ObjectiveTo clarify the expression of TRIM28 in non-M3 acute myeloid leukemia （AML） and its correlation with clinical indicators and prognosis， and to further explore the effect of TRIM28 expression levels on the proliferation and apoptosis of AML cells using small interfering RNA. MethodsThe GSE34577 dataset was analyzed using R software to compare TRIM28 expression between healthy controls and non-M3 acute myeloid leukemia （AML） patients. Clinical samples from non-M3 AML patients were collected， with TRIM28 expression levels measured using real-time quantitative PCR （qPCR）. The analysis focused on correlations between TRIM28 expression and various clinical indicators， treatment efficacy， and patient prognosis. Furthermore， small interfering RNA （siRNA） technology was employed to downregulate TRIM28 expression in human primary AML cells （HL60 cell line）. The effects on cell proliferation and apoptosis were then assessed through CCK-8 assays and flow cytometry， respectively. ResultsThe results showed that TRIM28 was up-regulated in non-M3 AML of both online database GSE34577 and clinical samples （P<0.000 1）， TRIM28 expression of new diagnosis group and relapsed refractory group was higher than iron deficiency anemia group （P<0.01）， and there was no significance between different French-American-British classification systems subtype. TRIM28 expression was higher in non-M3 AML patients with a poor genetic prognosis stratified as moderate than in the good prognosis group， and TRIM28 expression was associated with NPM1 combined with the FLT3-ITD mutation， positively correlated with age， bone marrow blast， peripheral blood blast and white blood cell， negatively correlated with hemoglobin. In addition， interference TRIM28 greatly inhibited cell proliferation and promoted cell apoptosis. ConclusionThis study reveals that TRIM28 is highly expressed in non-M3 AML and associated with prognosis， and plays a key role in the proliferation and apoptosis of AML cells， suggesting that TRIM28 may serve as a novel therapeutic target for non-M3 AML.

ObjectiveTo humanize the variable region of the IL9-2G1 rabbit monoclonal antibody （IL9 2G1 mAb） and to evaluate its biological activity. MethodsThe humanized sequences of IL9-2G1 mAb were designed. The heavy chain variable region （VH） and light chain variable region （VL） of the antibody were humanized and cloned into vector backbones containing the constant region frameworks of human IgG1 and Ig Kappa subtypes. Protein purification was carried out using the Chinese hamster ovary cell （CHO） expression system. Enzyme-linked immunosorbent assay （ELISA） was used to qualitatively analyze the antigen-binding activity of the antibodies. After screening the optimal humanized antibody candidate， surface plasmon resonance （SPR） was performed to detect molecular affinity. Western blot was used to examine the effects of the rIL-9 group and the rIL-9 + hIL-9 mAb group on the phosphorylation of Janus kinase 1/signal transducer and activator of transcription 3 （JAK1/STAT3）， while ELISA was employed to assess the impact of the Th9 group and the Th9 + hIL-9 mAb group on interleukin-9 （IL-9） secretion. ResultsPurification and ELISA results showed that the heavy chain OH and light chain FL exhibited good compatibility and expression performance. SPR results indicated that the equilibrium dissociation constant （KD） of M501042-OHFL against the analyte M30903 was 1.33×10^-7 M， which was superior to the parental 2G1 antibody. The Western blot results showed that the levels of phosphorylated JAK1 （p-JAK1） and phosphorylated STAT3 （p-STAT3） were significantly decreased in the rIL-9 + hIL-9 mAb group compared with the rIL-9 group （P<0.01）. The ELISA results indicated that the secretion of IL-9 was significantly reduced in the Th9 + hIL-9 mAb group compared with the Th9 group （P<0.01）. ConclusionHumanization modification is performed on the variable region of IL9-2G1 rabbit monoclonal antibody， and the humanized antibody hIL-9 mAb OHFL with high affinity and strong binding stability is successfully constructed， which provides an experimental basis for subsequent research.

ObjectiveTo construct an interleukin-9 （Il9） gene knockout mouse model on a C57BL/6N background， establish a reliable genotyping method， and provide a standardized animal model for investigating Il9-related immune mechanisms and diseases. MethodsIl9 knockout mouse strain （C57BL/6N-Il9^em1Cya） was generated using conventional gene knockout technology. The method for DNA extraction from mouse tails was optimized to ensure template quality. Two sets of specific primers （F1/R1 and F1/R2） were designed to amplify target fragments by PCR， and mouse genotypes were determined by agarose gel electrophoresis. Specifically， primer pair F1/R1 targeted the knockout sequence with an expected product of 465 bp， while F1/R2 targeted the wild-type sequence with an expected product of 703 bp. Subsequently， Western blot was performed to verify the knockout efficiency of Il9 in primary immune cells and key organs of the offspring mice， and flow cytometry was used to assess the effects of Il9 deficiency on the function of the immune system in major immune cell populations. ResultsIl9 knockout mice were successfully bred and genotyped. PCR identification showed that homozygous mice （-/-） exhibited only a 465 bp band， wild-type mice （+/+） displayed only a 703 bp band， and heterozygous mice （+/-） showed both bands simultaneously. Meanwhile， Western blot analysis revealed significantly decreased IL-9 expression in various organs of the offspring mice （P<0.001）. Flow cytometric results demonstrated that Il9 deficiency exerted no obvious effects on the proportions of peripheral blood lymphocytes， splenic lymphocytes， and peritoneal macrophages. ConclusionAn Il9 gene knockout mouse model on a C57BL/6N background is successfully constructed. The established PCR genotyping system is highly efficient and accurate， which can effectively distinguish mice of different genotypes. This model provides a stable and reliable experimental basis for subsequent functional studies of Il9 and exploration of the mechanisms underlying Il9-related diseases.

Objective : To construct a humanized mouse model of the interleukin-9 receptor(IL-9R) coding DNA sequence(CDS) gene and to verify the genotype and IL-9R expression in mice. Methods : The CRISPR/Cas9 genome editing technology was used to replace the exon 2-7 fragment of the il-9r gene in mouse embryonic stem cells with the corresponding human IL-9R sequence. After verifying the completion of the gene fragment replacement, tetraploid embryos were constructed and microinjected back into the oviducts of surrogate mice. Through surrogacy by female mice, homozygous humanized mice were obtained. DNA was extracted from the homozygous humanized mice IL-9R CDS gene, and their genotypes were identified by agarose gel electrophoresis after PCR amplification. Western blot was used to detect the expression of IL-9R in the spleen and thymus of homozygous humanized mice with either wild-type(WT) or IL-9R gene humanization. Results : Gel electrophoresis after PCR amplification showed that mice with only a 1 805 bp band amplified using WT primers were wild-type, while mice with 2 553 bp and 2 340 bp bands amplified using 5KI and 3KI primers, respectively, were homozygous humanized mice with IL-9R CDS gene. Western blot results indicated that the tissues of homozygous humanized mice model with IL-9R CDS gene expressed IL-9R significantly. Conclusion The humanized mouse model with IL-9R CDS gene has been successfully constructed and characterized.

OBJECTIVE: To investigate the effect of irradiation dose rate of ⁶⁰Co γ-ray on hematopoietic and immune cells in total body irradiation (TBI) mice. METHODS: After TBI with 8 Gy ⁶⁰Co γ-ray at three irradiation dose rates of 0.027, 0.256 and 0.597 Gy/min, the survival and change of body weight of C57BL/6J mice were observed within 30 days. The peripheral blood parameters were examined at each time point within 30 days post-irradiation. The hematopoietic stem/progenitor cell counts of mice were examined on the 10^th and 30^th day post-irradiation by flow cytometry, as well as the proportions of immune cells in peripheral blood, bone marrow and spleen of mice on the 30^th day post-irradiation. RESULTS: After TBI with 8 Gy ⁶⁰Co γ-ray, the 30-day survival rate of high dose-rate group was 0, which was significantly lower than 90% of medium dose-rate group and 100% of low dose-rate group (both P < 0.001). The peripheral blood parameters of all three groups showed a sharp decline → low value → gradually recovering trend. The count of white blood cell, neutrophil, lymphocyte, red blood cell, platelet and hemoglobin level in the high dose-rate and medium dose-rate groups were significantly lower than those in the low dose-rate group on day 7-18 post-irradiation (all P < 0.05), but there were no significant differences between the high dose-rate and medium dose-rate groups (P >0.05). On the 10^th day after irradiation, the proportion and number of bone marrow hematopoietic stem/progenitor cells (including LK, LSK, LT-HSC, ST-HSC, and MPP cells) in the low dose-rate and medium dose-rate groups were significantly decreased compared to those in the normal group (all P < 0.05), but there were no significant differences between the two groups (P >0.05). On the 30^th day after irradiation, LSK, LT-HSC, ST-HSC and MPP cells in the low dose-rate group recovered to normal levels, while those in the medium dose-rate group were still significantly lower than those in the low dose-rate group (all P < 0.001). The results of bone marrow and peripheral immune cell tests on the 30^th day after irradiation showed that the ratios of T and B lymphocytes in the low dose-rate and medium dose-rate groups were reduced compared to that in the normal group (both P < 0.05), while the ratio of neutrophils was increased (P < 0.01). The trend of changes in the spleen and peripheral blood was consistent. CONCLUSION The degree of hematopoietic and immune cell damage in mice after TBI with 8 Gy ⁶⁰Co γ-ray is related to the dose rate, and low dose-rate irradiation can reduce the damage in the animal model. Therefore, choosing the appropriate dose rate of irradiation is a key factor in establishing an objective and reliable experimental animal model of irradiation.
Animals ; Mice ; Whole-Body Irradiation ; Gamma Rays ; Mice, Inbred C57BL ; Hematopoietic Stem Cells/radiation effects* ; Cobalt Radioisotopes ; Dose-Response Relationship, Radiation ; Male

Mycophenolic acid (MPA), the active moiety of both mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS), serves as a primary immunosuppressant for maintaining solid organ transplants. Therapeutic drug monitoring (TDM) enhances treatment outcomes through tailored approaches. This study aimed to develop an evidence-based guideline for MPA TDM, facilitating its rational application in clinical settings. The guideline plan was drawn from the Institute of Medicine and World Health Organization (WHO) guidelines. Using the Delphi method, clinical questions and outcome indicators were generated. Systematic reviews, Grading of Recommendations Assessment, Development, and Evaluation (GRADE) evidence quality evaluations, expert opinions, and patient values guided evidence-based suggestions for the guideline. External reviews further refined the recommendations. The guideline for the TDM of MPA (IPGRP-2020CN099) consists of four sections and 16 recommendations encompassing target populations, monitoring strategies, dosage regimens, and influencing factors. High-risk populations, timing of TDM, area under the curve (AUC) versus trough concentration (C₀), target concentration ranges, monitoring frequency, and analytical methods are addressed. Formulation-specific recommendations, initial dosage regimens, populations with unique considerations, pharmacokinetic-informed dosing, body weight factors, pharmacogenetics, and drug‍-‍drug interactions are covered. The evidence-based guideline offers a comprehensive recommendation for solid organ transplant recipients undergoing MPA therapy, promoting standardization of MPA TDM, and enhancing treatment efficacy and safety.
Mycophenolic Acid/administration & dosage* ; Drug Monitoring/methods* ; Humans ; Organ Transplantation ; Immunosuppressive Agents/administration & dosage* ; Delphi Technique

Cannabidiol (CBD), a non-psychoactive cannabinoid, shows great promise in treating methamphetamine (METH) addiction. Nonetheless, the molecular target and the mechanism through which CBD treats METH addiction remain unexplored. Herein, CBD was shown to counteract METH-induced locomotor sensitization and conditioned place preference. Additionally, CBD mitigated the adverse effects of METH, such as cristae loss, a decline in ATP content, and a reduction in membrane potential. Employing an activity-based protein profiling approach, a target fishing strategy was used to uncover CBD's direct target. ATP5A1, a subunit of ATP synthase, was identified and validated as a CBD target. Moreover, CBD demonstrated the ability to ameliorate METH-induced ubiquitination of ATP5A1 via the D376 residue, thereby reversing the METH-induced reduction of ATP5A1 and promoting the assembly of ATP synthase. Pharmacological inhibition of the ATP efflux channel pannexin 1, blockade of ATP hydrolysis by a CD39 inhibitor, and blocking the adenosine A1 receptor (A1R) all attenuated the therapeutic benefits of CBD in mitigating METH-induced behavioral sensitization and CPP. Moreover, the RNA interference of ATP5A1 in the ventral tegmental area resulted in the reversal of CBD's therapeutic efficacy against METH addiction. Collectively, these data show that ATP5A1 is a target for CBD to inhibit METH-induced addiction behaviors through the ADO-A1R signaling pathway.

A previous study showed that the length of the foreskin plays a role in the risk of sexually transmitted infections and chronic prostatitis, which can lead to poor quality of sexual life. Here, the association between foreskin length and sexual dysfunction was evaluated. A total of 5700 participants were recruited from the andrology clinic at The First Affiliated Hospital of University of Science and Technology of China (Hefei, China). Clinical characteristics, including foreskin length, were collected, and sexual function was assessed by the International Index of Erectile Function-5 (IIEF-5) and Premature Ejaculation Diagnostic Tool (PEDT) questionnaires. Men with sexual dysfunction were more likely to have redundant foreskin than men without sexual dysfunction. Among the 2721 erectile dysfunction (ED) patients and 1064 premature ejaculation (PE) patients, 301 (11.1%) ED patients and 135 (12.7%) PE patients had redundant foreskin, respectively. Men in the PE group were more likely to have redundant foreskin than men in the non-PE group ( P = 0.004). Logistic regression analyses revealed that the presence of redundant foreskin was associated with increased odds of moderate/severe ED (adjusted odds ratio [aOR] = 1.31, adjusted P = 0.04), moderate PE (aOR = 1.38, adjusted P = 0.02), and probable PE (aOR = 1.37, adjusted P = 0.03) after adjusting for confounding variables. Our study revealed a positive correlation between the presence of redundant foreskin and the risk of sexual dysfunction, especially in PE patients. Assessment of the length of the foreskin during routine clinical diagnosis may provide information for patients with sexual dysfunction.
Humans ; Male ; Foreskin ; Cross-Sectional Studies ; Adult ; Erectile Dysfunction/epidemiology* ; Premature Ejaculation/epidemiology* ; Middle Aged ; China/epidemiology* ; Surveys and Questionnaires ; Sexual Dysfunction, Physiological/epidemiology* ; Young Adult

Objective: To explore the breeding and genotyping of CCR2 knockout mice, and to verify the applicability of the polymerase chain reaction(PCR) method for genotype detection of CCR2 knockout mice. Methods: The introduced CCR2 pure male mice and wild-type female mice were mated and bred to produce the offspring generation, the obtained F1 generation heterozygous mice were continued to be mated. DNA was extracted by clipping the tail tissues of the mice at the age of 2 weeks, the target gene fragment was amplified by PCR, and the genotypic results were determined by agarose gel electrophoresis. The proportion of purebred progeny carrying the CCR2 knockout gene was increased by genetic crosses, the effect of CCR2 knockout in the progeny mice was verified by using Western blot against major immune cells and key organs, and flow cytometry was used to detect whether the knockout of the CCR2 gene had any effect on the function of the immune system by targeting the major immune cells. Results: CCR2 knockout mice were successfully bred and characterized, and three genotypes of F2 generation mice were obtained: CCR2+/+, CCR2+/-, and CCR2-/-. The offspring genotypes were identified by PCR, and Western blot showed extremely low CCR2 protein expression in CCR2 knockout mice. Flow analysis showed that CCR2 knockdown reduced the expression of CD4+T and Th1 cells in mouse spleen-derived T cells, but did not affect macrophage function. Conclusion Correct breeding and identification are important ways to get the pure CCR2 knockout mice, and PCR method for identifying mouse genotypes is simple, fast and reliable.