An effective method has been developed for laboratory scale production of IgG. Hybridomas were cultured in serum-free media with 2% IgG-free ascites. Cell density of up to 3.55 × 10 6cells/ml and antibody concentration of 135μ g/ml after purification were abtained, which is four time more than total production of that of IgG concentration in serum-free media. This in vitro method allows great improvement in antibodies production in batch tissue culture. The method reported here is easy to handle and is economical and universally adaptable.

Objective: To compare the biotype and genetic similarity between groups of commensal and pathogenic strains of candida species. Methods:Random amplified polymorphic DNA (RAPD) was used to analyse the type of Candida albicans. Results: 12 pathogenic isolates of Candida albicans, 3 commensal isolates of Candida abicans and 3 isolates of pathogenic non-albicans were obtained. The similarity coefficient of albicans with non-albicans was 51.7% by RAPD. Intra-candida similarity coefficient was more than 70%. Both the commensal and pathogenic isolates showed the genetically similar to C. albicans. Intro-isolate DNA polymorphism was observed by RAPD. Conclusion: Both the commensal and pathogenic groups contain a major cluster of genetically similar C. albicans isolates.

OBJECTIVETo test whether folic acid offers protection of the brain tissue against acute cerebral infarction in rats.

METHODSSprague-Dawley rats were divided into control (n=8), pre-treatment (n=12) and treatment (n=16) groups, all having routine feed for 7 days. The rats in the control and treatment groups were given normal saline daily, and those in the pre-treatment group received folic acid suspension daily. All the rats were then subject to middle cerebral artery occlusion (MCAO) for 24 h followed by reperfusion. On and after the operation day, the rats in the control group were given normal saline and those in the other two groups were given folic acid suspension daily. Neural function deficiency was evaluated on a daily basis after the operation, and on day 6 after the operation, brain biopsy was performed for examination with TTC staining. Monocyte chemokine -1 (MCP-1) in both normal and infarct tissues was measured by ELISA.

RESULTSOn day 6 after the operation, the neural function deficiency scores of the control, pre-treatment and treatment groups were 4.56∓3.63, 2.94∓2.94 and 1.00∓1.00, and the percentages of the infarct area (to the whole brain area) were (44.23∓10.06)%, (20.64∓6.78)% and (14.61∓13.51)%, respectively. The contents of MCP-1 in the infarct area of the brain tissues were 168.58∓107.21 ng/L, 152.91∓64.78 ng/L, and 97.74∓46.19 ng/L in the 3 groups, respectively.

CONCLUSIONFolic acid can protect brain tissue against acute cerebral infarction in rats.

Animals ; Cerebral Infarction ; drug therapy ; Female ; Folic Acid ; therapeutic use ; Infarction, Middle Cerebral Artery ; drug therapy ; Neuroprotective Agents ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; Thrombolytic Therapy

Objective: To analyze the associations among body mass index (BMI), learning screen/gaming screen exposure and executive function in preschool children in Anhui Province, so as to provide a basis for promoting the development of executive function in preschool children. Methods: In June 2022, a stratified cluster sampling and convenience sampling methods were used to survey 3 534 mothers of preschool children in Wuhu City, Luan City, and Fuyang City, Anhui Province. The Behavior Rating Inventory of Executive Function-Preschool Version (BRIEF-P) was used to assess the preschool childrens executive function abnormalities. Binary Logistic regression was conducted to examine the relationships among BMI, learning screen/gaming screen exposure, and their combined effects on executive function abnormalities. Results: The detection rate of abnormal executive function in preschool children was 9.65%. Logistic regression analysis showed that after adjusting for the confounding factors such as pregnancyinduced hypertension, primary caregivers, family per capita monthly income and family structure, the risk of abnormal executive function of children in overweight/obesity group and high learning screen/gaming screen exposure group increased significantly (overweight/obesity:OR=1.78, 95%CI=1.31-2.42, learning screen exposure:OR=1.48, 95%CI=1.18-1.86, gaming screen exposure:OR=1.50, 95%CI=1.18-1.91,P<0.05). Compared with children with normal BMI and low learning screen/gaming screen screen exposure, those with both overweight/obesity and high learning screen/gaming screen exposure had a significantly greater risk of executive function abnormalities (OR=2.07, 95%CI=1.29-3.31; OR=2.42, 95%CI=1.59-3.68,P<0.05). Conclusions Overweight/obesity and high learning screen/gaming screen exposure are important risk factors for executive function abnormalities in preschool children. Therefore, actively guiding preschool children to develop healthy life habits to promote the normal development of their executive functions is essential.

Background/Aims: The occurrence and development of hepatitis B virus-associated acute-onchronic liver failure (HBV-ACLF) is closely related to the immune pathway. We explored the heterogeneity of peripheral blood T cell subsets and the characteristics of exhausted T lymphocytes, in an attempt to identify potential therapeutic target molecules for immune dysfunction in ACLF patients. Methods: A total of 83,577 T cells from HBV-ACLF patients and healthy controls were screened for heterogeneity by single-cell RNA sequencing. In addition, exhausted T-lymphocyte subsets were screened to analyze their gene expression profiles, and their developmental trajectories were investigated. Subsequently, the expression of exhausted T cells and their capacity in secreting cytokines (interleukin 2, interferon γ, and tumor necrosis factor α) were validated by flow cytometry. Results: A total of eight stable clusters were identified, among which CD4 + TIGIT + subset and CD8 + LAG-3 + subset, with high expression of exhaust genes, were significantly higher in the HBV-ACLF patients than in normal controls. As shown by pseudotime analysis, T cells experienced a transition from naïve T cells to effector T cells and then exhausted T cells. Flow cytometry confirmed that the CD4 + TIGIT + subset and CD8 + LAG-3 + subset in the peripheral blood of the ACLF patients were significantly higher than those in the healthy controls. Moreover, in vitro cultured CD8 + LAG-3 + T cells were significantly fewer capable of secreting cytokines than CD8 + LAG-3- subset. Conclusions Peripheral blood T cells are heterogeneous in HBV-ACLF. The exhausted T cells markedly increase during the pathogenesis of ACLF, suggesting that T-cell exhaustion is involved in the immune dysfunction of HBV-ACLF patients.

Objective:To evaluate the developmental toxicity of Cry1Ab protein by studying its effects on cell proliferation and differentiation ability using a developmental toxicity assessment model based on embryonic stem-cell.Methods:Cry1Ab protein was tested in seven dose groups(31.25,62.50,125.00,250.00,320.00,1 000.00,and 2 000.00 μg/L)on mouse embryonic stem cells D3(ES-D3)and 3T3 mouse fibroblast cells,with 5-fluorouracil(5-FU)used as the positive control and phos-phate buffer saline(PBS)as the solvent control.Cell viability was detected by CCK-8 assay to calculate the 50％inhibitory concentration(IC50)of the test substance for different cells.Additionally,Cry1 Ab protein was tested in five dose groups(125.00,250.00,320.00,1 000.00,and 2 000.00 μg/L)on ES-D3 cells,with PBS as the solvent control and 5-FU used for model validation.After cell treatment,cardiac differentiation was induced using the embryonic bodies(EBs)culture method.The growth of EBs was observed under a microscope,and their diameters on the third and fifth days were measured.The proportion of EBs differentiating into beating cardiomyocytes was recorded,and the 50％inhibition con-centration of differentiation(ID50)was calculated.Based on a developmental toxicity discrimination func-tion,the developmental toxicity of the test substances was classified.Furthermore,at the end of the cul-ture period,mRNA expression levels of cardiac differentiation-related markers(Oct3/4,GATAA-4,Nkx2.5,and β-MHC)were quantitatively detected using real-time quantitative polymerase chain reaction(qPCR)in the collected EBs samples.Results:The IC50 of 5-FU was determined as 46.37 μg/L in 3T3 cells and 32.67 μg/L in ES-D3 cells,while the ID50 in ES-D3 cells was 21.28 μg/L.According to the discrimination function results,5-FU was classified as a strong embryotoxic substance.There were no sta-tistically significant differences in cell viability between different concentrations of Cry 1 Ab protein treat-ment groups and the control group in both 3T3 cells and ES-D3 cells(P＞0.05).Moreover,there were no statistically significant differences in the diameter of EBs on the third and fifth days,as well as their morphology,between the Cry1Ab protein treatment groups and the control group(P＞0.05).The cardi-ac differentiation rate showed no statistically significant differences between different concentrations of Cry1Ab protein treatment groups and the control group(P＞0.05).5-FU significantly reduced the mRNA expression levels of β-MHC,Nkx2.5,and GATA-4(P＜0.05),showing a dose-dependent trend(P＜0.05),while the mRNA expression levels of the pluripotency-associated marker Oct3/4 exhibited an increasing trend(P＜0.05).However,there were no statistically significant differences in the mRNA expression levels of mature cardiac marker β-MHC,early cardiac differentiation marker Nkx2.5 and GATA-4,and pluripotency-associated marker Oct3/4 between the Cry1Ab protein treatment groups and the control group(P＞0.05).Conclusion:No developmental toxicity of Cry1Ab protein at concen-trations ranging from 31.25 to 2 000.00 μg/L was observed in this experimental model.

Cryptococcal meningitis is a common and refractory central nervous system infection,with high rates of mortality and disability.The experts of the Society of Infectious Diseases of Chinese Medical Association have reached this consensus after a thorough discussion.Based on the current situation of cryptococcal meningitis in China,the management of cryptococcal meningitis includes 6 aspects:introduction,microorganism identification,clinical manifestations and diagnosis,principles of antifungal therapy,treatment of refractory and recurrent meningitis,treatment of intracranial hypertension.There is not a separate consensus on human immunodeficiency virus (HIV) infection in patients with cryptococcal meningitis.This article focuses on different antifungal regimens and reducing intracranial pressure by reference to Infectious Disease Society of America (IDSA) guidelines.The importance of early diagnosis,combined long-term antifungal therapy,control of intracranial hypertension are emphasized.

Objective:Trigeminal neuralgia(TN)is a severe chronic neuropathic pain that mainly affects the distribution area of the trigeminal nerve with limited treating efficacy.There are numerous treatments for TN,but currently the main clinical approach is to suppress pain by carbamazepine(CBZ).Brain-derived neurotrophic factor(BDNF)is closely related to chronic pain.This study aims to determine the effects of CBZ treatment on BDNF expression in both the trigeminal ganglion(TG)and serum of TN via a chronic constriction injury of the infraorbital nerve(ION-CCI)rat model. Methods:The ION-CCI models were established in male Sprague-Dawley rats and were randomly divided into a sham group,a TN group,a TN+low-dose CBZ treatment group(TN+20 mg/kg CBZ group),a TN+medium-dose CBZ treatment group(TN+40 mg/kg CBZ group),and a TN+high-dose CBZ treatment group(TN+80 mg/kg CBZ group).The mechanical pain threshold in each group of rats was measured regularly before and after surgery.The expressions of BDNF and tyrosine kinase receptor B(TrkB)mRNA in TGs of rats in different groups were determined by real-time PCR,and the expression of BDNF protein on neurons in TGs was observed by immunofluorescence.Western Blotting was used to detect the protein expression of BDNF,TrkB,extracellular regulated protein kinases(ERK),and phospho-extracellular regulated protein kinases(p-ERK)in TGs of rats in different groups.The expression of BDNF in the serum of rats in different groups was detected by enzyme-linked immunosorbent assay(ELISA). Results:The results of mechanical pain sensitivity showed that there was no significant difference in the mechanical pain threshold in the right facial sensory area of the experimental rats in each group before surgery(all P>0.05).From the 3rd day after operation,the mechanical pain threshold of rats in the TN group was significantly lower than that in the sham group(all P<0.01),and the mechanical pain threshold of rats in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 CBZ mg/kg group was higher than that in the TN group(all P<0.05).The BDNF and TrkB mRNA and protein expressions in TGs of rats in the TN group were higher than those in the sham group(all P<0.05),and those in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were lower than the TN group(all P<0.05).The p-ERK levels in TG of rats in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were significantly decreased compared with the TN group(all P<0.05).The BDNF and neuron-specific nuclear protein(NeuN)were mainly co-expressed in neuron of TGs in the TN group and they were significantly higher than those in the sham group(all P<0.05).The co-labeled expressions of BDNF and NeuN in TGs of the TN+ 80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were lower than those in the TN group(all P<0.05).The results of ELISA showed that the level of BDNF in the serum of the TN group was significantly higher than that in the sham group(P<0.05).The levels of BDNF in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were lower than those in the TN group(all P<0.05).Spearman correlation analysis showed that the BDNF level in serum was negatively correlated with mechanical pain threshold(r=-0.650,P<0.01). Conclusion:CBZ treatment can inhibit the expression of BDNF and TrkB in the TGs of TN rats,reduce the level of BDNF in serum of TN rats and the phosphorylation of ERK signaling pathway,so as to inhibit TN.The serum level of BDNF can be considered as an indicator for the diagnosis and prognosis of TN.

OBJECTIVE To investigate the effects and mechanism of luteolin on osteogenic repair of bone defects. METHODS The targets and potential pathways of luteolin in the treatment of bone defects were screened by network pharmacology method， and then the top 2 targets were selected by Hub gene screening for molecular docking verification， with binding energy as the evaluation standard. In vitro experiments were conducted on rat bone mesenchymal stem cells （BMSC） and rat umbilical vein endothelial cells （RUVEC）. Phenotypic validation was performed using alkaline phosphatase staining， alizarin red S staining， and in vitro angiogenesis experiments. Western blot assay was used to detect the protein expressions of phosphatidylinositol 3 kinase （PI3K） and protein kinase 1 （Akt1）， so as to validate the mechanism of luteolin on osteogenic differentiation of BMSC and angiogenesis of RUVEC in vitro. RESULTS The results of network pharmacology showed that the effects of luteolin on vascular formation and bone repair in bone defects were mainly related to Akt1， SRC， estrogen receptor 1， epidermal growth factor receptor， cyclooxygenase 2， matrix metalloproteinase 9 targets， and were closely related to PI3K-Akt signaling pathway. The results of molecular docking showed that luteolin binding to Akt1 and SRC proteins was stable. The results of in vitro experiments showed that luteolin could significantly improve the expressions and activities of alkaline phosphatase in BMSC， increased the number of calcium salt deposits and calcified nodules， and promoted calcification of BMSC. Compared with luteolin 0 μmol/L group， the angiogenesis ability of RUVEC was enhanced significantly in luteolin 1， 10 μmol/L groups， the length of blood vessels and the protein expressions of PI3K and Akt1 were significantly increased （P＜0.05 or P＜0.01）； the higherthe concentration， the better the effect. CONCLUSIONS Luteolin may play a role in promoting angiogenesis and bone repair at the fracture site by activating PI3K/Akt signaling pathway and promoting the protein expressions of PI3K and Akt1.

A 6-year-old girl presented with recurrent skin rash at the initial stage, recent joint pain, and neutrophilia was found during a routine blood test. After a multidisciplinary case discussion, she was diagnosed with chronic neutrophil leukemia, and the symptoms were relieved after hydroxyurea and luxolitinib treatment. She received the allogeneic hematopoietic stem cell transplantation subsequently. At present, she is in stable condition and under follow-up. Chronic neutrophil leukemia is a rare disease, which rarely occurs in children. It is more difficult to diagnose in patients with skin rash as the first manifestation. The diagnosis and treatment of this case reflects the important role of multidisciplinary cooperation in the diagnosis and treatment of difficult and rare diseases.