OBJECTIVE: To identify the gene mutation types of 4 suspected β-thalassemia patients in Yunnan Province, and to analyze the genotypes and hematological phenotypes. METHODS: Whole genome sequencing was performed on the samples of 4 suspected β-thalassemia patients from the Dai ethnic group in a thalassemia endemic area of Yunnan Province, whose hematological phenotypes were not consistent with the results of common thalassemia gene mutations. The mutations of β-globin gene clusters were confirmed by polymerase chain reaction (PCR) and Sanger DNA sequencing technology. RESULTS: The 3.5 kb deletion in β-globin gene cluster (NC_000011.10: g. 5224302-5227791del3490bp) was detected in 4 patients' samples, of which 1 case was also detected with HbE mutation and 1 case with CD17 mutation. These 2 patients displayed moderate anemia phenotype, while the two patients with only the 3.5 kb deletion presented with other mild anemia phenotype. CONCLUSION Heterozygous carriers with rare 3.5 kb deletion of the β-globin gene cluster may develop mild anemia, compound mutations of the 3.5 kb deletion with other mutations may led to intermediate thalasemia with moderate to sever anemia. In areas with a high incidence of thalassemia, suspected patients should undergo genetic testing to avoid missing or misdiagnosing rare mutations.
Humans ; beta-Globins/genetics* ; Multigene Family ; beta-Thalassemia/genetics* ; Mutation ; Genotype ; Sequence Deletion ; Phenotype ; Male ; Female

Objective: To investigate the clinical characteristics and related factors of corticosteroid induced adrenal crisis (AC) in children with primary nephrotic syndrome (NS). Methods: Case control study. The case group included 7 children aged 1 to 18 years with NS combined with AC hospitalized in Peking University First Hospital from January 2016 to May 2021 (AC group). According to the ratio of case group: control group 1: 4, 28 children aged 1 to 18 years who were diagnosed with NS without AC during the same period were matched as controls (non-AC group). Clinical data were collected. The clinical characteristics of AC were described. The clinical parameters were compared between the 2 groups by t test, Mann-Whitney U test or Fisher's test. Receiver operating characteristic (ROC) curve was used to analyze the cutoff values of clinical parameters for prediction of AC. Results: The AC group included 4 boys and 3 girls aged 6.9 (4.6, 10.8) years. The non-AC group included 20 boys and 8 girls aged 5.2 (3.3, 8.4) years. All AC events occurred during the relapse of NS with infection. Seven children had gastrointestinal symptoms such as nausea, vomiting and abdominal pain. Six children had poor mental state or impaired consciousness. No significant differences in NS course, corticosteroid treatment course, corticosteroid type, steroid dosage, steroid medication interval, the proportion of gastroenteritis and fever existed between the two groups (all P>0.05). Compared with the non-AC group, the duration from the onset of the relapse of NS until hospitalization in the AC group was significantly shorter (0.2 (0.1, 0.6) vs. 1.0 (0.4, 5.0) month,U=25.50, P=0.005). The 24 h urinary total protein (UTP) level was significantly higher in the AC group (193 (135, 429) vs. 81 (17, 200) mg/kg, U=27.00,P=0.036) than the non-AC group. The serum albumin level in the AC group was significantly lower((13.1±2.1) vs. (24.5±8.7) g/L,t=-6.22,P<0.001) than the non-AC group. There were significantly higher total white blood cell counts ((26±9)×10⁹ vs. (11±5)×10⁹/L,t=4.26,P=0.004), percentage of neutrophils (0.71±0.08 vs. 0.60±0.19,t=2.56,P=0.017) and the proportion of children with C reactive protein level≥8 mg/L (3/7 vs. 0,P=0.005) in the AC group than in the non-AC group. ROC curve analysis showed that the cutoff value of 24 h UTP was 122 mg/(kg·d) with a sensitivity of 100.0% and specificity of 70.4%. The cutoff value of serum albumin was 17.0 g/L with a sensitivity of 100.0% and specificity of 82.1%. Conclusions: Gastrointestinal symptoms and poor mental state were prominent manifestations of AC in children with NS. High 24 h UTP level, low serum albumin level, high peripheral white blood cell counts, high neutrophils percentage, and high C-reactive protein level during the early stage of NS relapse may be related to the occurrence of AC in children with NS.
Nephrotic Syndrome/drug therapy* ; Humans ; Child ; Adolescent ; Male ; Female ; Gastrointestinal Diseases/diagnosis* ; Adrenal Cortex Hormones/therapeutic use* ; Nausea/chemically induced* ; Vomiting/chemically induced* ; Abdominal Pain/chemically induced* ; Mental Processes/drug effects* ; China

Objective : To analyze the epidemiological characteristics and pathogen spectrum of hand，foot mouth disease (HFMD) in Anhui province from 2015 to 2022，and to provide scientific evidence for prevention and control measures of HFMD． Methods : The surveillance data of hand，foot and mouth disease in Anhui province from 2015 to 2022 were analyzed by descriptive epidemiology. Real-time PCR was used to detect and classify HFMD samples. Results : A total of 650 590 HFMD cases were reported in Anhui province from 2015 to 2022，including 1 406 se- vere cases and 17 deaths．The annual reported incidence was 131. 45 /100 000．The epidemic features of“low incidence in odd years and high incidence in even years”were presented from 2015 to 2019．The incidence showed a continuous decline from 2020 to 2022．The monthly distribution showed the characteristics of bimodal epidemic，and the main peak was not obvious in 2020．Hefei，Fuyang，Bozhou，Chuzhou and Suzhou ranked the top five cities in terms of cumulative incidence．The age of onset was mainly distributed in children aged 5 years and below，accounting for 89. 26% of the total cases．The male to female ratio was 1. 48 ∶ 1．A total of 28 657 laboratory-confirmed cases had been reported from 2015 to 2022．EV71 cases accounted for 10. 57% ，Cox A16 cases accounted for 24. 90% ，and other enterovirus cases accounted for 64. 53%．The dominant pathogens showed dynamic changes in different years．Since 2018，the proportion of EV71 decreased significantly，and the proportion of other enteroviruses gradually increased to become the dominant pathogens．Among other enteroviruses，Cox A6 strain was dominant (80. 48% ) ． Conclusion This study suggests that the prevention and control of HFMD in Anhui province should be paid more attention from April to July and from October to December．The focus areas are the cities in northern Anhui and Hefei where the floating population is large．The focus of prevention and control is on children aged 5 years and below．Other enteroviruses have become the dominant pathogens of hand-foot-mouth disease in Anhui province，Cox A6 strain is dominant.

Objective:To determine the expression of Brahma-related gene 1 (BRG1) in cutaneous squamous cell carcinoma (cSCC) tissues and cells, and to investigate molecular mechanisms underlying the regulatory effect of its interaction with activating transcription factor 2 (ATF2) on the proliferation, migration and invasion of cSCC cells.Methods:From 2015 to 2021, 66 paraffin-embedded actinic keratosis (AK) tissue samples and 80 paraffin-embedded cSCC (including squamous cell carcinoma in situ) tissue samples were collected from the Department of Dermatology, Affiliated Hospital 2 of Nantong University, and the diagnoses of all the cases were confirmed histopathologically; at the same time, 35 paraffin-embedded normal skin tissue samples obtained by cosmetic surgery served as normal control group. Immunohistochemical staining was performed to determine the BRG1 expression in cSCC, AK, and normal skin tissues, and correlations between BRG1 expression and clinicopathological parameters of cSCC patients were analyzed. Fresh tissue samples were collected from 12 cSCC patients and 12 healthy controls, and cSCC cell lines A431 and Scl-1 and a human immortalized keratinocyte cell line HaCaT were routinely cultured; real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to determine the mRNA expression of BRG1 in tissues and cells, and co-immunoprecipitation assay and cellular immunofluorescence staining were conducted to analyze the interaction between BRG1 and ATF2. The expression of BRG1 (BRG1 siRNA1 - 5 groups) and ATF2 (ATF2-shRNA group) in A431 and Scl-1 cells was knocked down by RNA interference, and cells transfected with negative control siRNA or shNC served as controls (control siRNA group and shNC group, respectively), cell counting kit-8 (CCK8) assay, colony formation assay, cell scratch assay, and Transwell assay were conducted to evaluate effects of knocking down BRG1 and ATF2 on the proliferation, migration, and invasion of cSCC cells. Comparisons of measurement data among multiple groups were conducted using one-way analysis of variance, and multiple comparisons were conducted using Dunnett- t test. Results:Immunohistochemical staining showed that the expression intensity of BRG1 protein was significantly lower in the cSCC and AK tissues than in the normal skin tissues ( χ2 = 44.40, P < 0.001). qRT-PCR showed that the mRNA expression level of BRG1 was significantly lower in the cSCC tissues (1.345 ± 0.956) than in the normal skin tissues (2.499 ± 1.501, t = 2.25, P = 0.035), and also significantly lower in A431 and Scl-1 cells (0.041 ± 0.002, 0.026 ± 0.003, respectively) than in HaCaT cells (0.135 ± 0.033, t = 4.95, 5.73, P = 0.008, 0.005, respectively). The low expression of BRG1 was associated with tumors at sun-exposed sites ( P = 0.041), low tumor differentiation ( P = 0.001), and high Broder′s grade ( P < 0.001) in the cSCC patients. In both A431 cells and Scl-1 cells, the BRG1 siRNA1 group and BRG1 siRNA2 group showed significantly increased numbers of cell colonies, migratory cells and invasive cells, as well as cell migration rates compared with the control siRNA group (all P < 0.05). Co-immunoprecipitation assay showed that BRG1 protein could bind to ATF2 protein in A431 and Scl-1 cells, and immunofluorescence staining showed that the two proteins were co-localized; compared with the control siRNA group, the BRG1 siRNA1 group (both A431 and Scl-1 cells) and BRG1 siRNA2 group (A431 cells) both showed increased phosphorylation and activation of ATF2 (all P < 0.05) ; in both A431 cells and Scl-1 cells, the shATF2 group showed significantly decreased numbers of cell colonies (both P = 0.001), cellular proliferative activity at 24 - 96 hours (all P < 0.001), and numbers of migratory cells and invasive cells compared with the shNC group (all P ≤ 0.001) . Conclusion:BRG1 was lowly expressed in the cSCC and AK tissues, and could inhibit the proliferation, migration, and invasion of cSCC cells; ATF2 could promote the proliferation, migration, and invasion of cSCC cells; BRG1 may exert an anti-tumor effect by interacting with ATF2 protein and inhibiting phosphorylation-dependent activation of ATF2.

Lysine decarboxylase is a key enzyme involved in the upstream biosynthesis of lycopodium alkaloids (LAs) such as huperzine A, contributing to the decarboxylation of lysine to 1,5-pentanediamine (cadaverine). Three lysine decarboxylase genes (HsLDC-L1, HsLDC-L2, HsLDC-L3) were successfully cloned from Huperzia serrata using transcriptomic sequence data mining strategy combined with reverse transcription PCR. The physicochemical properties, secondary and tertiary structures, amino acid identities, and evolutionary relationship of the three LDCs were analyzed by online bioinformatics analysis platforms and DNAMAN, MEGA 7.0 software, revealing that all of these proteins had the conserved PLP binding domain and active site residues were completely conserved in LDCs. Phylogenetic analysis showed that these LDCs were located in the same branch as other known LDCs from LA-producing plants. Accordingly, the ORFs of these three HsLDCs were inserted into different expression plasmids for further expression in E. coli. However, only HsLDC-L1 was successfully expressed in E. coli BL21 (DE3) by inserting into a pCold TF vector. The recombinant protein was purified by Ni²⁺ affinity chromatography purification. HsLDC-L1 contains 469 amino acid residues, with a calculated molecular weight of 50.50 kDa. HsLDC-L1 expectedly catalyzed the decarboxylation of lysine to produce cadaverine. In addition, HsLDC-L1 can also catalyze the generation of putrescine from ornithine. However, it cannot catalyze the decarboxylation of tyrosine, phenylalanine, tryptophan and histidine. The results not only provide insight into the biosynthesis of LAs including huperzine A, but also provide a critical genetic element for the overproduction of Δ¹-piperideine and pelletierine, the essential biosynthetic precursors of LAs, using synthetic biology strategies.

Objective:To investigate the perfection and improvement of the execution of integrative medicine therapy in severe tetanus therapy, to successfully control tetanus severe spasms, autonomic dysfunction and prevent lethal side-effect of prolong and high-dosage sedative-muscle-relaxant therapy, resulted in significant reduction of mortality of tetanus.Methods:Symptoms, treatments and outcome of tetanus patients admitted to Peking University Third Hospital from 1965 to 2020 were reviewed. Patients were classified with Ablett classification. The cases of Ablett grade Ⅲ and Ⅳ were severe tetanus. The patients were divided into two groups according to whether they were treated together with traditional Chinese medicine (TCM) simultaneously during the standard tetanus treatment; the patients in the TCM group were divided into the tetanus TCM medication group and the non tetanus TCM medication group according to the medicine provided whether was in accord with the conventional tetanus TCM prescriptions. The mortality of each group was calculated. In addition, one survived and one deceased case with severe convulsion, autonomic nerve dysfunction (Ablett grade Ⅳ) were selected, combined with the treatment methods and curative effects, the types, use methods and outcomes of Chinese and Western medicine were analyzed.Results:The 46 tetanus cases were treated with Western medicine. Twenty-two of them, TCM were applied. Fifteen of the 22 cases took the TCM prescription which was accord with the conventional tetanus prescription. The mortality of the 46 cases was 21.7% (10/46). The number of non-TCM group was 24 cases, with mortality of 20.8% (5/24); 1 case was Ablett Ⅱ, 1 was Ablett Ⅲ and 3 were Ablett Ⅳ. The number of the TCM group was 22 cases, with mortality of 22.7% (5/22), 2 cases were Ablett Ⅲ, 3 were Ablett Ⅳ. The TCM prescription of these 5 deceased cases was not directed towards tetanus. The tetanus TCM medication group was 15 cases, with no mortality. Case analyses: case 1 was intubated because of severe spasms. Autonomic dysfunction occurred on the 8th day after admission. Esmolol with increasing the dosage of the sedatives and muscle relaxant, was not effective. Tetanus TCM was applied after 2 days of autonomic dysfunction happened. Autonomic dysfunction was then under controlled on the 2nd day post-TCM. She was recovery and discharged after 4 weeks. Case 2, also was intubated because of severe spasms. Autonomic dysfunction happened on the 3rd day after admission, and failed to be controlled by large-dose sedatives, muscle relaxant, and Esmolol. After 8 days of persistent autonomic dysfunction, tetanus TCM was applied and autonomic dysfunction was under controlled on the 2nd day post-TCM administration. Large dosage of muscle-relaxant was applied continuously. After 5 days' administration of TCM, the TCM was withdrew. One day after the withdrawal of TCM, respiratory and cardiac arrest happened because of the diffused bronchiole obstruction with pulmonary secretions loading.Conclusion:Based on the precise and real-time diagnosis of the state of the disease, integrative medicine therapy with an overall analysis tetanus TCM prescription, is the key of declining tetanus mortality.

OBJECTIVE: To analyze the hematological characteristics of HbE homozygotes. METHODS: Complete blood cells count and hemoglobin electrophoresis were used for phenotypic analysis of 78 cases with HbE homozygotes from Yunnan province, China. The PCR-fluorescence hybridization was used to detect the common gene mutation of thalassemia. The hematological indexes, including MCV, MCH, Hb, HbA2, HbF and HbE were statistically analyzed between groups with different sex, ages and compound α thalassemia status. RESULTS: In HbE homozygotes (HbEE), 89.5% (17/19) children presented mild to moderate microcytic hypochromic anemia, and 10.5% of them presented moderate anemia. 39.6% (19/48) of women with HbEE developed mild anemia ,while 11 cases of male with HbE homozygotes were asymptomatic. The levels of MCV and MCH in HbE homozygotes increased by co-inheritance of α thalassemia mutation. CONCLUSION The clinical phenotype of HbE homozygote shows highly heterogeneous, which is relates with age, sex and co-inheriting α-globin genotypes. In Hb EE women and children are more likely to develop mild to moderate anemia. The microcytic hypochromic anemia degree is relieved when HbEE combined with α- thalassemia.
Child ; China ; Female ; Genotype ; Hemoglobin E ; genetics ; Homozygote ; Humans ; Male ; Phenotype ; alpha-Thalassemia

Objective To identify molecular differences between EBVaGC and EBCnGC by omics to provide a basis for treatment options of EBVaGC. Methods Chromogenic in situ hybridization was used to detect EBV RNA status of surgical specimens and PDXs. Targeted capture sequencing and protein mass spectrometry were implemented to analyze the different molecules, verify the PD-L1 expression by immunochemistry in EBV positive and negative tis-sues. Results Compared with EBVnGC, the higher PIK3CA mutation rate and lower TP53 mutation rate were found in EBVaGC. Post-transcriptional regulation molecules were up-regulated and molecules associated with me-tabolism and oxidative phosphorylation were down-regulated in EBVaGC. PD-L1 expression in EBV positive PDXs was significantly higher than that in EBV negative (76.92% vs 25.0%, P＜0.05). Conclusions There is a great difference between EBVaGC and EBVnGC on genetic variation and expression, which provides the basis for further exploration of the molecular mechanism and treatment strategy of EBVaGC.

Objective:To investigate the role of pro-inflammatory cytokine IL-17 involved in the pathogenesis of cytomegalovirus hepatitis in vivo.Methods:First of all,disseminated infection model was established.Then,mice were randomly divided into 4 groups:normal control group,MCMV-infected control group,IL-17 blockade group,and isotype control group.Mice were sacrificed on day 7 after infection.The levels of IL-17 protein were detected by Western blot.Hematoxylin eosin (HE) staining was performed to evaluate the pathologic change of the liver.Serum ALT levels were detected by a Roche DPPI biochemical analyzer.The level of serum IL-17 was measured by double antibody sandwich ELISA.The expressions of mRNA of IL-17R,IFN-γand IL-10 in liver were detected by RT-PCR.Results:Compared with MCMV-infected mice and isotype control,the blockade of IL-17 inhibited the expression of IL-17 protein in liver (P＜0.05).The degree of liver damage reduced obviously.The serum ALT was significantly lower [(146±15)vs (102±11)vs (37±12),P＜0.05].The level of serum IL-17 was relatively reduced[(719.76±6.06)vs (722.1±4.62) vs (707.53 ±8.58),P＜0.05].The expression of IFN-γmRNA [(0.56± 0.06)vs (0.55±0.13)vs (0.96±0.2),P＜0.05] and IL-10 mRNA[(0.55±0.073) vs (0.51 ±0.07) vs (0.903 ±0.18),P＜0.05] increased significantly,while that of IL-17R did not change apparently[(0.81±0.16)vs (0.89±0.38) vs (0.87±0.23),P＞0.05].Conclusion:The increased expression of pro-inflammatory cytokine IL-17 is involved in the pathogenesis of immune injury in cytomegalovirus hepatitis.The blockade of IL-17 is helpful to relieve the liver damage and improve the liver function.

Objective To investigate the effect of nicotine on proliferation and chemosensitivity of A549 cells in vitro. Methods A549 cells was assessed by MTT assay to measure cell proliferation and was assessed by RT-PCR tomeasure chemosensitivity. Results 0.01~100μmol/L nicotine could promote the proliferation of A549 cells, the most marked proliferation at 1μmol/L, compared with the control group, the activity of A549 cells was increased by 1.85 times (P<0.01). When the concentration of nicotine above 1μmol/L, the proliferation of A549 cells had an decreasing tendency. When the concentration above 1000μmol/L, the proliferation of A549 cells can be inhibited. Nicotine can also reduce chemosensitivity of A549 cells to 5-FU, with the addition of nicotine, A549 cells survival rate increased significantly, the most marked at 1μmol/L, compared with the control group, the inhibitory rate of A549 cells was 9 % (P< 0.01). Nicotine significantly increased the expression level of α7 nAChR in A549 cells and decreased the expression of PTEN , in a concentration dependent manner. Compared with the control group, 1μmol/L of nicotine could increase the expression levels of α7 nAChR by 3.4 fold, and decrease the expression levels of PTEN by 60.36 % (P< 0.01). Conclusion Nicotinecan promote the growth of A549 cells and reduce chemosensitivity of A549 cells to 5-FU.