Successive Rearrangements Research Articles

Experimental and computational studies of steric factors in the isomerization of internal alkynes within the coordination environment of half-sandwich metal complexes.

The isomerization of internal alkynes Ar1C≡CAr2 within the coordination environment of low-valent half-sandwich [Ru(dppe)Cp]+ complexes via a 1,2-migration process affords vinylidene species [Ru{=C=C(Ar1)Ar2}(dppe)Cp]+. The rearrangement reactions of symmetrically and asymmetrically substituted substrates featuring different electron-donating and -withdrawing groups and of varying steric bulk were modelled using density functional theory (DFT), and the conclusions supported by experimental observations. Examination of the reaction pathway and associated activation barriers reveal a high solvent dependency for the generation of the key intermediate species [Ru(dppe)Cp]+ from [RuCl(dppe)Cp] by Na+ induced halide abstraction , with the lattice enthalpy of the NaCl by-product playing a critical role in the overall thermochemical balance of the reaction. The activation barriers associated with the reaction of [Ru(dppe)Cp]+ with Ar1C≡CAr2, and the relative energies of the alkyne complexes [Ru(h2-Ar1C≡CAr2)(dppe)Cp]+, are sensitive to the electron density of the alkyne and conformational changes associated with the 'bend-back' of the substrate. The latter differs by up to 66.1 kJ/mol, which in turn impacts the barrier height of the subsequent 1,2-migration step . The relative importance of these factors is evinced by the successful rearrangement of the very sterically congested 1(9-anthryl)-2(9-phenanthryl) acetylene into the fully characterized diaryl vinylidene complex, which was isolated in 89% yield.

Synthesis of Long‐Chain Polyamides via Main‐Chain Modification of Polyethyleneketones

AbstractLong‐chain polyamides (polyethyleneamides) were prepared from polyethylenes bearing in‐chain carbonyl groups (polyethyleneketones) by the oxime formation and successive Beckmann rearrangement. (Diethylamino)sulfur trifluoride (DAST) was utilized as a promoter, which allowed mild conversion of the oxime group in spite of low solubility of the polymers. The polyethyleneamide exhibited different tensile property compared to a commercial HDPE.

Synthesis of Long-Chain Polyamides via Main-Chain Modification of Polyethyleneketones.

Long-chain polyamides (polyethyleneamides) were prepared from polyethylenes bearing in-chain carbonyl groups (polyethyleneketones) by the oxime formation and successive Beckmann rearrangement. (Diethylamino)sulfur trifluoride (DAST) was utilized as a promoter, which allowed mild conversion of the oxime group in spite of low solubility of the polymers. The polyethyleneamide exhibited different tensile property compared to a commercial HDPE.

ScRNA-seq revealed the special TCR β & α V(D)J allelic inclusion rearrangement and the high proportion dual (or more) TCR-expressing cells

Allelic exclusion, one lymphocyte expresses one antigen receptor, is a fundamental mechanism of immunological self-tolerance and highly specific immune responses to pathogens. However, the phenomenon of V(D)J allelic inclusion (incomplete allelic exclusion or allelic escape) rearrangement and dual TCR T cells have been discovered by multiple laboratories. Despite continuous new discoveries, the proportion and underlying mechanism of dual TCR has been puzzling immunologists. In this study, we observed the presence of single T cells expressing multiple TCR chains in all samples, with the proportion of 15%, 10%, and 20% in the human thymus, human peripheral blood, and mouse lymphoid organs, respectively. The proportion of T cells possessing multiple T-cell receptors (TCR) varied significantly in different physiological states and developmental stages. By analyzing RSS category, RSS direction, and V(D)J gene position at TR locus of T cells which contain multiple TCR chains, we creatively found that one of TCR β (or TCR α) should originate from the transcription of V(D)J combination in T-cell receptor excision circle (TREC) formed after the twice successful rearrangement in the same chromosome. Moreover, human V30 (or mouse V31) gene may participate in reverse recombination and transcription to prevent allelic exclusion. In general, high proportion of T cells with multiple TCR at the transcriptome level was first made public, and we proposed a novel mechanism of secondary (or more) TCR rearrangement on a single chromosome. Our findings also indicated that the single-cell sequencing data should be classified according to the single, multiple, and abnormal TCR when analyzing the T-cell repertoire.

Hydroxyl transfer versus cyclization reaction in the gas phase: Sequential loss of NH3 and CH2CO from protonated phenylalanine derivatives.

Collisional activation of protonated phenylalanine derivatives deamination products leads to hydroxyl skeletal rearrangement versus cyclization reaction, and to form hydroxylbenzyl cation via elimination of CH2CO. To better clarify this unusual fragmentation reaction, accurate mass measurements experiments, native isotope experiments, multiple-stage mass spectrometry experiments, different substituents experiments, and density functional theory (DFT) calculations were carried out to investigate the dissociation mechanistic pathways of protonated phenylalanine derivatives deamination products. In route 1, a three-membered ring-opening reaction and a 1,3-hydroxyl transfer (from the carbonyl carbon atom to the interposition carbon atom of carbonyl) occurs to form 3-hydroxy-1-oxo-3-phenylpropan-1-ylium, followed by dissociation to lose CH2CO to give hydroxy (phenyl)methylium. In route 2, a successive cyclization rearrangement reaction and proton transfer occur to form a 2-hydroxylphenylpropionyl cation or protonated 2-hydroxy-4H-benzopyran, followed by dissociation to lose CH2CO or CH≡COH to give 2-hydroxylbenzyl cation. In route 3, a successive hydroxyl transfer (from the carbonyl carbon atom to the ortho carbon atom on benzene) and two stepwise proton transfer (1,2-proton transfer to the ipso-carbon atom of the phenyl ring followed by 1,3-proton transfer to the ortho carbon atom of carbonyl) occurs to form a 2-hydroxylphenylpropionyl cation, which subsequently dissociates to form 2-hydroxylbenzyl cation by elimination of CH2CO. DFT calculations suggested that route 1 was more favorable than route 2 and route 3 from a thermodynamic point of view.

Chromosomal Rearrangements and Chromothripsis: The Alternative End Generation Model

Chromothripsis defines a genetic phenomenon where up to hundreds of clustered chromosomal rearrangements can arise in a single catastrophic event. The phenomenon is associated with cancer and congenital diseases. Most current models on the origin of chromothripsis suggest that prior to chromatin reshuffling numerous DNA double-strand breaks (DSBs) have to exist, i.e., chromosomal shattering precedes rearrangements. However, the preference of a DNA end to rearrange in a proximal accessible region led us to propose chromothripsis as the reaction product of successive chromatin rearrangements. We previously coined this process Alternative End Generation (AEG), where a single DSB with a repair-blocking end initiates a domino effect of rearrangements. Accordingly, chromothripsis is the end product of this domino reaction taking place in a single catastrophic event.

Loss of Zfp335 triggers cGAS/STING-dependent apoptosis of post-β selection thymocytes

Production of a functional peripheral T cell compartment typically involves massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment of double-negative (DN) 2 cells and after successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes the transition of DN4 cells to the DP stage. The signals driving the expansion of DN2 thymocytes are well studied. However, factors regulating the proliferation and survival of DN4 cells remain poorly understood. Here, we uncover an unexpected link between the transcription factor Zfp335 and control of cGAS/STING-dependent cell death in post-β-selection DN4 thymocytes. Zfp335 controls survival by sustaining expression of Ankle2, which suppresses cGAS/STING-dependent cell death. Together, this study identifies Zfp335 as a key transcription factor regulating the survival of proliferating post-β-selection thymocytes and demonstrates a key role for the cGAS/STING pathway in driving apoptosis of developing T cells.

Poor-Quality Vβ Recombination Signal Sequences and the DNA Damage Response ATM Kinase Collaborate to Establish TCRβ Gene Repertoire and Allelic Exclusion.

The monoallelic expression (allelic exclusion) of diverse lymphocyte Ag receptor genes enables specific immune responses. Allelic exclusion is achieved by asynchronous initiation of V(D)J recombination between alleles and protein encoded by successful rearrangement on the first allele signaling permanent inhibition of V rearrangement on the other allele. The ATM kinase that guides DNA repair and transiently suppresses V(D)J recombination also helps impose allelic exclusion through undetermined mechanisms. At the TCRβ locus, one Vβ gene segment (V31) rearranges only by inversion, whereas all other Vβ segments rearrange by deletion except for rare cases in which they rearrange through inversion following V31 rearrangement. The poor-quality recombination signal sequences (RSSs) of V31 and V2 help establish TCRβ gene repertoire and allelic exclusion by stochastically limiting initiation of Vβ rearrangements before TCRβ protein-signaled permanent silencing of Vβ recombination. We show in this study in mice that ATM functions with these RSSs and the weak V1 RSS to shape TCRβ gene repertoire by restricting their Vβ segments from initiating recombination and hindering aberrant nonfunctional Vβ recombination products, especially during inversional V31 rearrangements. We find that ATM collaborates with the V1 and V2 RSSs to help enforce allelic exclusion by facilitating competition between alleles for initiation and functional completion of rearrangements of these Vβ segments. Our data demonstrate that the fundamental genetic DNA elements that underlie inefficient Vβ recombination cooperate with ATM-mediated rapid DNA damage responses to help establish diversity and allelic exclusion of TCRβ genes.

Loss of Zfp335 triggers cGAS/STING-dependent apoptosis of post-β selection pre-T cells

Abstract Production of a diverse peripheral T cell compartment requires massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment at the DN2 stage and following successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes development of DN4 cells to the DP stage. Signals driving expansion of DN2 thymocytes are well studied, however, factors regulating the proliferation and survival of DN4 cells remain poorly understood. Here, we uncover an unexpected link between the transcription factor Zfp335 and control of cGAS/STING-dependent cell death in post-β-selection DN4 thymocytes. Zfp335 controls survival by sustaining expression of Ankle2 and regulating phosphorylation of Baf which functions to promote nuclear envelope integrity and suppress cGAS/STING-dependent cell death. Together, this study identifies Zfp335 as a key transcription factor controlling the survival of proliferating post-β-selection thymocytes and demonstrates a key role for the cGAS/STING pathway driving apoptosis of developing T cells. Supported by grants from NIH R01-GM059638, P01-AI102853, RO1-AI099100, RO1-NS120417, and RO1-AI160737

Cutting Edge: TCR-β Selection Is Required at the CD4+CD8+ Stage of Human T Cell Development.

T cell development is predicated on the successful rearrangement of the TCR gene loci, which encode for Ag-specific receptors. Recombination-activating gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T cell development. In this study, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCR β-chain in mediating β-selection during human T cell development. In stark contrast to mice, human RAG2-KO T lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRβ rearrangements. Nonetheless, RAG2-KO DPs retrovirally transduced to express a rearranged TCR β-chain showed increased survival and proliferation as compared with control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRβ- and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide important insights as to the distinct requirement for the TCR β-chain during human T cell development.

Chloro- and Trifluoromethyl Derivatives of a Pentagon-Fused C60: 1810C60Cl24, 1810C60Cl20, and 1810C60(CF3)14.

The carbon cage of Ih-C60, obeying the isolated-pentagon rule (IPR), can be transformed to the non-IPR D2h-1810C60 cage via two successive Stone-Wales rearrangements in the course of high-temperature chlorination of C60 with SbCl5. Two chloro derivatives, C2v-1810C60Cl24 and C2v-1810C60Cl20, have been isolated by high-performance liquid chromatography (HPLC). High-temperature trifluoromethylation of the chlorination products with CF3I, followed by HPLC separation, afforded a non-IPR CF3 derivative, Cs-1810C60(CF3)14. Structural elucidation of the isolated compounds revealed that all eight sites of pentagon-pentagon fusions on the carbon cage are preferentially occupied by Cl atoms or CF3 groups. According to density functional theory calculations, chloro and CF3 derivatives of 1810C60 are more stable than the isomeric derivatives of 1809C60 or IPR 1812C60, possessing respectively four or no sites of pentagon fusion in their carbon cages.

Assessing Reaction Mechanisms of Graphite Negative Electrodes Based on Operando Synchrotron Radiation Diffraction Data

Since the commercialization of rechargeable Li ion batteries in the early 1990 s, the performance of these devices has continually improved. In such batteries, graphite is typically used as the negative electrode and the present work examined the reaction mechanisms at graphite negative electrodes based on operando synchrotron X-ray diffraction analyses during charge/discharge. The resulting in-plane diffraction patterns of the Li-intercalated graphite permitted a detailed analysis of changes in the three-dimensional structure of the electrode. As the intercalation proceeded from a dilute stage 1 (with less Li intercalation) to a final stage 1 (the formation of LiC6), the material transitioned from a random in-plane structure to a p(√3 × √3)R30° in-plane structure via a superlattice based on a p(3 × 3)R0° in-plane structure. The data also indicate that a series of superlattices was formed during the reaction of the electrode as a result of successive rearrangements, depending on the amount of Li intercalated into the graphite.

EPEN-34. THE CRISPR-Cas9 SYSTEM-MEDIATED ENDOGENOUS GENE REARRANGEMENT INDUCED C11orf95-RELA FUSION IN VITRO AND IN VIVO THAT LED TO THE DEVELOPMENT OF EPENDYMOMA-LIKE TUMOR

Recent large-scale genomic studies of ependymal tumors have identified recurrent RELA and YAP1 fusion genes in supratentorial ependymomas. The formation of the C11orf95-RELA fusion gene has been attributed to massive genomic rearrangement involving chromosome 11q termed Chromothripsis in many cases. However, the causal relationship has not been clarified experimentally. In this study, we developed a system to reproduce the oncogenic gene rearrangement using the CRISPR-Cas9 system and examined whether consequent endogenous ependymoma fusion genes are competent to form brain tumors in mice. Initially, to investigate whether C11orf95-RELA fusion can be formed by inducing the relevant gene rearrangement in vitro, we designed multiple guide RNAs on the human and mouse genomic loci and introduced them into cultured cells. RT-PCR and immunoblot analyses detected endogenous C11orf95-RELA fusion transcript and protein in both human and mouse cultured cells. Subsequently, we lentivirally introduced the gRNAs into a mouse brain. Brain tumor formation was observed from around 2 months after the lentivirus injection, thus indicating successful gene rearrangement followed by C11orf95-RELA fusion expression in vivo. Analysis of the tumor tissue confirmed the expression of the endogenous C11orf95-RELA fusion gene. These results suggested that a gene rearrangement is a primary mechanism to form the C11orf95-RELA fusion which is the direct driver of tumorigenesis. Our system to simulate a genomic event will provide significant insights into the understanding of the tumorigenic mechanism in ependymomas.

Ambient Temperature Graphitization Based on Mechanochemical Synthesis

AbstractGraphite has become a critical material because of its high supply risk and essential applications in energy industries. Its present synthesis still relies on an energy‐intensive thermal treatment pathway (Acheson process) at about 3000 °C. Herein, a mechanochemical approach is demonstrated to afford highly crystalline graphite nanosheets at ambient temperature. The key to the success of our methodology lies in the successive decomposition and rearrangement of a carbon nitride framework driven by a denitriding reaction in the presence of magnesium. The afforded graphite features high crystallinity, a high degree of graphitization, a thin nanosheet architecture, and a small flake size, which endow it with superior efficiency in lithium‐ion batteries as an anode material in terms of rate capacity and cycle stability. The mild and cost‐effective pathway used in this study could be a promising alternative for graphite production.

Ambient Temperature Graphitization Based on Mechanochemical Synthesis.

Graphite has become a critical material because of its high supply risk and essential applications in energy industries. Its present synthesis still relies on an energy-intensive thermal treatment pathway (Acheson process) at about 3000 °C. Herein, a mechanochemical approach is demonstrated to afford highly crystalline graphite nanosheets at ambient temperature. The key to the success of our methodology lies in the successive decomposition and rearrangement of a carbon nitride framework driven by a denitriding reaction in the presence of magnesium. The afforded graphite features high crystallinity, a high degree of graphitization, a thin nanosheet architecture, and a small flake size, which endow it with superior efficiency in lithium-ion batteries as an anode material in terms of rate capacity and cycle stability. The mild and cost-effective pathway used in this study could be a promising alternative for graphite production.

Cross-Species BAC Mapping Highlights Conservation of Chromosome Synteny across Dragon Lizards (Squamata: Agamidae).

Dragon lizards (Squamata: Agamidae) comprise about 520 species in six subfamilies distributed across Asia, Australasia and Africa. Only five species are known to have sex chromosomes. All of them possess ZZ/ZW sex chromosomes, which are microchromosomes in four species from the subfamily Amphibolurinae, but much larger in Phrynocephalus vlangalii from the subfamily Agaminae. In most previous studies of these sex chromosomes, the focus has been on Australian species from the subfamily Amphibolurinae, but only the sex chromosomes of the Australian central bearded dragon (Pogona vitticeps) are well-characterized cytogenetically. To determine the level of synteny of the sex chromosomes of P. vitticeps across agamid subfamilies, we performed cross-species two-colour FISH using two bacterial artificial chromosome (BAC) clones from the pseudo-autosomal regions of P. vitticeps. We mapped these two BACs across representative species from all six subfamilies as well as two species of chameleons, the sister group to agamids. We found that one of these BAC sequences is conserved in macrochromosomes and the other in microchromosomes across the agamid lineages. However, within the Amphibolurinae, there is evidence of multiple chromosomal rearrangements with one of the BACs mapping to the second-largest chromosome pair and to the microchromosomes in multiple species including the sex chromosomes of P. vitticeps. Intriguingly, no hybridization signal was observed in chameleons for either of these BACs, suggesting a likely agamid origin of these sequences. Our study shows lineage-specific evolution of sequences/syntenic blocks and successive rearrangements and reveals a complex history of sequences leading to their association with important biological processes such as the evolution of sex chromosomes and sex determination.

Nonlinear dynamics of dewetting thin films

Fluid films spreading on hydrophobic solid surfaces exhibit complicated dynamics that describe transitions leading the films to break up into droplets. For viscous fluids coating hydrophobic solids this process is called "dewetting". These dynamics can be represented by a lubrication model consisting of a fourth-order nonlinear degenerate parabolic partial differential equation (PDE) for the evolution of the film height. Analysis of the PDE model and its regimes of dynamics have yielded rich and interesting research bringing together a wide array of different mathematical approaches. The early stages of dewetting involve stability analysis and pattern formation from small perturbations and self-similar dynamics for finite-time rupture from larger amplitude perturbations. The intermediate dynamics describes further instabilities yielding topological transitions in the solutions producing sets of slowly-evolving near-equilibrium droplets. The long-time behavior can be reduced to a finite-dimensional dynamical system for the evolution of the droplets as interacting quasi-steady localized structures. This system yields <i>coarsening</i>, the successive re-arrangement and merging of smaller drops into fewer larger drops. To describe macro-scale applications, mean-field models can be constructed for the evolution of the number of droplets and the distribution of droplet sizes. We present an overview of the mathematical challenges and open questions that arise from the stages of dewetting and how they relate to issues in multi-scale modeling and singularity formation that could be applied to other problems in PDEs and materials science.

PDTM-27. THE CRISPR-Cas9 SYSTEM-MEDIATED ENDOGENOUS GENE-REARRANGEMENT INDUCED C11orf95-RELA FUSION IN VITRO AND IN VIVO THAT LED TO DEVELOPMENT OF BRAIN TUMORS

Abstract Recent large-scale genomic studies of ependymal tumors have identified recurrent RELA and YAP1 fusion genes in supratentorial ependymomas. We have so far shown that C11orf95-RELA fusion was a potent oncogene capable of inducing human ependymoma-like tumors in the RCAS/tv-a retroviral gene transfer system. However, molecular mechanism underlying the tumor formation has not been fully investigated. Given that C11orf95-RELA fusion genes are caused by gene rearrangement between C11orf95 and RELA gene due to chromothripsis involving 11q, it has been suggested that expression of these fusion genes is regulated under the C11orf95 promoter. In this study, we developed a system to reproduce the oncogenic gene rearrangement using the CRISPR-Cas9 system and examined if consequent endogenous ependymoma fusion genes are competent to form brain tumor in mice. Initially, we designed guide RNAs on the human and mouse genomic loci corresponding to breakpoints observed in human RELA fusion positive ependymomas and introduced them into HEK293T or NIH3T3 cells. RT-PCR and immunoblot analyses revealed that a combination of multiple guide RNAs efficiently induced gene rearrangement between C11orf95 and RELA gene, resulting in C11orf95-RELA fusion protein. Subsequently, we introduced the same guide RNAs using a lentiviral gene transfer system into mouse brain. Brain tumor formation was observed around 2 months after the guide RNA delivery, indicating successful gene rearrangement followed by C11orf95-RELA fusion expression in vivo. These results thus firmly establish that a gene rearrangement is the mechanism to form the C11orf95-RELA fusion which is the direct driver of tumorigenesis. Furthermore, we found that endogenous YAP1-FAM118B fusion gene can be also induced with a similar experimental strategy to that of C11orf95-RELA fusion. Our system to simulate genomic event will provide insights into the tumorigenic mechanism of ependymomas that harbor various fusion genes.

Ni(NHC) Catalyzed Rearrangement of 1‐Acyl‐2‐vinylcyclopropanes: Tackling a Mechanistic Puzzle by Combined Experimental and Computational Studies

The Ni(NHC) catalyzed rearrangement of 1‐acyl‐2‐vinylcyclopropanes to the corresponding 4‐acyl‐cyclopent‐1‐enes is highly promising for the synthesis of keto‐functionalized annelated bi‐ and tricyclic subunits of natural products. Therefore, we investigated the catalytic activity of Ni(NHC) complexes in the rearrangement of 1‐acyl‐2‐vinylcyclopropanes with different ring sizes and substitution patterns. Surprising effects regarding substrate scope and stereoselectivity of the Ni(NHC) catalyzed vinylcyclopropane‐cyclopentene rearrangement were observed. Only vinylcyclopropanes with 1‐methyl, 1‐phenyl, 1,2‐dialkyl or 2‐phenyl‐substitution at the vinyl moiety could be rearranged successfully. Moreover, an endo‐configuration on the cyclopropane ring was required for successful rearrangement. By treatment of the vinylcyclopropanes with Rh catalysts or Lewis acids, the involvement of Lewis acid catalysis could be ruled out. In order to understand these experimental results and to rationalize the reactivity of the Ni(NHC) complexes computational studies were performed, which provided insights into mechanistic details.

Oxidative Polymerization of 3,6-Phenylenediamino-2,5-dichlorobenzoquinone

The oxidative polymerization of 3,6-phenylenediamino-2,5-dichlorobenzoquinone is studied for the first time, and the monomer is shown to exhibit a high reactivity in this process. The rate of its polymerization is much higher than that of aniline and 3,6-dianiline-2,5-dichlorobenzoquinone, and the induction period of oxidation is absent. The kinetic features of the oxidative polymerization of 3,6-phenylenediamino-2,5-dichlorobenzoquinone are investigated by measuring variation in the redox potential of the reaction mixture in the course of time. It is found that the kinetic curves of 3,6-phenylenediamino-2,5-dichlorobenzoquinone polymerization do not follow the S-shaped pattern typical of aniline polymerization. The mechanism of polymerization proceeding through a number of successive tautomeric rearrangements is advanced. The structure of the polymers is studied by FTIR and X-ray photoelectron spectroscopy. During the reaction of oxidative polymerization, cyclization processes occur to form phenazine and phenoxazine rings which give rise to a ladder polyconjugated polymer. The effect of reaction conditions, such as the concentration of reagents, their ratio, and the pH of the reaction medium, on the process is investigated.