in DEC1+/+ but not DEC1-/- mice. Interestingly, these very cytokines strongly induced DEC1, and such a dual role of DEC1, as an inducer for and being induced by proinflammatory cytokines, constitutes a DEC1-amplifying circuit for inflammation. Knockdown of DEC1 in human MH7A cells strongly decreased cell migration and invasion as well as the expression of genes related to RA phenotypes. The combination of DEC1-directed migration and invasion in vitro with synovial hyperplasia in vivo mechanistically establishes cellular bases on how DEC1 is involved in the development of RA phenotypes. In addition to inflammatory signaling, DEC1 functionally interacted with PI3KCA(p110α)/Akt/GSK3ß, Wnt/ß-catenin, and NFATc1. Such engagement in multiple signaling pathways suggests that DEC1 plays coordinated and integral roles in developing RA, one of the most common autoimmune diseases.

Knockdown of Tet2 Inhibits the Myogenic Differentiation of Chicken Myoblasts Induced by Ascorbic Acid.

Ascorbic acid (also called Vitamin C, VC) strengthens the function of Tets families and directly increases DNA demethylation level to affect myogenic differentiation. However, the precise regulatory mechanism of DNA methylation in chicken myogenesis remains unclear. Results of present study showed that the mRNA expression of MyoD significantly decreased and MyoG and MyHC increased in myoblasts treated with 5 µM 5-azacytidine (5-AZA) and 5 µM VC (p < 0.05). Results also indicated the formation of myotubes was induced by 5-AZA or VC, but this effect was attenuated after knockdown of Tet2. In addition, the protein expression of TET2, DESMIN and MyHC was remarkable increased by the addition of 5-AZA or VC, and the upregulation was inhibited after knockdown of Tet2 (p < 0.05). DNA dot blot and immunofluorescence staining results suggested that the level of 5hmC was significantly increased when treated with 5-AZA or VC, even by Tet2 knockdown (p < 0.05). Moreover, 5-AZA and VC reduced the level of dimethylation of lysine 9 (H3K9me2) and trimethylation of lysine 27 of histone 3 (H3K27me3), and this inhibitory effect was eliminated after Tet2 knockdown (p < 0.05). These data indicated that Tet2 knockdown antagonized the increased levels of 5hmC and H3K27me3 induced by 5-AZA and VC, and eventually reduced myotube formation by modulating the expression of genes involved in myogenic differentiation. This study provides insights that epigenetic regulators play essential roles in mediating the myogenic program of chicken myoblasts.

Chemical Bonding as a New Avenue for Controlling Excited-State Properties and Excitation Energy-Transfer Processes in Zinc Phthalocyanine-Fullerene Dyads.

Whether chemical bonding can regulate the excited-state and optoelectronic properties of donor-acceptor dyads has been largely elusive. In this work, we used electronic structure and nonadiabatic dynamics methods to explore the excited-state properties of covalently bonded zinc phthalocyanine (ZnPc)-fullerene (C60 ) dyads with a 6-6 (or 5-6) bonding configuration in which ZnPc is bonded to two carbon atoms shared by the two hexagonal rings (or a pentagonal and a hexagonal ring) in C60 . In both cases, the locally excited (LE) states on ZnPc are spectroscopically bright. However, their different chemical bonding differentiates the electronic interactions between ZnPc and C60 . In the 5-6 bonding configuration, the LE states on ZnPc are much higher in energy than the LE states on C60 . Thus, the excitation energy transfer from ZnPc to C60 is thermodynamically favorable. On the other hand, in the 6-6 bonding configuration, such a process is inhibited because the LE states on ZnPc are the lowest ones. More detailed mechanisms are elucidated from nonadiabatic dynamics simulations. In the 6-6 bonding configuration, no excitation energy transfer was observed. In contrast, in the 5-6 bonding configuration, several LE and charge-transfer (CT) excitons were shown to participate in the energy-transfer process. Further analysis reveals that the photoinduced energy transfer is mediated by a CT exciton, such that electron- and hole-transfer processes take place in a concerted but asynchronous manner in the excitation energy transfer. It is also found that high-level electronic structure methods including exciton effects are indispensable to accurately describe photoinduced energy- and electron-transfer processes. Furthermore, this work opens up new avenues for regulating the excited-state properties of molecular donor-acceptor dyads by means of chemical bonding.

Importance of Appropriately Regularizing the ML-MCTDH Equations of Motion.

The multiconfiguration time-dependent Hartree (MCTDH) method and its generalization, the multilayer MCTDH (ML-MCTDH), result in equations of motion (EOMs) that are singular when there are virtual orbitals-the unoccupied single-particle functions-in the wave function expansion. For decades this singularity had been numerically removed by regularizing the reduced density matrix. In this Perspective we discuss our recent proposal to regularize the coefficient tensor instead, which has significant impact on both the efficiency and correctness of the EOMs in MCTDH and ML-MCTDH for challenging problems. We further demonstrate that when the system becomes large such that it is necessary to use ML-MCTDH with many layers, it is much more important to employ this new regularization scheme. We illustrate this point by studying a spin-boson model with a large bath that contains up to 100 000 modes. We show that even in the weak coupling regime the new regularization scheme is required to quickly rotate the virtual orbitals into the correct directions in Hilbert space. We argue that this situation can be common for applying a time-dependent tensor network approach to any large enough system.

Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation.

Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region for compaction of chromatin in vitro, promote the condensate formation both in vitro and in vivo We showed that the CBX2 condensates concentrate DNA and nucleosomes. Using genetic engineering, we report that trimethylation of Lys-27 at histone H3 (H3K27me3), a marker of heterochromatin formation produced by PRC2, had minimal effects on the CBX2 condensate formation. We further demonstrated that the CBX2 condensate formation does not require CBX2-PRC1 subunits; however, the condensate formation of CBX2-PRC1 subunits depends on CBX2, suggesting a mechanism underlying the assembly of CBX2-PRC1 condensates. In summary, our results reveal that PcG condensates assemble through liquid-liquid phase separation (LLPS) and suggest that phase-separated condensates can organize PcG-bound chromatin.

Experimental and theoretical rationalization for the base pairing abilities of inosine, guanosine, adenosine, and their corresponding 8-oxo-7,8-dihydropurine, and 8-bromopurine analogues within A-form duplexes of RNA.

Inosine is an important RNA modification, furthermore RNA oxidation has gained interest due, in part, to its potential role in the development/progression of disease as well as on its impact on RNA structure and function. In this report we established the base pairing abilities of purine nucleobases G, I, A, as well as their corresponding, 8-oxo-7,8-dihydropurine (common products of oxidation at the C8-position of purines), and 8-bromopurine (as probes to explore conformational changes), derivatives, namely 8-oxoG, 8-oxoI, 8-oxoA, 8-BrG, and 8-BrI. Dodecamers of RNA were obtained using standard phosphoramidite chemistry via solid-phase synthesis, and used as models to establish the impact that each of these nucleobases have on the thermal stability of duplexes, when base pairing to canonical and noncanonical nucleobases. Thermal stabilities were obtained from thermal denaturation transition (Tm ) measurements, via circular dichroism (CD). The results were then rationalized using models of base pairs between two monomers, via density functional theory (DFT), that allowed us to better understand potential contributions from H-bonding patterns arising from distinct conformations. Overall, some of the important results indicate that: (a) an anti-I:syn-A base pair provides thermal stability, due to the absence of the exocyclic amine; (b) 8-oxoG base pairs like U, and does not induce destabilization within the duplex when compared to the pyrimidine ring; (c) a U:G wobble-pair is only stabilized by G; and (d) 8-oxoA displays an inherited base pairing promiscuity in this sequence context. Gaining a better understanding of how this oxidatively generated lesions potentially base pair with other nucleobases will be useful to predict various biological outcomes, as well as in the design of biomaterials and/or nucleotide derivatives with biological potential.

DoRWA3 from Dendrobium officinale Plays an Essential Role in Acetylation of Polysaccharides.

The acetylation or deacetylation of polysaccharides can influence their physical properties and biological activities. One main constituent of the edible medicinal orchid, Dendrobium officinale, is water-soluble polysaccharides (WSPs) with substituted O-acetyl groups. Both O-acetyl groups and WSPs show a similar trend in different organs, but the genes coding for enzymes that transfer acetyl groups to WSPs have not been identified. In this study, we report that REDUCED WALL ACETYLATION (RWA) proteins may act as acetyltransferases. Three DoRWA genes were identified, cloned, and sequenced. They were sensitive to abscisic acid (ABA), but there were no differences in germination rate and root length between wild type and 35S::DoRWA3 transgenic lines under ABA stress. Three DoRWA proteins were localized in the endoplasmic reticulum. DoRWA3 had relatively stronger transcript levels in organs where acetyl groups accumulated than DoRWA1 and DoRWA2, was co-expressed with polysaccharides synthetic genes, so it was considered as a candidate acetyltransferase gene. The level of acetylation of polysaccharides increased significantly in the seeds, leaves and stems of three 35S::DoRWA3 transgenic lines compared to wild type plants. These results indicate that DoRWA3 can transfer acetyl groups to polysaccharides and is a candidate protein to improve the biological activity of other edible and medicinal plants.

Functional Characterization of a Dendrobium officinale Geraniol Synthase DoGES1 Involved in Floral Scent Formation.

Floral scent is a key ornamental trait that determines the quality and commercial value of orchids. Geraniol, an important volatile monoterpene in orchids that attracts pollinators, is also involved in responses to stresses but the geraniol synthase (GES) responsible for its synthesis in the medicinal orchid Dendrobium officinale has not yet been identified. In this study, three potential geraniol synthases were mined from the D. officinale genome. DoGES1, which was localized in chloroplasts, was characterized as a geraniol synthase. DoGES1 was highly expressed in flowers, especially in petals. DoGES1 transcript levels were high in the budding stage of D. officinale flowers at 11:00 a.m. DoGES1 catalyzed geraniol in vitro, and transient expression of DoGES1 in Nicotiana benthamiana leaves resulted in the accumulation of geraniol in vivo. These findings on DoGES1 advance our understanding of geraniol biosynthesis in orchids, and lay the basis for genetic modification of floral scent in D. officinale or in other ornamental orchids.

Heat Transport in a Spin-Boson Model at Low Temperatures: A Multilayer Multiconfiguration Time-Dependent Hartree Study.

Extending our previous work, quantum dynamic simulations are performed to study low temperature heat transport in a spin-boson model where a two-level subsystem is coupled to two independent harmonic baths. Multilayer multiconfiguration time-dependent Hartree theory is used to numerically evaluate the thermal flux, for which the bath is represented by hundreds to thousands of modes. The simulation results are compared with the approximate Redfield theory approach, and the physics is analyzed versus different physical parameters.

Photocycloaddition of S,S-Dioxo-benzothiophene-2-methanol, Reactivity in the Solid State and in Solution: Mechanistic Studies and Diastereoselective Formation of Cyclobutyl Rings.

The formation of cyclobutane rings is a promising strategy in the development of potential drugs and/or synthetic intermediates, typically challenging to obtain due to their constrained nature. In this work, the [2 + 2] photocycloaddition reaction of S,S-dioxobenzothiophene-2-methanol was explored in microcrystalline powders and its outcome was compared to that observed in solution. It was found that the molecular constraints inherited within the crystal lattice provide an optimal environment that leads to photodimer 4 as the major product in ca. 9.6:0.4 diastereomeric ratios with conversions >95%. The photoreaction was analyzed via X-ray, displaying a crystalline-to-amorphous transformation and showing that units of monomer 2 align to generate the corresponding dimer with a syn-head-to-tail regio- and diastereoselectivity. This result contrasted with that obtained in solution, where the diastereomeric ratio varied as a function of the excited state that is generated, to yield mixtures of dimers 4 and 5 (anti-head-to-tail), or exclusively 5 in the triplet-sensitized photoreaction, in the presence of benzophenone. Density functional theory was used to elucidate a plausible detailed mechanism for the phototransformation, which aided in justifying the results that led to the corresponding dimers. X-ray crystallography allowed us to establish the stereochemical assignment of the obtained cyclobutyl rings. Thus, the use of solid-state or solution photochemistry can be used to gain control of diastereo- and regioselectivities in the formation of this important moiety.

Quantum Phase Transition in the Spin-Boson Model: A Multilayer Multiconfiguration Time-Dependent Hartree Study.

The multilayer improved relaxation is applied to study the delocalization-localization transition in the spin-boson model at zero temperature-a well-known example of quantum phase transition. Calculations of energy eigenstates are obtained by iteratively diagonalizing the matrix of the Boltzmann operator in the top layer representation, using a Lanczos/Arnoldi method while relaxing the single particle functions of all layers using the multilayer multiconfiguration time-dependent Hartree imaginary time propagation. Two properties are used to examine the quantum phase transition: the energy splitting for the lowest pair of eigenstates and the magnetic susceptibility. Consistent findings are obtained with appropriate scaling parameters.

A unified view of hierarchy approach and formula of differentiation.

The stochastic differential equation is a powerful tool for describing the dynamics of a dissipative system in which noise characterizes the influence of the environment. For the Ornstein-Uhlenbeck noise, both the formula of differentiation and the hierarchy approach provide efficient numerical simulations, with the stochastic differential equation transformed into a set of coupled, linear ordinary differential equations. We show that while these two deterministic schemes result in different sets of equations, they can be regarded as two representations of an underlying linear-dynamics. Moreover, by manipulating the involved Ornstein-Uhlenbeck noise, we propose a unified algorithm that may reduce to the hierarchy approach or the formula of differentiation in different limits. We further analyze the numerical performance of this algorithm and find that the hierarchy approach appears to be more efficient for our numerical model studies.

Approximate DFT-based methods for generating diabatic states and calculating electronic couplings: models of two and more states.

Four types of density functional theory (DFT)-based approaches are assessed in this work for the approximate construction of diabatic states and the evaluation of electronic couplings between these states. These approaches include the constrained DFT (CDFT) method, the constrained noninteracting electron (CNE) model to post-process Kohn-Sham operators, the approximate block-diagonalization (BD) of the Kohn-Sham operators, and the generalized Mulliken-Hush method. It is shown that the first three approaches provide a good description for long-distance intermolecular electron transfer (ET) reactions. On the other hand, inconsistent results were found when applying these approaches to intramolecular ET in strongly coupled, mixed-valence systems. Model analysis shows that this discrepancy is caused by the inappropriate use of the two-state model rather than the defects of the approaches themselves. The situation is much improved when more states are included in the model electronic Hamiltonian. The CNE and BD approaches can thus serve as efficient and robust alternatives for building ET models based on DFT calculations.

On regularizing the ML-MCTDH equations of motion.

In a recent paper [H.-D. Meyer and H. Wang, J. Chem. Phys. 148, 124105 (2018)], we have examined the regularization of the equations of motion (EOMs) of the multiconfiguration time-dependent Hartree (MCTDH) approach. We could show that the standard regularization scheme used by almost all researchers in the field is not optimal. The improved regularization allows for larger values of the regularization parameter ϵ, is less sensitive to the actual choice of ϵ, and performs the rotation of initially unoccupied single-particle functions into the "correct" direction in Hilbert space much faster than the old scheme. The latter point increases both the accuracy and efficiency of time propagation for challenging problems. For simple problems, the new scheme requires some additional numerical work as compared with the old scheme, ranging from negligible to almost doubling the total numerical labor. For demanding problems, on the other hand, the additional numerical work of the new scheme is often overcompensated by less steps taken by the integrator. In the present paper, we generalize the new regularization scheme to the multi-layer (ML) extension of MCTDH. Although the principle idea of the new regularization scheme remains unaltered, it was not obvious how the new scheme should be implemented into ML-MCTDH. The ML-MCTDH EOMs are much more complicated than the MCTDH ones, and for optimal numerical performance it was necessary to derive a recursive algorithm for implementing the new regularization scheme.

On regularizing the MCTDH equations of motion.

The Multiconfiguration Time-Dependent Hartree (MCTDH) approach leads to equations of motion (EOM) which become singular when there are unoccupied so-called single-particle functions (SPFs). Starting from a Hartree product, all SPFs, except the first one, are unoccupied initially. To solve the MCTDH-EOMs numerically, one therefore has to remove the singularity by a regularization procedure. Usually the inverse of a density matrix is regularized. Here we argue and show that regularizing the coefficient tensor, which in turn regularizes the density matrix as well, leads to an improved performance of the EOMs. The initially unoccupied SPFs are rotated faster into their "correct direction" in Hilbert space and the final results are less sensitive to the choice of the value of the regularization parameter. For a particular example (a spin-boson system studied with a transformed Hamiltonian), we could even show that only with the new regularization scheme could one obtain correct results. Finally, in Appendix A, a new integration scheme for the MCTDH-EOMs developed by Lubich and co-workers is discussed. It is argued that this scheme does not solve the problem of the unoccupied natural orbitals because this scheme ignores the latter and does not propagate them at all.

On the memory kernel and the reduced system propagator.

We relate the memory kernel in the Nakajima-Zwanzig-Mori time-convolution approach to the reduced system propagator which is often used to obtain the kernel in the Tokuyama-Mori time-convolutionless approach. The connection provides a robust and simple formalism to compute the memory kernel for a generalized system-bath model circumventing the need to compute high order system-bath observables, thus streamlining the use of numerically exact solvers for calculating the memory kernel. We illustrate this for a model system with electron-electron and electron-phonon couplings, driven away from equilibrium.

Direct Aryloxylation/Alkyloxylation of Dialkyl Phosphonates for the Synthesis of Mixed Phosphonates.

A strategy for the direct functionalization strategy of inertial dialkyl phosphonates with hydroxy compounds to afford diverse mixed phosphonates with good yields and functional-group tolerance has been developed. Mechanistic investigations involving both NMR studies and DFT studies suggest that an unprecedented highly reactive PV species (phosphoryl pyridin-1-ium salt), a key intermediate for this new synthetic transformation, is generated in situ from dialkyl phosphonate in the presence of Tf2 O/pyridine.

A multilayer multiconfiguration time-dependent Hartree simulation of the reaction-coordinate spin-boson model employing an interaction picture.

The multilayer multiconfiguration time-dependent Hartree method is applied in an interaction picture to simulate dynamics of the spin-boson model in the reaction-coordinate representation. The use of the interaction picture allows a more effective description of correlation effects, especially when the coupling strength between the reaction coordinate and the bath is very strong. Examples show that in most physical regimes the efficiency is improved significantly, in some cases up to several orders of magnitude. This opens up new avenues for studying quantum dynamical problems.

Quantum kinetic expansion in the spin-boson model: Matrix formulation and system-bath factorized initial state.

Within the framework of the hierarchy equation of motion (HEOM), the quantum kinetic expansion (QKE) method of the spin-boson model is reformulated in the matrix representation. The equivalence between the two formulations (HEOM matrices and quantum operators) is numerically verified from the calculation of the time-integrated QKE rates. The matrix formulation of the QKE is extended to the system-bath factorized initial state. Following a one-to-one mapping between HEOM matrices and quantum operators, a quantum kinetic equation is rederived. The rate kernel is modified by an extra term following a systematic expansion over the site-site coupling. This modified QKE is numerically tested for its reliability by calculating the time-integrated rate and non-Markovian population kinetics. For an intermediate-to-strong dissipation strength and a large site-site coupling, the population transfer is found to be significantly different when the initial condition is changed from the local equilibrium to system-bath factorized state.

Synthesis, Thermal Stability, Biophysical Properties, and Molecular Modeling of Oligonucleotides of RNA Containing 2'-O-2-Thiophenylmethyl Groups.

Dodecamers of RNA [CUACGGAAUCAU] were functionalized with C2'-O-2-thiophenylmethyl groups to obtain oligonucleotides 10-14 and 17. The modified nucleotides were incorporated into RNA strands via solid-phase synthesis. The biophysical properties of these ONs were used to quantify the effects of this modification on RNA:RNA and RNA:DNA duplexes. A combination of UV-vis and circular dichroism were used to determine thermal stabilities of all strands, which hybridized into A-form geometries. Destabilization of the double stranded RNA was measured as a function of number of consecutive modifications, reflected in decreased thermal denaturation values (ΔTm, ca. 2.5-11.5 °C). Van't Hoff plots on a duplex containing one modification (10:15) displayed a ca. ΔΔG° of +4 kcal/mol with respect to its canonical analogue. Interestingly, hybridization of two modified strands (13:17, containing a total of eight modifications) resulted in increased stability and a distinct secondary structure, reflected in its CD spectrum. Molecular modeling based on DFT calculations shed light on the nature of this stability, with induced changes in the torsional angle δ (C5'-C4'-C3'-O3) and phosphate-phosphate distances that are in agreement with a compacted structure. The described synthetic methodology and structural information will be useful in the design of thermodynamically stable structures containing chemically reactive modifications.