Objective To investigate the molecular basis of ABO gene in a patient with serologic ABO blood group discrepancy.Methods Serologic blood group identification,Coombs' test and antibody screening were detected with DG Gel Confirm cards,Neutral cards,Coombs cards by WADiana/8XT Compact Analyzer (from Diagnostic Grifols,S.A).The enhancer,promoter,exon 1 ～ 7 and their adjacent intron region of ABO gene were amplified by using polymerase chain reaction (PCR) method.Results The patient's red blood cells was determined as weak B phenotype showing two groups in gel and mixed field in tube with monoclonal anti-B,and A phenotype with monoclonal anti-A.DNA sequencing showed nine variants in ABO gene.One heterozygous variation in exon 6 (297A＞G) and eight heterozygous variations in exon 7 (467C＞T,526C ＞G,657C＞T,703G＞A,796C＞A,803G＞C,829G＞T 930G＞A) were identified and 829G＞T was the novel variant.Compared with Blood Group Antigen Gene Mutation Database,genotype of the patient was weak expression of A102/B101.Conclusion The novel variation of B allele is the main reason of Bweak phonetype in A102/B101 genotype.Serological and molecular biological detection help to understand the characteristics of blood group phenotype and genotype,provide the guidance for clinical transfusion strategies.

Objective To determine the detergent-enzymatic cycles and evaluate the biomechanical characteristics as well as extracellular matrix integrity of the decellularized tracheal scaffold in rabbit.Methods Forty tracheal segments were harvested from New Zealand white rabbits.Thirty-five of tracheas were subjected to a detergent-enzymatic method of decellularization for 1/3/5/6/7/8/9 cycles,respectively,and other five were stored in phosphate-buffered saline at 4℃ as a control.Comparative examinations were performed by the macroscopic view,histological view(hematoxylin and eosin stain,Movat Pentachrome stain,4-6-diamidino-2-phenylindole),scanning electron microscope (SEM) and biomechanical properties between decellularized groups and control group.Results After 7 detergent-enzymatic cycles,almost complete decellularized tracheae,retaining the hierarchical and mechanical properties of the native tissues,could be obtained.Histological and molecular biology analysis demonstrated that all cellular components and nuclear material were removed.SEM analysis revealed that the decellularized matrices retained the hierarchical structures of native trachea,and biomechanical tests showed that decellularization approach did not led to any influence on tracheal morphological and mechanical properties.Immunofluorescence analysis show a significant reduction of nuclear material in decellularized tracheas (P ＜ 0.05).Conclusion In conclusion,this work suggests that 7 cycles of the modified DEM generates a bioengineered rabbit tracheal matrix that is structurally and mechanically similar to native trachea which could be a better selection for tracheal reconstruction with tissue engineering method.

Objective:To investigate the risk factors for postoperative early recurrence of patients with single large hepatocellular carcinoma (HCC) (tumor diameter≥5cm).Methods:Clinical data of 135 single large HCC patients who underwent radical resection from Jan 2015 to Sep 2020 in Ningbo Medical Centre Lihuili Hospital were analyzed.Results:Seventy-five HCC patients suffered recurrence,among those 42 patients had early recurrence(within 12 months). Multivariate analysis showed that alpha-fetoprotein (AFP)≥400 ng/ml ( OR=3.510,95% CI: 1.528-8.064; P=0.003) and tumor microvascular invasion (MVI) ( OR=2.769,95% CI: 1.143-6.706; P=0.024) were independent risk factors for early recurrence of single large hepatocellular carcinoma. Survival analysis showed that early recurrence risk factors significantly reduced recurrence free survival (RFS)(AFP≥400 ng/ml, χ 2=23.038, P<0.001; MVI positive , χ 2=10.554, P=0.001) and overall survival (OS) (AFP≥400 ng/ml, χ 2=14.336, P<0.001; MVI positive, χ 2=10.481, P=0.001) in single large hepatocellular carcinoma patients. Conclusion:AFP≥400 ng/ml and MVI positive are independent risk factors for postoperative early recurrence in single large hepatocellular carcinoma patients.

Objective: To investigate the in vitro interaction of amphotericin B (AmB) and fluconazole (FLC) at different time points and provide a reference for clinical combined treatment therapy of polyenes and azoles. Methods: Candida albicans ATCC SC5314 was used in the study. The minimum inhibitory concentration (MIC) of antifungal drugs was determined using the double microdilution broth method. The same amount of DMSO and low concentration drugs were added to the DMSO treatment group at different time points (0, 2, 4, 6 h) to determine whether the solvent background environment affected the growth of Candida albicans. In the experimental group, to observe the effect of low concentration AmB on the antifungal effect of FLC, the experimental group was administered a low concentration of AmB (0.25 μg/mL or 0.125 μg/mL) added to FLC at different time points (0, 2, 4, 6 h), and the same amount of DMSO was added to FLC at different time points in the single drug control group. In the experimental group, to observe the effect of low concentration of FLC on the antifungal effect of AmB, the experimental group was administered a low concentration of FLC (0.06 μg/mL or 0.03 μg/mL) in AmB at different time points (0, 2, 4, 6 h), and the same amount of DMSO was used at different time points as the single drug control group. In the solvent group, the same amounts of DMSO and low concentration drugs were added at different time points. After resuscitation, the colony growth of each solvent control group, single-drug control group and experimental group was observed to evaluate the interaction between drug concentration and time. Compared with the AmB single-drug control group, there was no significant change in the experimental group with added low concentrations of FLC at 0 h (F=0.27, P=0.775), which was 1.74-1.93 times that of the control group at 2-4 h (P < 0.001), and there was no significant difference in colony count after 6 h (P > 0.05). Results: Under the treatment of FLC at an inhibitory concentration (0.25 μg/ml), adding low concentration AMB did not affect the antifungal effect of FLC, and the multiple of colony count differences were not significant (P > 0.05). Conclusion The interaction between AmB and FLC was time-dependent. At the early stage (0 h), the interaction effect between fluconazole and amphotericin B was not clear. The fungicidal effect of AmB could be weakened when FLC was supplied at 2-4 h, and the effect of FLC on AmB was absent after 6 h.

Candida albicans is the most abundant fungal species in oral cavity. As a smart opportunistic pathogen, it increases the virulence by switching its forms from yeasts to hyphae and becomes the major pathogenic agent for oral candidiasis. However, the overuse of current clinical antifungals and lack of new types of drugs highlight the challenges in the antifungal treatments because of the drug resistance and side effects. Anti-virulence strategy is proved as a practical way to develop new types of anti-infective drugs. Here, seven artemisinins, including artemisinin, dihydroartemisinin, artemisinic acid, dihydroartemisinic acid, artesunate, artemether and arteether, were employed to target at the hyphal development, the most important virulence factor of C. albicans. Artemisinins failed to affect the growth, but significantly inhibited the hyphal development of C. albicans, including the clinical azole resistant isolates, and reduced their damage to oral epithelial cells, while arteether showed the strongest activities. The transcriptome suggested that arteether could affect the energy metabolism of C. albicans. Seven artemisinins were then proved to significantly inhibit the productions of ATP and cAMP, while reduced the hyphal inhibition on RAS1 overexpression strain indicating that artemisinins regulated the Ras1-cAMP-Efg1 pathway to inhibit the hyphal development. Importantly, arteether significantly inhibited the fungal burden and infections with no systemic toxicity in the murine oropharyngeal candidiasis models in vivo caused by both fluconazole sensitive and resistant strains. Our results for the first time indicated that artemisinins can be potential antifungal compounds against C. albicans infections by targeting at its hyphal development.
Animals ; Mice ; Candida albicans ; Candidiasis, Oral/drug therapy* ; Antifungal Agents/pharmacology* ; Hyphae ; Artemisinins/pharmacology*