Antipsychotic-induced epigenomic reorganization in frontal cortex of individuals with schizophrenia.

Genome-wide association studies have revealed >270 loci associated with schizophrenia risk, yet these genetic factors do not seem to be sufficient to fully explain the molecular determinants behind this psychiatric condition. Epigenetic marks such as post-translational histone modifications remain largely plastic during development and adulthood, allowing a dynamic impact of environmental factors, including antipsychotic medications, on access to genes and regulatory elements. However, few studies so far have profiled cell-specific genome-wide histone modifications in postmortem brain samples from schizophrenia subjects, or the effect of antipsychotic treatment on such epigenetic marks. Here, we conducted ChIP-seq analyses focusing on histone marks indicative of active enhancers (H3K27ac) and active promoters (H3K4me3), alongside RNA-seq, using frontal cortex samples from antipsychotic-free (AF) and antipsychotic-treated (AT) individuals with schizophrenia, as well as individually matched controls (n=58). Schizophrenia subjects exhibited thousands of neuronal and non-neuronal epigenetic differences at regions that included several susceptibility genetic loci, such as NRG1, DISC1, and DRD3. By analyzing the AF and AT cohorts separately, we identified schizophrenia-associated alterations in specific transcription factors, their regulatees, and epigenomic and transcriptomic features that were reversed by antipsychotic treatment; as well as those that represented a consequence of antipsychotic medication rather than a hallmark of schizophrenia in postmortem human brain samples. Notably, we also found that the effect of age on epigenomic landscapes was more pronounced in frontal cortex of AT-schizophrenics, as compared to AF-schizophrenics and controls. Together, these data provide important evidence of epigenetic alterations in the frontal cortex of individuals with schizophrenia, and remark for the first time on the impact of age and antipsychotic treatment on chromatin organization.

Epigenomic tomography for probing spatially defined chromatin state in the brain.

Spatially resolved epigenomic profiling is critical for understanding biology in the mammalian brain. Single-cell spatial epigenomic assays were developed recently for this purpose, but they remain costly and labor intensive for examining brain tissues across substantial dimensions and surveying a collection of brain samples. Here, we demonstrate an approach, epigenomic tomography, that maps spatial epigenomes of mouse brain at the scale of centimeters. We individually profiled neuronal and glial fractions of mouse neocortex slices with 0.5 mm thickness. Tri-methylation of histone 3 at lysine 27 (H3K27me3) or acetylation of histone 3 at lysine 27 (H3K27ac) features across these slices were grouped into clusters based on their spatial variation patterns to form epigenomic brain maps. As a proof of principle, our approach reveals striking dynamics in the frontal cortex due to kainic-acid-induced seizure, linked with transmembrane ion transporters, exocytosis of synaptic vesicles, and secretion of neurotransmitters. Epigenomic tomography provides a powerful and cost-effective tool for characterizing brain disorders based on the spatial epigenome.

Classification and Quantitative Characterization of Archean Metamorphic Buried Hill Reservoirs in the Bohai Sea.

Based on productivity test data and physical property test results from multiple wells, a classification scheme of Archean metamorphic buried hill reservoirs in the Bohai Sea is established by means of mathematical function fitting. By combining data from cores, casting thin sections, scanning electron microscopy, imaging logging, and high-pressure mercury injection and nitrogen adsorption tests, we clarified the reservoir composition and pore structure characteristics of different types of reservoirs are clarified. Furthermore, taking the BZ19-6 and 13-2 wells in the Archean metamorphic buried hills as an example, the development sites of different types of reservoirs are analyzed and the reservoir development model is established. The results show that the Archean metamorphic buried hill reservoirs in the Bohai Sea can be divided into three categories and six subcategories, including type I reservoirs with porosities greater than 8% or permeabilities greater than 1 × 10-3 µm2 and type II reservoirs with porosities of 5-8% or permeabilities in the range of 0.1-1 × 10-3 µm2. Reservoirs with porosities of 2-5% and permeabilities of 0.01-0.1 × 10-3 µm2 are type III reservoirs. Each type of reservoir can be further divided into a fracture-pore type and a fracture type according to the relative contribution of the porosity and permeability to the reservoir. From type I to type III, the dissolution degree and fracture development gradually weaken, the pore size gradually decreases, and the pore volume gradually decreases. The distribution of favorable reservoirs is comprehensively controlled by weathering and tectonic transformation. The presence of a weathered glutenite zone, weathered leaching zone, or weathered disintegration zone is favorable for the development of type I reservoirs in the weathering crust. In the inner part of the buried hill, the presence of a fracture zone with a thickness of more than 10 m or a dense fracture zone with a thickness of more than 40 m is favorable for the formation of type I reservoirs.

Copper-catalyzed intermolecular formal (5 + 1) annulation of 1,5-diynes with 1,2,5-oxadiazoles.

One-carbon homologation reactions based on one-carbon insertion into the N-O bond of heterocycles have received tremendous interest over the past decades. However, these protocols have to rely on the use of hazardous and not easily accessible diazo compounds as precursors, and examples of the relevant asymmetric catalysis have not been reported. Here we show that a copper-catalyzed intermolecular formal (5 + 1) annulation of 1,5-diynes with 1,2,5-oxadiazoles involving one-carbon insertion into the heterocyclic N-O bond via non-diazo approach. This method enables practical and atom-economic synthesis of valuable pyrrole-substituted oxadiazines in generally moderate to good yields under mild reaction conditions. In addition, the possibility of such an asymmetric formal (5 + 1) annulation also emerges.

Droplet-based bisulfite sequencing for high-throughput profiling of single-cell DNA methylomes.

The genome-wide DNA methylation profile, or DNA methylome, is a critical component of the overall epigenomic landscape that modulates gene activities and cell fate. Single-cell DNA methylomic studies offer unprecedented resolution for detecting and profiling cell subsets based on methylomic features. However, existing single-cell methylomic technologies are based on use of tubes or well plates and these platforms are not easily scalable for handling a large number of single cells. Here we demonstrate a droplet-based microfluidic technology, Drop-BS, to construct single-cell bisulfite sequencing libraries for DNA methylome profiling. Drop-BS takes advantage of the ultrahigh throughput offered by droplet microfluidics to prepare bisulfite sequencing libraries of up to 10,000 single cells within 2 days. We apply the technology to profile mixed cell lines, mouse and human brain tissues to reveal cell type heterogeneity. Drop-BS offers a promising solution for single-cell methylomic studies requiring examination of a large cell population.

Droplet-based bisulfite sequencing for high-throughput profiling of single-cell DNA methylomes.

Genome-wide DNA methylation profile, or DNA methylome, is a critical component of the overall epigenomic landscape that modulates gene activities and cell fate. Single-cell DNA methylomic studies offer unprecedented resolution for detecting and profiling cell subsets based on methylomic features. However, existing single-cell methylomic technologies are all based on use of tubes or well plates and these platforms are not easily scalable for handling a large number of single cells. Here we demonstrate a droplet-based microfluidic technology, Drop-BS, to construct single-cell bisulfite sequencing libraries for DNA methylome profiling. Drop-BS takes advantage of the ultrahigh throughput offered by droplet microfluidics to prepare bisulfite sequencing libraries of up to 10,000 single cells within 2 d. We applied the technology to profile mixed cell lines, mouse and human brain tissues to reveal cell type heterogeneity. Drop-BS will pave the way for single-cell methylomic studies requiring examination of a large cell population.

Copper-Catalyzed Tandem Cyclization/Direct C(sp2)-H Annulation of Azide-Ynamides via α-Imino Copper Carbenes: Access to Azepino[2,3-b:4,5-b']diindoles.

A novel copper-catalyzed tandem cyclization/direct C(sp2)-H annulation of phenyl azide-ynamides via α-imino copper carbenes has been developed, which provides a concise and flexible approach for the construction of a range of valuable azepino[2,3-b:4,5-b']diindoles in mostly good to excellent yields with high chemoselectivities. This tandem reaction also exhibits a broad substrate scope, excellent functional group tolerance, simple operation, and mild reaction conditions.

I2 -Catalyzed Cycloisomerization of Ynamides: Chemoselective and Divergent Access to Indole Derivatives.

Herein, an I2 -catalyzed unprecedented cycloisomerization of ynamides is developed, furnishing various functionalized bis(indole) derivatives in generally good to excellent yields with wide substrate scope and excellent atom-economy. This protocol not only represents the first molecular-iodine-catalyzed tandem complex alkyne cycloisomerizations, but also constitutes the first chemoselective cycloisomerization of tryptamine-ynamides involving distinctively different C(sp3 )-C(sp3 ) bond cleavage and rearrangement. Moreover, chiral tetrahydropyridine frameworks containing two stereocenters are obtained with moderate to excellent diastereoselectivities and excellent enantioselectivities. Meanwhile, cycloisomerization and aromatization of ynamides produce pyrrolyl indoles with high efficiency enabled by I2 . Additionally, control experiments and theoretical calculations reveal that this reaction probably undergoes a tandem 5-exo-dig cyclization/rearrangement process.

Highly Site-Selective Oxidative Cyclization of Ene-ynamides via Non-Noble-Metal Catalysis: Access to Functionalized Lactams.

Herein, an unprecedented non-noble-metal-catalyzed oxidation/cyclization of ene-ynamides is developed, allowing the synthesis of diversely functionalized lactams in moderate to good yields with excellent diastereoselectivities without the observation of typical cyclopropanation products. In combination with Ellman's tert-butylsulfinimine chemistry, chiral γ-lactams containing three contiguous stereocenters are obtained with high diastereo- and enantioselectivity. Moreover, density functional theory (DFT) calculations indicate that this protocol probably undergoes a carbon cation or proton transfer process.

Enantioselective Rh-Catalyzed Azide-Internal-Alkyne Cycloaddition for the Construction of Axially Chiral 1,2,3-Triazoles.

Significant advances have been achieved for the construction of chiral skeletons containing 1,2,3-triazoles via transition-metal-catalyzed asymmetric azide-alkyne cycloaddition; however, most of them have been limited to terminal alkynes in the synthesis of central chirality via desymmetrization and dynamic/dynamic kinetic resolution. Enantioselective transition-metal-catalyzed azide-internal-alkyne cycloaddition is extremely limited. Moreover, the construction of a challenging five-membered (hetero)biaryl axially chiral molecule via transition-metal-catalyzed asymmetric azide-internal-alkyne cycloaddition is still underexplored. Herein, we first report an atroposelective and atom-economical synthesis of axially chiral 1,4,5-trisubstituted 1,2,3-triazoles, directly acting as core chiral units of challenging five-membered atropisomers, via the enantioselective Rh-catalyzed azide-alkyne cycloaddition (E-RhAAC) of internal alkynes and azides. The reaction demonstrates excellent functional group tolerance, forging a variety of C-C axially chiral 1,2,3-triazoles under mild conditions with moderate to excellent yields (up to 99% yield) and generally high to excellent enantioselectivities (up to 99% ee) along with specific regiocontrol. The origin of regio- and enantioselectivity control is disclosed by density functional theory (DFT) calculations, providing new guidance for the facile construction of axially chiral compounds.

nMOWChIP-seq: low-input genome-wide mapping of non-histone targets.

Genome-wide profiling of interactions between genome and various functional proteins is critical for understanding regulatory processes involved in development and diseases. Conventional assays require a large number of cells and high-quality data on tissue samples are scarce. Here we optimized a low-input chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology for profiling RNA polymerase II (Pol II), transcription factor (TF), and enzyme binding at the genome scale. The new approach produces high-quality binding profiles using 1,000-50,000 cells. We used the approach to examine the binding of Pol II and two TFs (EGR1 and MEF2C) in cerebellum and prefrontal cortex of mouse brain and found that their binding profiles are highly reflective of the functional differences between the two brain regions. Our analysis reveals the potential for linking genome-wide TF or Pol II profiles with neuroanatomical origins of brain cells.

Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice.

Clinical evidence suggests that rapid and sustained antidepressant action can be attained with a single exposure to psychedelics. However, the biological substrates and key mediators of psychedelics' enduring action remain unknown. Here, we show that a single administration of the psychedelic DOI produces fast-acting effects on frontal cortex dendritic spine structure and acceleration of fear extinction via the 5-HT2A receptor. Additionally, a single dose of DOI leads to changes in chromatin organization, particularly at enhancer regions of genes involved in synaptic assembly that stretch for days after the psychedelic exposure. These DOI-induced alterations in the neuronal epigenome overlap with genetic loci associated with schizophrenia, depression, and attention deficit hyperactivity disorder. Together, these data support that epigenomic-driven changes in synaptic plasticity sustain psychedelics' long-lasting antidepressant action but also warn about potential substrate overlap with genetic risks for certain psychiatric conditions.

Complete mitochondrial genome sequence of Aspergillus flavus SRRC1009: insight of intraspecific variations on A. flavus mitochondrial genomes.

The mitogenome of Aspergillus flavus SRRC1009 was sequenced to investigate intraspecific variations on mitochondrial genomes of A. flavus. It shows 29,202 bp with a typical configuration of Aspergillus mitogenome. Sixteen SNPs and 22 INDELs and 17 SNPs and 27 INDELs were identified against AflaGuard® and JQ355000, respectively. Phylogenetic trees present in the three A. flavus mitochondrial genomes were clustered with A. oryzae mitochondrial genome in one clade.

The velvet Regulator VosA Governs Survival and Secondary Metabolism of Sexual Spores in Aspergillus nidulans.

The velvet regulator VosA plays a pivotal role in asexual sporulation in the model filamentous fungus Aspergillus nidulans. In the present study, we characterize the roles of VosA in sexual spores (ascospores) in A. nidulans. During ascospore maturation, the deletion of vosA causes a rapid decrease in spore viability. The absence of vosA also results in a lack of trehalose biogenesis and decreased tolerance of ascospores to thermal and oxidative stresses. RNA-seq-based genome-wide expression analysis demonstrated that the loss of vosA leads to elevated expression of sterigmatocystin (ST) biosynthetic genes and a slight increase in ST production in ascospores. Moreover, the deletion of vosA causes upregulation of additional gene clusters associated with the biosynthesis of other secondary metabolites, including asperthecin, microperfuranone, and monodictyphenone. On the other hand, the lack of vosA results in the downregulation of various genes involved in primary metabolism. In addition, vosA deletion alters mRNA levels of genes associated with the cell wall integrity and trehalose biosynthesis. Overall, these results demonstrate that the velvet regulator VosA plays a key role in the maturation and the cellular and metabolic integrity of sexual spores in A. nidulans.

Microfluidic Platform for Next-Generation Sequencing Library Preparation with Low-Input Samples.

Advances in next-generation sequencing (NGS) have made available a wealth of information that had previously been inaccessible to researchers and clinicians. NGS has been applied to understand genomic, transcriptomic, and epigenomic changes and gained traction as a significant tool capable of accelerating diagnosis, prognosis, and biomarker discovery. However, these NGS assays have yet to be practical methods for patient stratification or diagnosis because of the gap between the tiny quantities of biomaterials provided by a clinical sample and the large DNA input required by most of these assays. Current library preparation methodologies typically require large input amounts of DNA and a long and complicated manual process. Here, we present a microfluidic droplet-based system for NGS library preparation, capable of reducing the number of pipetting steps significantly, reducing reagent consumption by 10×, and automating much of the process, while supporting an extremely low DNA input requirement (10 pg per library). This semiautomated technology will allow for low-input preparations of 8 libraries simultaneously while reducing batch-to-batch variation and operator hands-on time.

MOWChIP-seq for low-input and multiplexed profiling of genome-wide histone modifications.

Epigenetic mechanisms such as histone modifications play critical roles in adaptive tuning of chromatin structures. Profiling of various histone modifications at the genome scale using tissues from animal and human samples is an important step for functional studies of epigenomes and epigenomics-based precision medicine. Because the profile of a histone mark is highly specific to a cell type, cell isolation from tissues is often necessary to generate a homogeneous cell population, and such operations tend to yield a low number of cells. In addition, high-throughput processing is often desirable because of the multiplexity of histone marks of interest and the large quantity of samples in a hospital setting. In this protocol, we provide detailed instructions for device fabrication, setup, and operation of microfluidic oscillatory washing-based chromatin immunoprecipitation followed by sequencing (MOWChIP-seq) for profiling of histone modifications using as few as 100 cells per assay with a throughput as high as eight assays in one run. MOWChIP-seq operation involves flowing of chromatin fragments through a packed bed of antibody-coated beads, followed by vigorous microfluidic oscillatory washing. Our process is semi-automated to reduce labor and improve reproducibility. Using one eight-unit device, it takes 2 d to produce eight sequencing libraries from chromatin samples. The technology is scalable. We used the protocol to study a number of histone modifications in various types of mouse and human tissues. The protocol can be conducted by a user who is familiar with molecular biology procedures and has basic engineering skills.

A general framework for solving inverse dynamics problems in multi-axis motion control.

An inverse dynamics compensation (IDC) scheme for the execution of curvilinear paths by multi-axis motion controllers is proposed. For a path specified by a parametric curve r(ξ), the IDC scheme computes a real-time path correction Δr(ξ) that (theoretically) eliminates path deviations incurred by the inertia and damping of the machine axes. To exploit the linear time-invariant nature of the dynamic equations, the correction term is computed as a function of elapsed time t, and the corresponding curve parameter values ξ are only determined as the final step of the IDC scheme, through a real-time interpolator algorithm. It is shown that, in general, the correction term for P, PI, and PID controllers consists of derivative, natural, and integral terms (the integrand of the latter involving only the path r(ξ), and not its derivatives). The use of lead segments to minimize transient effects associated with the initial conditions is also discussed, and the performance of the method is illustrated by simulation results. The IDC scheme is expressed in terms of a linear differential operator formalism to provide a clear, general, and systematic development, amenable to further adaptations and extensions.

Facile synthesis of α-alkoxyl amides via scandium-catalyzed oxidative reaction between ynamides and alcohols.

A novel and efficient scandium-catalyzed oxidative reaction between ynamides and alcohols for the facile synthesis of various α-alkoxyl amides is reported in this paper. The reaction avoids the need for the use of α-diazo carbonyls which are unstable and may cause some safety concerns. Instead, by using alkynes as the starting materials, this protocol features readily available substrates, compatibility with a broad range of functional groups, simple procedure, mild reaction conditions, and high chemoselectivity.

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites.

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs - polysiloxane-based and epoxy-based - are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 °C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

Electromechanically Responsive Liquid Crystal Elastomer Nanocomposites for Active Cell Culture.

Liquid crystal elastomers (LCEs) are unique among shape-responsive materials in that they exhibit large and reversible shape changes and can respond to a variety of stimuli. However, only a handful of studies have explored LCEs for biomedical applications. Here, we demonstrate that LCE nanocomposites (LCE-NCs) exhibit a fast and reversible electromechanical response and can be employed as dynamic substrates for cell culture. A two-step method for preparing conductive LCE-NCs is described, which produces materials that exhibit rapid (response times as fast at 0.6 s), large-amplitude (contraction by up to 30%), and fully reversible shape changes (stable to over 5000 cycles) under externally applied voltages (5-40 V). The electromechanical response of the LCE-NCs is tunable through variation of the electrical potential and LCE-NC composition. We utilize conductive LCE-NCs as responsive substrates to culture neonatal rat ventricular myocytes (NRVM) and find that NRVM remain viable on both stimulated and static LCE-NC substrates. These materials provide a reliable and simple route to materials that exhibit a fast, reversible, and large-amplitude electromechanical response.