CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function.

CTCF and the associated cohesin complex play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and ß-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context, the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. These findings reveal how 3D chromosome architecture can be encoded by linear genome sequences.

Confluence and convergence of Dscam and Pcdh cell-recognition codes.

The ability of neurites of the same neuron to avoid each other (self-avoidance) is a conserved feature in both invertebrates and vertebrates. The key to self-avoidance is the generation of a unique subset of cell-surface proteins in individual neurons engaging in isoform-specific homophilic interactions that drive neurite repulsion rather than adhesion. Among these cell-surface proteins are fly Dscam1 and vertebrate clustered protocadherins (cPcdhs), as well as the recently characterized shortened Dscam (sDscam) in the Chelicerata. Herein, we review recent advances in our understanding of how cPcdh, Dscam, and sDscam cell-surface recognition codes are expressed and translated into cellular functions essential for neural wiring.

Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion.

Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site in vivo and in vitro, generating double-strand break ends with 5' overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.

Simple and Safe Synthesis of Yolk-Shell-Structured Silicon/Carbon Composites with Enhanced Electrochemical Properties.

With its substantial theoretical capacity, silicon (Si) is a prospective anode material for high-energy-density lithium-ion batteries (LIBs). However, the challenges of a substantial volume expansion and inferior conductivity in Si-based anodes restrict the electrochemical stability. To address this, a yolk-shell-structured Si-carbon composite, featuring adjustable void sizes, was synthesized using tin (Sn) as a template. A uniform coating of tin oxide (SnO2) on the surface of nano-Si particles was achieved through a simple annealing process. This approach enables the removal of the template with concentrated hydrochloric acid (HCl) instead of hydrofluoric acid (HF), thereby reducing toxicity and corrosiveness. The conductivity of Si@void@Carbon (Si@void@C) was further enhanced by using a high-conductivity carbon layer derived from pitch. By incorporating an internal void, this yolk-shell structure effectively enhanced the low Li+/electron conductivity and accommodated the large volume change of Si. Si@void@C demonstrated an excellent electrochemical performance, retaining a discharge capacity of 735.3 mAh g-1 after 100 cycles at 1.0 A g-1. Even at a high current density of 2.0 A g-1, Si@void@C still maintained a discharge capacity of 1238.5 mAh g-1.

Design method for a small F-number two-material uniform dispersion immersion grating imaging spectrometer.

Immersion gratings have high dispersion efficiency and have important application value in miniaturized imaging spectrometers, but its serious dispersion nonlinearity causes difficulties in calibration and image processing, which limits its application range. To solve this, this paper presents a design method for a two-material linear dispersion immersion grating device design method, and a compact small F-number immersion grating spectrometer based on it. First the vector form dispersion equation of the two-material immersion grating is derived and the linear spectral dispersion immersion grating design process is given, then a compact small F-number uniform dispersion imaging spectrometer is given as a design example using the proposed method. The results show that when the operating band of the system is 1590-1675 nm, the spectral resolution is better than 0.25 nm, and F-number can achieve better than 2. Compared with traditional single-material immersion grating imaging spectrometer, the designed imaging spectrometer dispersion linearity is significantly improved. Finally, the influence of prism materials, structure parameters and grating parameters on dispersion nonlinearity is analyzed. Design and analysis results show that the proposed two-material immersion grating device has much better spectral dispersion nonlinearity correction ability, and its design method can provide reference to the compact spectrometer design based on immersion gratings.

Quantitative analysis of SO2, NO2 and NO mixed gases based on ultraviolet absorption spectrum.

SO2, NO2 and NO are the main atmospheric pollutants produced by the combustion of fossil fuel. Detecting these gases is of great significance for atmospheric protection and the online concentration detection of pollutants. In this study, the concentration retrieval methods of NO, NO2 and SO2 and their mutual effects were studied in the wavelength range of 192.3-254.4 nm. In this band, NO, NO2 and SO2 have large absorption cross-sections; however, their spectrum superpositions were serious. A novel method was proposed to separate the superposed absorption spectra of NO and SO2 or NO2. The advantage of this method is that it can remove the influence of SO2 and NO2 on NO concentration retrieval. The fast Fourier transform (FFT) amplitude method was used to calculate the concentrations of SO2 and NO2, and the direct absorption spectroscopy method was used to calculate NO concentration. Via these methods, the gas concentrations of SO2, NO2 and NO can be calculated in ternary-gas mixtures. The experimental results show that these methods can effectively remove the mutual interferences between the concentration retrieval of NO, NO2 and SO2. The maximum absolute values of the relative deviations for the concentration retrieval of SO2, NO2 and NO in ternary-gas mixtures are 3.868%, 4.740% and 5.008%, respectively. These methods have high detection precision and good adaptability and are suitable for online flue detection equipment.

Polarization-selective absorptive and transmissive metamaterials.

A polarization sorting metamaterial with polarization filtering and absorption is proposed. When unpolarized incident light strikes the metamaterial, one polarization component is completely absorbed, and the other polarization component is completely transmitted. We achieved an absorption extinction ratio of up to 350 and a transmission extinction ratio of 425 simultaneously in the LWIR. Unlike the 50% energy utilization limit of other polarization absorbers due to the complete reflection of another polarization component, our proposed metamaterial can be composed of layered polarization selective absorption devices to achieve more than 90% energy utilization. Therefore our design can provide a new solution for real-time polarization detection.

Lithographic pattern quality enhancement of DMD lithography with spatiotemporal modulated technology.

In this paper, we propose spatiotemporal modulation projection lithography (STPL) technology, which is a spatiotemporal modulation technology applied to the conventional digital micromirror device (DMD) projection lithography system. Through coordinating the micro-movement of the piezoelectric stage, the flexible pattern generation of DMD, and the exposure time, the proposed STPL enables us to fabricate a microstructure with smooth edges, accurate linewidth, and accurate line position. Further application on fabricating a diffraction lens has been implemented. The edge sawtooth of the Fresnel zone plate fabricated by using the STPL is reduced to 0.3 µm, the error between the actual measured linewidth and the ideal linewidth is only within ±0.1µm, and the focal length is 15 mm, which is basically consistent with the designed focal length. These results indicated that STPL can serve a significant role in the micromanufacturing field for achieving high-fidelity microdevices.

[Combinatorial CRISPR inversions of CTCF sites in HOXD cluster reveal complex insulator function].

CTCF (CCCTC-binding factor) is a zinc-finger protein which plays a vital role in the three-dimensional (3D) genome architecture. A pair of forward-reverse convergent CTCF binding sites (CBS elements) mediates long-distance DNA interactions to form chromatin loops with the assistance of the cohesin complex, while CBS elements at the chromatin domain boundaries show reverse-forward divergent patterns and function as insulators to discriminate against DNA interaction between chromatin domains. However, there are still many unresolved problems regarding CTCF-mediated insulation function. In order to study the connections between chromatin loops and the insulation function of CBS elements, we combinatorically inverted CBS elements at the HOXD locus by using CRISPR/Cas9 DNA-fragment editing methods in the HEK293T cell line and obtained five different kinds of single-cell CRISPR clones. By performing quantitative high-resolution chromosome conformation capture copy (QHR-4C) experiments, we found that boundary CBS inversions abolish original chromatin loops and establish new loops from the opposite direction, thus shifting the insulator boundary to the new divergent CTCF sites. Furthermore, tandem CBS elements block cohesin permeated from the opposite orientation to function as insulators. RNA-seq experiments showed that alterations of local three-dimensional genome architecture would further influence gene expression of the HOXD cluster. In conclusion, a pair of divergent CBS elements function as insulators by forming chromatin loops within chromatin domains to block cohesin sliding.

[Serial deletions of tandem reverse CTCF sites reveal balanced HOXD regulatory landscape of enhancers].

The genome architectural protein CTCF (CCCTC-binding factor) not only mediates long-distance chromatin interactions between distal enhancers and target promoters, but also functions as an important insulator-binding factor to block improper enhancer activation of non-target promoters, and is thus of great significance to transcriptional regulation of developmental genes. The Hox (Homeobox) gene family plays an important role in the development of the brain, bones, and limbs. The spatiotemporal colinear expression of the HOXD cluster along the proximal-distal axis of limbs is regulated by two clusters of enhancers known as super-enhancers located in the flanking regulatory regions. We focused on the HOXD cluster to explore the architectural role of CTCF in transcriptional regulation of developmental genes. The HOXD cluster contains 9 paralogous genes intermixed with a series of CBS (CTCF-binding site) elements. Using the CRISPR DNA-fragment editing system, we generated a series of single-cell HEK293T clones with deletion of increasing numbers of reverse CBS elements. RNA-seq experiments revealed decreased levels of HOXD gene expression. In addition, chromosome conformation capture experiments revealed increased long-distance chromatin interactions between HOXD and the upstream enhancer cluster and corresponding decreased interactions between HOXD and the downstream enhancer cluster. Thus, tandem reverse CTCF sites function as insulators to maintain HOXD regulatory balance between the upstream and downstream enhancer clusters. This study has interesting implications on the precise gene expression control of the Hox family during animal development.

Graphene oxide induced assembly and crumpling of Co3O4 nanoplates.

Cobalt (II, III) oxide (Co3O4) has been widely studied and applied in various fields, however, it suffers from slow mass and electron transfer during applications. Herein, crumpled Co3O4 and Co3O4/reduced graphene oxide (rGO) with tunable 2D-in-3D structures were prepared by combining spray pyrolysis with a graphene oxide (GO) template. The 2D Co3O4 nanoplates were interconnected with each other to form a 3D ball with many wrinkles, resulting in defect enrichment on the abundant boundaries of the nanosheets, which provided more active sites for catalytic reactions. In addition, the unique 2D-in-3D structure allowed fast mass transfer and structural stability. Furthermore, the assembled structure could be understood as being composed of uniformly distributed oxygen-containing functional groups pinning metal cations on the GO surface through electrostatic interaction, and the 2D structure of the GO enabled the in situ converted Co3O4 to grow along the GO surface with excellent dispersion. Taking advantage of the above, the Co3O4/rGO balls demonstrated an excellent oxygen evolution reaction performance, an overpotential of 298 mV at a current density of 10.0 mA cm-2 and a current density of 115.9 mA cm-2 at the overpotential of η = 500 mV.

User-defined microstructures array fabricated by DMD based multistep lithography with dose modulation.

A flexible and efficient strategy, digital micromirror devices (DMD) based multistep lithography (DMSL), is proposed to fabricate arrays of user-defined microstructures. Through the combination of dose modulation, flexible pattern generation of DMD, and high-resolution step movement of piezoelectrical stage (PZS), this method enables prototyping a board range of 2D lattices with periodic/nonperiodic spatial distribution and arbitrary shapes and the critical feature size is down to 600 nm. We further explore the use of DMSL to fabricate microlens array by combining with the thermal reflowing process. The square shape and hexagonal shape microlens with customized distribution are realized and characterized. The results indicate that the proposed DMSL can be a significant role in the microfabrication techniques for manufacturing functional microstructures array.

Thermally enhanced optical contrast of graphene oxide for thickness identification.

We present a simple, but rapid and accurate approach to identify the layer number of graphene oxide (GO) by using its thermally enhanced optical contrast via vacuum heating. As expected, changes have been observed both in the thicknesses and chemical structures of the material upon the thermal treatment, which can be attributed to the reduction of the amount of intercalated water and oxygen content. This results in the increase of refractive index and absorption coefficient approaching the values for intrinsic graphene. Finally, we achieve an almost complete recovery of optical contrast of GO compared with the one of graphene. The method would be made suitable for the thickness identification of mass-produced GO since it can greatly facilitate sample evaluation and manipulation, and provide immediate feedback to improve synthesis and processing strategies.

Illumination uniformity improvement in digital micromirror device based scanning photolithography system.

Illumination uniformity in photolithography systems determines the dimensional difference across the entire lithographic substrate. However, traditional lithography system relies on expensive and complex illumination system for achieving uniform illumination. In this paper, we propose a simple and cost-effective method based on the modulation of digital micromirror device to improve illumination uniformity. The modulation according to a digital mask achieved via an iteration program improves the uniformity to be above 95%. We demonstrate the effectiveness of the method by experimentally fabricating a linear grating. By implementing this method, the maximum dimensional difference is decreased from 3.3µm to 0.3µm. Further simulations indicate that higher uniformity is achievable once the field of view on the DMD is divided into smaller subregions.

Edge smoothness enhancement in DMD scanning lithography system based on a wobulation technique.

The resolution of digital micro-mirror device (DMD) scanning lithography is limited in the transverse direction (the scanning direction is vertical) as a result of the compacted size of the DMD micro-mirror and the low magnification of the projection lens. Above-stated restrictions lead to an unsatisfactory saw-tooth edge (size ~one DMD pixel) after the lithography process within all directions except for the scanning orientation. In order to smooth the edge, an optimized sub-pattern construction method, described as the combination of wobulation techniques and the continuous scanning lithography process, is proposed. Afterward, lithography experiments were implemented by introducing the wobulation techniques within the DMD scanning lithography system. The experimental results show that the saw-tooth edge is reduced to nearly 0.5 pixel size after 1/2 pixel dislocation superposition exposure, and is even scaled down to less than 0.1 pixel after 1/4 pixel dislocation superposition exposure. At this point, the edge of the lithography pattern is appropriately smoothed. The effectiveness of the above-mentioned method that improves the edge smoothness of the lithography pattern is demonstrated.

Selective photo-oxidation induced bi-periodic plasmonic structures for high-density data storage.

Stable and controllable optical memory is necessary for the development of current information technology. In this context, Ag/TiO2 films have received much attention for their photosensitivity in wavelength and polarization, which can be applied to high-density optical storage. Here, we carried out dual-wavelength holographic recording using 403.4 nm and 532 nm lasers, and obtained mixed microfringes based on selective photodissolution of Ag nanoparticles of various sizes in TiO2 nanoporous films. Two recording methods of simultaneous and sequential multiplexing were investigated. It was found that using simultaneous irradiation it is easier to obtain equivalent efficiency in both spectral hole burning and multiplexed grating diffraction, compared with the sequential one. The results can be explained by the Time-accumulation effect during Ag+ ion diffusion and migration in holographic recordings. Based on such properties, multiplexed-holographic fringes with uniform contrast were reserved by simultaneous recording in Ag/TiO2 films. This work provides a new strategy for fabrication of photonic devices with complex microstructures.

DNA fragment editing of genomes by CRISPR/Cas9.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system from bacteria and archaea emerged recently as a new powerful technology of genome editing in virtually any organism. Due to its simplicity and cost effectiveness, a revolutionary change of genetics has occurred. Here, we summarize the recent development of DNA fragment editing methods by CRISPR/Cas9 and describe targeted DNA fragment deletions, inversions, duplications, insertions, and translocations. The efficient method of DNA fragment editing provides a powerful tool for studying gene function, regulatory elements, tissue development, and disease progression. Finally, we discuss the prospects of CRISPR/Cas9 system and the potential applications of other types of CRISPR system.

[Correlation between children's dental decay and the contents of saliva CCL28 and secretory immunoglobulin A].

OBJECTIVE: To explore the association of the dental decay of children with the contents of chemokine CCL28 and secretory immunoglobulin A (sIgA) in saliva. METHODS: A total of 108 children in 2 kindergartens of Changsha, with age from 3 to 5 years old, were enrolled for this study. The saliva was collected from these children when they were in the examination of mouth. Th e children were divided into 3 groups: A non-caries group [dynamical mean-field theory (DMFT)=0], a low caries group (DMFT=1-4) and a high caries group (DMFT ≥ 5). Th e contents of CCL28 and sIgA were measured by ELISA. RESULTS: The contents of CCL28 and sIgA in saliva were (121.22 ± 32.63) pg/mL and (16.49 ± 8.02) µg/mL, respectively. A positive linear correlation was found between the CCL28 content and sIgA content in saliva (r=0.734). Th e CCL28 and sIgA contents in saliva were positively correlated with the degree of dental caries in children (P<0.05). CONCLUSION: The dental decay of children leads to the secretion of chemokine CCL28, which promotes the secretion of sIgA in saliva.

Short tandem repeats of human genome are intrinsically unstable in cultured cells in vivo.

Short tandem repeats (STRs) are a class of abundant structural or functional elements in the human genome and exhibit a polymorphic nature of repeat length and genetic variation within human populations. Interestingly, STR expansions underlie about 60 neurological disorders. However, "stutter" artifacts or noises render it difficult to investigate the pathogenesis of STR expansions. Here, we systematically investigated STR instability in cultured human cells using GC-rich CAG and AT-rich ATTCT tandem repeats as examples. We found that triplicate bidirectional Sanger sequencing with PCR amplification under proper conditions can reliably assess STR length. In addition, we found that next-generation sequencing with paired-end reads bidirectionally covering STR regions can accurately and reliably assay STR length. Finally, we found that STRs are intrinsically unstable in cultured human cell populations and during single-cell cloning. Our data suggest a general method for accurately and reliably assessing STR length and have important implications in investigating pathogenesis of STR expansion diseases.

Redefining the Glyphosate Sector: Harmonizing Inventiveness and Sustainable Practices for a Better World.

The glyphosate industry has long been a critical player in global agriculture, providing effective and economical solutions for weed control. However, growing concerns over environmental and health impacts have led to increased scrutiny and calls for more sustainable practices. This Viewpoint focuses on the scientific aspects of greener glyphosate synthesis strategies, discussing recent advancements in biobased pathways and catalytic methods, challenges such as scalability and technical hurdles, and future prospects for the herbicide industry. By embracing these new techniques, we can ensure a more sustainable future for herbicide production and contribute to a healthier world.