BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

In situ bioimaging is a powerful tool for directly observing the localization, expression, and interactions of nucleic acids or protein targets within cells, providing essential insights into cell function and disease mechanisms. In recent years, the CRISPR/Cas9 system, a revolutionary gene-editing tool, has been applied to develop efficient in situ imaging techniques. This paper reviews recent CRISPR/Cas9-based imaging methods utilizing Cas9 protein, engineered single-guide RNA (sgRNA), and coupled fluorescent tags, and compares their application in living and fixed cells. It focuses on the specificity, signal amplification efficiency, and multi-modal imaging capabilities of these methods, with further discussion based on current research, aiming to offer a comprehensive overview of CRISPR/Cas9-based in situ bioimaging techniques, with some valuable reference and guidance for research in related fields.

ObjectiveTo systematically explore the underlying mechanisms of Huashi Baidu prescription （HBP） against myocardial injury through a multidimensional network analysis of "transcriptome-putative target-phenotype gene". MethodPutative targets of compounds in HBP were predicted using the Encyclopedia of Traditional Chinese Medicine （ETCM 2.0，http：//www.tcmip.cn/ETCM2/front/） and the Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine （TCMIP v2.0，http：//www.tcmip.cn/TCMIP/index.php/Home/Login/login.html）. Clinical predominant symptom phenotypes for HBP against coronavirus disease 2019 （COVID-19） were collected from CNKI and PubMed databases， while clinical side effects of doxorubicin were gathered from the CTD database. Phenotype-related genes were collected from the HPO database and SoFDA data platform （http：//www.tcmip.cn/Syndrome/front/#/）. An interaction network of "transcriptome-putative target-phenotype gene" was constructed. Key nodes were identified through topological feature calculations， and functional enrichment analysis was conducted to explain the underlying mechanism. Pharmacodynamic evaluation and mechanism verification were performed using a doxorubicin-induced myocardial injury mouse model. Sixty-five SPF-grade C57BL/6J male mice were randomly divided into a control group， a doxorubicin model group， and low-， medium-， and high-dose HBP groups （HBP-L/M/H）， with 13 mice per group. Histopathological severity was assessed using hematoxylin-eosin （HE） and Masson staining. Fibrosis markers were measured by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and protein expression was detected by Western blot. ResultA total of 1 044 putative targets for HBP were obtained from ETCM 2.0 and TCMIP v2.0. From the CTD， HPO， and SoFDA databases， 1 223 potential effect-related targets were identified. Cardiac transcriptome sequencing （RNA-seq） yielded 214 genes related to doxorubicin-induced myocardial injury and 58 genes associated with the effects of HBP， using criteria of P-value<0.05 and FC > 1.5/< 0.667. Analysis of the multidimensional molecular network revealed that the mechanisms of HBP on myocardial injury were mainly related to immune-inflammation imbalance， cellular metabolism， myocardial cell function and fibrosis， angiogenesis， contraction and platelet activation， DNA synthesis， metabolism and repair， as well as cell death and oxidative stress. HBP improved cardiovascular abnormalities such as reduced ejection fraction， myocardial fibrosis， myocarditis， hypertriglyceridemia， and arterial/venous thrombosis. Animal experiments demonstrated that HBP reduced the abnormal high expression of Nod-like receptor heat protein domain Protein 3 （NLRP3）， Caspase-1， apoptosis-associated speck-like protein （ASC）， and Gasdermin D （GSDMD） in myocardial tissue induced by doxorubicin （P<0.05）， and improved the elevated mRNA levels of fibrosis markers transforming growth factor-β₁ （TGF-β₁）， Smad3， and α-smooth muscle actin （α-SMA） （P<0.05）. ConclusionHBP can improve myocardial injury through multiple components， targets， and effects， with the inhibition of the NLRP3 inflammasome activation pathway being a key pharmacodynamic mechanism. This study is expected to provide new insights for the development of targeted therapeutic drugs.

Abstract: The differential expression and subcellular localization of single-cell proteins are closely related to the physiological state and pathological mechanisms of the body. The development of single-cell protein in situ imaging methods provides powerful tools for spatial single-cell proteomics research and single-cell protein profiling. This article summarizes the single-cell protein imaging methods developed in recent years, including the circulating immunofluorescence imaging methods based on ordered multi-round antibody incubation, mass spectrometry imaging based on metal element labeled antibodies, fluorescence imaging based on DNA-barcoded antibody, gene encoded fluorescence protein imaging and spectral imaging based on Raman spectroscopy or X-ray spectroscopy, with brief explanation of the imaging principles of these methods. It focuses on the multiple performance, imaging resolution and signal amplification performance of these methods, and analyzes their application characteristics in practical scientific research and clinical work, in the hope of providing some reference for the development of more revolutionary single-cell imaging methods, and promoting the development of biomedical and precision medicine.

【Objective】 To investigate the relationship between preoperative albumin-to-alkaline phosphatase ratio (AAPR) and postoperative recurrence of localized penile cancer (T1-3N0M0). 【Methods】 Clinical data of patients with limited penile cancer admitted to our hospital during Jan.2012 and Jan.2017 were collected to compare the differences in age, body mass index (BMI), AAPR, hypertension, diabetes mellitus, tumor diameter, postoperative pathological grading and staging between the postoperative recurrence group and the non-recurrence group. 【Results】 The differences in AAPR(P=0.001), WHO/ISUP pathological grading(P=0.018), and pathological stage(P=0.012)between the recurrence and non-recurrence groups were statistically significant. Cox regression results showed that AAPR was an independent risk factor for recurrence (P=0.041). Survival curve results showed that patients in the high and low AAPR groups had an inverse relationship with recurrence-free survival (P=0.028). 【Conclusion】 Preoperative AAPR is an independent risk factor for recurrence after surgery for limited penile cancer and is associated with recurrence-free survival. As AAPR increases, the incidence of recurrence decreases and progression-free survival increases.

Objective:We employ different regimens of induction therapy in living donor ABO-incompatible kidney transplantation(ABOi-KT) recipients to compare their clinical outcomes during 6 months post-KT.Methods:A retrospective analysis was conducted for the relevant clinical data of 41 ABOi-KT recipients from June 2018 to September 2022.Thirteen recipients on induction therapy of anti-human T lymphocyte porcine immunoglobin(pATG)were enrolled in pATG group; 19 recipients on induction therapy of basiliximab in basiliximab group; 9 recipients on induction therapy of rabbit anti-human thymocyte immunoglobulin(rATG)in rATG group.Differences in age, gender, body mass index(BMI), dialysis modality/duration, sideness of donor kidney, frequency of blood group antibody treatment, dose of rituximab, basic blood group antibody titers of IgG/IgM, and the gender and BMI of recipient's donor were compared for three groups.Immune status was assessed by comparing absolute lymphocyte count before pre-treatment and within 6 months post-KT in recipients under different induction regimens among 3 groups by one-way analysis of variance.Transplant kidney function was assessed by comparing the levels of serum creatinine, estimated glomerular filtration rate(eGFR)and serum urea nitrogen using one-way analysis of variance.The incidence of delayed graft function(DGF), acute rejection(AR)and infection was compared among three groups.Results:Regarding baseline profiles, except for donor age pATG group[(60.23±6.10)years]versus basiliximab group[(51.95±6.97)years]was statistically significant( P=0.002), the differences in the remaining parameters were not statistically significant among three groups(all P>0.05). At Day 1/3/7/10/14 post-KT, absolute lymphocyte counts were(0.17±0.07)×10 9/L, (0.27±0.14)×10 9/L, (0.85±0.40)×10 9/L, (1.05±0.56)×10 9/L and(1.10±0.56)×10 9/L in pATG group and(0.69±0.04)×10 9/L, (0.18±0.21)×10 9/L, (0.57±0.44)×10 9/L, (0.67±0.45)×10 9/L and(0.81±0.46)×10 9/L in rATG group respectively.They were all higher than those in basiliximab group[(0.46±0.18)×10 9/L, (0.67±0.26)×10 9/L, (1.29±0.48)×10 9/L, (1.56±0.49)×10 9/L, (1.75±0.53)×10 9/L]and the differences were statistically significant(all P<0.05). No statistically significant difference existed in absolute lymphocyte count among 3 groups before pre-treatment and after Day 21 post-KT(all P>0.05). At Week 1/2/4/12/24 post-KT, the differences in serum levels of creatinine and urea nitrogen were not statistically significant( P>0.05). At Month 1/3 post-KT, eGFR was(47.24±14.51)and(49.94±14.31)ml·min -1·(1.73 2) -1 in rATG group and they were lower than(67.36±21.60)and(65.00±14.67)ml·min -1·(1.73 2) -1 in basiliximab group with a statistically significant difference( P<0.05). However, at Week 1/2/24 post-KT, no statistically significant difference existed in eGFR among 3 groups( P>0.05). In ATG, basiliximab and rATG groups, DGF(1 case, 1 case, 1 case), AR(2 cases, 2 cases, 1 case)and infection(4 cases, 7 cases, 3 cases)occurred during 6 months post-KT. Conclusions:Through a limited sample of single centers, no statistically significant difference existed in graft function recovery for ABOi-KT recipients on induction therapies of pATG, basiliximab and rATG.And DGF, AR and infections occurred in all three groups.However, there were little inter-group differences.

Abstract OBJECTIVE To analyze and summarize the research history, development status, trends and hotspots of Codonopsis Radix by bibliometrics. METHODS Search and screen the related literatures of Codonopsis Radix from the CNKI database and the Web of Science core collection database from Januar 1, 1992 to June 30, 2022. VOSviewer and CiteSpace softwares were used for visual analysis of the number of publications, authors, institutions, countries, funding funds, published journals, literature citation frequency, keywords, clustering and emergent word, and a visual graph was drew. RESULTS A total of 594 effective literature, 484 in Chinese and 110 in English were included. GAO Jianping and ZOU Yuanfeng were the scholars with the largest amount of Chinese and English literature. China was the country with the most published researches on Codonopsis Radix. The School of Pharmaceutical Science of Shanxi Medical University and the Chinese Academy of Sciences were the institutions with the largest number of Chinese and English literature publications respectively. The National Science Foundation of China was the largest fund to support the research of Codonopsis Radix. The journal that received the most Chinese and English literature were Journal of Chinese Medicinal Materials and International Journal of Biological Macromolecules respectively. The most frequently cited Chinese literature was a summary of the chemical constituents and pharmacological effects of Codonopsis Radix. The most frequently cited English literature was a study on the structure identification and pharmacological effects of Codonopsis Radix polysaccharides. The map of key words, clustering and emergent words in Chinese literature showed that the main research directions of Codonopsis Radix were compatibility formula, resource planting, pharmacological action and quality control. The map of key words, clustering and emergent words in English literature indicated that the study of Codonopsis Radix always focused on the material basis and mechanism of its active ingredients. CONCLUSION The annual publications on of Codonopsis Radix show an increasing trend, and the main research institutions are distributed in many universities and research institutes in China. Both Chinese and English literature have their own research directions, and the pharmacological effects of active ingredients are the common trend hotspots.

Bioluminescence is a widespread phenomenon in nature, and luminescent organisms can be found both on land and in the ocean. Among them, luciferase based bioluminescence systems have been widely studied, inspiring the exploration of genetic and epigenetic aspects and the development of a series of related assays for in vivo and in vitro studies. This paper summarizes the recent developments of luciferase based bioluminescence assays in terms of bioluminescence systems, types of luciferases, and the development and application of luciferase bioluminescence assays.

Objective:To analyze the short and mid-term efficacy of aortic valvuloplasty with autopericardium on children with aortic valve diseases.Methods:A total of 26 children with aortic valve diseases (stenosis or regurgitation) who underwent aortic valvuloplasty with autopericardium in Fuwai Central China Cardiovascular Hospital from September 2017 to June 2021 were retrospectively analyzed.The short-term and mid-term follow-up data were collected.The maximum aortic valve pressure gradient, subaortic regurgitation area, left ventricular end-diastolic volume (LVEDV) and left ventricular ejection fraction (LVEF) were compared before and after operation.Paired t test was used to analyze the short-term and mid-term efficacy of aortic valvuloplasty with autopericardium on children with aortic valve diseases. Results:All 26 cases were successfully operated, and there were no deaths and serious complications during the follow-up period of (22.96±6.45) months.There was a significant difference between the preoperative and postoperative maximum aortic valve pressure gradient at 1 month ( t=7.85, P<0.05), 6 months ( t=6.43, P<0.05), 1 year ( t=6.16, P<0.05) and 2 years postoperatively ( t=4.22, P<0.05) in children with aortic stenosis or that combined with mild-to-moderate closure.The follow-up data of 9 children with simple aortic stenosis showed that there was a significant difference between the preoperative (8.87±3.57) cm 2 and postoperative aortic regurgitation area at 1 month ( t=6.85, P<0.05), 6 months ( t=5.13, P<0.05), 1 year ( t=6.62, P<0.05) and 2 years postoperatively ( t=5.41, P<0.05). The LVEDV of 26 children was significantly lower at 6 months[(63.54±27.61) mL], 1 year [(53.61±20.20) mL] and 2 years postoperatively [(64.39±17.78) mL] compared with that of preoperative level[(89.42±45.89) mL]( t=3.89, 4.67, 3.58, all P<0.05). The left ventricular pressure and volume decreased, the enlarged heart was narrowed down, and the geometry of the heart was restored.The LVEF of 26 patients also from (61.65±9.67)% before surgery increased to (67.88±4.69)% 6 months after surgery( t=3.68, P<0.05), and increased to (68.62±4.46)% 1 year after surgery( t=4.01, P<0.05), and increased to (67.55±3.09)% 2 years after operation( t=3.01, P<0.05), and the heart function was improved. Conclusions:Aortic valvuloplasty with autopericardium presents an effective short and mid-term efficacy on children with aortic valve diseases, which prevents or delays the aortic valve replacement.

Objective To evaluate the efficacy and safety of tacrolimus extended-release (Tac-ER) in the early stage after kidney transplantation. Methods Clinical data of 68 recipients undergoing kidney transplantation from 34 pairs of renal allografts were retrospectively analyzed. Two recipients who received bilateral kidneys from the same donor were treated with Tac-ER (Tac-ER group) and tacrolimus immediate-release (Tac-IR) (Tac-IR group) as one of the basic immunosuppressant. The changes of tacrolimus dosage and blood concentration, intra-patient variability (IPV), renal function, incidence of acute rejection, recipient and allograft survival rates and adverse events were statistically compared between two groups. Results The average daily dose of tacrolimus in the Tac-ER group was significantly higher than that in the Tac-IR group (F=8.386, P=0.005). In the Tac-ER group, the mean trough concentration at postoperative 4 d was (6.14±4.04) ng/mL, did not reach the target concentration, significantly lower than (9.41±5.47) ng/mL in the Tac-IR group (F=7.854, P=0.007). In the Tac-ER group, the IPV of trough concentration of tacrolimus within postoperative 1 month was significantly higher than that in the Tac-IR group (0.44±0.15 vs. 0.36±0.12, P=0.032). At postoperative 6 months, there was no significant difference in the renal function between two groups [serum creatinine level was (126±26) μmol/L vs. (120±28) μmol/L, and the estimated glomerular filtration rate was (56±13) mL/(min·1.73 m²) vs. (60±15) mL/(min·1.73 m²), both P > 0.05]. The allograft and recipient survival rates were 100% in both groups. The incidence of acute rejection within postoperative 1 month was 18% in the Tac-ER group and 3% in the Tac-IR group, with no significant difference (P > 0.05). The overall incidence of adverse events was 94% in the Tac-ER group and 97% in the Tac-IR group, with no significant difference (P > 0.05). Conclusions The efficacy and safety of Tac-ER are equivalent to those of Tac-IR, whereas a higher dose of Tac-ER should be orally given to reach the blood concentration similar to that of Tac-IR. During early-stage drug treatment, Tac-ER should be orally given before kidney transplantation or inittally with loading dose, aiming to increase the systemic exposure to tacrolimus early after kidney transplantation and prevent acute rejection caused by insufficient exposure.