BACKGROUND:Primary cortical neurons and microglial cells play a crucial role in exploring cell therapies for neurological disorders,and most of the current methods for obtaining the two types of cells are cumbersome and require separate extraction.It is therefore crucial to find a convenient and rapid method to extract both types of cells simultaneously. OBJECTIVE:To explore a novel method for simultaneous extraction of primary cortical neurons and microglial cells. METHODS:Newborn suckling SD rats were taken within 24 hours.The brain was removed and placed in a dish with DMEM,and the pia mater was removed for later use.Primary neurons were extracted from the same brain tissue,and then the remaining brain tissue was used to extract microglial cells.The whole process was performed on ice.Extraction and culture steps of primary cortical neurons:The cerebral cortex was taken 2.0-3.0 mm with forceps,and the tissue was digested with papain for 20 minutes.After aborting digestion,the blown tissue presented an adherent tissue suspension.The supernatant cell suspension was obtained,filtered,and dispensed into 15 mL centrifuge tubes.After centrifugation and re-suspension,the cells were inoculated onto 6-well plate crawls coated with L-polylysine.Neuronal morphology was observed at 1-day intervals,and staining could be performed for identification using immunofluorescence staining of MAP2 and β-Tubulin by day 7.Microglia extraction and culture steps:The remaining brain tissue at 8-10 mm thick was subjected to microglial cell extraction,digested by trypsin for 20 minutes.After digestion was stopped,the tissue was blown to a homogenate,and then the homogenate was transferred to the culture bottle for culture.On day 14,the culture flasks were sealed and subjected to constant temperature horizontal shaking for 2 hours.Microglial cells were shed in the supernatant.Purified microglial cells were taken and continued to be cultured for 3 days for identification by Iba1 immunofluorescence staining. RESULTS AND CONCLUSION:(1)After 24 hours of culture,the neurons were adherent to the wall,the cytosol was enlarged,and some neurons developed synapses.After 3 and 5 days of culture,the cytosol was further enlarged,and most of the neurons were in the form of synapses,and some neurons were growing in clusters.On day 7,neuronal synapses were prolonged and thickened,and they were connected with each other to form a network.The neurons were identified by β-Tubulin and MAP2 immunofluorescence staining.(2)The cells grew close to the wall on day 1 of culture.On days 3,5,and 7,the density of microglial cells was small,and the cell morphology was bright oval or round,but the cells basically grew in clumps on the upper layer of other cells.On day 10,the density of microglial cells increased significantly.On day 14,microglial cells grew in dense clumps on the upper layer of other cells,and then they could be isolated and purified.The isolated and purified cells were taken and re-cultured to day 3 and identified as microglial cells by Iba1 immunofluorescence;their purity was greater than 95%.(3)The results show that primary cortical neurons and microglial cells obtained by this method after extraction and culture are of high purity,good morphology,and high viability.

ObjectiveTo analyze the adverse reactions associated with the clinical use of Banxia （Rhizoma Pinelliae）- Wutou （Aconitum） in the same formula， with the aim of providing a reference for the safety of their clinical application. MethodsLiterature on the clinical application of antagonistic herbs "Banxia-Wutou" used in the same formula， published from January 1st， 2014， to June 30th， 2023， was retrieved from databases including CNKI， VIP， Wanfang， SinoMed， PubMed， Cochrane Library， and Embase. A database was established， and information related to adverse reactions was extracted， including descriptions， classifications， specific manifestations， management and outcomes， patients' primary diseases （western medicine diseases and traditional Chinese medicine diagnoses and syndromes）， and medication information （dosage， ratio， administration routes， and dosage forms）. ResultsA total of 79 researches simultaneously used antagonistic herbs Banxia-Wutou in the same formula and reported associated advers reactions. Gastrointestinal adverse reactions were the most common， with 8 studies reporting management of adverse reactions and 3 studies reporting improvement with no intervention. Among the 11 researches， the adverse reaction relieved to extant， while other 69 researches didn't report the managment of adverse reaction and its prognosis. For the primary disease in western medicine system， chronic bronchitis and chronic obstructive pulmonary disease （COPD） were most common， while gastric pain was the most common symptom in traditional Chinese medicine with spleen and kidney deficiency and spleen stomach cold deficiency being the most frequent syndromes. The most common Banxia dosage was 10 g， while for the Wutou， Fuzi （Radix Aconiti Lateralis Praeparata） was predominant with the highest dose at 15 g. The most frequent herbal combination was Banxia-fuzi， with a 1∶1 ratio. The main administration route was oral， and the primary dosage form was decoction. ConclusionGastrointestinal adverse reactions are the most common in the clinical use of Banxia-Wutou antagonistic herb combinations. Research on the safety of "Banxia-Wutou" combinations should focus on respiratory system diseases and spleen-stomach related conditions.

Objective To determine the prognostic biomarkers and new therapeutic targets of the lung adenocarcinoma (LUAD), based on which to establish a prediction model for the survival of LUAD patients. Methods An integrative analysis was conducted on gene expression and clinicopathologic data of LUAD, which were obtained from the UCSC database. Subsequently, various methods, including screening of differentially expressed genes (DEGs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA), were employed to analyze the data. Cox regression and least absolute shrinkage and selection operator (LASSO) regression were used to establish an assessment model. Based on this model, we constructed a nomogram to predict the probable survival of LUAD patients at different time points (1-year, 2-year, 3-year, 5-year, and 10-year). Finally, we evaluated the predictive ability of our model using Kaplan-Meier survival curves, receiver operating characteristic (ROC) curves, and time-dependent ROC curves. The validation group further verified the prognostic value of the model. Results The different-grade pathological subtypes' DEGs were mainly enriched in biological processes such as metabolism of xenobiotics by cytochrome P450, natural killer cell-mediated cytotoxicity, antigen processing and presentation, and regulation of enzyme activity, which were closely related to tumor development. Through Cox regression and LASSO regression, we constructed a reliable prediction model consisting of a five-gene panel (MELTF, MAGEA1, FGF19, DKK4, C14ORF105). The model demonstrated excellent specificity and sensitivity in ROC curves, with an area under the curve (AUC) of 0.675. The time-dependent ROC analysis revealed AUC values of 0.893, 0.713, and 0.632 for 1-year, 3-year, and 5-year survival, respectively. The advantage of the model was also verified in the validation group. Additionally, we developed a nomogram that accurately predicted survival, as demonstrated by calibration curves and C-index. Conclusion We have developed a prognostic prediction model for LUAD consisting of five genes. This novel approach offers clinical practitioners a personalized tool for making informed decisions regarding the prognosis of their patients.

Objective:To prepare recombinant protein of group 36 allergen of Dermatophagoides farinae(Der f 36),to deter-mine its immunogenicity and bioinformatics analysis was performed.Methods:Nucleic acid sequence coding for Der f 36 was obtained and artificially synthesized,pET-28a(+)was inserted to construct pET-28a(+)-Der f 36 plasmid and transformed into BL21(DE3)receptor cells.After expressed and purified in BL21(DE3),recombinant allergen rDer f 36 was obtained,recombinant protein rDer f 36 was identified by SDS-PAGE and Western blot.Serum IgE binding rates of rDer f 36 was determined by IgE-ELISA.Cross-reactivity between rDer f 36 and rDer p 36 was detected by IgE-ELISA inhibition assay.HNEpC cells were cultured with rDer f 36 for 24 h and cytokines were detected.Bioinformatics softwares were used to analyze physicochemical properties and structures of Der f 36 and Der p 36.Results:Coding gene for Der f 36 was obtained with a total length of 690 bp,whose molecular weight was 25.6 kD;serum IgE binding rate of rDer f 36 was 42.1%by IgE-ELISA;IgE-ELISA inhibition assay showed that rDer p 36 had 40%(8/20)inhibition rate(>50%)for rDer f 36,with an average inhibition rate of 52.98%.Compared with control group,HNEpC cells cultured with rDer f 36 showed that IL-6,IL-8,IL-33,IL-25 and TSLP expressions had increased;bioinformatics analyses show that sequence consistency of Der f 36 and Der p 36 was 77.63%,with similar physicochemical properties.Secondary structure analysis showed that both of them contained α helix,β-turn and random coils,and content of random coils was the highest;structure of C2 domains was also highly overlapping(RMSD=0.046).Conclusion:Recombinant allergen rDer f 36 is successfully prepared with good immunogenicity,laying foundation for diagnosis and treatment of dust mite allergen single component.High similarity in physical and chemical properties,secondary structure,and tertiary structure between Der f 36 and Der p 36 determines their cross reactivity.

Objective:To prepare recombinant protein of group 27 allergen of Dermatophagoides farinae(Der f 27),and to determine its immunoactivity.Methods:pET-28a(+)-Der f 27 plasmid was constructed and inserted into E.coli BL21(DE3)cells.After being expressed and purified,recombinant allergen rDer f 27 was obtained.IgE binding rates of rDer f 27 with sera from patients with allergic rhinitis induced by Dermatophagoides farinae was determined by ELISA and Western blot.PBMC from patients with allergic rhinitis and BESA-2B cells were cultured with rDer f 27 for 24 h,respectively,and cytokine expression was measured.Bioinformatics softwares were used to analyze physicochemical properties and structures of Der f 27.Results:pET-28a(+)-Der f 27 plasmids were prepared successfully and transformed into BL21(DE3)cells.After expressed and purified with PTG,SDS-PAGE and Western blot identified a band about 48 kD.IgE binding rates of rDer f 27 were 39.5%and 45.5%with sera from patients with allergic rhinitis allergic to Dermatophagoides farinae by IgE-ELISA and IgE-Western blot,respectively.Compared with control group,IL-6 and IL-8 expressions were increased in PBMC from patients with allergic rhinitis being cultured with rDer f 27(P<0.05);expressions of IL-10 and TGF-β were decreased in BESA-2B cells being cultured with rDer f 27,while IL-17A and IL-23A expressions were increased.Bioinformatics analysis showed that Der f 27 belong to serine protease inhibitor family and had universal structure and func-tion of this family.Secondary structure of Der f 27 was mainly composed of α-helix(42.62%)and random coil(35.60%).Conclusion:Recombinant allergen rDer f 27 has been prepared successfully with good immunoreactivity and immunogenicity,becoming one of important allergens of allergic rhinitis.

Objective:To statistically analyze the application and funding status in the endocrine system/metabolism and nutritional support(H07) since the establishment of the Department of Medical Sciences of the National Natural Science Foundation of China(NSFC). To understand the current states and development trends in the field of endocrine and metabolic disorders in China, providing a reference basis for future research.Methods:Based on the NSFC data platform, a statistical analysis was conducted on applications and funding of projects in the field of H07 in the Department of Medical Sciences of the NSFC from 2010 to 2023. The analysis covered funding rate, application code, and host institutions, summarizing the current status of basic research in the H07 field.Results:From 2010 to 2023, the total number of applications for grants under the H07 code was 22 566, with a total of 3 703 projects funded, resulting in an overall funding rate 16.4%, and the total funding amount of 187 687 million yuan. Applications and funded projects were primarily concentrated in General Projects and the Young Scientist Fund Projects. Over the 14 years, the total number of applications for General Projects, the Young Scientist Fund Projects, and Regional Science Fund Projects showed an overall upward trend, while the number of funded projects remained relatively stable and the funding rate declined year by year. Under H07 code, a total of 23 projects of the National Science Foundation for Outstanding Young Scholars Projects and 11 Distinguished Young Scholars Projects were funded, and the funding rates were 12.1% and 12.5% respectively. The annual funding volume of Key Program fluctuated from 1 to 7, and the average funding rate in the past 14 years was 25.5%. The top four secondary codes in terms of application volume were H0708(30.8%), H0709(11.6%), H0712(11.4%), H0710(9.1%), and the top four secondary codes in terms of funding volume were H0708(27.9%), H0709(13.5%), H0712(10.9%), H0710(10.8%).Conclusions:With the support of the NSFC, basic research in the H07 discipline has made significant strides. The funded research spans diverse directions, talent nurturing, and extensive regional coverage. Diabetes continues to dominate as a primary research focus, alongside obesity, abnormal energy metabolism, and lipid metabolism disorders, which represent critical research areas within the field.

Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.

Objective:To explore the optimum opportunity for lumbodorsal muscles exercises of patients undergoing posterior lumbar decompression and instrumentation, and investigate its effect on the rehabilitation outcomes and kinesiophobia.Methods:A randomized controlled trial was used. By convenient sampling method, a total of 120 lumbar disc herniation patients were prospectively selected from Affiliated Nantong Hospital of Shanghai(Nantong Sixth People′s Hospital) from February 2020 to December 2021. The paitients were assigned to early group, middle group and late group, with 40 cases in each group. All patients were given routine postoperative care and lumbodorsal muscles exercises. The early group started to exercise 10th day after operation, the middle group started to exercise 3 weeks after operation, and the late group started to exercise 6 weeks after operation. The intervention effect was respectively evaluated by Japanese Orthopaedics Association (JOA) and Tampa Scale for Kinesiophobia (TSK).Results:There were 3, 1 and 1 missing cases in the early, middle and late group respectively, the age in the 3 groups were (56.05 ± 11.77), (57.33 ± 14.64) and (54.23 ± 15.73) years old in turn. Three months after exercising, the total score of JOA in the early, middle and late group were (25.32 ± 2.45), (24.44 ± 2.19) and (22.13 ± 1.58) in turn, the difference was significant ( F=23.64, P<0.05); the score of TSK in the early, middle and late group were (37.95 ± 6.81), (34.18 ± 6.39) and (33.33 ± 7.36) in turn, the difference was significant ( F=4.82, P<0.05). Conclusions:Lumbodorsal muscles exercises start at 3 weeks after operation can significantly improve the rehabilitation outcome of lumbar disc herniation patients undergoing posterior lumbar decompression and instrumentation, and will not increase the level of kinesiophobia, its can be consider as optimum opportunity for lumbodorsal muscles exercises.

At the end of 2022,the Fifth International Workshop on Primary Hyperparathyroidism(PHPT)released the most recent guidelines for the evaluation and management of PHPT.These guidelines offer a comprehensive overview and provide guideline recommendations on various aspects of PHPT,including epidemiology,pathophysiology,genetics,clinical manifestations,as well as both surgical and non-surgical management.This paper focuses on surgical management issues concerning PHPT as addressed in these guidelines,with the aim of further improving the diagnosis and treatment of PHPT in China and promoting the standardization of surgical management for PHPT.

BACKGROUND:Studies have found that activation of nuclear factor-erythroid 2-related factor 2/heme oxidase-1(Nrf2/HO-1)pathway can alleviate oxidative stress caused by cerebral ischemia-reperfusion injury,but whether human bone marrow mesenchymal stem cells(hBMSC)can activate Nrf2/HO-1 pathway to alleviate cerebral ischemia-reperfusion injury is still lacking relevant studies. OBJECTIVE:To investigate whether intracranial transplantation of hBMSC alleviates oxidative stress injury in cerebral ischemia-reperfusion animal models by activating Nrf2/HO-1 pathway. METHODS:Totally 40 male SPF SD rats were randomly divided into sham operation group,model group,hBMSC transplantation group,hBMSC+solvent group and hBMSC+Nrf2 inhibitor group.Each group consisted of eight animals.In the model group and the hBMSC transplantation group,middle cerebral artery occlusion model was prepared by thread embolization method.The thread embolization was removed 1 hour later,and 30 μL PBS or hBMSC cultured to at least passage 5 was injected into the right cortex and striatum of rats.In the hBMSC+Nrf2 inhibitor group and hBMSC+solvent group,the left ventricle was injected with Nrf2 inhibitor Brusatol and its solvent dimethyl sulfoxide respectively 24 hours before model establishment,then the middle cerebral artery occlusion model was prepared,and hBMSC was injected.Relevant indexes were detected 3 days after transplantation. RESULTS AND CONCLUSION:(1)CT and TTC staining showed the same area and volume of cerebral infarction:model group>hBMSC+Nrf2 inhibitor group>hBMSC+solvent group>hBMSC transplantation group>sham operation group.(2)Hematoxylin-eosin staining and Nissl's staining showed that the ischemic brain tissue was intact and the neurons were normal in the sham operation group.Compared with the model group,the pathological morphology and neuronal injury of the hBMSC transplantation group and the hBMSC+solvent group were significantly improved.Compared with the hBMSC+solvent group,the hBMSC+Nrf2 inhibitor group had more serious pathological morphology and neuronal damage.(3)Western blot assay and oxidative stress index detection results showed that compared with the sham operation group,Nrf2 and HO-1 proteins were decreased(all P<0.05),malondialdehyde was increased and superoxide dismutase was decreased(all P<0.05)in the model group.Compared with the model group,the expression levels of Nrf2 and HO-1 proteins were increased(all P<0.05),malondialdehyde was decreased and superoxide dismutase was increased(all P<0.05)in the hBMSC transplantation group and the hBMSC+solvent group.Compared with the hBMSC+solvent group,the expression levels of Nrf2 and HO-1 proteins were simultaneously decreased(all P<0.05),and malondialdehyde was increased and superoxide dismutase was decreased(all P<0.05)in the hBMSC+Nrf2 inhibitor group.(4)These results indicate that hBMSC can alleviate cerebral ischemia-reperfusion injury possibly by activating Nrf2/HO-1 pathway.