Genome editing in rice using CRISPR/Cas12i3.

The CRISPR/Cas type V-I is a family of programmable nuclease systems that prefers a T-rich protospacer adjacent motif (PAM) and is guided by a short crRNA. In this study, the genome-editing application of Cas12i3, a type V-I family endonuclease, was characterized in rice. We developed a CRIPSR/Cas12i3-based Multiplex direct repeats (DR)-spacer Array Genome Editing (iMAGE) system that was efficient in editing various genes in rice. Interestingly, iMAGE produced chromosomal structural variations with a higher frequency than CRISPR/Cas9. In addition, we developed base editors using deactivated Cas12i3 and generated herbicide-resistant rice plants using the base editors. These CRIPSR/Cas12i3-based genome editing systems will facilitate precision molecular breeding in plants.

Discovery Small-Molecule p300 Inhibitors Derived from a Newly Developed Indazolone-Focused DNA-Encoded Library.

The DNA-encoded library (DEL) is a robust tool for chemical biology and drug discovery. In this study, we developed a DNA-compatible light-promoted reaction that is highly efficient and plate-compatible for DEL construction based on the formation of the indazolone scaffold. Employing this high-efficiency approach, we constructed a DEL featuring an indazolone core, which enabled the identification of a novel series of ligands specifically targeting E1A-binding protein (p300) after DEL selection. Taken together, our findings underscore the feasibility of light-promoted reactions in DEL synthesis and unveil promising avenues for developing p300-targeting inhibitors.

Trends in Venous Diameter and Brachial Artery Volume Flow of arteriovenous fistula in 12 Weeks Postoperatively.

BACKGROUND: To explore the trends of venous diameter and brachial artery volume flow (VF) in 12 weeks after arteriovenous fistula (AVF) and the influence of preoperative arterial diameter on this trend. Our goal was to clarify the maturation process within 12 weeks after AVF surgery. METHODS: Clinical data of 257 patients with end-stage renal disease who had their first radial-cephalic AVF established at our institution from February 1, 2023, to February 1, 2024, were included. The patients were divided into group A (radial artery diameter <1.5 mm), group B (radial artery diameter 1.5-2.0 mm), and group C (radial artery diameter >2.0 mm) according to the preoperative radial artery diameter. After AVF surgery, the artery and vein diameter and brachial artery VF were recorded at 1 day, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, and 12 weeks. RESULTS: The venous diameter and brachial artery VF of AVF showed an upward trend and increased significantly in 1 day-6 weeks postoperatively (P < 0.05), especially between 1 day and 2 weeks, while no significant difference in the increases at 6-12 weeks. Groups B and C were in line with the above trend, whereas the patients in group A showed best growth in 2-4 weeks postoperatively. The natural maturation rates of AVF in groups B and C were significantly better than that of group A at all postoperative time (P＜0.05). CONCLUSIONS: The AVF was in a developmentally dominant stage at 6 weeks postoperatively, with 1 day-2 weeks being particularly prominent. The postoperative natural maturation rate of AVF with arteries diameter of <1.5 mm was low; the direct use of such arteries to establish AVF needs careful consideration.

Optimized Radio Frequency Footprint Identification Based on UAV Telemetry Radios.

With the widespread use of unmanned aerial vehicles (UAVs), the detection and identification of UAVs is a vital security issue for the safety of airspace and ground facilities in the no-fly zone. Telemetry radios are important wireless communication devices for UAVs, especially in UAVs beyond the visual line of sight (BVLOS) operating mode. This work focuses on the UAV identification approach using transient signals from UAV telemetry radios instead of the signals from UAV controllers that the former research work depended on. In our novel UAV Radio Frequency (RF) identification system framework based on telemetry radio signals, the EC-α algorithm is optimized to detect the starting point of the UAV transient signal and the detection accuracy at different signal-to-noise ratios (SNR) is evaluated. In the training stage, the Convolutional Neural Network (CNN) model is trained to extract features from raw I/Q data of the transient signals with different waveforms. Its architecture and hyperparameters are analyzed and optimized. In the identification stage, the extracted transient signals are clustered through the Self-Organizing Map (SOM) algorithm and the Clustering Signals Joint Identification (CSJI) algorithm is proposed to improve the accuracy of RF fingerprint identification. To evaluate the performance of our proposed approach, we design a testbed, including two UAVs as the flight platform, a Universal Software Radio Peripheral (USRP) as the receiver, and 20 telemetry radios with the same model as targets for identification. Indoor test results show that the optimized identification approach achieves an average accuracy of 92.3% at 30 dB. In comparison, the identification accuracy of SVM and KNN is 69.7% and 74.5%, respectively, at the same SNR condition. Extensive experiments are conducted outdoors to demonstrate the feasibility of this approach.

Mutation of Basic Residues R283, R286, and K288 in the Matrix Protein of Newcastle Disease Virus Attenuates Viral Replication and Pathogenicity.

The matrix (M) protein of Newcastle disease virus (NDV) contains large numbers of unevenly distributed basic residues, but the precise function of most basic residues in the M protein remains enigmatic. We previously demonstrated that the C-terminus (aa 264-313) of M protein interacted with the extra-terminal (ET) domain of chicken bromodomain-containing protein 2 (chBRD2), which promoted NDV replication by downregulating chBRD2 expression and facilitating viral RNA synthesis and transcription. However, the key amino acid sites determining M's interaction with chBRD2/ET and their roles in the replication and pathogenicity of NDV are not known. In this study, three basic residues-R283, R286, and K288-in the NDV M protein were verified to be responsible for its interaction with chBRD2/ET. In addition, mutation of these basic residues (R283A/R286A/K288A) in the M protein changed its electrostatic pattern and abrogated the decreased expression of endogenic chBRD2. Moreover, a recombinant virus harboring these mutations resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chickens due to the decreased viral RNA synthesis and transcription. Our findings therefore provide a better understanding of the crucial biological functions of M's basic residues and also aid in understanding the poorly understood pathogenesis of NDV.

The association of ribosomal protein L18 with Newcastle disease virus matrix protein enhances viral translation and replication.

Numerous studies have shown that viruses can utilize or manipulate ribosomal proteins to achieve viral protein biosynthesis and replication. In our recent studies using proteomics analysis of virus-infected cells, we found that ribosomal protein L18 (RPL18) was the highest up-regulated differentially expressed protein, along with the increasingly expressed viral proteins later in Newcastle disease virus (NDV) infection. However, the association of RPL18 with viral protein biosynthesis and NDV replication remains unclear. In this study, we found that the expression and transcription levels of RPL18 was reduced early in NDV infection but increased later in NDV infection. In addition, the presence of cytoplasmic NDV matrix (M) protein was responsible for the increased expression of RPL18 in both virus-infected cells and plasmid-transfected cells. Moreover, cytoplasmic M protein increased RPL18 expression in a dose-dependent manner, even though they did not interact with each other. Furthermore, siRNA-mediated knockdown of RPL18 or overexpression of RPL18 dramatically reduced or enhanced NDV replication by decreasing or increasing viral protein translation rather than viral RNA synthesis and transcription. Taken together, these results suggested that the increased expression of RPL18 might be associated with the physical clumping together of the M protein, which in turn promoted viral protein biosynthesis and NDV replication. RESEARCH HIGHLIGHTSThe increased expression of RPL18 is associated with the presence of cytoplasmic M protein.Cytoplasmic M protein increases RPL18 expression in a dose-dependent manner.Knockdown of RPL18 reduces NDV replication by decreasing viral protein translation.Overexpression of RPL18 enhances NDV replication by increasing viral protein translation.

Cas12a-based on-site, rapid detection of genetically modified crops.

A CRISPR/LbCas12a-based nucleic acid detection method that uses crude leaf extracts as samples and is rapid (≤40 min for a full run) and highly sensitive (0.01%) can be used to monitor genetically modified organisms in the field.

Chicken bromodomain-containing protein 2 interacts with the Newcastle disease virus matrix protein and promotes viral replication.

Bromodomain-containing protein 2 (BRD2) is a nucleus-localized serine-threonine kinase that plays pivotal roles in the transcriptional control of diverse genes. In our previous study, the chicken BRD2 (chBRD2) protein was found to interact with the Newcastle disease virus (NDV) matrix (M) protein using a yeast two-hybrid screening system, but the role of the chBRD2 protein in the replication of NDV remains unclear. In this study, we first confirmed the interaction between the M protein and chBRD2 protein using fluorescence co-localization, co-immunoprecipitation and pull-down assays. Intracellular binding studies indicated that the C-terminus (aa 264-313) of the M protein and the extra-terminal (ET) domain (aa 619-683) of the chBRD2 protein were responsible for interactions with each other. Interestingly, although two amino acids (T621 and S649) found in the chBRD2/ET domain were different from those in the human BRD2/ET domain and in that of other mammals, they did not disrupt the BRD2-M interaction or the chBRD2-M interaction. In addition, we found that the transcription of the chBRD2 gene was obviously decreased in both NDV-infected cells and pEGFP-M-transfected cells in a dose-dependent manner. Moreover, small interfering RNA-mediated knockdown of chBRD2 or overexpression of chBRD2 remarkably enhanced or reduced NDV replication by upregulating or downregulating viral RNA synthesis and transcription, respectively. Overall, we demonstrate for the first time that the interaction of the M protein with the chBRD2 protein in the nucleus promotes NDV replication by downregulating chBRD2 expression and facilitating viral RNA synthesis and transcription. These results will provide further insight into the biological functions of the M protein in the replication of NDV.

Comparative phosphoproteomic analysis of compatible and incompatible pollination in Brassica napus L.

Self-incompatibility (SI) promotes outbreeding and prevents self-fertilization to promote genetic diversity in angiosperms. Several studies have been carried to investigate SI signaling in plants; however, protein phosphorylation and dephosphorylation in the fine-tuning of the SI response remain insufficiently understood. Here, we performed a phosphoproteomic analysis to identify the phosphoproteins in the stigma of self-compatible 'Westar' and self-incompatible 'W-3' Brassica napus lines. A total of 4109 phosphopeptides representing 1978 unique protein groups were identified. Moreover, 405 and 248 phosphoproteins were significantly changed in response to SI and self-compatibility, respectively. Casein kinase II (CK II) phosphorylation motifs were enriched in self-incompatible response and identified 127 times in 827 dominant SI phosphorylation residues. Functional annotation of the identified phosphoproteins revealed the major roles of these phosphoproteins in plant-pathogen interactions, cell wall modification, mRNA surveillance, RNA degradation, and plant hormone signal transduction. In particular, levels of homolog proteins ABF3, BKI1, BZR2/BSE1, and EIN2 were significantly increased in pistils pollinated with incompatible pollens. Abscisic acid and ethephon treatment partially inhibited seed set, while brassinolide promoted pollen germination and tube growth in SI response. Collectively, our results provided an overview of protein phosphorylation during compatible/incompatible pollination, which may be a potential component of B. napus SI responses.

The Brassica napus GATA transcription factor BnA5.ZML1 is a stigma compatibility factor.

Self-incompatibility (SI) is a genetic mechanism that rejects self-pollen and thus prevents inbreeding in some hermaphroditic angiosperms. In the Brassicaceae, SI involves a pollen-stigma recognition system controlled by a single locus known as the S locus, which consists of two highly polymorphic genes that encode S-locus cysteine-rich protein (SCR) and S-receptor kinase (SRK). When self-pollen lands on the stigma, the S-haplotype-specific interaction between SCR and SRK triggers SI. Here, we show that the GATA transcription factor BnA5.ZML1 suppresses SI responses in Brassica napus and is induced after compatible pollination. The loss-of-function mutant bna5.zml1 displays reduced self-compatibility. In contrast, overexpression of BnA5.ZML1 in self-incompatible stigmas leads to a partial breakdown of SI responses, suggesting that BnA5.ZML1 is a stigmatic compatibility factor. Furthermore, the expression levels of SRK and ARC1 are up-regulated in bna5.zml1 mutants, and they are down-regulated in BnA5.ZML1 overexpressing lines. SRK affects the cellular localization of BnA5.ZML1 through direct protein-protein interaction. Overall, our findings highlight the fundamental role of BnA5.ZML1 in SI responses in B. napus, establishing a direct interaction between BnA5.ZML1 and SRK in this process.

Nuclear localization of Newcastle disease virus matrix protein promotes virus replication by affecting viral RNA synthesis and transcription and inhibiting host cell transcription.

Nuclear localization of paramyxovirus proteins is crucial for virus life cycle, including the regulation of viral replication and the evasion of host immunity. We previously showed that a recombinant Newcastle disease virus (NDV) with nuclear localization signal mutation in the matrix (M) protein results in a pathotype change and attenuates viral pathogenicity in chickens. However, little is known about the nuclear localization functions of NDV M protein. In this study, the potential functions of the M protein in the nucleus were investigated. We first demonstrate that nuclear localization of the M protein could not only promote the cytopathogenicity of NDV but also increase viral RNA synthesis and transcription efficiency in DF-1 cells. Using microarray analysis, we found that nuclear localization of the M protein might inhibit host cell transcription, represented by numerous up-regulating genes associated with transcriptional repressor activity and down-regulating genes associated with transcriptional activator activity. The role of representative up-regulated gene prospero homeobox 1 (PROX1) and down-regulated gene aryl hydrocarbon receptor (AHR) in the replication of NDV was then evaluated. The results show that siRNA-mediated knockdown of PROX1 or AHR significantly reduced or increased the viral RNA synthesis and viral replication, respectively, demonstrating the important roles of the expression changes of these genes in NDV replication. Together, our findings demonstrate for the first time that nuclear localization of NDV M protein promotes virus replication by affecting viral RNA synthesis and transcription and inhibiting host cell transcription, improving our understanding of the molecular mechanism of NDV replication and pathogenesis.

Polyhydric polymer-loaded pyrene composites as powerful adsorbents and fluorescent probes: highly efficient adsorption and test strips-based fluorimetric analysis of curcumin in urine and plant extracts.

Polyhydric poly (vinyl alcohol) was covalently loaded with a 1-pyrenecarboxyaldehyde fluorophore. The yielded PVA-Pyr composites can serve as powerful adsorbents and strong fluorescent probes for the highly efficient adsorption and sensitive fluorimetric detection with test strips of curcumin in samples of urine and plant extracts.

Mechanism of Salt-Induced Self-Compatibility Dissected by Comparative Proteomic Analysis in Brassica napus L.

Self-incompatibility (SI) in plants genetically prevents self-fertilization to promote outcrossing and genetic diversity. Its hybrids in Brassica have been widely cultivated due to the propagation of SI lines by spraying a salt solution. We demonstrated that suppression of Brassica napus SI from edible salt solution treatment was ascribed to sodium chloride and independent of S haplotypes, but it did not obviously change the expression of SI-related genes. Using the isobaric tags for relative and absolute quantitation (iTRAQ) technique, we identified 885 differentially accumulated proteins (DAPs) in Brassica napus stigmas of un-pollinated (UP), pollinated with compatible pollen (PC), pollinated with incompatible pollen (PI), and pollinated with incompatible pollen after edible salt solution treatment (NA). Of the 307 DAPs in NA/UP, 134 were unique and 94 were shared only with PC/UP. In PC and NA, some salt stress protein species, such as glyoxalase I, were induced, and these protein species were likely to participate in the self-compatibility (SC) pathway. Most of the identified protein species were related to metabolic pathways, biosynthesis of secondary metabolites, ribosome, and so on. A systematic analysis implied that salt treatment-overcoming SI in B.napus was likely conferred by at least five different physiological mechanisms: (i) the use of Ca2+ as signal molecule; (ii) loosening of the cell wall to allow pollen tube penetration; (iii) synthesis of compatibility factor protein species for pollen tube growth; (iv) depolymerization of microtubule networks to facilitate pollen tube movement; and (v) inhibition of protein degradation pathways to restrain the SI response.

Characterization of the nuclear import pathway for BLM protein.

Numerous studies have shown that nuclear localization of BLM protein, a member of the RecQ helicases, mediated by nuclear localization signal (NLS) is critical for DNA recombination, replication and transcription, but the mechanism by which BLM protein is imported into the nucleus remains unknown. In this study, the nuclear import pathway for BLM was investigated. We found that nuclear import of BLM was inhibited by two dominant-negative mutants of importin ß1 and NTF2/E42K, which lacks the ability to bind Ran and RanGDP, respectively, but was not inhibited by the Ran/Q69L, which is deficient in GTP hydrolysis. Further studies revealed that nuclear import of BLM was reconstituted using importin ß1, RanGDP and NTF2 in digitonin-permeabilized HeLa cells. Moreover, BLM had direct binding to importin ß1 through its NLS domain with the 14-16 HEAT repeats of importin ß1. Furthermore, importin ß1, Ran or NTF2 depletion by siRNA disrupted the accumulation of BLM protein in the nucleus. These results showed that BLM enters the nucleus via the importin ß1, RanGDP and NTF2 dependent pathway, demonstrating for the first time the nuclear trafficking mechanism of a DNA helicase.

[The nuclear import of Newcastle disease virus matrix protein depends on KPNB1 and Ran protein].

Objective: The aim of this study was to identify the transport proteins that mediates the nuclear import of Newcastle disease virus (NDV) matrix (M) protein. Methods: Chicken KPNA1 to KPNA6 gene and KPNB1 gene were cloned from DF-1 cells and then inserted into eukaryotic expression vectors. The constructed recombinant plasmids with a combination of grouping were transfected into HEK-293T cells to identify the transport proteins interacting with NDV M protein by co-immunoprecipitation (Co-IP) assay. Moreover, fluorescent co-localization assay was used to verify the transport proteins by co-expressing M and Ran protein mutant or M and its interactive protein deletant. Results: The recombinant proteins could normally express in plasmid-transfected HEK-293T cells. Indirect immunofluorescence detection showed that the recombinant proteins except for Myc-KPNA2 displayed the same nuclear localization as NDV M protein. The results of Co-IP revealed that M protein could interact with KPNA1 and KPNB1. Further fluorescent co-localization indicated that co-expression of M and DN-KPNA1 did not change the nuclear localization of M, whereas co-expression of M and DN-KPNB1 or M and Ran-Q69L disrupted the nuclear localization of M, demonstrating that the nuclear import of M protein was dependent on KPNB1 and Ran protein. Conclusion: KPNB1 and Ran protein jointly mediated the nuclear import of NDV M protein, showing that KPNB1 protein interacted with NDV M protein to form binary complex and then entered into the nucleus with the assistance of Ran protein.

Simultaneous mutation of G275A and P276A in the matrix protein of Newcastle disease virus decreases virus replication and budding.

The matrix (M) protein of Newcastle disease virus (NDV) is a highly conserved hydrophobic viral protein. In some paramyxoviruses (measles virus and Sendai virus), the paired glycine (G) near the C terminus of the M protein may form a turn that mediates the specific interaction with the cell membrane. Similar amino acids (glycine-proline [GP], at position 275-276) exist in the M protein of NDV. However, the role of these residues in the replication and pathogenicity of NDV is unknown. In this study, recombinant NDV with the sequence GP/AA or LGP/GGL in the M protein was generated to investigate the role of this conserved sequence. Budding experiments on the mutant viruses revealed that the GP/AA mutation reduced virus budding and virus replication in DF-1 cells; biological characterization revealed attenuated virulence and pathogenicity in chickens, indicating that the GP sequence plays a critical role in the life cycle of the virus.

Effective light absorption and its enhancement factor for silicon nanowire-based solar cell.

Although nanowire (NW) antireflection coating can enhance light trapping capability, which is generally used in crystal silicon (CS) based solar cells, whether it can improve light absorption in the CS body depends on the NW geometrical shape and their geometrical parameters. In order to conveniently compare with the bare silicon, two enhancement factors E(T) and E(A) are defined and introduced to quantitatively evaluate the efficient light trapping capability of NW antireflective layer and the effective light absorption capability of CS body. Five different shapes (cylindrical, truncated conical, convex conical, conical, and concave conical) of silicon NW arrays arranged in a square are studied, and the theoretical results indicate that excellent light trapping does not mean more light can be absorbed in the CS body. The convex conical NW has the best light trapping, but the concave conical NW has the best effective light absorption. Furthermore, if the cross section of silicon NW is changed into a square, both light trapping and effective light absorption are enhanced, and the Eiffel Tower shaped NW arrays have optimal effective light absorption.

[Function comparison of the matrix protein between Newcastle disease virus and other paramyxoviruses - A review]

Recent studies have found that the matrix protein of paramyxoviruses is a multifunctional viral protein. In addition to inhibiting the transcription and translation of cell genes, regulating the replication and transcription of viral genome and recruiting cellular proteins to facilitate viral assembly and budding, the matrix protein can enhance the replication of paramyxoviruses through its ubiquitination and phosphorylation. However, as a member of paramyxoviruses, the matrix protein of Newcastle disease virus (NDV) is only demonstrated to participate in viral assembly and budding. Moreover, the functions of matrix protein identified in other paramyxoviruses still remain unknown in NDV. This review compares the functions of matrix protein between NDV and other paramyxoviruses, and focuses on the relationship of matrix protein to the virulence, replication and pathogenicity of NDV. Meanwhile, challenges and research prospects of NDV matrix protein are also discussed.

Unravelling the complex trait of harvest index in rapeseed (Brassica napus L.) with association mapping.

BACKGROUND: Harvest index (HI), the ratio of grain yield to total biomass, is considered as a measure of biological success in partitioning assimilated photosynthate to the harvestable product. While crop production can be dramatically improved by increasing HI, the underlying molecular genetic mechanism of HI in rapeseed remains to be shown. RESULTS: In this study, we examined the genetic architecture of HI using 35,791 high-throughput single nucleotide polymorphisms (SNPs) genotyped by the Illumina BrassicaSNP60 Bead Chip in an association panel with 155 accessions. Five traits including plant height (PH), branch number (BN), biomass yield per plant (BY), harvest index (HI) and seed yield per plant (SY), were phenotyped in four environments. HI was found to be strongly positively correlated with SY, but negatively or not strongly correlated with PH. Model comparisons revealed that the A-D test (ADGWAS model) could perfectly balance false positives and statistical power for HI and associated traits. A total of nine SNPs on the C genome were identified to be significantly associated with HI, and five of them were identified to be simultaneously associated with HI and SY. These nine SNPs explained 3.42% of the phenotypic variance in HI. CONCLUSIONS: Our results showed that HI is a complex polygenic phenomenon that is strongly influenced by both environmental and genotype factors. The implications of these results are that HI can be increased by decreasing PH or reducing inefficient transport from pods to seeds in rapeseed. The results from this association mapping study can contribute to a better understanding of natural variations of HI, and facilitate marker-based breeding for HI.

Two amino acid substitutions in the haemagglutinin of the 2009 pandemic H1N1 virus decrease direct-contact transmission in guinea pigs.

The 2009 pandemic H1N1 influenza A virus spread across the globe and caused the first influenza pandemic of the 21st century. Many of the molecular factors that contributed to the airborne transmission of this pandemic virus have been determined; however, the direct-contact transmission of this virus remains poorly understood. In this study, we report that a combination of two mutations (N159D and Q226R) in the haemagglutinin (HA) protein of the representative 2009 H1N1 influenza virus A/California/04/2009 (CA04) caused a switch in receptor binding preference from the α2,6-sialoglycan to the α2,3-sialoglycan receptor, and decreased the binding intensities for both glycans. In conjunction with a significantly decreased replication efficiency in the nasal epithelium, this limited human receptor binding affinity resulted in inefficient direct-contact transmission of CA04 between guinea pigs. Our findings highlight the role of the HA gene in the transmission of the influenza virus.