Robust miniature Cas-based transcriptional modulation by engineering Un1Cas12f1 and tethering Sso7d.

The miniature V-F CRISPR-Cas12f system has been repurposed for gene editing and transcription modulation. The small size of Cas12f satisfies the packaging capacity of adeno-associated virus (AAV) for gene therapy. However, the efficiency of Cas12f-mediated transcriptional activation varies among different target sites. Here, we developed a robust miniature Cas-based transcriptional activation or silencing system using Un1Cas12f1. We engineered Un1Cas12f1 and the cognate guide RNA and generated miniCRa, which led to a 1,319-fold increase in the activation of the ASCL1 gene. The activity can be further increased by tethering DNA-binding protein Sso7d to miniCRa and generating SminiCRa, which reached a 5,628-fold activation of the ASCL1 gene and at least hundreds-fold activation at other genes examined. We adopted these mutations of Un1Cas12f1 for transcriptional repression and generated miniCRi or SminiCRi, which led to the repression of ∼80% on average of eight genes. We generated an all-in-one AAV vector AIOminiCRi used to silence the disease-related gene SERPINA1. AIOminiCRi AAVs led to the 70% repression of the SERPINA1 gene in the Huh-7 cells. In summary, miniCRa, SminiCRa, miniCRi, and SminiCRi are robust miniature transcriptional modulators with high specificity that expand the toolbox for biomedical research and therapeutic applications.

Orthogonal Control of Nucleic Acid Function via Chemical Caging-Decaging Strategies.

The ability to precisely control the function of nucleic acids plays an important role in biosensing and biomedicine. In recent years, novel strategies employing biological, physical, and chemical triggers have been developed to modulate the function of nucleic acids spatiotemporally. These approaches commonly involve the incorporation of stimuli-responsive groups onto nucleic acids to block their functions until triggers-induced decaging restore activity. These inventive strategies deepen our comprehension of nucleic acid molecules' dynamic behavior and provide new techniques for precise disease diagnosis and treatment. Focusing on the spatiotemporal regulation of nucleic acid molecules through the chemical caging-decaging strategy, we here present an overview of the innovative triggered control mechanisms and accentuate their implications across the fields of chemical biology, biomedicine, and biosensing.

De Novo Assembly of 20 Chicken Genomes Reveals the Undetectable Phenomenon for Thousands of Core Genes on Microchromosomes and Subtelomeric Regions.

The gene numbers and evolutionary rates of birds were assumed to be much lower than those of mammals, which is in sharp contrast to the huge species number and morphological diversity of birds. It is, therefore, necessary to construct a complete avian genome and analyze its evolution. We constructed a chicken pan-genome from 20 de novo assembled genomes with high sequencing depth, and identified 1,335 protein-coding genes and 3,011 long noncoding RNAs not found in GRCg6a. The majority of these novel genes were detected across most individuals of the examined transcriptomes but were seldomly measured in each of the DNA sequencing data regardless of Illumina or PacBio technology. Furthermore, different from previous pan-genome models, most of these novel genes were overrepresented on chromosomal subtelomeric regions and microchromosomes, surrounded by extremely high proportions of tandem repeats, which strongly blocks DNA sequencing. These hidden genes were proved to be shared by all chicken genomes, included many housekeeping genes, and enriched in immune pathways. Comparative genomics revealed the novel genes had 3-fold elevated substitution rates than known ones, updating the knowledge about evolutionary rates in birds. Our study provides a framework for constructing a better chicken genome, which will contribute toward the understanding of avian evolution and the improvement of poultry breeding.

Leaf Transcriptome and Weight Gene Co-expression Network Analysis Uncovers Genes Associated with Photosynthetic Efficiency in Camellia oleifera.

Camellia oleifera Abel. (C. oleifera) as an important economic tree species in China has drawn growing attention because of its highly commercial, medic, cosmetic, and ornamental value. To deepen our understanding about the photosynthetic characters during the whole developmental stage as well as the molecular basis of photosynthesis, a comparative analysis of the leaf transcriptome of two C. oleifera cultivars, 'Guoyou No.13' (GY13) and 'Xianglin No.82' (XL82), with different photosynthetic characteristics from May to September has been conducted. In this study, a group of genes related to photosynthesis, hormone regulation, circadian clock and transcription factor, involved in the photosynthetic advantage. Photosynthetic parameters from May to September of these two cultivars provided evidence supporting photosynthetic advantage of GY13 compared to XL82. In addition, expression levels of 12 differentially expressed genes (DEGs) were validated using real-time PCR (RT-PCR). To screen gene clusters and hub genes that might directly regulated the photosynthetic differences between cultivars, a Weight Gene Co-expression Network Analysis (WGCNA) was conducted. Three co-expression network (module) and top ten connected genes (hub genes) were identified that might play crucial role in the regulatory network of photosynthesis. The results not only showed multiple functional genes that might involve in the differences of photosynthetic characteristics between cultivars, but also provide some evidences for the heat tolerance might be an important character which helps GY13 kept higher photosynthetic parameters than XL82 during the developmental stage. In summary, our transcriptomic approach together with RT-PCR tests allowed us to expand our understanding of the characters of C. oleifera cultivars with different photosynthetic efficiency during the developmental stage and to further exploring new candidate genes involve in high photosynthetic efficiency in molecular-assisted breeding program of C. oleifera.

"Repaired and Activated" DNAzyme Enables the Monitoring of DNA Alkylation Repair in Live Cells.

Direct measurement of DNA repair is critical for the annotation of their clinical relevance and the discovery of drugs for cancer therapy. Here we reported a "repaired and activated" DNAzyme (RADzyme) by incorporating a single methyl lesion (O6 MeG, 3MeC, or 1MeA) at designated positions through systematic screening. We found that the catalytic activity of the RADzyme was remarkably suppressed and could be restored via enzyme-mediated DNA repair. Benefit from these findings, a fluorogenic RADzyme sensor was developed for the monitoring of MGMT-mediated repair of O6 MeG lesion. Importantly, the sensor allowed the evaluation of MGMT repair activity in different cells and under drugs treatment. Furthermore, another RADzyme sensor was engineered for the monitoring of ALKBH2-mediated repair of 3MeC lesion. This strategy provides a simple and versatile tool for the study of the basic biology of DNA repair, clinical diagnosis and therapeutic assessment.

Assessment of genetic diversity in Camellia oleifera Abel. accessions using morphological traits and simple sequence repeat (SSR) markers.

Camellia oleifera Abel. (C. oleifera) is a cultivable plant with important economic value. It is very helpful for the scientific utilization, cultivation and preservation of germplasm resources through evaluating the genetic diversity. In this study, we estimated the genetic relationship of 150 accessions of C. oleifera using morphological and economic traits, as well as SSR molecular marker. Through the variation and cluster analysis of 17 morphological and economic traits, the germplasm was divided into a candidate core breeding group with higher economic traits and a core breeding group with higher morphological traits. The genetic similarity coefficients of SSR markers ranged from 0.05 to 0.91, and the germplasm materials were divided into five groups. The results demonstrated that C. oleifera germplasms perform a rich genetic variation. This is the first report to evaluate the genetic diversity of different C. oleifera germplasms using the morphological and economic traits, together with SSR molecular marker, and the results allow us to find evidence for the origin of varieties, establish core breeding populations and its fingerprint.

Genomic, molecular evolution, and expression analysis of NOX genes in soybean (Glycine max).

Reactive oxygen species (ROS) are versatile signaling molecules in sensing stresses and play critical roles in signaling and development. Plasma membrane NADPH oxidases (NOXs) are key producers of ROS, and play important roles in the regulation of plant-pathogen interactions. Here, we performed a comprehensive analysis of the NOX gene family in the soybean genome (Glycine max) and 17 NOX (GmNOX) genes were identified. Structural analysis revealed that the GmNOX proteins in soybean were as conserved as those in other plants. 8 duplicated gene pairs were formed by a Glycine-specific whole-genome duplication (WGD) event approximately 13 million years ago (Mya). The Ka/Ks ratios of GmNOX genes ranged from 0.04 to 0.28, suggesting that the GmNOX family had undergone purifying selection in soybean. Gene expression patterns showed different expression of these duplicate genes, suggesting that the GmNOXs were retained by substantial subfunctionalization during the soybean evolutionary processes. Subsequently, the expression of GmNOXs in response to drought and phytohormones were characterized via qPCR. Importantly, four GmNOXs showed strong expression in nodules, pointing to their probable involvement in nodulation. Thus, our results shed light on the evolutionary history of this family in soybean and contribute to the functional characterization of GmNOX genes in soybean.

Detection of Macromolecular Content in a Mixed Solution of Protein Macromolecules and Small Molecules Using a Weak Measurement Linear Differential System.

In this paper, we propose a linear differential detection system based on a frequency domain weak measurement. The system can be used for detecting optical substances. Moreover, we completed an experiment to detect himan serum albumin (HSA) content in a mixture of human serum albumin and l-proline via dialysis. This work also proves the differential function of the system. This experiment can be further extended to detecting protein content in a mixed solution that contains protein macromolecules and various small molecules. It is very important for detecting molecules without photomarking in solutions of complex biological samples. In this paper, the system has an optical resolution of 1.39 × 10-5, and resolution of 4.06 × 10-8 mol/L for himan serum protein solution.

Diastereoselective Synthesis of Oxazoloisoindolinones via Cascade Pd-Catalyzed ortho-Acylation of N-Benzoyl α-Amino Acid Derivatives and Subsequent Double Intramolecular Cyclizations.

The first cascade diastereoselective synthesis of oxazoloisoindolinones via the palladium-catalyzed decarboxylative ortho-acylation of N-benzoyl α-amino acid derivatives followed by double intramolecular cyclizations has been demonstrated. This reaction, using α-amino acids as directing groups and α-oxocarboxylic acids as the acylation source, features a broad substrate scope, good functional group tolerance, high regioselectivity, and excellent diastereoselectivity.

Identification of a novel 43-bp insertion in the heparan sulfate 6-O-sulfotransferase 3 (HS6ST3) gene and its associations with growth and carcass traits in chickens.

Previous studies have revealed a significant association between SNPs found within the heparan sulfate 6-O-sulfotransferase 3 (HS6ST3) gene and obesity. This study identified a novel 43-bp indel polymorphism in intron 1 of HS6ST3 in 1963 chickens from nine different breeds, and three genotypes, designated II, ID and DD, were observed. The frequency of the 'I' (0.62-0.87) allele was higher than that of the 'D' (0.13-0.38) allele. A total of 777 individuals of the Gushi-Anka F2 resource population were used for the analysis of associations according to growth traits, carcass traits, serum variables and meat quality traits. The results showed that the 43-bp indel polymorphism was significantly associated with the body weight at 4 and 6 weeks of age, chest depth at 4 and 12 weeks of age and shank girth at 12 weeks of age (P < 0.05). In terms of the carcass traits, the indel polymorphism was significantly associated with breast muscle weight, heart weight and leg weight (P < 0.05). These findings suggested that this indel polymorphism has the potential to become a new target for the marker-assisted selection of chicken growth and carcass traits.

A limiting Current Oxygen Sensor Constituted of(CeO2)0.95(Y2O3)0.05 as Solid Electrolyte Layer and(CeO2)0.75(ZrO2)0.25 as Dense Diffusion Barrier Layer.

Using the co-precipitation method to synthesize (CeO2)0.95(Y2O3)0.05 (YDC) and solidreaction method to synthesize (CeO2)0.75(ZrO2)0.25 (ZDC), and the crystal structure, micro-structure,total conductivity and electronic conductivity of the two materials was measured with X-raydiffraction (XRD), scanning electron microscope (SEM), DC van der Pauw and Hebb-Wagnermethods. A limiting current oxygen sensor was prepared with YDC solid electrolyte and a ZDCdense diffusion barrier layer by employing platinum pasting bonding. Sensing characteristics ofthe sensor were obtained at different conditions, including temperature (T), oxygen concentration(x(O2)) and water vapor pressure (p(H2O)), and the influence of various conditions on sensingperformance was studied. The long-term stability of the sensor was measured in an oxygen concentration of 1.2% and at a temperature of 800 °C for 120 h. XRD results show that the phase structure of both YDC and ZDC belongs to the cubic phase. SEM results show that both YDC and ZDC layers are dense layers, which are then qualified to be the composition materials of the sensor. The limiting current (IL) of the sensor is obtained and the sensor exhibits good sensing characteristics to satisfy the Knudsen model. Log(IL·T) depends linearly on 1000/T with a squared correlation coefficient (R2) of 0.9904; IL depends linearly on x(O2) with an R2 of 0.9726; and sensing characteristics are not affected by p(H2O). It was found that the oxygen sensor has good long-term stability.

Branched Hybridization Chain Reaction Circuit for Ultrasensitive Localizable Imaging of mRNA in Living Cells.

Hybridization chain reaction (HCR) circuits are valuable approaches to monitor low-abundance mRNA, and current HCR is still subjected to issues such as limited amplification efficiency, compromised localization resolution, or complicated designs. We report a novel branched HCR (bHCR) circuit for efficient signal-amplified imaging of mRNA in living cells. The bHCR can be realized using a simplified design by hierarchically coupling two HCR circuits with two split initiator fragments of the secondary HCR circuit incorporated in the probes for the primary HCR circuit. The bHCR circuit enables one to generate a hyperbranched assembly seeded from a single target initiator, affording the potential for localizing single target molecules in live cells. In vitro assays show that bHCR offers very high amplification efficiency and specificity in single mismatch discrimination with a detection limit of 500 fM. Live cell studies reveal that bHCR displays intense fluorescence spots indicating mRNA localization in living cells with improved contrast. The bHCR method can provide a useful platform for low-abundance biomarker detection and imaging for cell biology and diagnostics.

Cell Surface-Anchored DNA Nanomachine for Dynamically Tunable Sensing and Imaging of Extracellular pH.

DNA nanodevices that mimic natural biomolecular machines changing configurations in response to external inputs have enabled smart sensors to live cell imaging. We report for the first time the development of a dynamic DNA nanomachine that is anchored on a cell's surface and undergoes pH-responsive triplex-duplex conformation switching, allowing tunable sensing and imaging of extracellular pH. Results reveal that the DNA nanomachine can be stably anchored on the cell surface via multiple anchors, and the adjustment of C+G-C content in the switch element confers tunability of pH response windows. The anchored DNA nanomachine also demonstrates desirable sensitivity, excellent reversibility, and quantitative ability for extracellular pH detection and imaging. This cell surface-anchored pH-responsive DNA nanomachine can provide a useful platform for pH sensing in extracellular microenvironments and diagnostics of different pH-related diseases.

Optimization of a quantum weak measurement system with its working areas.

Phase-sensitive weak measurement systems have been receiving an increasing amount of attention. In this paper, we introduce a series of weak measurement working areas. By adjusting the pre-selection and post-selection states and the total phase difference between vertically polarized light and horizontally polarized light, the measurement of the weak value is amplified by several times in one system. Its applicability is verified in a label-free total internal reflection system. The original sensitivity and resolution are improved at different working areas, reaching 1.85 um/refractive index unit (RIU) and 6.808 × 10-7 RIU, respectively.

Fast phase retrieval in off-axis digital holographic microscopy through deep learning.

Traditional digital holographic imaging algorithms need multiple iterations to obtain focused reconstructed image, which is time-consuming. In terms of phase retrieval, there is also the problem of phase compensation in addition to focusing task. Here, a new method is proposed for fast digital focus, where we use U-type convolutional neural network (U-net) to recover the original phase of microscopic samples. Generated data sets are used to simulate different degrees of defocused image, and verify that the U-net can restore the original phase to a great extent and realize phase compensation at the same time. We apply this method in the construction of real-time off-axis digital holographic microscope and obtain great breakthroughs in imaging speed.

Optimization of a quantum weak measurement system with digital filtering technology.

In this paper, we propose a post-Gaussian filtering theory for weak measurement in the frequency domain, and propose a highly deformed digital filtering technique that can be used to optimize sensors based on weak-frequency measurement techniques. We completed the experimental verification based on the weak measurement total internal reflection sensor. The experimental results show that digital filtering technology can optimize the system in the working range, sensitivity, and resolution of the frequency domain weak measurement system, so that it can reach 0.210 rad, 3210.9 nm/RIU, and 7.12×10-7 RIU, respectively.

Determination of Tumor Marker Carcinoembryonic Antigen with Biosensor Based on Optical Quantum Weak Measurements.

A phase-sensitive weak measurement biosensor was proposed for the detection of carcinoembryonic antigen (CEA), one common category of tumor markers. The total internal reflection (TIR) at the interface of the prism without precious metal coating was exploited to introduce the phase delay between horizontal and vertical polarizations, which can be determined through the central wavelength shift of output spectra for the sensing of the refractive index of the sample. In the weak measurement analysis, the specific binding reaction of tumor markers with a refractive index change on the surface of the prism can be monitored in real time through the central wavelength shift. With the specific absorption measurement, the feasibility of this weak measurement-based biosensor was experimentally demonstrated. We provide a low cost and convenient approach for tumor marker detection.

Rapid Separation of Enantiomeric Impurities in Chiral Molecules by a Self-Referential Weak Measurement System.

We propose a self-referential fast detection scheme for a frequency domain weak measurement system for the detection of enantiomeric impurities in chiral molecules. In a transmissive weak measurement system, the optical rotation (OR) is used to modify the pre-selected polarization state and the post-selection polarization state. We obtained the sum and difference of the optical rotations produced by the sample and the standard by rotating the quarter wave plate in the system. Then, we estimate the ratio of chiral molecules to enantiomeric impurities using the ratio of the central wavelength shifts caused by the addition and subtraction states described above. In this paper, our system has an optical resolution of 1.88 × 10-5°. At the same time, we completed the detection of the ratio of the two substances in the mixture of L-proline and D-proline in different proportions, which proved that our system can quickly detect the content of enantiomeric impurities in chiral molecules.

Theoretical study on second-harmonic generation in two-dimensional nonlinear photonic crystals.

We theoretically study second-harmonic generation in two-dimensional nonlinear photonic crystals and obtain a unified expression that combines nonlinear Raman-Nath diffraction, Cerenkov-type second-harmonic generation, and nonlinear Bragg diffraction. The analytical solution is deduced, and the theoretical result coincides well with the nonlinear Raman-Nath, nonlinear Cerenkov, and nonlinear Bragg diffraction phase-matching conditions. This method has potential applications in second-harmonic generation of more complicated two-dimensional and even three-dimensional nonlinear photonic crystals.

Surface Enhanced Laser Desorption Ionization of Phospholipids on Gold Nanoparticles for Mass Spectrometric Immunoassay.

High-throughput and sensitive detection of proteins are essential for clinical diagnostics and biomarker discovery. We develop a novel high-throughput, multiplexed, sensitive mass spectrometric (MS) immunoassay method, which utilizes antibody-modified phospholipid bilayer coated gold nanoparticles (PBL-AuNPs) as the detection label and antibody-immobilized magnetic beads as the capture reagent. This method enables magnetic enrichment of the PBL-AuNPs label specific to target protein, allowing sensitive surface enhanced laser desorption ionization (SELDI)-TOF MS detection of the protein via its specific label. AuNPs act as not only the support but also the matrix for the phospholipids in SELDI TOF MS detection. Moreover, with phospholipids with varying molecular weights as the encoded MS reporters, this method allows multiplexed detection of multiple proteins. With the use of a predefined phospholipids internal standard, this method also affords excellent reproducibility in protein quantification. We have demonstrated this method using the assays of two tumor biomarkers, and the results reveal that it provides a sensitive platform for multiplexed protein detection with detection limits in the picomolar ranges. This method may provide a useful platform for high-throughput and sensitive detection of protein biomarkers for clinical diagnostics.