Objective To determine the best method to detect thermo-acidophilic bacteria in apple juice concentrate. Methods L 16 (4 5) orthogonal test was applied to analyse the four factors, including culture medium, ways of heat shock, dilution degrees of AJC and pH value. The results were comprehensively scored according to the number of colonies. Results The isolated bacteria were polymorphous. A better method was as follows: after being diluted to 1∶10, the AJC was submitted to a heat shock at 80 ℃ for 10 min, and then spread on k agar with a pH value of 3.5. Samples were incubated at 45 ℃ for 3 days, and then enumerated. Conclusion This method can considerably improve the sensitivity of detecting thermo-acidophilic bacteria in apple juice concentrate.

It remains controversial whether tumor necrosis factor (TNF)-α antagonism is effective for asthma. This meta-analysis was performed to evaluate efficacy of TNF-α antagonism in treatment of patients with asthma. MEDLINE, EMBASE, LILACS, and CINAHL databases were searched for English-language studies published through January 3, 2010. Randomized-controlled trials comparing TNF-α antagonism with control therapy were selected. For each report, data were extracted in relation to the outcomes analyzed: asthma exacerbation, asthma quality of life questionnaire scores, and forced expiratory volume in 1 second. Four assessable trials were identified including 641 patients with asthma. TNF-α antagonism therapy was superior to control therapy in preventing exacerbations in asthmatics [pooled odds ratio 0.52 (95% confidence interval 0.29-0.88), P=0.02]; however, there was a nonsignificant reduction in asthma quality of life questionnaire scores [0.23 (0 to 0.47), P=0.05], forced expiratory volume in 1 second [0.03, (-0.14 to 0.10), P=0.74] when analyzed using standardized mean differences. TNF-α antagonism was superior to control chemotherapy in terms of asthma exacerbation, but not asthma quality of life questionnaire scores or forced expiratory volume in 1 second.

OBJECTIVE:To investigate the effects of TanshinoneⅡA sodium sulfonate injection on levels of P-selectin,glial fi-brillary acidic protein (GFAP),vascular endothelial growth factor (VEGF) and neurological function in patients with acute cere-bral infarction. METHODS:A total of 114 patients with acute cerebral infarction selected from Lianyungang First People's Hospi-tal during Apr. 2013-Apr. 2016 were divided into control group and observation group according to random number table,with 57 cases in each group. Control group was given routine treatment. Observation group was additionally given Tanshinone ⅡA sodium sulfonate injection 40 mg 0.9％ sodium chlonride injection 250 mL,ivgtt,qd. A treatment course lasted for 7 d,and both received 2 courses of treatment. NIHSS scores,the levels of serum P-selectin,GFAP and VEGF were compared between 2 groups before treatment and after 7,14 d of treatment. The occurrence of ADR was also compared. RESULTS:Before treatment,there was no statistical significance in above indexes between 2 groups(P>0.05). Compared to before treatment,NIHSS score,the levels of se-rum P-selectin and GFAP in 2 groups were decreased significantly after 7,14 d of treatment,while the serum level of VEGF was increased significantly. These indexes of 2 groups after 14 d of treatment were significantly better than 7 d of treatment,except for NIHSS score. Above indexes of observation group was significantly better than those of control group during corresponding period, with statistical significance (P<0.05). No obvious ADR was found in 2 groups. CONCLUSIONS:For acute cerebral infarction, Tanshinone ⅡA sodium sulfonate injection can significantly reduce the levels of serum P-selectin and GFAP,improve VEGF level and promote the recovery of neurological damage with good safety.

Objective To investigate the expression of a proliferation-inducing ligand (APRIL) in urothelial carcinoma tissue and its clinical significance. Methods We investigated the expression of APRIL mR-NA in urothelial carcinoma tissue of 33 patients in urothelial carcinoma in Cangzhou Central Hospital from July 2013 to March 2015, Among of them, 23 cases of adjacent pericancerous tissues were detected by qRT-PCR. Immunohistochemistry and western blot were used to detect the APRIL protein expression level in urothelial carci-noma tissue and adjacent pericancerous tissues. We analyzed the relationship between APRIL expression and clinical pathology in patients with urothelial carcinoma by statistical methods. Results The qRT-PCR revealed that the expression of APRIL in urothelial carcinoma tissue was significantly higher than that in the adjacent peri-cancerous tissues (P < 0.05). Immunohistochemistry and western blot showed that the expression of APRIL in urothelial carcinoma tissue was higher than that in the adjacent pericancerous tissues. Correlation analysis found that the high expression of APRIL in urothelial carcinoma tissue was not significantly related to gender , age , size of tumor, lymph node metastasizing or not, and clinical stages (P > 0.05). Conclusion The APRIL is a high abnormal expression in urothelial carcinoma tissues , and maybe related to the occurrence and development of urothelial carcinoma.

_ Objective_ To evaluate the efficacy of unilateral subtotal adrenalectomy in the treatment of bilateral adrenal solitary neoplasma causing Cushing's syndrome and to elaborate the therapeutic principle. Methods From 2007 to 2013, a total of ten patients were diagnosed with Cushing's syndrome caused by bilateral solitary adrenal neoplasma. We compared patients'clinical symptoms, hormone profiles, biochemical and metabolic parameters, and imaging data before and after the surgery. Five of them chose the optimal neoplasma based on the lateralization ratio of adrenal venous sampling result and the other 5 patients chose the optimal neoplasma based on the diameter of the mass reflected by the computed tomography result and were then operated. Results After the unilateral subtotal adrenalectomy,the24-hour urinary free cortisol decreased significantly(P<0.05)and the midnight serum cortisol level also significantly reduced(P<0. 01). Plasma adrenocorticotropic hormone level increased significantly(P<0. 01). Nine patients of them did not need contralateral adrenalectomy and one patient received contralateral adrelectomy because of the remnant of Cushingnoid symptoms. Conclusion Unilateral subtotal adrenalectomy is an effective and safe way to treat Cushing's syndrome caused by bilateral solitary neoplasma.

Objective:To detect the expression of Shank1 protein in renal cell carcinoma (RCC), to investigate its difference between the tumor and carcinoma adjacent tissue, and to analyze its correlation with RCC clinicopathological characteristics and prognosis. Methods:The renal carcinoma and carcinoma adjacent tissues of 120 patients were selected from Cangzhou Central Hospital and Ji'nan Central Hospital from May 2008 to December 2014. The expression level of Shank1 was determined by immunohistochemistry and Western blot. Statistical analysis was performed to determine the relationship between the expression of Shank1 and the clinicopathological features of RCC patients. Results:Results of immunohistochemistry showed that the expression level of Shank1 in renal cancer tissue was significantly higher than that in carcinoma adjacent tissue, and the difference was statistically significant (P<0.05). Western blot results showed that the expression level of Shank1 protein in renal cancer tissue was also significantly higher than in carcinoma adjacent tissue. Correlation analysis found that the high expression level of Shank1 in renal cancer tissue was not significantly related to gender, age, tumor size, and TNM stage, but was significantly associated with the histological differentiation of RCC (P<0.05). Conclusion:Shank1 is abnormal y expressed in RCC renal cancer tissues and is correlated with the histological differentiation of RCC.

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe²⁺ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.
Arabidopsis/metabolism* ; Rhododendron/metabolism* ; Amino Acid Sequence ; Anthocyanins/metabolism* ; Phylogeny ; Flavonoids/metabolism* ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism*

Isopeptide bond-mediated molecular superglue is the irreversible covalent bond spontaneously formed by the side chains of lysine (Lys) and asparagine/aspartic acid (Asn/Asp) residues. The peptide-peptide interaction is specific, stable, and can be achieved quickly without any particular physicochemical factor. In the light of recent progress by domestic and foreign researchers, here we summarize the origin, assembly system and mechanism of isopeptide bond reaction, as well as the molecular cyclization and protein topological structure mediated by it. The prospect for its application in synthetic vaccine, hydrogel and bacterial nanobiological reactor is further discussed.
Cyclization ; Lysine ; Peptides ; chemistry ; Proteins

Objective To investigate the role and mechanism of B7-H4, a negative costimulatory molecule, in mediating the immunomodulatory effects of mesenchymal stem cells C3H10T1/2 (C3H10) on T cell polarization. Methods The lentiviral vectors that carried the shRNA targeting mouse B7-H4 were transfected into mouse mesenchymal stem cells (C3H10-B7-H4). The cells were co-cultured with PHA-acti-vated mice spleen lymphocytes before and after the transfection. ELISA was performed to detect the concen-trations of cytokines in supernatants of cell culture in order to elucidate the effects of B7-H4 expressed by C3H10 on T cell polarization. A mouse model of experimental allergic encephalitis (EAE) was established. Fifty C57BL/6 mice were divided into five groups including control group, EAE group, C3H10 group (injec-ting EAE mice with C3H10 cells), C3H10-NC group ( injecting EAE mice with C3H10-NC cells) and C3H10-B7-H4 group (injecting EAE mice with C3H10-B7-H4 cells). ELISA was performed to detect the soluble form of IL-2, IL-17, IFN-γ and IL-4 in plasma samples. Results Knocking down the B7-H4 gene with shRNA significantly decreased the expression of B7-H4 on C3H10 cells, which weakened the inhibitory effects of C3H10 cells on the secretion of IL-2, IL-17 and IFN-γ by spleen lymphocytes. The therapeutic effects of C3H10-B7-H4 cells on mice with EAE were weakened after silencing the B7-H4 gene expression, which was manifested as higher nerve function score and earlier onset and bring forwarded peak time of EAE than those of the C3H10 group. Treating EAE mice with C3H10-B7-H4 cells was less efficient in inhibiting the expression of IL-2, IL-17 and IFN-γin plasma. However, knocking down the B7-H4 gene had no signif-icant effect on the expression of IL-4 in terms of treating EAE with C3H10 cells. Conclusion The co-inhib-itor molecule B7-H4 expressed on C3H10 cells mediated the treatment of EAE with C3H10 cells by regula-ting Th1 and Th17 effector T cells.

Objective: To evaluate the effect of low-dose hormone replacement therapy（HRT）on menopausal-related symptoms in young patients with cervical squamous cell carcinoma. Methods: From March 2016 to September 2018,eighty patients aged 45 years below with cervical squamous cell carcinoma of stage Ⅰ-Ⅱ and iatrogenic menopause were recruited,who were diagnosed at Zhejiang Cancer Hospital and Women's Hospital Affiliated to Medical College of Zhejiang University. Fourty of them performed low dose HRT（1 mg estradiol valerate a day or 1.25 mg tibolone a day）,twenty performed standard dose HRT（1 mg estradiol valerate and 1.25 mg tibolone a day）,another twenty do not perform HRT as a control group. The levels of follicle stimulating hormone（FSH）,estradiol（E2）,menopausal symptoms（the improved Kupperman score）and incidence of side effects were assessed before and 1,3,6 months after the intervention. Results: There were significant differences in E2 levels,FSH levels and improved Kupperman scores among the low dose group,the standard dose group and the control group（all P<0.05）. With the extension of intervention time,E2 levels in the low dose group and the standard dose group increased first and then decreased,FSH levels and improved Kupperman scores decreased（all P<0.05）. Compared with the control group,E2 levels,FSH levels and improved Kupperman scores in the standard dose group changed more significantly（all P<0.05）. E2 and FSH levels in the low dose group changed less than that in the standard dose group（all P<0.05）,while improved Kupperman scores was close to that in the standard dose group. The incidence rate of side effects in the low dose group was 2.50%,which was lower than 20.00% in the standard dose group（P<0.05）. Conclusion For young patients with cervical squamous cell carcinoma,using low dose HRT may less affects E2 and FSH levels than using the standard dose,but could achieve similar effects in treatment of menopausal-related symptoms.