To design and construct chimeric antigen receptor (CAR) T cells targeting Trop2, establish an in vitro cell exhaustion model through continuous antigen stimulation, and investigate their anti-tumor activity and exhaustion characteristics.

The second-generation CAR plasmid was constructed based on the single-chain variable fragment (scFv) sequence of Sacituzumab Govitecan targeting Trop2. The viral vector titer was determined by retroviral vector packaging and gradient dilution. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using Ficoll density gradient centrifugation, and CAR virus vectors were transduced into PBMCs activated with OKT-3/IL-2 to generate Trop2-targeted CAR T cells. CAR expression levels were assessed by flow cytometry using MYC tags. In vitro 3 tumor cell models were established, including human ovarian cancer cells (SKOV3), human breast cancer cells (MDA-MB-453), and human lung cancer cells (A549). The expression of the Trop2 antigen in these models was confirmed using flow cytometry. Additionally, luciferase assay was employed to evaluate the cytotoxic efficiency of Trop2-targeted therapy at various effector-to-target ratios. An in vitro CAR-T exhaustion model was developed, and the long-term killing ability of CAR-T cells was dynamically monitored using the Incucyte live-cell imaging system. The PD-1/TIM-3 phenotype of CAR-T cells was analyzed by flow cytometry, and cytokine secretion levels were quantified using the cytometric bead array (CBA). Transcriptomic sequencing and RT-qPCR were employed to validate the differentially expressed genes associated with exhaustion.

The second-generation CAR T cells targeting Trop2 were successfully constructed. Compared to the P-T group, in vitro experiments demonstrated that these CAR T cells exhibited antigen-specific and dose-dependent cytotoxic effects against tumor cells with high Trop2 expression, such as MDA-MB-453 and SKOV3. A CAR-T cell exhaustion model established through repeated tumor antigen stimulation in vitro revealed that, compared to the initial state, the exhausted Trop2 CAR-T cells exhibited significantly reduced tumor-killing capacity while P-T cells showed almost no killing effect, the expression of inhibitory receptors (PD-1 and TIM-3) was up-regulated on the surface of exhausted CAR-T cells, and the secretion of effector cytokines was diminished. Transcriptomic analysis identified multiple differentially expressed genes in the exhausted CAR-T cells. Pathways related to immune response and T cell receptor signaling were down-regulated, while apoptosis-related pathways were activated. RT-qPCR further confirmed abnormal expression of immunoregulatory genes, including IL3, IL5, and IL13 (P<0.05).

During continuous in vitro tumor antigen stimulation, the second-generation CAR-T cells targeting the Trop2 antigen demonstrate declined anti-tumor activity, weakened effector function and up-regulated expression of exhaustion-related molecules.

To develop novel metformin carbon dots (MCDs) with superior performance to enhance the osteogenic differentiation potential of human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions.

Three types of MCDs-MCDsCA, MCDsAA, and MCDsCS were synthesized via a one-step hydrothermal method using metformin (Met) in combination with citric acid (CA), ascorbic acid (AA), and carboxymethyl chitosan (CS), respectively. Their physicochemical properties were characterized by transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), and UV-visible spectrophotometry. The biocompatibility and cellular uptake of the MCDs were assessed using CCK-8 assay and confocal laser scanning microscopy. Alkaline phosphatase (ALP) staining and ALP activity assay were conducted to identify the most effective MCDs type in promoting osteogenic differentiation of hPDLSCs under inflammatory conditions. Subsequently, ALP and Alizarin Red S (ARS) staining, qRT-PCR, and Western blotting were performed to assess the effects of the optimal concentration of MCDsCS and its raw material (Met+CS) on osteogenic differentiation under inflammatory conditions.

All 3 types of nanoscale MCDs exhibited favorable physicochemical characteristics and biocompatibility, and could be effectively internalized by hPDLSCs. Both MCDsCA and MCDsAA significantly promoted hPDLSCs proliferation at lower concentrations (0~0.50 mg/mL), while MCDsCS exhibited excellent cytocompatibility over a broader range (0~2.00 mg/mL). Under inflammatory conditions, MCDsCA and MCDsAA showed no significant effect on ALP expression in hPDLSCs, whereas MCDsCS significantly enhanced ALP expression in a dose-dependent manner, with the optimal effect observed at 0.10 mg/mL (P<0.05). Compared to Met+CS, 0.10 mg/mL MCDsCS significantly enhanced ALP expression and calcium nodule formation under inflammatory conditions. It also upregulated the expression of osteogenesis-related genes (ALP, RUNX2, COL-1, OCN) and proteins (ALP, RUNX2, OCN) (P<0.01), while reducing the levels of inflammatory cytokines IL-1β and IL-6 (P<0.01).

A novel MCDsCS with sound biocompatibility is successfully developed, and can effectively promote osteogenic differentiation of hPDLSCs and exerts anti-inflammatory effects under inflammatory conditions, indicating its potential as a nanotherapeutic agent for periodontitis-associated bone regeneration.

To investigate whether remimazolam attenuates intestinal ischemia-reperfusion (I/R) injury in mice by regulating ferroptosis through connexin-43 (CX43).

Molecular docking was applied to predict the binding affinity of remimazolam to CX43. A total of 72 SPF-grade adult male C57BL/6J mice (6~8 weeks old, weighing 20~25 g) were subjected. Thirty of them were randomly divided into sham operation group (Sham group), I/R group 1, and I/R+10, 20 and 40 mg/kg remimazolam groups (RM10, RM20 and RM40 groups), with 6 mice in each group. Another 30 mice were randomly assigned into 5 groups (n=6), I/R group 2, erastin group (E group), I/R+40 mg/kg remimazolam group 2 (RM40 group 2), I/R+Fer-1 group (Fer-1 group), and erastin +40 mg/kg remimazolam group (ERM group). The left 12 mice were randomly and equally grouped into I/R+RM+oe-NC group (oe-NC group) and I/R+RM+oe-CX43 group (oe-CX43 group). The Fer-1 group was given an intraperitoneal injection of 5 mg/kg Fer-1 in 1 h prior to reperfusion, the E group was given 10 mg/kg erastin intraperitoneally 1 d before modeling, and all the remimazolam groups, the oe-NC group and the oe-CX43 group were injected intravenously with corresponding doses of remimazolam 30 min pre-modeling, while the oe-NC and oe-CX43 groups were injected with empty vector virus and overexpression of CX43 vector virus, respectively, 48 h before the administration of remimazolam. A mouse intestinal I/R injury model was constructed by clamping the superior mesenteric artery for 45 min and reperfusion for 30 min. The small intestine tissues were harvested and observed for pathological changes, and the intestinal mucosal damage was assessed with Chiu’s score. The contents of Fe²⁺, total iron, malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were detected by colorimetric assay; the production of reactive oxygen species (ROS) was determined by DHE probe; the expression of ferroptosis-related genes was determined by RT-qPCR; and the expression levels of CX43, GPX4, and SLC7A11 were detected by Western blotting.

Molecular docking indicated that remimazolam had a binding energy of -6.699 kcal/mol with CX43 protein, suggesting good binding affinity between them. Compared with the Sham group, the I/R group 1 showed increases in Chiu’s scores and CX43 expression (P<0.05), along with pathological damage to intestinal tissues, and elevated contents of Fe²⁺, total iron, ROS and MDA (P<0.05), and down-regulated GPX4 and SLC7A11 (P<0.05). Compared with the I/R group 1, Chiu’s score was reduced in the RM40 group, and CX43 was significantly down-regulated (P<0.05), contents of Fe²⁺, total iron, ROS, and MDA were decreased (P<0.05), and expression levels of GPX4 and SLC7A11 were enhanced (P<0.05), and severity of intestinal histological damage was attenuated in both the RM40 and Fer-1 groups. Compared with the E group, the ERM group had the decreases in CX43 expression level (P<0.05), Fe²⁺, total iron, ROS, and MDA contents (P<0.05), and increases in GPX4 and SLC7A11 expression levels (P<0.05), with the improvement in intestinal tissue. Compared with the oe-NC group, overexpression of CX43 resulted in the increased CX43 expression, elevated contents of Fe²⁺, total iron, ROS and MDA (P<0.05) and decreased expression of GPX4 and SLC7A11 (P<0.05), leading to the exacerbated injury in intestinal tissue.

Remimazolam attenuates intestinal I/R injury by inhibiting ferroptosis through down-regulating CX43 expression.

To prepare humanized monoclonal antibodies (Mabs) targeting Acinetobacter baumannii (Ab) based on single memory B cell sequencing technology, construct the immune repertoire of the core protein of Ab, hemolysin-coregulated protein (Hcp), and express its Mabs with binding activity.

E. coli BL21 harboring the recombinant plasmid pGEX-6p-1-Hcp was constructed. Hcp protein was obtained using protein expression and affinity chromatography. Female SPF BALB/c mice (6~8 weeks old, weighing 18~20 g) were immunized intramuscularly with antigen Hcp to generate specific memory B cells. Single antigen-specific memory B cells were sorted using flow cytometry. The immune repertoire of Hcp was constructed using single-cell sequencing technology, and bioinformatics analysis was performed on the sequencing results. Mabs were obtained using antibody humanization techniques. The in vitro binding activity of the antibodies was detected by ELISA.

The target protein Hcp with a purity >95% was obtained after expression and purification. The immune repertoire of Hcp was successfully constructed, and the results of BCR clonotype identification and analysis, CDR3 region characteristic analysis, and V-J gene pairing characteristic analysis were achieved. Antibody humanization got 7 Mabs, that is, IgG1-1, IgG1-2, IgG2-1, IgG2-2, IgG3-1, IgG4-1 and IgG4-2. ELISA results showed IgG1-1, IgG3-1, IgG4-1, and IgG4-2 had an antibody binding titer of 1∶1 280, IgG2-2 of 1∶10 240, IgG2-1 of 1∶5 120, and IgG1-2 of 1∶160.

Single-cell sequencing technology enables rapid, accurate, and efficient construction of an Hcp protein immune repertoire containing extensive antibody information. Utilizing this immune repertoire allows for the expression of Mabs with binding activity.

To evaluate the predictive value of triglyceride-glucose body mass index (TyG-BMI), atherogenic index of plasma (AIP), and postprandial glucose excursion (PPGE) for sarcopenia in patients with type 2 diabetes mellitus (T2DM).

An observational cohort trial was conducted on 590 hospitalized T2DM patients (322 males and 268 females) admitted to Department of Endocrinology of the First Affiliated Hospital of Chongqing Medical University between January 2023 and December 2024. General demographic data and key metabolic indicators, including fasting plasma glucose (FPG), postprandial 2-hour plasma glucose (PPG), triglyceride (TG) were collected, and then TyG-BMI, AIP, and PPGE were calculated. Based on the Asian Working Group for Sarcopenia (AWGS) 2019 criteria, they were divided into a sarcopenia group (n=140) and a non-sarcopenia group (n=450). After 1∶1 propensity score matching (PSM), 102 patients with sarcopenia were matched with 102 without. Differences in metabolic indicators FPG, PPG, TyG-BMI, AIP and PPGE were compared between the 2 groups. Spearman correlation analysis was conducted to assess correlations of these indicators with sarcopenia. Conditional logistic regression analysis was performed to identify independent risk factors, and receiver operating characteristic (ROC) curves were plotted to assess predictive performance. An external validation cohort consisting of 192 T2DM patients from Department of Endocrinology of Jiangbei Branch of First Affiliated Hospital of Army Medical University between January 2023 and December 2024 was included to validate the prediction model.

After PSM, the baseline data of the 2 groups was balanced (P>0.05). The sarcopenia group showed higher levels of PPG, AIP, and PPGE, but lower TyG-BMI value than the non-sarcopenia group (all P=0.001). Spearman correlation analysis revealed that the TyG-BMI was negatively correlated (r=-0.404, P=0.001), whereas AIP and PPGE were positively correlated with concomitant sarcopenia (r=0.280,P=0.001;r=0.372, P=0.001) in the T2DM patients. Conditional logistic regression analysis identified AIP (OR=14.367) and PPGE (OR=1.245) as independent risk factors, while TyG-BMI (OR=0.966) as independent protective factors. ROC curve analysis revealed that the area under the curve (AUC) of the combined model of TyG-BMI, AIP and PPGE in predicting sarcopenia was 0.918, and the AUC value was 0.954 in external validation, with good sensitivity and specificity.

TyG-BMI, AIP, and PPGE are important metabolic predictors in T2DM patients with sarcopenia. Their combination has good screening value for sarcopenia, and can be applied in external prediction for T2DM patients.

To compare the effects of main artery clamping (MAC) and selective artery clamping (SAC) strategies on postoperative renal function in patients with chronic renal insufficiency undergoing robot-assisted partial nephrectomy.

A retrospective cohort study was conducted on 231 patients with preoperative chronic renal insufficiency [eGFR <90 mL/(min·1.73 m²) with renal injury markers or eGFR <60 mL/(min·1.73 m²)] who underwent robot-assisted partial nephrectomy in the Department of Urology of the First Affiliated Hospital of Army Medical University from February 2018 to February 2024. According to intraoperative renal artery clamping strategy, they were divided into a MAC group (n=129) and a SAC group (n=102). Preoperatively, individualized renal artery clamping strategies were developed using a machine learning-based multimodal holographic 3-D reconstruction technique. Serum creatinine (Scr) level was measured at 3 d and 3 months after surgery, and estimated glomerular filtration rate (eGFR) was calculated using the chronic kidney disease epidemiology collaboration equation (CKD-EPI) formula. Renal dynamic imaging with 99mTc-DTPA or 99mTc-MAG3 was used to assess the GFR of the affected kidney.

At 3 d after surgery, the decrease in GFR of the affected kidney was significantly lower [(8.3±7.7) vs (16.0±10.2) mL/(min·1.73 m²), 95%CI: -10.2~-5.2, P<0.001] in the SAC group than the MAC group. Scr increment analysis showed that the SAC group exhibited notably lower Scr increase [8.2 (2.5, 18.7) vs 15.5 (5.8, 28.3) μmol/L, 95%CI: -12.3~-1.8, P=0.027], and milder eGFR decline [3.0 (0.5, 7.8) vs 7.5 (2.0, 14.3) mL/(min·1.73 m²), 95%CI: -6.2~-0.8, P=0.015]. And, in 3 months after surgery, the SAC group had lower Scr level [(89.2±23.1) vs (95.3±22.1) μmol/L, 95%CI: -11.9~-0.3, P=0.042], and higher GFR of the affected kidney [(33.5±10.5) vs (26.1±10.9) mL/(min·1.73 m²), 95%CI: 4.6~10.2, P<0.001].

For patients with chronic renal insufficiency undergoing robot-assisted partial nephrectomy, SAC strategy is superior to MAC strategy in protecting postoperative renal function without increasing surgical risk.

To investigate the protective effects of remimazolam (Remi), a novel benzodiazepine sedative, on intestinal barrier function in septic mice.

A mouse model of sepsis was established using cecal ligation and puncture (CLP). A total of 96 SPF-grade adult male C57BL/6 mice were randomized into sham operation (Sham), sepsis (Sepsis), and sepsis with Remi intervention (Sepsis+Remi) groups. Survival rate and survival time were recorded within 72 h after modeling. Intestinal pathological alterations, barrier functional indicators, ZO-1 expression, and macrophage polarization status were observed and detected to evaluate the effects of Remi. Lipopolysaccharide (LPS) was used to treat RAW264.7 cells for 24 h to simulate in vitro sepsis model. The cells were divided into control (Control), LPS, and LPS+Remi groups. Immunofluorescence staining was performed to assess macrophage phenotype, mitochondrial morphology, and mitochondrial reactive oxygen species (MtROS), and Western blotting was applied to detect the protein expression of iNOS and CD206.

Compared with the sepsis group, Remi intervention significantly improved the survival rate of septic mice from 12.50% to 68.75% and markedly prolonged survival duration (P<0.05). Histopathological analysis demonstrated partial restoration of intestinal villus architecture, accompanied with attenuated interstitial edema and reduced inflammatory cell infiltration after Remi intervention. Furthermore, the intervention group demonstrated significant improvement in functional indicators. Both in vivo and in vitro experiments demonstrated elevated iNOS and decreased CD206 expression in the septic mice and LPS-stimulated macrophages（P<0.05）, which were partially reversed after Remi intervention. Furthermore, LPS-stimulated macrophages exhibited fragmented mitochondria and elevated MtROS level, whereas Remi intervention ameliorated these conditions（P<0.05）.

Remi protects intestinal barrier function in septic mice by mitigating mitochondrial dynamics imbalance-induced oxidative damage and ameliorating inflammatory macrophage activation.

To investigate the effects of long-term radiofrequency (RF) radiation at 2.65 GHz on behavior, inflammatory response, and intestinal microecology in mice in order to provide a basis for the safety assessment of long-term RF exposure.

One hundred and eight male C57BL/6N mice (17~21 g, 6~8 weeks old) were randomly assigned to a control group (Con) and a RF exposure group. The mice of the RF exposure group were subjected to whole-body uniform exposure to 2.65 GHz RF radiation in an electromagnetic reverberation chamber for 3 h/day for 28 consecutive days. RF field distribution and changes in core body temperature were monitored using an electromagnetic radiation analyzer and a fiber-optic temperature probe, respectively. Cognitive function was assessed using the Y-maze and novel object recognition (NOR) test. Anxiety-like behaviors were evaluated through open field test (OFT) and elevated plus maze (EPM), while depressive-like behaviors were examined with sucrose preference test (SPT) and tail suspension test (TST). HE staining was used to observe the histopathological changes in mouse tissues. Radioimmunoassay (RIA) was employed to detect the expression of pro-inflammatory cytokines, TNF-α and IL-1β, as well as anti-inflammatory cytokines, IL-4 and IL-10 in the serum, brain, jejunum, and spleen samples. Additionally, metagenomic sequencing was performed to assess alterations in the gut microbiota composition.

Long-term RF radiation led to a maximal increase of 0.59 ℃ in the core body temperature, but had no significant effects on cognitive function, anxiety-like behaviors, or depressive-like behaviors, or apparent damage of the hippocampal or jejunal tissues in the exposed mice. However, RF exposure significantly up-regulated the expression of the pro-inflammatory cytokine TNF-α in serum (P<0.05), and did not significantly alter the concentrations of other cytokines (IL-1β，IL-4，IL-10), caused significant decrease in α-diversity of the intestinal microbiota (P<0.01), with reduced relative abundances of Ligilactobacillus murinus and Acetatifactor muris (P<0.05), while elevated abundances of Lachnospiraceae bacterium (P<0.01).

Long-term exposure to 2.65 GHz RF radiation induces systemic inflammatory responses and disrupts gut microbiota homeostasis in mice.

To construct an automatic segmentation model to segment female urine control anatomy on MRI images based on deep learning methods in order to improve the segmentation efficiency and accuracy.

A dataset comprising 49 female pelvic floor muscle MRI images [30 women with varying degrees of pelvic organ prolapse (POP) and 19 healthy individuals], obtained from Faculty of Biomedical Engineering and Medical Imaging in Army Medical University, was used for model training and testing. The dataset was split into a training set (17 normal cases and 22 POP cases) and a testing set (4 normal cases and 6 POP cases) in a ratio of 8∶2. The training set was used to train UNet, UNet+++, Dense UNet, and UNet++ models separately, and then input into each network. The model achieving the highest testing accuracy was selected as the backbone network.

Under the training of UNet, UNet+++, Dense UNet, and UNet++, the 4 models achieved average Dice similarity coefficients of 61.82%, 57.94%, 57.63%, and 62.76%, respectively, for the segmentation of 5 anatomical structures (compressor urethrae, urethra sphincter body, bladder wall, bladder cavity and urethra submucosa). The corresponding Intersection over Union (IoU) score was 49.74%, 46.59%, 46.07%, and 49.44%, while the accuracy rate was 61.74%, 55.03%, 59.23%, and 61.91%, respectively for the 4 models. Notably, UNet++ consistently outperformed UNet, UNet+++, and Dense UNet across the 3 metrics, indicating that UNet++ achieved the highest overall segmentation accuracy.

In UNet, UNet++, Dense UNet and UNet++ for automatic segmentation of 5 female urine control anatomical elements, UNet++ achieves the best overall segmentation accuracy.