Objective:To investigate the improved performance of hepatic elastography combined with the serum biomarkers for the diagnosis of biliary atresia.Methods:A total of 193 patients with suspected biliary atresia in Beijing Children′s Hospital from March 2019 to November 2020 were consecutively collected. All patients were randomly divided into the training cohort and validation cohort at a ratio of 7∶3. LASSO regression analysis was used for the selection of the model index based on the data set from the training cohort including the serum biomarkers, demographic features (age and sex) and hepatic elastic measurement, and a diagnostic model for biliary atresia was subsequently developed by weighting on the basis of the dominance ration. The performance of the model was respectively evaluated with respect to the discrimination and calibration in each cohort.Results:Alanine aminotransferase (ALT), glutamyl transferase (GGT) and hepatic elastic measurement were selected to build the model. The area under the ROC curve of the final diagnostic model was 0.943 with a sensitivity of 90.9% and a specificity of 85.7% in the training cohort, and 0.955 in the validation cohort. Hosmer-Lemeshow test ( P=0.292, P=0.951) and calibration curves further validated its satisfactory calibration in both cohorts. As demonstrated by Delong et al.test, employing the model in the training cohort achieved the best diagnostic performance compared with using single model index ( P<0.001, P=0.016, P<0.001). In the validation cohort, the decision curve analysis showed the model had a higher overall net benefit over using hepatic elastography alone in every predicted probability. Conclusions:The diagnostic model for biliary atresia, which incorporates ALT, GGT and hepatic elastic measurement, can improve the performance of hepatic elastography with a higher clinical value.

Trichoderma reesei Rut-C30 is widely used in industrial cellulase production, and development of cellulase hyper-producer is of great importance for economic lignocellulosic biorefinery. In this study, T. reesei Rut-C30 was engineered with an artificial zinc finger proteins (AZFPs) library. Two mutants T. reesei M1 and M2 with improved cellulase production were obtained. Compared to the parent strain, the filter paper activity (FPase) of T. reesei M1 and M2 increased 100% and 53%, respectively. In addition, the total amount of extracellular protein from the M1 mutant increased 69%, whereas the endo-β-glucanase (CMCase) activity of the M2 mutant is 64% higher compared to the parental strain. Furthermore, RT-qPCR analysis showed that the major cellulase genes exhibited significantly increased expression in both mutants, but different patterns were observed in the two mutants. On the other hand, the cellulase transcriptional repressor ace1 was down-regulated in both mutants, but the transcription level of the activator xyr1 was only up-regulated in the strain M1. These results demonstrated that different AZFPs exert diverse regulatory mechanisms on cellulase production in T. reesei. Analysis of the target genes of AZFPs from T. reesei M1 and M2 will not only benefit further exploration of the regulatory mechanisms of cellulase biosynthesis in T. reesei, but also enable development of cellulase hyper-producing strains by metabolic engineering.
Cellulase ; Gene Library ; Transcription Factors ; Trichoderma ; Zinc Fingers

Objective To observe the clinical efficacy of radiofrequency ablation for treatment of discogenic lumbar spinal nerve posterior branch neuralgia after vertebral column endoscope operation. Methods Thirty-six patients with discogenic lumbar spinal nerve posterior branch neuralgia after vertebral column endoscope surgery admitted to the Affiliated Hospital of Logistics University of People's Armed Police from December 2011 to December 2017 were enrolled. According to difference in therapeutic methods, they were randomly divided into two groups, 18 cases in each group. The radiofrequency ablation group was treated with X-ray imaging guided lumbar spinal nerve posterior branch radiofrequency thermo-coagulation; the drug group received oral diclofenac sodium conservative treatment, 75 mg twice daily for 3 weeks. Both groups were followed up for 6 months, visual analogue scores (VAS) were used to evaluate the pain before and after treatment, the Oswestry dysfunction index was used to assess the degree of lumbar function recovery, and the surgical complications and adverse drug reactions were observed. Results The VAS scores in the two groups were similar before treatment; after treatment for 1 month, the VAS scores in both groups were significantly lower than those before treatment (radiofrequency ablation group: 1.83±0.71 vs. 5.67±0.77; drug group: 2.22±0.43 vs. 5.28±0.67, both P < 0.05); after treatment for 3 months and 6 months, the VAS scores were increased gradually, however, the scores of radiofrequency ablation group were significantly lower than those in the drug group (3 months was 2.00±0.59 vs. 3.39±0.70, 6 months was 2.17±0.51 vs. 3.61±0.50, both P < 0.05), moreover, the excellent and good rates of postoperative pain efficacy and of Oswestry dysfunction index improvement in the radiofrequency ablation group were significantly higher than those in the drug group [excellent and good rates of postoperative pain efficacy: 94.44% (17/18) vs. 22.22% (4/18), excellent and good rates of Oswestry dysfunction index improvement: 77.78% (14/18) vs. 44.44% (8/18), both P < 0.05]. There were no complications of infection and spinal nerve anterior branch injury in the radiofrequency ablation group, and 6 patients in the drug group presented mild gastric discomfort, which was relieved after symptomatic treatment. Conclusion The radiofrequency ablation is an effective method for treatment of discogenic lumbar neuralgia after vertebral column operation, compared with the conservative therapy, the ablation is more effective to relieve pain for a long time, promote the recovery of neural function, and the operation is safe with very few adverse reactions.

Kluyveromyces marxianus, as unconventional yeast, attracts more and more attention in the biofuel fermentation. Although this sort of yeasts can ferment pentose sugars, the fermentation capacity differs largely. Xylose and arabinose fermentation by three K. marxianus strains (K. m 9009, K. m 1911 and K. m 1727) were compared at different temperatures. The results showed that the fermentation performance of the three strains had significant difference under different fermentation temperatures. Especially, the sugar consumption rate and alcohol yield of K. m 9009 and K. m 1727 at 40 ℃ were better than 30 ℃. This results fully reflect the fermentation advantages of K. marxianus yeast under high-temperature. On this basis, five genes (XR, XDH, XK, AR and LAD) coding key metabolic enzymes in three different yeasts were amplified by PCR, and the sequence were compared by Clustalx 2.1. The results showed that the amino acid sequences coding key enzymes have similarity of over 98% with the reference sequences reported in the literature. Furthermore, the difference of amino acid was not at the key site of its enzyme, so the differences between three stains were not caused by the gene level, but by transcribed or translation regulation level. By real-time PCR experiment, we determined the gene expression levels of four key enzymes (XR, XDH, XK and ADH) in the xylose metabolism pathway of K. m 1727 and K. m 1911 at different fermentation time points. The results showed that, for thermotolerant yeast K. m 1727, the low expression level of XDH and XK genes was the main factors leading to accumulation of xylitol. In addition, according to the pathway of Zygosaccharomyces bailii, which have been reported in NCBI and KEGG, the xylose and arabinose metabolic pathways of K. marxianus were identified, which laid foundation for further improving the pentose fermentation ability by metabolic engineering.

BACKGROUND:Oligodendrocytes are mostly differentiated from oligodendrocyte precursor cel s. A suitable medium and cel seeding density have a significant impact on the process of the isolation of oligodendrocyte precursor cel s to obtain oligodendrocytes. OBJECTIVE:To explore the optimization of oligodendrocyte culture conditions. METHODS:Oligodendrocyte precursor cel s isolated from the newborn rats 48 hours after birth were cultured in DMEM/high glucose medium or DMEM/F12 medium using seeding densities of 2×104 cel s/cm2, 4×104 cel s/cm2, 8×104 cel s/cm2, 16×104 cel s/cm2, 32×104 cel s/cm2, and 64×104 cel s/cm2, respectively. Oligodendrocyte precursor cel s were induced to differentiate into oligodendrocytes at 72 hours after cel adhesion. Morphology of differentiated oligodendrocyte precursor cel s were observed under a light microscope, and the differentiation results were identified by immunofluorescence staining after 7-day induced differentiation. RESULTS AND CONCLUSION:Morphology of oligodendrocyte precursor cel s were recognized when cultured in DMEM/high glucose medium or DMEM/F12 medium using seeding densities of 2×104 cel s/cm2, 4×104 cel s/cm2, and 8×104 cel s/cm2, respectively. Immunofluorescence staining showed that myelin basic protein-positive cel s were found after 7-day induced differentiation, and the positive cel number were 16.40±3.30, 49.95±2.33, and 76.95±4.86 in DMEM/F12 medium, and 12.65±2.53, 32.10±1.17, and 54.05±1.56 in DMEM/high glucose medium (P<0.05). These findings indicate that DMEM/F12 medium is more suitable for culturing oligodendrocyte precursor cel s compared with DMEM/high glucose medium to some extent. The number of differentiated oligodendrocytes was gradual y increased with the enhanced seeding density of oligodendrocyte precursor cel s, and the seeding densities from 4×104 to 8×104 cel s/cm2 were appropriate for the observation of cel morphology.

Objective To analyze the etiology of fever of unknown origin (FUO).Methods A total of 372 patients with FUO who hospitalized in Capital Medical University Affiliated Beijing Friendship Hospital were retrospectively analyzed from January 2003 to August 2013.All the patients were divided into two groups:group A (January 2003-December 2007) and group B (January 2008-August 2013).Diagnosis rate,duration of hospitalization (days) and time to diagnosis between the two groups were artificially compared.Results Of the 372 FUO cases,336 were positively diagnosed with a diagnosis rate of 90.3％.Infectious diseases were still the primary causes of FUO (60.2％),including 72 cases (32.1％) of tuberculosis.Connective tissue diseases accounted for 12.9％ of the FUO cases,malignancies were 8.3％,and miscellaneous diseases were 8.9％.Yet thirty six patients (9.7％) could not be confirmed until they were discharged from hospital.The duration of fever in patients with malignancies was longer than that with infectious diseases [60.0 (30.0,90.0) days vs 30.0 (20.0,60.0) days,P =0.003].Time to diagnosis of connective tissue disease and malignancies was longer than infectious diseases [(12.0 (7.3,18.8) days and 11.0 (7.0,18.0) vs 5.0 (3.0,8.0) days,both P values =0.000].The duration of hospitalization in group A was longer than that of group B [17.0(12.0,30.0) days vs 14.0(10.0,20.0) days,P =0.000].The diagnosis rate and time to diagnosis of group A were similar with those of group B.The proportion of connective tissue diseases in group A was higher than group B(18.1％ vs 9.2％,x2 =6.201,P =0.013).The proportion of infectious disease,malignancies and miscellaneous diseases was not significantly different between the two groups.Conclusions Infectious diseases are the major causes of FUO,and the most common cause is tuberculosis.Connective tissue diseases and malignancies are the second and third causes of FUO.The duration of fever and time to diagnosis are significantly different between the different origins.

Breeding of robust industrial Saccharomyces cerevisiae strains with high ethanol tolerance is of great significance for efficient fuel ethanol production. Zinc finger proteins play important roles in gene transcription and translation, and exerting control on the regulation of multiple genes. The sequence and localization of the zinc finger motif can be designed and engineered, and the artificial zinc finger protein can be used to regulate celluar metabolism. Stress tolerance of microbial strains is related to multiple genes. Therefore, it is possible to use artificially-designed zinc finger proteins to breed stress tolerant strains. In this study, a library containing artificial zinc finger protein encoding genes was transformed into the model yeast strain S288c. A recombinant strain named M01 with improved ethanol tolerance was obtained. The plasmid in M01 was isolated, and then transformed into the industrial yeast strain Sc4126. Ethanol tolerance of the recombinant strain of Sc4126 were significantly improved. When high gravity ethanol fermentation using 250 g/L glucose was performed, comparing with the wild-type strain, fermentation time of the recombinant strain was decreased by 24 h and the final ethanol concentration was enhanced by 6.3%. The results of this study demonstrate that artificial zinc finger proteins are able to exert control on stress tolerance of yeast strains, and these results provide basis to construct robust industrial yeast strains for efficient ethanol fermentation.
Adaptation, Physiological ; drug effects ; Drug Resistance, Fungal ; genetics ; Ethanol ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Industrial Microbiology ; Mutation ; genetics ; Peptide Library ; Saccharomyces cerevisiae ; genetics ; growth & development ; Zinc Fingers

Ethanol tolerance is related to the expression of multiple genes, and genome-based engineering approaches are much more efficient than manipulation of single genes. In this study, ultraviolet (UV) mutagenesis, dielectric barrier discharge (DBD) air plasma mutagenesis, and artificial transcription factor (ATF) technology were adopted to treat an industrial yeast strain S. cerevisiae Sc4126 to obtain mutants with improved ethanol tolerance. Mutants with high ethanol tolerance were obtained, and the ratio of positive mutants was compared. Among the three approaches, the rate of positive mutation obtained by ATF technology was 10- to 100-folds of that of the two other methods, with highest genetic stability, suggesting the ATF technology promising for rapid alteration of phenotypes of industry yeast strains for efficient ethanol fermentation.
Adaptation, Physiological ; drug effects ; Drug Resistance, Fungal ; genetics ; Ethanol ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Industrial Microbiology ; methods ; Mutagenesis ; Saccharomyces cerevisiae ; drug effects ; genetics ; growth & development

Ethanol fermentation from Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae strains expressing the inulinase gene (inu) from Kluyveromyces marxianus was investigated. The inu native and pgk promoters were used to drive the expression of the inu gene, and the inulinase was expressed as an extracellular enzyme. All positive clones (confirmed by PCR) were able to express inulinase as measured by enzyme activity in the culture supernatant, among which two clones HI6/6 and HPI6/3 were selected, and their inulinase activity and ethanol fermentation performance were compared with their wild type. The inulinase activities of 86 and 23.8 U/mL were achieved, which were 4.6-fold and 1.5-fold higher than that of the wild type. Furthermore, ethanol fermentation was carried out with the recombinants and medium containing 200 g/L raw Jerusalem artichoke meal, and ethanol concentrations of 55 g/L and 52 g/L were obtained, with ethanol yields of 0.495 and 0.453, respectively, equivalent to 96.9% and 88.6% of the theoretical value.
Ethanol ; metabolism ; Fermentation ; Glycoside Hydrolases ; genetics ; secretion ; Helianthus ; metabolism ; Kluyveromyces ; genetics ; Metabolic Engineering ; methods ; Plant Tubers ; metabolism ; Recombination, Genetic ; Saccharomyces cerevisiae ; enzymology ; genetics

Directed evolution of transcription factors can be employed to effectively improve the phenotypes which are controlled by multiple genetic loci. In this study, we used error-prone PCR for the directed evolution of SPT3, which is the component of yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex responsible for the transcription of stress-related genes, and studied its effect on the improvement of ethanol tolerance. Mutant library was constructed by ligating the error-prone PCR products with a modified pYES2.0 plasmid, and the expression plasmids were subsequently transformed to yeast industrial strain Saccharomyces cerevisiae 4126. One mutant strain M25 showing superior growth in presence of 10% ethanol was selected. M25 produced 11.7% more ethanol than the control strain harboring the empty vector when 125 g/L glucose was used as substrate. This study revealed that SPT3 is an important transcription factor for the metabolic engineering of yeast ethanol tolerance.
Directed Molecular Evolution ; methods ; Drug Resistance, Fungal ; Drug Tolerance ; Ethanol ; metabolism ; pharmacology ; Industrial Microbiology ; methods ; Saccharomyces cerevisiae ; drug effects ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; Trans-Activators ; genetics ; Transcription Factors ; genetics