Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

OBJECTIVE: To identify the underlying molecular mechanism of Modified Hu-Lu-Ba-Wan (MHW) in alleviating renal lesions in mice with diabetic kidney disease (DKD). METHODS: The db/db mice were divided into model group and MHW group according to a random number table, while db/m mice were settled as the control group (n=8 per group). The control and model groups were gavaged daily with distilled water [10 mL/(kg·d)], and the MHW group was treated with MHW [17.8 g/(kg·d)] for 6 weeks. After MHW administration for 6 weeks, indicators associated with glucolipid metabolism and urinary albumin were tested. Podocytes were observed by transmission electron microscopy. Kidney transcriptomics was performed after confirming therapeutic effects of MHW on DKD mice. The relevant target of MHW' effect in DKD was further determined by enzyme-linked immunosorbent assay, Western blot analysis, immunohistochemistry, and immunofluorescence staining. RESULTS: Compared with the model group, MHW improved glucose and lipid metabolism (P<0.05), and reduced lipid deposition in the kidney. Meanwhile, MHW reduced the excretion of urinary albumin (P<0.05) and ameliorated renal damage. Transcriptomic analysis revealed that the inflammation response, particularly the interleukin-17 (IL-17) signaling pathway, may be responsible for the effect of MHW on DKD. Furtherly, our results found that MHW inhibited IL-17A and alleviated early fibrosis in the diabetic kidney. CONCLUSION MHW ameliorated renal damage in DKD via inhibiting IL-17A, suggesting a potential strategy for DKD therapy.
Animals ; Diabetic Nephropathies/genetics* ; Interleukin-17/antagonists & inhibitors* ; Drugs, Chinese Herbal/therapeutic use* ; Male ; Kidney/ultrastructure* ; Podocytes/metabolism* ; Mice ; Albuminuria ; Lipid Metabolism/drug effects* ; Mice, Inbred C57BL

Genetic transformation is a fundamental tool in molecular biology research of medicinal plants. Tailoring transgenic technologies to each distinct medicinal plant would necessitate a substantial investment of time and effort. Here, we present a simple hairy root transformation method that does not require sterile conditions, utilizing Agrobacterium rhizogenes strain K599 and the visible RUBY reporter system. Transgenic hairy roots were obtained for six tested medicinal plant species, roots or rhizomes of which have recognized medicinal value, spanning four botanical families and six genera (Platycodon grandiflorus, Atractylodes macrocephala, Scutellaria baicalensis, Codonopsis pilosula, Astragalus membranaceus, and Glycyrrhiza uralensis). Furthermore, two previously identified Glycyrrhiza uralensis UGTs that convert liquiritigenin into liquiritin in heterologous systems were studied in planta using the method. Our results indicate that overexpression of GuUGT1 but not GuUGT10 and Cas9-mediated knockout of GuUGT1 profoundly influenced the accumulation of liquiritin and isoliquiritin in licorice roots. Therefore, the method described here represents a simple, rapid and widely applicable hairy root transformation method that enables fast gene functional study in medicinal plants.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Humans ; Urinary Bladder Neoplasms/pathology* ; Carcinoma, Transitional Cell/pathology* ; Genetic Markers ; Biomarkers, Tumor/genetics* ; Urothelium/pathology*

Cells not only contain membrane-bound organelles (MBOs), but also membraneless organelles (MLOs) formed by condensation of many biomacromolecules. Examples include RNA-protein granules such as nucleoli and PML nuclear bodies (PML-NBs) in the nucleus, as well as stress granules and P-bodies in the cytoplasm. Phase separation is the basic organizing principle of the form of the condensates or membraneless organelles (MLOs) of biomacromolecules including proteins and nucleic acids. In particular, liquid-liquid phase separation (LLPS) compartmentalises and concentrates biological macromolecules into liquid condensates. It has been found that phase separation of biomacromolecules requires some typical intrinsic characteristics, such as intrinsically disordered regions, modular domains and multivalent interactions. The phase separation of biomacromolecules plays a key role in many important cell activities. In recent years, the phase separation of biomacromolecules phase has become a focus of research in gene transcriptional regulation. Transcriptional regulatory elements such as RNA polymerases, transcription factors (TFs), and super enhancers (SEs) all play important roles through phase separation. Our group has previously reported for the first time that long-term inactivation or absence of assembly factors leads to the formation of condensates of RNA polymerase II (RNAPII) subunits in the cytoplasm, and this process is reversible, suggesting a novel regulatory model of eukaryotic transcription machinery. The phase separation of biomacromolecules provides a biophysical understanding for the rapid transmission of transcriptional signals by a large number of TFs. Moreover, phase separation during transcriptional regulation is closely related to the occurrence of cancer. For example, the activation of oncogenes is usually associated with the formation of phase separation condensates at the SEs. In this review, the intrinsic characteristics of the formation of biomacromolecules phase separation and the important role of phase separation in transcriptional regulation are reviewed, which will provide reference for understanding basic cell activities and gene regulation in cancer.

The quality evaluation of traditional Chinese medicine is one of the key issues related to the modernization of traditional Chinese medicine. The quality evaluation technology system of traditional Chinese medicine mainly includes traditional evaluation (traits, microscopic and physicochemical identification), chemical evaluation and biological evaluation. Due to the complex composition of traditional Chinese medicine, the single detection method in the above evaluation technology system usually cannot obtain sufficient quality information. The multi-source information fusion strategy can organically integrate data from multiple analysis and detection technologies to obtain more comprehensive information of samples and improve the quality evaluation effect. At present, multi-source information fusion strategy has been widely used in the fields of military, industrial and food, and it is still in its infancy in the field of quality evaluation of traditional Chinese medicine. This research introduces the definition, structure, method (algorithm) and fusion level of multi-source information fusion, summarizes its research progress in the origin traceability, variety identification and pharmaceutical analysis of traditional Chinese medicine, and sorts out the specific methods of data fusion in each literature. Finally, we summarized, prospected and discussed the application, development and existing problems of information fusion technology and its application in the quality evaluation of traditional Chinese medicine, in order to provide reference for broadening the application of this technology in the field of traditional Chinese medicine.

Objective To explore the correlation between the genetic variations of methylenetetrahydrofolate reductase(MTHFR),transcobalamin Ⅱ(TCN2),and reduced folate carrier(RFC)genes and the incidence of ventricular septal defect(VSD)congenital heart disease(CHD)in children.Methods Children with VSD CHD were selected as the treatment group,and healthy children who underwent physical examination in our hospital during the same period were selected as the control group.Detection of the MTHFR C677T,MTHFR A1298C,TCN2 rs1801198,RFC rs1051266 polymorphisms were performed by the polymerase chain reaction.Binary logistic regression was used to analyze the relationship between CHD and the above gene polymorphisms.Results A total of 108 cases were included in both the treatment and control groups.The proportions of TT at MTHFR C677T locus in the treatment and control groups were 44.44％and 19.44％,CC at MTHFR A1298C locus were 51.85％and 37.04％,GG at TCN2 rs1801198 locus were 55.56％and 33.33％,with statistically significant differences(all P＜0.05).Binary regression analysis showed that the polymorphisms at MTHFR C677T and TCN2 rs1801198 were risk factors for VSD CHD(both P＜0.05).Conclusion The occurrence of VSD CHD is closely related to the polymorphisms at MTHFR C677T and TCN2 rs 1801198 loci.

Objective: To analyze the survival of newly diagnosed malignant tumors in cancer registration areas of Hubei Province from 2013 to 2015. Methods: From January 1, 2013 to December 31, 2015, all newly diagnosed malignant tumors were collected from cancer registration areas in Hubei Province, and patients were followed up using a combination of active and passive methods. Cancer survival was analyzed using the strs package in Stata software. Observed and expected survival were calculated using the life table and Ederer Ⅱ methods, and the difference in survival rate of patients with different sex, age, urban and rural areas and different cancer species was compared. Results: From 2013 to 2015, 83 987 new malignant tumors were diagnosed in cancer registration areas in Hubei Province, including 45 742 males (54.46%) and 38245 females (45.54%). The overall 5-year relative survival rate was 41.46%, 34.43% for men and 49.63% for women. With the increase of age, the observed survival rate and relative survival rate of patients of different genders showed a decreasing trend. The 5-year relative survival rate of patients with malignant tumors was 47.58% in urban areas and 26.58% in rural areas. The observed survival rate and relative survival rate in rural areas were significantly lower than those in urban areas. The overall 5-year relative survival rates for common malignancies were 20.61% for lung cancer, 15.36% for liver cancer, 22.89% for esophageal cancer, 34.92% for gastric cancer, and 54.87% for colorectal cancer. In addition, the 5-year relative survival rates of common malignant tumors in women were 78.65% for breast cancer and 52.55% for cervical cancer. Conclusions: In Hubei Province, the survival rate of malignant tumors is different among different genders, regions, age groups and cancer species. Prevention and treatment and health education should be strengthened for malignant tumor patients in rural areas and those with high incidence and low survival rate such as liver cancer and lung cancer, and relevant strategies should be formulated according to the gender and age distribution characteristics of different cancer species.
Humans ; Female ; Male ; Liver Neoplasms ; Uterine Cervical Neoplasms/epidemiology* ; Lung Neoplasms ; China/epidemiology* ; Urban Population ; Incidence ; Survival Analysis ; Rural Population ; Registries

Objective: To analyze the survival of newly diagnosed malignant tumors in cancer registration areas of Hubei Province from 2013 to 2015. Methods: From January 1, 2013 to December 31, 2015, all newly diagnosed malignant tumors were collected from cancer registration areas in Hubei Province, and patients were followed up using a combination of active and passive methods. Cancer survival was analyzed using the strs package in Stata software. Observed and expected survival were calculated using the life table and Ederer Ⅱ methods, and the difference in survival rate of patients with different sex, age, urban and rural areas and different cancer species was compared. Results: From 2013 to 2015, 83 987 new malignant tumors were diagnosed in cancer registration areas in Hubei Province, including 45 742 males (54.46%) and 38245 females (45.54%). The overall 5-year relative survival rate was 41.46%, 34.43% for men and 49.63% for women. With the increase of age, the observed survival rate and relative survival rate of patients of different genders showed a decreasing trend. The 5-year relative survival rate of patients with malignant tumors was 47.58% in urban areas and 26.58% in rural areas. The observed survival rate and relative survival rate in rural areas were significantly lower than those in urban areas. The overall 5-year relative survival rates for common malignancies were 20.61% for lung cancer, 15.36% for liver cancer, 22.89% for esophageal cancer, 34.92% for gastric cancer, and 54.87% for colorectal cancer. In addition, the 5-year relative survival rates of common malignant tumors in women were 78.65% for breast cancer and 52.55% for cervical cancer. Conclusions: In Hubei Province, the survival rate of malignant tumors is different among different genders, regions, age groups and cancer species. Prevention and treatment and health education should be strengthened for malignant tumor patients in rural areas and those with high incidence and low survival rate such as liver cancer and lung cancer, and relevant strategies should be formulated according to the gender and age distribution characteristics of different cancer species.
Humans ; Female ; Male ; Liver Neoplasms ; Uterine Cervical Neoplasms/epidemiology* ; Lung Neoplasms ; China/epidemiology* ; Urban Population ; Incidence ; Survival Analysis ; Rural Population ; Registries