ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

Objective: To analyze the effects of blue light on the formation of kidney stones. Methods: A total of 40 rats were randomly divided into 4 groups：A，B，C，and D，with 10 rats in each group.Rats in groups C and D were administered with a mixture of 10 g/L ethylene glycol，20 g/L ammonium chloride，and 100 g/L calcium gluconate via gavage (2 mL per mouse)，while rats in groups A and B received an equal volume of physiological saline via gavage.From the second day after gavage，rats in groups A and C were subjected to twice-daily blue light irradiation (one hour per session) as an intervention，while rats in groups B and D were subjected to fluorescent lamp irradiation using the same method.After 4 weeks of intervention，the 24-hour urine samples were collected，and the rats were then euthanized for the collection of blood and kidney tissue samples.Serum levels of antidiuretic hormone (ADH)，urinary Ca ，and urinary oxalate (Oxa) were measured.Levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in kidney tissues were detected using ELISA.Von Kossa staining was performed to observe pathological changes in kidney tissues and the presence of calcium salt crystals in the kidneys. Results: Compared with groups A and B，groups C and D showed higher accumulation of calcium salt crystals in renal tissues，as well as elevated levels of ADH，urinary Ca ，urinary Oxa，and MDA in renal tissues, additionally，the SOD level in renal tissues was lower (P<0.05).Compared with group D，group C exhibited higher accumulation of calcium salt crystals in renal tissues，along with increased levels of ADH，urinary Ca ，urinary Oxa，and MDA in renal tissues；conversely，the SOD level in renal tissues was lower (P<0.05). Conclusion: Blue light may increase the formation of kidney stones in rats by promoting the secretion of ADH in serum and oxidative stress in kidney tissues through the brain-kidney axis.

ObjectiveThis study explores the methods of lung tissue extraction and fixation required for pathological studies of fetal rats, based on the unique physiological structure of fetal rat lung tissue and existing lung tissue fixation techniques for adult rats. MethodsSix pregnant adult SD rats at 20.5 days of gestation were subjected to cesarean section to obtain fetal rats. Four healthy fetal rats with similar body weight, vital signs, and respiratory status were selected from each pregnant rat, and they were randomly divided into the following groups using a random number table: direct lung infiltration group, lung infiltration group after intratracheal infusion, whole-body infiltration group of fetal rats, and whole-body infiltration group after intratracheal infusion of fetal rats. To systematically compare and analyze the anatomical morphology under different fixation methods, lung tissues from four groups of fetal rats were harvested, perfused, and fixed, and the gross morphology of lung tissues in each group was observed. Paraffin sections were prepared and stained with Hematoxylin-Eosin (H&E). The histological morphology of the whole lung, alveoli, and bronchi was further examined under optical microscopy. ResultsIn the direct lung infiltration group, the hilar structures were unclear, lung lobation was indistinct, the shape was irregular, lung cavities were small, and alveoli and bronchi were shrunken. In the lung infiltration group after intratracheal infusion, the hilar structures were clear, lobation was pronounced, the shape was regular, lung cavities were large, and alveoli and bronchi were full. Both the whole-body infiltration group and whole-body infiltration group after intratracheal infusion of fetal rats exhibited visible lungs, hearts, skins, and other organs. The lung tissues of both groups showed obvious lobulation, irregular shape, and damage at the margins of lung lobes. In the whole-body infiltration group, the thoracic cavities of the fetus were flattened, lung cavities were small, and alveoli and bronchi were shrunken. In the whole-body infiltration group after intratracheal infusion of fetal rats, the fetal thoracic cavities were full, lung cavities were large, and alveoli and bronchi were relatively full. ConclusionThe lung infiltration after intratracheal infusion method for fetal rat lung tissue fixation outperforms direct lung infiltration, whole-body infiltration of fetal rats, and whole-body infiltration after intratracheal infusion of fetal rats in terms of preservation of the lung tissue's original morphology, paraffin sectioning, staining, and pathological observation and analysis. The embedding, sectioning, and staining processes are also simple and save consumables. Therefore, intratracheal infusion followed by lung infiltration method is recommended for fixation in histopathological observation of fetal rat lung tissue.

In recent years,organs-on-chips(OOCs)technology has gradually matured and been recognized by regulatory agencies,playing a key role in the construction of pathophysiological models,pharmacodynamic screening,toxicological evaluation and other fields,providing guidance for the research and development of innovative drugs of Chinese materia medica and the clinical application of classic prescriptions.This article reviewed the application progress of OOCs technology in the research of Chinese materia medica from four dimensions:pharmacodynamic and toxicological evaluation,quality control,active component analysis and innovative drug research and development,in order to provide a reference for the precise application of this technology in the field of Chinese materia medica.

The purpose of this study is to develop a shark single domain antibody(SdAb)targeting a unique B cell epitope in the SARS-CoV-2 spike protein,and explore its role in the immunological detection targeting SARS-CoV-2 spike protein S.A u-nique peptide S9 was artificially synthesized based on the sequence of a unique B cell epitope of SARS-CoV-2 spike protein,then it was conjugated to the carrier protein KLH.It was used as an immunogen for subcutaneous injection into shark back and boosted according to the standard immunization protocol.Blood collected from shark tail vein and peripheral blood lymphocytes(PBL)were isolated.Total RNA was purified from PBL and transcribed to cDNA by reverse transcription.Shark vNAR frag-ments were amplified from cDNA templates and cloned into pComb3XSS vector to obtain phage library.A positive clone named T01 was obtained through screening the phage library by indirect ELISA.Then its gene was cloned into the expression vector pET-28a.The SdAb T01 was then prokaryotically expressed and purified,and its specific recognition of the SARS-CoV-2 spike protein S was indentified by Western-blot(WB),indirect ELISA and IF A.T01 binds well with peptide S9 at EC50 value of 2.050±0.064 nmol/L.The purified SdAb T01 was proven by WB to be able to selectively detect recombinant spike protein sub-unit 1(S1)of SARS-CoV-2,with no cross-reactive to recombinant spike protein subunit 1 of other six human coronavirus.It was showed by ELISA that SdAb T01 can sensitively detect the recombinant N terminal domain(NTD)of SARS-CoV-2 pro-tein.Moreover,it also specifically recognizes the spike protein of SARS-CoV-2 that was transiently expressed in transfected HEK293 cells by IFA.Therefore,a shark single domain antibody targeting a unique B cell epitope in the spike protein of SARS-CoV-2 was successfully developed,and has shown potential immunodiagnostic value by WB,ELISA and IFA.Thus,it provides an effective tool for unique antigen detection of SARS-CoV-2.

Congenital generalized lipodystrophic diabetes(CGLD) is a rare form of diabetes caused by the genetic defects affecting insulin action. This article describes two cases of CGLD within a family. The patients, siblings, exhibited systemic fat atrophy, severe hypertriglyceridemia, insulin resistance and hepatomegaly. Genetic analysis revealed a shared homozygous nonsense mutations in the BSCL2 gene c. 757G>T: p.E253*. Case 1(elder brother) presented with more severe hypertriglyceridemia, pronounced microvascular complications of diabetes, and abnormalities in bone metabolism. In contrast, Case 2(younger brother) demonstrated lower body fat content and reduced limb muscle strength. Despite sharing the same pathogenic mutation, the siblings displayed notable differences in clinical presentation, underscoring the phenotypic heterogeneity of CGLD. These findings highlight the importance of recognizing variable clinical manifestations in CGLD. Endocrinologists should consider this heterogeneity to ensure accurate diagnosis and tailored treatment strategies for affected individuals.

Objective:To understand the management of children with pediatric acute liver failure (PALF) in pediatric intensive care unit (PICU).Methods:A retrospective case-control study was conducted. A total of 101 children with PALF hospitalized in PICU of Beijing Children′s Hospital from July 2017 to October 2022 were included. Demographic, clinical management and prognosis data were collected. According to whether PALF was the main diagnosis, the patients were divided into primary diagnosis group and complication group. The primary diagnosis group was subdivided into effective group and ineffective group with routine treatment (except liver transplantation). The intergroup comparisons were performed using independent samples t-test, Mann-Whitney U test, χ2 test or Fisher exact test. Multivariate Logistic regression analysis was employed to identify risk factors associated with prognosis. Results:Among the 101 children with PALF, 58 were male and 43 were female, with an age of 30 (10, 103) months, 60 cases in primary diagnosis group and 41 cases in complication group. There were no significant differences in prothrombin time (PT) and international normalized ratio (INR) between the two groups (both P>0.05), while the total bilirubin, direct bilirubin and blood ammonia were all significantly higher in the primary diagnosis group (all P<0.05). Unoriginal liver failure (25 cases (42%)) and poisoning (13 cases (22%)) were the most common causes of PALF in the primary diagnosis group, while shock (17 cases, 43%) and hemophagocytic syndrome (14 cases (34%)) in the complication group. The mortality rate of the main diagnosis group was significantly lower than that of the complication group (25% (15/60) vs. 61% (25/41), χ2=13.18, P<0.001), as well as the incidence of combined organ function injury, while the amount of plasma used and the ratio of plasma exchange times to PICU hospitalization days were significantly higher (all P<0.05). In the primary diagnosis group, there were 32 cases (53%) in the effective group and 28 cases (47%) in the ineffective group. In the ineffective group, 15 cases (54%) died and 13 cases (46%) were transferred to another site for liver transplantation assessment. The hospitalization time of PICU in the effective group was significantly longer than that in the ineffective group, while the ratio of plasma exchange times to PICU hospitalization days, the average daily hours of continuous renal replacement therapy (CRRT), the rate of CRRT and the average daily plasma dosage in the effective group were all significantly lower than those in the ineffective group (all P<0.05). The worst PT, INR and blood ammonia, and the stage 4 hepatic encephalopathy morbidity and significant bleeding rate in the effective group were all significantly lower than those in the ineffective group (all P<0.05). Multivariate Logistic regression analysis showed that after adjusting for age, sex, total bilirubin, INR and blood ammonia, stage 4 hepatic encephalopathy was the independent risk factor for the failure of routine treatment of PALF ( OR=84.16,95% CI 4.04-1752.37, P=0.004). Conclusions:PT and INR could not specifically represent liver synthetic function in some PICU patients, so current PALF diagnostic criteria for PICU children has limitations. Complicated with stage 4 hepatic encephalopathy was an independent risk factor of the failure of conventional treatment in patients with PALF.

Objective:To explore the impact of different blood transfusion thresholds on clinical outcomes in children with severe traumatic brain injury (TBI).Methods:A retrospective cohort study was conducted. Clinical data was collected from 64 children with severe TBI who received red blood cell transfusions and were admitted to the Pediatric Intensive Care Unit (PICU) of Beijing Children′s Hospital between January 2020 and December 2024. Data included basic clinical characteristics, mortality rate, neurological recovery (measured by Glasgow coma scale (GCS) at discharge, pediatric cerebral performance category (PCPC) score), length of stay in the PICU, duration of mechanical ventilation, and incidence of complications. Patients were divided into a liberal transfusion group (hemoglobin >70-<100 g/L at first transfusion) and a restrictive transfusion group (hemoglobin ≤70 g/L at first transfusion). Stratified analysis was performed based on age (children >5 and children ≤5 years old). Comparisons between groups were conducted using the independent samples t test, Mann-Whitney U test, χ2 test or Fisher′s exact test. Results:Among the 64 children with severe TBI (43 males and 21 females), the age was 4.9 (2.3, 10.0) years. There were 33 cases in the liberal transfusion group and 31 cases in the restrictive transfusion group. No statistically significant differences were observed in baseline data, including gender, age, trauma mechanism, GCS at admission, surgical intervention, presence of multiple injuries, or comorbidities (sepsis, shock, ventilator-associated pneumonia and acute kidney injury) between the 2 groups (all P>0.05). There were no statistically significant differences between the liberal and restrictive transfusion groups in mortality rate, GCS and PCPC score at discharge, length of PICU stay, duration of mechanical ventilation, or transfusion volume (all P>0.05). In the stratified analysis, 38 children aged over 5 years were included. The restrictive transfusion subgroup, which included 19 children, had a significantly longer PICU length of stay compared to the liberal transfusion subgroup, which also included 19 children (29.5 (18.0, 36.3) vs. 17.0 (6.3, 25.8) d, Z=2.11, P=0.035). Conclusions:There were no significant differences in PICU mortality or neurological functional recovery between the liberal and restrictive blood transfusion strategies in children with severe TBI. However, among children aged over than 5 years, a restrictive transfusion strategy might be associated with a longer length of PICU stay.

OBJECTIVE: To determine risk factors for cutout failure in geriatric intertrochanteric fracture patients after cephalomedullary nail fixation. METHODS: A retrospective review of 518 elderly patients who underwent cephalomedullary nail fixation for intertrochanteric fractures between January 2008 and August 2018 was conducted, including 167 males and 351 females, age from 65 to 97 years old. All patients were followed up for at least one year after surgery and divided into a healed group and a cutout group based on whether the hip screw cutout occurred. Among all patients, 10 cases experienced hip screw cutout. The general information, surgical data, and radiological data of the two groups were compared, and risk factors influencing hip screw cutout were analyzed. Propensity score matching was then performed on the cutout group based on gender, age, body mass index(BMI), and American Society of Anesthesiologists(ASA), and 40 patients from the healed group were matched at a ratio of 1∶4. Key risk factors affecting hip screw cutout were further analyzed. Multivariable logistic regression analysis was conducted to evaluate associations between variables and cutout failure. RESULTS: There were no statistically significant differences between the healed group and the cutout group in terms of age, gender, BMI, ASA, and AO classification. However, statistically significant differences were observed between the two groups in terms of reduction quality(P=0.003) and tip-apex distance(TAD), P<0.001. Multivariate analysis identified poor reduction quality OR=23.138, 95%CI(2.163, 247.551), P=0.009 and TAD≥25 mm OR=30.538, 95%CI(2.935, 317.770), P=0.004 as independent risk factors for cutout failure. CONCLUSION The present study identified poor reduction quality and TAD≥25 mm as factors for cutout failure in geriatric intertrochanteric fractures treated with cephalomedullary nails. Further studies are needed to calculate the optimal TAD for cephalomedullary nails.
Humans ; Male ; Female ; Hip Fractures/surgery* ; Aged, 80 and over ; Aged ; Risk Factors ; Retrospective Studies ; Fracture Fixation, Intramedullary/adverse effects* ; Bone Nails ; Bone Screws