Transfusion and Anemia Expertise Initiative-Control/Avoidance of Bleeding developed a strategy for platelet and plasma infusion management in critically ill children based on systematic reviews and consensus meetings of international multidisciplinary experts. One good practice statement and six expert consensus statements were proposed for plasma and platelet transfusions in critically ill children following severe trauma, traumatic brain injury, and/or intracranial hemorrhage. This article introduces the specific methods and basis for the formation of recommendations in this part of the guide.

Objective: To investigate the association between metabolic dysfunction associated steatotic liver disease (MASLD) and bone mineral density among children and adolescents, so as to provide evidence for the early prevention and intervention of bone health in this population. Methods: In September 2022, a method combining convenience sampling with cluster sampling was used to select 5 089 children and adolescents aged 6-18 years in 9 schools from kindergarten to senior high school in Tongzhou District, Beijing, for physical measurements, ultrasound measurements, blood biochemical index testing, and questionnaire surveys. Participants were categorized into three groups: the normal control group ( n =1 515), the metabolic abnormality group (MA, n = 3 007 ), and the MASLD group ( n =567). Multivariable linear regression model was applied to examine the association between MASLD and bone speed of sound (SOS), while multivariable Logistic regression model was used to assess the association between MASLD and low bone mineral density. Subgroup analysis was conducted by sex and age groups. Results: Compared with the normal control group, the MASLD group showed significantly lower SOS values ( β =-6.31, 95% CI =-9.63 to -2.99), lower SOS Z scores ( β = -0.21, 95% CI =-0.32 to -0.10), and higher susceptibility to low bone mineral density( OR =1.56, 95% CI =1.25-1.96)（all P <0.05）. No significant differences in SOS or odds of low bone density were observed between the MA and normal control groups (all P > 0.05). In sex stratified analyses, males with MASLD exhibited significantly lower SOS Z scores ( β =-0.35, 95% CI =-0.49 to -0.20 , P <0.05), whereas no significant difference was observed in females with MASLD ( β =-0.03, 95% CI =-0.21-0.15; P >0.05). When hepatic steatosis grade (0, 1, 2, and 3) was treated as a continuous variable, each one grade increase was associated with a 31% higher odds of low bone mineral density ( OR =1.31, 95% CI =1.13 to 1.53, P <0.05). Conclusions MASLD is significantly associated with low bone mineral density among children and adolescents, with a stronger association in males. Moreover, children and adolescents with hepatic steatosis have a higher risk of impaired bone health compared with those with metabolic abnormalities alone.

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.

[摘 要] 目的：探讨叉头框K1（FOXK1）在喉鳞状细胞癌（LSCC）中的表达、临床意义及对细胞增殖、迁移、侵袭的影响，并阐明其分子机制。方法：RT-qPCR、免疫组化检测LSCC组织及LSCC细胞TU177中FOXK1表达，分析其与临床病理参数及预后的相关性；通过瞬时转染构建FOXK1过表达与敲低细胞模型，通过MTS法、克隆形成实验、划痕愈合实验和Transwell实验检测细胞恶性生物学行为的变化；采用RT-qPCR检测上皮-间充质转化（EMT）相关因子mRNA的表达；采用WB法检测PI3K/AKT信号通路相关蛋白的表达；结合生物信息学预测与功能回复实验，验证FOXK1对AKT2的调控作用。结果：FOXK1在LSCC组织及细胞中高表达（P < 0.01），且主要定位于细胞核；FOXK1高表达与较晚TNM分期相关（P < 0.05），且FOXK1高表达患者5年生存率较低（P < 0.05）；过表达FOXK1显著增强TU177细胞增殖、迁移与侵袭能力并诱导EMT（P < 0.01或P < 0.05），敲低FOXK1则产生相反的效应；FOXK1可上调AKT2表达并激活PI3K/AKT信号通路；敲低AKT2可部分逆转FOXK1过表达对细胞增殖、迁移、侵袭及EMT进程的促进作用（P < 0.01或P < 0.05）。结论：FOXK1在LSCC中高表达，与肿瘤进展及不良预后密切相关。FOXK1可能通过上调AKT2激活PI3K/AKT信号通路，诱导EMT，从而促进LSCC细胞的恶性生物学行为。

Clinical trial registration， as a cornerstone for transparency in medical scientific research， aims to systematically and comprehensively disclose key information such as trial design and implementation protocols before trial initiation. This reduces selective reporting and outcome bias， avoids unnecessary duplicate research， thereby protects research participants’ legitimate rights and interests and ethical compliance， and ultimately upholds research integrity and public trust. However， despite the widespread international recognition of its importance and necessity， registration practices worldwide still exhibit significant disparities. From an ethical perspective， this paper deeply analyzed the current status of clinical trial registration and the challenges it confronted， including issues such as insufficient awareness of prospective registration， inconsistent registration standards， persistent difficulties in data openness， sharing， and result disclosure， as well as imbalanced global governance of registration. It further explored multi-dimensional paths involving institutional coordination， technological empowerment， global collaboration， and ethical guidance， to promote clinical trial registration to realize full-cycle closed-loop management from registration and implementation to result reporting. These will provide solid theoretical support and practical references for establishing a more ethically resilient， highly transparent， and sustainably operational clinical trial registration system.

Macrophages are innate immune cells connected with the development of inflammation. Retinoic acid has previously been proved to have anti-inflammatory and anti-arthritic properties. However, the exact mechanism through which retinoic acid modulates arthritis remains unclear. This study aimed to investigate whether retinoic acid ameliorates rheumatoid arthritis by modulating macrophage polarization. This study used retinoic acid to treat mice with adjuvant arthritis and evaluated anti-inflammatory effects by arthritis score, thermal nociceptive sensitization test, histopathologic examination and immunofluorescence assays. In addition, its specific anti-arthritic mechanism was investigated by flow cytometry, cell transfection and inflammatory signaling pathway assays in RAW264.7 macrophages in vitro. Retinoic acid significantly relieved joint pain and attenuated inflammatory cell infiltration in mice. Furthermore, this treatment modulated peritoneal macrophage polarization, increased levels of arginase 1, as well as decreased inducible nitric oxide synthase expression. In vitro, we verified that retinoic acid promotes macrophage transition from the M1 to M2 type by upregulating mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) expression and inhibiting P38, JNK and ERK phosphorylation in lipopolysaccharide-stimulated RAW264.7 cells. Notably, the therapeutic effects of retinoic acid were inhibited by MKP-1 knockdown. Retinoic acid exerts a significant therapeutic effect on adjuvant arthritis in mice by regulating macrophage polarization through the MKP-1/MAPK pathway, and play an important role in the treatment of rheumatic diseases.

Macrophages are innate immune cells connected with the development of inflammation. Retinoic acid has previously been proved to have anti-inflammatory and anti-arthritic properties. However, the exact mechanism through which retinoic acid modulates arthritis remains unclear. This study aimed to investigate whether retinoic acid ameliorates rheumatoid arthritis by modulating macrophage polarization. This study used retinoic acid to treat mice with adjuvant arthritis and evaluated anti-inflammatory effects by arthritis score, thermal nociceptive sensitization test, histopathologic examination and immunofluorescence assays. In addition, its specific anti-arthritic mechanism was investigated by flow cytometry, cell transfection and inflammatory signaling pathway assays in RAW264.7 macrophages in vitro. Retinoic acid significantly relieved joint pain and attenuated inflammatory cell infiltration in mice. Furthermore, this treatment modulated peritoneal macrophage polarization, increased levels of arginase 1, as well as decreased inducible nitric oxide synthase expression. In vitro, we verified that retinoic acid promotes macrophage transition from the M1 to M2 type by upregulating mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) expression and inhibiting P38, JNK and ERK phosphorylation in lipopolysaccharide-stimulated RAW264.7 cells. Notably, the therapeutic effects of retinoic acid were inhibited by MKP-1 knockdown. Retinoic acid exerts a significant therapeutic effect on adjuvant arthritis in mice by regulating macrophage polarization through the MKP-1/MAPK pathway, and play an important role in the treatment of rheumatic diseases.

Prescription optimization is a crucial aspect in the study of traditional Chinese medicine （TCM） compounds. In recent years， the introduction of mathematical methods， data mining techniques， and artificial neural networks has provided new tools for elucidating the compatibility rules of TCM compounds. The study of TCM compounds involves numerous variables， including the proportions of different herbs， the specific extraction parts of each ingredient， and the interactions among multiple components. These factors together create a complex nonlinear dose-effect relationship. In this context， it is essential to identify methods that suit the characteristics of TCM compounds and can leverage their advantages for effective application in new drug development. This paper provided a comprehensive review of the cutting-edge optimization experimental design methods applied in recent studies of TCM compound compatibilities. The key technical issues， such as the optimization of source material selection， dosage optimization of compatible herbs， and multi-objective optimization indicators， were discussed. Furthermore， the evaluation methods for component effects were summarized during the optimization process， so as to provide scientific and practical foundations for innovative research in TCM and the development of new drugs based on TCM compounds.

Objective To investigate the influence of the liver failing to convey and disperse on space navigation aging.Methods High and low neuroticism subjects screened by Eysenck personality questionnaire were included in liver failing to convey and disperse group and liver controlling conveyance and dispersion group,respectively.The two groups were then divided into youth and elderly groups based on age.Finally,spatial navigation task was conducted to record and analyze behavioral(reaction time and accuracy)and EEG data(amplitude and latency of P2 and N2 components)of all the four groups(30 subjects each group).Results Compared with liver controlling conveyance and dispersion group,the accuracy in subjects with liver failing to convey and disperse decreased significantly(P<0.001)and reaction time was prolonged significantly(P<0.05).The interaction effect between age and liver regulation status showed a marginal significant difference(P=0.078).The accuracy of elderly people was lower than that of youth people(P=0.002)for liver failing to convey and disperse subjects,while there was no significant difference between the two groups for subjects with liver controlling conveyance and dispersion.The P2 amplitude in the elderly group was significantly smaller than that in the youth group(P=0.027).The amplitude of P2 in group of liver failing to convey and disperse was significantly smaller than that in group of liver controlling conveyance and dispersion(P=0.042).The interaction effect of P2 amplitude between age and liver regulation status showed a marginal significance(P=0.073).For youth subjects,the P2 amplitude in group of liver controlling conveyance and dispersion was significantly larger than that in group of liver failing to convey and disperse(P=0.007),while there was no significant difference in P2 amplitude between the two groups for the elderly subjects.The N2 amplitude for tasks of allocentric frames of reference was significantly greater than that of egocentric frames of reference(P=0.024).Conclusion Liver failing to convey and disperse caused by long-term emotional disturbance accelerates spatial navigation aging,and selective attention feature inhibition disorder may be the underlying ERPs neuroelectrophysiological mechanism.