[Expression, purification and functional validation of phage depolymerase from hypervirulent Klebsiella pneumoniae serotype K1].

Objective: To express and purify the phage depolymerase from hypervirulent Klebsiella pneumoniae (hvKp) serotype K1 and validate its function. Methods: Phage that infected serotype K1-type hvKp was isolated from hospital sewage. The biology and morphology of the phage were determined by plaque assay and transmission electron microscopy. The whole genome of the phage was sequenced by the Illumina HiSeq 2500 platform. The presence of depolymerase was determined by observing the plaque halo. Bioinformatic analysis and prokaryotic protein expression system were further used to predict and identify phage depolymerase. The depolymerase gene fragment was obtained by PCR and cloned into the pET28a expression vector, and the expression and purification of the depolymerase were completed in strain BL21. The depolymerase activities on the capsular polysaccharide of serotype K1-type hvKp clinical isolates were detected by plaque assay and low-speed centrifugation assay. Results: A lytic phage (phiA2) that infected serotype K1-type hvKp clinical isolate was isolated from hospital sewage. It was typical of the Caudovirales order and Autographiviridae family, and its whole genome was 43 526 bp in length and contained 51 coding domain sequences. The phage phiA2-derived depolymerase phiA2-dep was predicted, expressed and purified. The plaque assay and low-speed centrifugation assay indicated that the depolymerase phiA2-dep had good lytic activity on the capsular polysaccharide of serotype K1-type hvKp clinical isolates. Conclusion: Depolymerase phiA2-dep can specifically degrade the capsular polysaccharide of serotype K1-type hvKp, which has potential application value in treating bacterial infection.

[The effect of wza gene deletion in Klebsiella pneumoniae on capsule formation and bacteriophage sensitivity].

Objective: To investigate the effects of wza gene deletion in Klebsiella pneumoniae on capsule formation ability and bacteriophage sensitivity. Methods: The wza deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination. The growth curves of W14 and Δwza were detected by measuring the optical density OD600. In order to analyze the effect of gene wza on bacterial capsule formation, wild-type strain W14 and Δwza mutant strain were detected by transmission electron microscope, and their capsule contents were measured by quantifying the uronic acid contents. The plaque assay was used to detect bacterial sensitivity to bacteriophage in wild-type strain W14 and Δwza mutant strain. The t test was used to compare whether there were differences in the contents of uronic acid in the capsules of wild-type strain W14 and Δwza mutant strain. Results: The PCR results revealed that the Δwza mutant strain was successfully constructed. Compared with wild-type strain W14, the growth curves of Δwza on the solid plates demonstrated a slightly slower growth. However, no difference in growth was observed among wild-type strain W14 and Δwza mutant strains in LB broth. The transmission electron microscope results showed that wza gene deletion resulted in the loss of capsule in bacteria. The uronic acid content assay suggested that the capsule content was significantly decreased in Δwza mutant strain (45.963±2.795) µg/ml compared with wild-type strain W14 (138.800±5.201) µg/ml. There was a statistical difference between the two groups (t=27.233, P<0.001). The plaque assay indicated that bacteria lost its sensitivity to bacteriophage when gene wza was deleted. Conclusion: Deletion of the wza gene impairs bacterial capsule formation ability and can affect bacterial sensitivity to bacteriophage phiW14.

[Predictive value of two-dimentional shear wave elastography in posthepatoectomy liver failure].

Objective: To investigate the value of two-dimensional shear wave elastography (2D SWE) combined with clinical biochemical data in predicting posthepatoectomy liver failure (PHLF) in patients with hepatocellular carcinoma (HCC). Methods: A total of 274 HCC patients who underwent hepatectomy in Zhongshan Hospital Fudan University from January 2015 to January 2016 were retrospectively collected, including 235 males and 39 females, age 19-80 (56±11) years. All patients were confirmed to be HCC by postoperative pathology. The preoperative 2D SWE examination, laboratory examination results and intraoperative indicators were analyzed. According to the occurrence of PHLF after surgery, single factor analysis and multiple logistic regression analysis were performed on the above indicators to obtain a binary logistic regression model, and evaluate the diagnostic effect of the model on PHLF. In addition, 103 HCC patients from October 2019 to January 2020 were retrospectively collected as an external validation set, including 89 males and 14 females, age 23-80 (55±11) years old. Results: The liver stiffness measurement (LSM) obtained from 2D SWE, INR and Laminin (LN) were independent predictors of PHLF. The formula of prediction model PM=-15.451+0.095×LSM+11.7×INR+0.012×LN was obtained by combining above three factors. The area under the curve (AUC) of PHLF was 0.82, which was higher than that of end-stage liver disease model (MELD) score and Child-Pugh grading diagnosis of PHLF. The AUC of PHLF predicted by PM in the external validation group was 0.81. Conclusion: 2D SWE is helpful for clinicians to evaluate liver reserve function preoperatively and to predict the occurrence of PHLF in patients with HCC.

[Diagnostic accuracy of liver and spleen stiffness by two dimensional shear wave elastography for portal hypertension in hepatitis B-related cirrhosis].

Objective: To assess the diagnostic accuracy of liver and spleen stiffness measured by two dimensional shear wave elastography (2D-SWE) in hepatitis B-related cirrhosis. Methods: The clinical data of fifty-eight hepatitis B-related cirrhosis patients were collected in Zhongshan Hospital, Fudan University from September 2017 to April 2018. Pearson's correlation analyses were used to assess the relationship between liver/spleen stiffness (L-SWE and S-SWE) and hepatic venous pressure gradient (HVPG), as well as the comparison with serological model. The SWE diagnostic performances of Liver (L-SWE), Spleen (S-SWE) were also evaluated. Results: Of all 58 patients, 47 were found HVPG ≥10 mmHg, diagnosed as clinically significant portal hypertension (CSPH) and severe portal hypertension (SPH), which patients are at increased risk of developing complications. Thirty-four patients were found HVPG≥12 mmHg, diagnosed as SPH, which patients were at increased risk of variceal bleeding. Moderate positive correlation was found between L-SWE and HVPG (r=0.42, P<0.01), and S-SWE were significantly correlated with HVPG (r=0.68, P<0.01), while serological models and HVPG were slightly correlated (r=0.36 and 0.28, all P<0.01). The area under the receiver operating characteristic curves of L-SWE, S-SWE and the combination for CSPH were 0.78, 0.88 and 0.89. When L-SWE was>12.86 kPa or S-SWE was>35.73 kPa, patients were at increased risk of developing complications. The area under the receiver operating characteristic curves for SPH were 0.68, 0.81 and 0.77 and the S-SWE had the highest specificity, so when S-SWE was>41.5 kPa, patients were at increased risk of variceal bleeding. Conclusion: L-SWE and S-SWE are reliable and promising non-invasive parameters to assess CSPH and SPH.

[Computer-aided assessment of liver fibrosis progression in patients with chronic hepatitis B: an exploratory research].

Objective: To establish automatic liver fibrosis classification models by using traditional machine learning and deep learning methods and preliminaryly evaluate the efficiency. Methods: Gray scale ultrasound images and corresponding elastic images of 354 patients, 247 males and 107 females, mean age (54±12) years undergoing partial hepatectomy in Zhongshan Hospital of Fudan University from November 2014 to January 2016 were enrolled in this study. By using traditional machine learning and deep learning methods, an automatic classification model of liver fibrosis stages (S0 to S4) were established through feature extraction and classification of ultrasound image data sets and the accuracy in different classification categories of each model were calculated, by using liver biopsy as the reference standard. Results: Pathological examination showed 73 cases in pathological stage S0, 40 cases in S1, 49 cases in S2, 41 cases in S3, and 151 cases in S4. The traditional machine classification model based on support vector machine (SVM) classifier and sparse representation classifier and the deep learning classification model based on LeNet-5 neural network, their accuracy rates in the two categories (S0/S1/S2 and S3/S4) were 89.8%, 91.8% and 90.7% respectively; the accuracy rates in the three categories (S0/S1 and S2/S3 and S4) were 75.3%, 79.4% and 82.8% respectively; the accuracy in the three categories (S0 and S1/S2/S3 and S4) were 79.3%, 82.7% and 87.2% respectively. Conclusions: Computer-aided assessment of liver fibrosis progression in patients with chronic hepatitis B has a high accuracy, and can achieve a more detailed classification. This method is expected to be applied in the non-invasive evaluation of liver fibrosis in patients with hepatitis B in clinical work in the future.

Comparison of batch and semicontinuous cultures for production of protein from mesquite wood by Brevibacterium sp. JM98A.

The production of protein by a Brevibacterium sp. JM98A using mesquite wood as the substrate was compared in batch and semicontinuous cultures. A 14 liter glass fermentor with automatic pH, temperature, and foam control was used for the study. A pH range of 6.6 to 7.2 was optimum for the growth of JM98A. The batch and semicontinuous cultures were compared on the basis of viable cell counts, protein production, CMC-Ase (beta-1,4-glucanase) activity, and filter paper cellulase (beta-1,4-glucan cellobiohydrolyase) activity. Total hexose, cellulose, and reducing sugar consumption were measured. The semicontinuous process yielded 2.97 times as much protein in 72 hr as the batch cultures. Most of the biomass resulted from the utilization of soluble sugars rather than from the degradation of cellulose during the semicontinuous process.