Constructing an efficient and easy-to-operate multigene expression system is currently a crucial part of plant genetic engineering. In this study, a fragment carrying three independent gene expression cassettes and the expression unit of the gene-silencing suppressor protein(RNA silencing suppressor 19 kDa protein, P19) simultaneously was designed and constructed. This fragment was cloned into the commonly used plant expression vector pCAMBIA300, and the plasmid pC1300-TP2-P19 was obtained. Each gene expression cassette consists of different promoters, fusion tags, and terminators. The target gene can be flexibly inserted into the corresponding site through enzymatic digestion and ligation or recombination and fused with different protein tags, which provides great convenience for subsequent detection. The enhanced green fluorescent protein(eGFP) reporter gene was individually constructed into each expression cassette to verify the feasibility of this vector system. The results of tobacco transient expression and laser-confocal microscopy showed that each expression cassette presented independent and normal expression. Meanwhile, the three key enzyme genes in the betanin synthesis pathway, BvCYP76AD, BvDODA1, and DbDOPA5GT, were constructed into the three expression cassettes. The results of tobacco transient expression phenotype, protein immunoblotting(Western blot), and chemical detection of product demonstrated that the three exogenous genes were highly expressed, and the target compound betanin was successfully produced. The above results indicated that the constructed multigene expression system for plants in this study was efficient and reliable and can achieve the co-transformation of multiple plant genes. It can provide a reliable vector platform for the analysis of plant natural product synthesis pathways, functional verification, and plant metabolic engineering.
Nicotiana/metabolism* ; Genetic Vectors/metabolism* ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Genetic Engineering/methods* ; Green Fluorescent Proteins/metabolism* ; Gene Expression

Dysregulated RNA splicing is a well-recognized characteristic of colorectal cancer (CRC); however, its intricacies remain obscure, partly due to challenges in profiling full-length transcript variants at the single-cell level. Here, we employ high-depth long-read scRNA-seq to define the full-length transcriptome of colorectal epithelial cells in 12 CRC patients, revealing extensive isoform diversities and splicing alterations. Cancer cells exhibited increased transcript complexity, with widespread 3'-UTR shortening and reduced intron retention. Distinct splicing regulation patterns were observed between intrinsic-consensus molecular subtypes (iCMS), with iCMS3 displaying even higher splicing factor activities and more pronounced 3'-UTR shortening. Furthermore, we revealed substantial shifts in isoform usage that result in alterations of protein sequences from the same gene with distinct carcinogenic effects during tumorigenesis of CRC. Allele-specific expression analysis revealed dominant mutant allele expression in key oncogenes and tumor suppressors. Moreover, mutated PPIG was linked to widespread splicing dysregulation, and functional validation experiments confirmed its critical role in modulating RNA splicing and tumor-associated processes. Our findings highlight the transcriptomic plasticity in CRC and suggest novel candidate targets for splicing-based therapeutic strategies.
Humans ; Colorectal Neoplasms/metabolism* ; RNA Isoforms/metabolism* ; Single-Cell Analysis ; RNA Splicing ; Gene Expression Regulation, Neoplastic ; RNA, Neoplasm/metabolism* ; Transcriptome

The 2-(2-phenylethyl)chromones were separated from agarwood of Aquilaria agallocha Roxb. and their anti-KRAS mutant non-small cell lung cancer (NSCLC) activities were evaluated. 2-(2-Phenyethyl)chromones in agarwood were separated and purified by silica gel, RP-18 reverse phase silica gel, MCI gel CH20P, Diol, and semi preparative HPLC chromatography techniques, while the structures of the compounds were identified by extensive spectroscopic analysis, such as 1D and 2D NMR and ESI-MS. The cell counting kit 8 (CCK-8) assay was used to screen the anti-tumor activity of the isolated monomeric compounds on three KRAS-mutant NSCLC cells. The cell proliferation, cloning formation, adhesion ability, and cell cycle arrest activity of compound 8 were analyzed. Molecular docking and Western blot experiments were used to study the mechanism of compound 8. The in vivo anti-tumor activity of the compound 8 was evaluated by zebrafish cell derived xenograft (CDX) model. Nineteen known 2-(2-phenylethyl)chromones were isolated from the ethyl acetate extract of agarwood. On A549 (KRAS G12S) cells, compounds 8, 10, and 19 showed good inhibitory activity, on H23 (KRAS G12C) cells, compounds 7, 8, and 19 showed good inhibitory activity, and on H358 (KRAS G12C) cells, compounds 8, 10, and 16 showed good inhibitory activity. Compound 8 had the best inhibitory activity in all three cell lines. It effectively inhibited cell proliferation, clone formation, adhesion ability, and arrested the H23 cell cycle at G2/M phase. Compound 8 could also inhibit the expression of c-Met and its downstream signaling pathways, effectively inhibiting tumor growth in zebrafish CDX model. In conclusion, among the nineteen known 2-(2-phenylethyl)chromones, compound 8 had the best activity and significantly inhibited the proliferation of KRAS-mutant NSCLC cells by arresting the cells in the G2/M phase.

Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.

Objective: Lingual fixed retainers, made from 0.0175-inch 3-strand twisted stainless steel wire (TW) and 0.016 × 0.022-inch straight rectangular wire (RW), are generally used in clinical practice. This study aimed to calculate their accuracy by comparing the discrepancy between computer-aided customized retainers made from these two types of wires. Methods: Eleven orthodontic patients were selected, resulting in 22 maxillary and mandibular threedimensional printing dental models. Two types of lingual fixed retainers were bonded from canine to canine. To determine the accuracy, five points were chosen for each model, resulting in 110 selected points. The absolute values of the distances on the x-, y-, and z-axes were measured to compare the accuracy of the two types of computer-aided retainers. Results: The accuracy of the two types of retainers did not differ significantly in the x- and z-axes, but only in the y-axis (P < 0.01), where RW-fixed retainers exhibited a slightly but significantly increased distance compared to the TW. Conclusions Both types of retainers showed high accuracy; however, RW had a slight but statistically significant difference along the y-axis compared with TW. This type of computer-aided design/computer-aided manufacturing bending machine is limited to two dimensions, and the dental arch is curved. Therefore, RW may require slight manual adjustment by the practitioner after manufacturing.

Objective: Lingual fixed retainers, made from 0.0175-inch 3-strand twisted stainless steel wire (TW) and 0.016 × 0.022-inch straight rectangular wire (RW), are generally used in clinical practice. This study aimed to calculate their accuracy by comparing the discrepancy between computer-aided customized retainers made from these two types of wires. Methods: Eleven orthodontic patients were selected, resulting in 22 maxillary and mandibular threedimensional printing dental models. Two types of lingual fixed retainers were bonded from canine to canine. To determine the accuracy, five points were chosen for each model, resulting in 110 selected points. The absolute values of the distances on the x-, y-, and z-axes were measured to compare the accuracy of the two types of computer-aided retainers. Results: The accuracy of the two types of retainers did not differ significantly in the x- and z-axes, but only in the y-axis (P < 0.01), where RW-fixed retainers exhibited a slightly but significantly increased distance compared to the TW. Conclusions Both types of retainers showed high accuracy; however, RW had a slight but statistically significant difference along the y-axis compared with TW. This type of computer-aided design/computer-aided manufacturing bending machine is limited to two dimensions, and the dental arch is curved. Therefore, RW may require slight manual adjustment by the practitioner after manufacturing.

Objective: Lingual fixed retainers, made from 0.0175-inch 3-strand twisted stainless steel wire (TW) and 0.016 × 0.022-inch straight rectangular wire (RW), are generally used in clinical practice. This study aimed to calculate their accuracy by comparing the discrepancy between computer-aided customized retainers made from these two types of wires. Methods: Eleven orthodontic patients were selected, resulting in 22 maxillary and mandibular threedimensional printing dental models. Two types of lingual fixed retainers were bonded from canine to canine. To determine the accuracy, five points were chosen for each model, resulting in 110 selected points. The absolute values of the distances on the x-, y-, and z-axes were measured to compare the accuracy of the two types of computer-aided retainers. Results: The accuracy of the two types of retainers did not differ significantly in the x- and z-axes, but only in the y-axis (P < 0.01), where RW-fixed retainers exhibited a slightly but significantly increased distance compared to the TW. Conclusions Both types of retainers showed high accuracy; however, RW had a slight but statistically significant difference along the y-axis compared with TW. This type of computer-aided design/computer-aided manufacturing bending machine is limited to two dimensions, and the dental arch is curved. Therefore, RW may require slight manual adjustment by the practitioner after manufacturing.

Objective: Lingual fixed retainers, made from 0.0175-inch 3-strand twisted stainless steel wire (TW) and 0.016 × 0.022-inch straight rectangular wire (RW), are generally used in clinical practice. This study aimed to calculate their accuracy by comparing the discrepancy between computer-aided customized retainers made from these two types of wires. Methods: Eleven orthodontic patients were selected, resulting in 22 maxillary and mandibular threedimensional printing dental models. Two types of lingual fixed retainers were bonded from canine to canine. To determine the accuracy, five points were chosen for each model, resulting in 110 selected points. The absolute values of the distances on the x-, y-, and z-axes were measured to compare the accuracy of the two types of computer-aided retainers. Results: The accuracy of the two types of retainers did not differ significantly in the x- and z-axes, but only in the y-axis (P < 0.01), where RW-fixed retainers exhibited a slightly but significantly increased distance compared to the TW. Conclusions Both types of retainers showed high accuracy; however, RW had a slight but statistically significant difference along the y-axis compared with TW. This type of computer-aided design/computer-aided manufacturing bending machine is limited to two dimensions, and the dental arch is curved. Therefore, RW may require slight manual adjustment by the practitioner after manufacturing.

Objective: Lingual fixed retainers, made from 0.0175-inch 3-strand twisted stainless steel wire (TW) and 0.016 × 0.022-inch straight rectangular wire (RW), are generally used in clinical practice. This study aimed to calculate their accuracy by comparing the discrepancy between computer-aided customized retainers made from these two types of wires. Methods: Eleven orthodontic patients were selected, resulting in 22 maxillary and mandibular threedimensional printing dental models. Two types of lingual fixed retainers were bonded from canine to canine. To determine the accuracy, five points were chosen for each model, resulting in 110 selected points. The absolute values of the distances on the x-, y-, and z-axes were measured to compare the accuracy of the two types of computer-aided retainers. Results: The accuracy of the two types of retainers did not differ significantly in the x- and z-axes, but only in the y-axis (P < 0.01), where RW-fixed retainers exhibited a slightly but significantly increased distance compared to the TW. Conclusions Both types of retainers showed high accuracy; however, RW had a slight but statistically significant difference along the y-axis compared with TW. This type of computer-aided design/computer-aided manufacturing bending machine is limited to two dimensions, and the dental arch is curved. Therefore, RW may require slight manual adjustment by the practitioner after manufacturing.