RNA-binding proteins (RBPs) are ubiquitous components within cells, fulfilling essential functions in a myriad of biological processes. These proteins interact with RNA molecules to regulate gene expression at various levels, including transcription, splicing, transport, localization, translation, and degradation. Understanding the intricate network of RBP-RNA interactions is crucial for deciphering the complex regulatory mechanisms that govern cellular function and organismal development. Ultravidet (UV) cross-linking and immunoprecipitation (CLIP) stands out as a powerful approach designed to map the precise locations where RBPs bind to RNA. By using UV light to create covalent bonds between proteins and RNA, followed by immunoprecipitation to isolate the protein-RNA complexes, researchers can identify the direct targets of specific RBPs. The advent of high-throughput sequencing technologies has revolutionized CLIP, enabling the identification of not only the types but also the exact sequences of RNA bound by RBPs on a genome-wide scale. The evolution of CLIP has led to the development of specialized variants, each with unique features that address specific challenges and expand the scope of what can be studied. High-throughput sequencing CLIP (HITS-CLIP) was one of the first advancements, significantly increasing the throughput and resolution of RNA-protein interaction mapping. Photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) introduced the use of photoactivatable ribonucleosides to enhance cross-linking efficiency and specificity, reducing background noise and improving the detection of low-abundance RNA-protein interactions. Individual-nucleotide resolution CLIP (iCLIP) further refined the technique, achieving unprecedented precision by resolving individual nucleotides involved in RBP binding, which is particularly valuable for studying the fine details of RNA structure and function. Despite the remarkable progress, there remains room for improvement in CLIP technology. Researchers continue to seek methods to increase sensitivity, reduce technical variability, and improve the reproducibility of results. Advances in sample preparation, data analysis algorithms, and computational tools are critical for addressing these challenges. Moreover, the application of CLIP to more diverse biological systems, including non-model organisms and clinical samples, requires the development of tailored protocols and the optimization of existing ones. Looking forward, the field of RNA biology is poised to benefit greatly from ongoing innovations in CLIP technology. The exploration of non-canonical RNA-protein interactions, such as those involving long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), promises to reveal new layers of cellular regulation and may lead to the discovery of novel therapeutic targets. Furthermore, integrating CLIP data with other omics approaches, such as proteomics and metabolomics, will provide a more comprehensive understanding of the dynamic interplay between RNA and its binding partners within the cell. In conclusion, the continuous refinement and expansion of CLIP techniques have not only deepened our knowledge of RNA biology but have also opened up new avenues for investigating the molecular underpinnings of health and disease. As the technology matures, it is expected to play an increasingly pivotal role in both basic and applied research, contributing to the advancement of medical science and biotechnology.

In this study, we constructed a GLP-2R-HEK293 cell line and established a method for the determination of the in vitro biological activity of teduglutide based on HTRF, after optimizing experimental conditions and methodological verification. We also carried out relative potency detection of teduglutide pharmaceutical products using this method. The result showed that there was a quantitative-efficient relationship between the teduglutide activity and cAMP contents in GLP-2R-HEK293 cells, which conformed to four-parameter model. Method verification results of five concentrations of teduglutide (64%, 80%, 100%, 125% and 156%) met the requirements of the General Rules of Chinese Pharmacopoeia, 2020 edition, Volume IV (9401). We then analyzed the relative potency of three batches of teduglutide drug substances and three batches of drug products. The linearity, regression and parallelism of the obtained curves all fit the system suitability requirements. The relative potency of six batches of teduglutide was from 83% to 105%. In summary, the biological activity detection method established in this study was accurate, precise, simple and time-saving, which can be used for quality control of teduglutide pharmaceutical products.

Objective:To evaluate the safety and efficiency of robotic visualization system(RVS)-assisted microsurgical re-construction of the reproductive tract in male rats and the satisfaction of the surgeons.Methods:We randomly divided 8 adult male SD rats into an experimental and a control group,the former treated by RVS-assisted microsurgical vasoepididymostomy(VE)or vaso-vasostomy(VV),and the latter by VE or VV under the standard operating microscope(SOM).We compared the operation time,me-chanical patency and anastomosis leakage immediately after surgery,and the surgeons'satisfaction between the two groups.Results:No statistically significant difference was observed the operation time between the experimental and the control groups,and no anasto-mosis leakage occurred after VV in either group.The rate of mechanical patency immediately after surgery was 100％in both groups,and that of anastomosis leakage after VE was 16.7％in the experimental group and 14.3％in the control.Compared with the control group,the experimental group achieved dramatically higher scores on visual comfort(3.00±0.76 vs 4.00±0.53,P＜0.05),neck/back comfort(2.75±1.16 vs 4.38±1.06,P＜0.01)and man-machine interaction(3.88±1.55 va 4.88±0.35,P＜0.05).There were no statistically significant differences in the scores on image definition and operating room suitability between the two groups.Conclusion:RVS can be used in microsurgical reconstruction of the reproductive tract in male rats and,with its advantages over SOM in ergonomic design and image definition,has a potential application value in male reproductive system micosurgery.

This study constructed a LHCGR-CRE-luc-HEK293 transgenic cell line according to the activation of the cAMP signaling pathway after recombinant human chorionic gonadotropin binding to the receptor. The biological activity of recombinant human chorionic gonadotropin was assayed using a luciferase assay system. The relative potency of the samples was calculated using four-parameter model. And the method conditions were optimized to validate the specificity, relative accuracy, precision and linearity of the method. The results showed that there was a quantitative potency relationship of human chorinonic gonadotropin (hCG) in the method and it was in accordance with the four-parameter curve. After optimization, the conditions were determined as hCG dilution concentration of 2.5 μg·mL^-1, dilution ratio of 1∶4, cell number of 10 000-15 000 cells/well, and induction time of 6 h. The method had good specificity, relative accuracy with relative bias ranging from -8.9% to 3.4%, linear regression equation correlation coefficient of 0.996, intermediate precision geometric coefficient of variation ranging from 3.3% to 15.0%, and linearity range of 50% to 200%. This study successfully established and validated a reporter gene method to detect hCG biological activity, which can be used for hCG biological activity assay and quality control.

AIM To investigate the variation rules of main secondary metabolites in Hedysari Radix before and after rubbing strip.METHODS UPLC-MS/MS was adopted in the content determination of formononetin,ononin,calycosin,calycosin-7-glucoside,medicarpin,genistein,luteolin,liquiritigenin,isoliquiritigenin,vanillic acid,ferulic acid,γ-aminobutyric acid,adenosine and betaine,after which cluster analysis,principal component analysis and orthogonal partial least squares discriminant analysis were used for chemical pattern recognition to explore differential components.RESULTS After rubbing strip,formononetin,calycosin,liquiritigenin and γ-aminobutynic acid demonstrated increased contents,along with decreased contents of ononin,calycosin-7-glucoside and vanillic acid.The samples with and without rubbing strip were clustered into two types,calycosin-7-glucoside,formononetin,γ-aminobutynic acid,vanillic acid,calycosin-7-glucoside and formononetin were differential components.CONCLUSION This experiment clarifies the differences of chemical constituents in Hedysari Radix before and after rubbing strip,which can provide a reference for the research on rubbing strip mechanism of other medicinal materials.

The Dionex CaboPac^TM PA10 BioLC^TM Analyical 2 mm × 250 mm column was used with a protective column (Dionex CaboPac^TM PA10 BioLC^TM Guard 2 mm × 50 mm). 100 mmol·L^-1 sodium hydroxide solution was used as eluent; the flow rate was 0.25 mL·min^-1. Sample tray temperature: 35 ℃. The pulse amperometric detector was adopted, and the waveform was Gold CWE, Ag-AgCl RE, Carbo, Quad. The samples were cultured with 8 concentrations of glycogen substrates (0.31, 1.25, 2.5, 5, 10, 20, 30, and 40 mg·mL^-1). D-Glucose concentrations were measured at 5 different time points (T0, T1, T2, T3 and T4). The glucose concentration from T1 to T4 minus the glucose concentration at T0. The reaction rate was calculated at different glycogen substrate concentrations. These reaction rates are plotted against substrate concentrations using Michaelis-Menten equation. The kinetic parameters were expressed as V_max (nmol·mg^-1·min^-1) and K_m (mg·mL^-1). The RSD of glucose standard curve R² (n = 6, linear range: 1.25-500 μmol·L^-1) was 0.1% and the RSD (n = 6) of the slope of the standard curve was 2.2%. The mean limit of quantitation was 0.14 μmol·L^-1, and the mean limit of detection was 0.05 μmol·L^-1. The RSD of K_m and V_max were 4.4% and 4.6% respectively in three separate experiments. The durability of the method was good. The method was developed for the on-line automatic determination of the hydrolysis kinetics of acid α-glucosidase (GAA) for injection by ion chromatography. The method has good precision, repeatability and durability, and can be used for the determination of glycogen hydrolysis kinetics of GAA for injection, and could reference value for the enzyme kinetics evaluation of recombinant enzyme replacement therapy.

According to the requirements of the regulatory authorities, degree of modification (DP) should be included in the characterisation of the PEGylated protein drug substance, and is one of the critical quality attributes for quality control. In this study, based on the fundamental assumption that the refractive index (RI) signal and the ultraviolet (UV) signal of PEGylated protein are equal to the sum of the corresponding signal produced by the polyethylene glycol (PEG) and protein parts of the conjugates in their uncoupled state, we developed a method to determine the DP of PEGylated recombinant human growth hormone (inpegsomatropin). In this method, 20 μL of 1 mg·mL^-1 human growth hormone (hGH) standard, 2 mg·mL^-1 PEG reference substance and 1 mg·mL^-1 drug substance solution were each injected to size exclusion chromatographic (SEC) column for separation, detected with ultraviolet and refractive index (UV-RI) detectors in series. Finally, the DP was calculated as the formula derived from the fundamental assumption. The developed SEC-UV-RI method showed good specificity, repeatability (RSD = 0.63%, n = 9) and accuracy, with a recovery of 100.0%, compared with the result obtained from the simultaneously established classical acid hydrolysis method, demonstrating pretty handleability and accessibility, and is appropriate for quality control test of DP for this drug substance.

In this study, we established a novel bioassay to determine the activity of polyethylene glycolated recombinant human growth hormone (PEG-rhGH) using Nb2-11 cells. We performed experimental condition optimization and methodological verification, and then detected the relative potency of PEG-rhGH products using this method. We demonstrated that the bioactivity of PEG-rhGH in promoting Nb2-11 cell proliferation displays a dose-response relationship, which conformed to the four-parameter model. Using PEG-rhGH reference as a control, we analyzed the relative potency of six batches of PEG-rhGH products, as well as linearity, regression and parallelism of the obtained curves. The relative potency of six batches of PEG-rhGH products was 95% to 105%. These results implied that the new bioassay established may be employed in quality control of PEG-rhGH products.

Based on transcriptome sequencing technology, the mouse model of prediabetes treated with Huangjing Qianshi Decoction was sequenced to explore the possible mechanism of treating prediabetes. First of all, transcriptome sequencing was performed on the normal BKS-DB mouse group, the prediabetic model group, and the Huangjing Qianshi Decoction treatment group(treatment group) to obtain differentially expressed genes in the skeletal muscle samples of mice. The serum biochemical indexes were detected in each group to screen out the core genes of Huangjing Qianshi Decoction in prediabetes. Gene Ontology(GO) database and Kyoto Encyclopedia of Genes and Genomes(KEGG) database were used to conduct signaling pathway enrichment analysis of differentially expressed genes, and real-time quantitative polymerase chain reaction(RT-qPCR) was used to verify them. The results showed that the levels of fasting blood glucose(FBG), fasting insulin(FINS), insulin resistance index(HOMA-IR), total cholesterol(TC), triglycerides(TG), and low-density lipoprotein cholesterol(LDL-C) in the mouse model were significantly decreased after treatment with Huangjing Qianshi Decoction. In the results of differential gene screening, there were 1 666 differentially expressed genes in the model group as compared with the normal group, and there were 971 differentially expressed genes in the treatment group as compared with the model group. Among them, interleukin-6(IL-6) and NR3C2 genes, which were closely related to the regulation of insulin resis-tance function, were significantly up-regulated between the model group and the normal group, and vascular endothelial growth factor A(VEGFA) genes were significantly down-regulated between the model group and the normal group. However, the expression results of IL-6, NR3C2, and VEGFA genes were adverse between the treatment group and the model group. GO functional enrichment analysis found that the biological process annotation mainly focused on cell synthesis, cycle, and metabolism; cell component annotation mainly focused on organelles and internal components; and molecular function annotation mainly focused on binding molecular functions. KEGG pathway enrichment analysis found that it involved the protein tyrosine kinase 6(PTK6) pathway, CD28-dependent phosphoinositide 3-kinase/protein kinase B(PI3K/AKT) pathway, p53 pathway, etc. Therefore, Huangjing Qianshi Decoction can improve the state of prediabetes, and the mechanism may be related to cell cycle and apoptosis, PI3K/AKT pathway, p53 pathway, and other biological pathways regulated by IL-6, NR3C2, and VEGFA.
Animals ; Mice ; Proto-Oncogene Proteins c-akt ; Phosphatidylinositol 3-Kinases ; Prediabetic State ; Vascular Endothelial Growth Factor A ; Interleukin-6 ; Transcriptome ; Tumor Suppressor Protein p53 ; Insulin ; Cholesterol

italic>Polygonatum franchetii Hua is a medicinal plant endemic to China from Polygonatum Mill. The chloroplast genomes of two P. franchetii individuals sampled from two different habitats were sequenced by using the DNBSEQ-T7 high-throughput sequencing platform. After assembly and annotation, the two complete chloroplast genomes were characterized, and then comparative and phylogenetic analyses were performed with other published chloroplast genome sequences from Polygonatum. The whole chloroplast genomes of the two P. franchetii individuals were 155 942 and 155 962 bp in length, with a large single copy region (LSC, 84 670 and 84 722 bp), a small single copy region (SSC, 18 564 and 18 566 bp) and a pair of reverse repeats (IRa/IRb, 26 354 and 26 337 bp), respectively. Both of them contained 113 genes, including 79 protein-coding genes (PCGs), 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. Comparative analyses showed that the genome length, the guanine and cytosine (GC) content, genes content and order were highly conserved between the two P. franchetii individuals and among different Polygonatum species. The detected repeat sequences, including dispersed repeats, tandem repeats and simple sequence repeats (SSRs), were also relatively similar in types and positions, though showing a slightly difference in number. No significant expansion or contraction of the inverted repeat regions was found. Sequences variation between the two P. franchetii individuals was lower than that among different Polygonatum species. Besides, coding sequences (CDS) showed less divergence than noncoding sequences, and sequence divergence of IRs regions was lower than that of the LSC and SSC regions, both intraspecifically and interspecifically. Eight sequences with high nucleotide diversity among different species were screened, all of which were found located in the LSC and SSC regions. Phylogenetic inference showed that all Polygonatum species clustered into a monophyletic clade with a 100% bootstrap value, within which, species in section Verticillata formed a distinct group, section Sibirica and section Polygonatum were sister groups. The two P. franchetii individuals grouped together and showed the closest phylogenetic affinity to P. stenophyllum Maxim., belonging to the section Verticillata. The chloroplast genome of P. franchetii and its phylogenetic position in Polygonatum were comprehensively investigated and clearly elucidated in this study, the results may lay a foundation for the resource development and utilization of P. franchetii, as well as further molecular identification and phylogenetic studies of medicinal Polygonatum species.