Efficacy of Chinese and Western Medical Techniques in Treating Diabetic Foot Ulcers With Necrotizing Fasciitis of the Lower Leg.

To analyze and evaluate the clinical efficacy of Chinese and Western medical techniques in the treatment of severe diabetic foot ulcers complicated with necrotizing fasciitis of the lower leg and summarize the treatment experience of such patients to identify a new method of limb salvage treatment. A total of 46 patients with severe diabetic foot ulcers and necrotizing fasciitis of the lower leg were treated with such techniques as surgical debridement, bone drilling, open joint fusion, and microskin implantation. Wounds were treated with moisture-exposed burn therapy (a regenerative medical treatment for burns, wounds, and ulcers) and moisture-exposed burn ointment (a traditional Chinese medicine); underlying diseases were also treated effectively. The wound healing time, rate of high amputation, and mortality of these patients were summarized, and the clinical efficacy of such treatments was evaluated. Of the 46 patients enrolled, 38 patients were cured, with a cure rate of 82.61%. The average wound healing time was 130 ± 74.37 days. Two patients underwent high amputations, with an amputation rate of 4.35%, and 4 deaths occurred, with a mortality rate of 8.70%. The combination of Chinese and Western medical techniques in the treatment of severe diabetic foot ulcers complicated with necrotizing fasciitis of the lower leg not only effectively saved patients' lives and promoted wound healing but also greatly reduced the rates of high amputation and disability.

MiR-150-5p Alleviates Renal Tubular Epithelial Cell Fibrosis by Activating Autophagy via ß-catenin Signaling.

OBJECTIVE: To verify whether miR-150-5p modulates the development of renal fibrosis and its mechanism. METHODS: Transforming growth factor (TGF)-ß1 was implemented on HK-2 cells to construct a renal fibrosis in vitro model. Inhibition of autophagy was performed on HK-2 cells by treating with 3-methyladenine (3-MA, an inhibitor of autophagy). HK-2 cells experienced transfection by miR-150-5p mimics/inhibitor and pcDNA-ß-catenin plasmids, and the negative controls. Dual luciferase reporter gene assay was applied to validate the relationship between miR-150-5p and ß-catenin. Cell apoptosis exploration was implemented by flow cytometry assay. The level detection of CoII, α-SMA, miR-150-5p and ß-catenin was executed by real-time quantitative reverse transcription-polymerase chain reaction. The expression of CoII, α-SMA, LC3I, LC3II, Bax, Cleaved Caspase 3, Beclin 1, Bcl-2 and ß-catenin proteins was monitored by western blot. RESULTS: Autophagy was inhibited in TGF-ß1-induced HK-2 cells. MiR-150-5p alleviated fibrosis, enhanced autophagy, and inhibited apoptosis in TGF-ß1-induced HK-2 cells. ß-catenin was a target of miR-150-5p. Autophagy inhibition or ß-catenin partially counteracted miR-150-5p effect on TGF-ß1-induced fibrosis in HK-2 cells. CONCLUSIONS: MiR-150-5p alleviates renal tubular epithelial cell fibrosis by activating autophagy via ß-catenin signaling.

EZH2 as a prognostic-related biomarker in lung adenocarcinoma correlating with cell cycle and immune infiltrates.

BACKGROUNDS: It has been observed that high levels of enhancer of zeste homolog 2 (EZH2) expression are associated with unsatisfactory prognoses and can be found in a wide range of malignancies. However, the effects of EZH2 on Lung Adenocarcinoma (LUAD) remain elusive. Through the integration of bioinformatic analyses, the present paper sought to ascertain the effects of EZH2 in LUAD. METHODS: The TIMER and UALCAN databases were applied to analyze mRNA and protein expression data for EZH2 in LUAD. The result of immunohistochemistry was obtained from the HPA database, and the survival curve was drawn according to the library provided by the HPA database. The LinkedOmics database was utilized to investigate the co-expressed genes and signal transduction pathways with EZH2. Up- and down-regulated genes from The Linked Omics database were introduced to the CMap database to predict potential drug targets for LUAD using the CMap database. The association between EZH2 and cancer-infiltrating immunocytes was studied through TIMER and TISIDB. In addition, this paper explores the relationship between EZH2 mRNA expression and NSCLC OS using the Kaplan-Meier plotter database to further validate and complement the research. Furthermore, the correlation between EZH2 expression and EGFR genes, KRAS genes, BRAF genes, and smoking from the Cancer Genome Atlas (TCGA) database is analyzed. RESULTS: In contrast to paracancer specimens, the mRNA and protein levels of EZH2 were higher in LUAD tissues. Significantly, high levels of EZH2 were associated with unsatisfactory prognoses in LUAD patients. Additionally, the coexpressed genes of EZH2 were predominantly associated with numerous cell growth-associated pathways, including the cell cycle, DNA replication, RNA transport, and the p53 signaling pathway, according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. The results of TCGA database revealed that the expression of EZH2 was lower in normal tissues than in lung cancer tissues (p < 0.05). Smoking was associated with elevated EZH2 expression (p < 0.001). EZH2 was highly expressed in lung cancers with positive KRAS expression, and the correlation was significant in lung adenocarcinoma (r = 0.3129, p < 0.001). CMap was applied to determine the top 15 positively correlated drugs/molecules and the top 15 negatively correlated drugs/molecules. MK-1775, MK-5108, fenbendazole, albendazole, BAY-K8644, evodiamine, purvalanol-a, mycophenolic-acid, PHA-793887, and cyclopamine are potential drugs for patients with lung adenocarcinoma and high EZH2 expression. CONCLUSIONS: Highly expressed EZH2 is a predictor of a suboptimal prognosis in LUAD and may serve as a prognostic marker and target gene for LUAD. The underlying cause may be associated with the synergistic effect of KRAS, immune cell infiltration, and metabolic processes.

Analysis of the correlation between Zeste enhancer homolog 2 (EZH2) mRNA expression and the prognosis of mesothelioma patients and immune infiltration.

Mesothelioma lies one of the most malignant tumors, in which the identification of the corresponding biomarkers is extremely critical. This study aims to investigate the prognostic value of enhancer homolog 2 (EZH2) mRNA expression in mesothelioma patients accompanied with its immune infiltration analysis. Gene expression, clinical information and enrichment analysis were obtained based on the Cancer Genome Atlas (TCGA), the immune infiltration analysis and bioinformatics analysis were performed. Clinical information and gene expression were obtained from 86 patients with mesothelioma based on TCGA database. Survival analysis, GSEA enrichment analysis, and immune infiltration analysis of EZH2 expression were carried out using R (version 3.6.3) (statistical analysis and visualization). The correlation of EZH2 expression with immune cell infiltration in mesothelioma was analyzed according to the TIMER database (Fig. https://cistrome.shinyapps.io/timer/ ). A univariate and multivariate analysis of general data obtained from the TCGA database was performed, involving age, gender, stage, pathological type, and whether they had received radiotherapy, the results indicated the association of high expression of EZH2 with poor prognosis in mesothelioma patients, with the worse prognosis in the High group (HR = 2.75, 95% CI 1.68-4.52, P < 0.010). Moreover, ROC curves showed that EZH2 expression predicted 1-year survival with an AUC of 0.740, 2-year survival with an AUC of 0.756, and 3-year survival with an AUC of 0.692, suggesting a robust predictive effect of EZH2 expression on prognosis. KEGG pathway analysis indicated five pathways showing the strongest positive correlation with EZH2 expression: cell cycle, DNA replication, Cell adhesion molecules cams, Primary immuno deficiency, Tsate transduction, and five pathways showing the strongest negative correlation with EZH2 expression: Glycolysis gluconeogenesis, Drug metabolism, cytochrome P450, retinol metabolism, fatty acid metabolism ribosome. We investigated the correlation between EZH2 expression and the level of immune infiltration in mesothelioma tissues. The results indicated that EZH2 expression played a critical role in immune infiltration, of which the high expression was correlated with the reduced number of NK cells, Mast cells, and Th17 cells. Moreover, mesothelioma patients with high EZH2 expression differ from those with low EZH2 expression in their tumor immune microenvironment. EZH2, as a new prognostic biomarker for mesothelioma, contributes to elucidating how changes in the immune environment promote the development of mesothelioma. Further analysis, EZH2 may serve as a biological test to predict the prognosis of mesothelioma.

[Analysis of glycan ratio of Chinese hamster ovary cell-cetuximab antigen-binding segment via rapid enzyme digestion with endo-ß-N-acetylglucosaminidase F].

The N-glycosylation of proteins is a typical post-translational modification. Compared with other monoclonal antibodies, N-glycosylation modification in cetuximab is more complicated. Because cetuximab contains two N-glycosylation sites, one is located on the antigen-binding fragment (Fab) and the other is on the crystallizable fragment (Fc) of the heavy chain (HC). Among the two, the glycosylation of the Fab segment is more complicated. As this segment is located in the hypervariable region (VH), it may affect the affinity of the antibody antigen and cause other issues. Therefore, it is necessary to study glycosylation modification at this site. This modification is particularly challenging, necessitating the development of specific glycan cutting technology and a stable glycan ratio analysis method. In this study, cetuximab expressed in Chinese hamster ovary (CHO) cell was used as the experimental research object. Based on the digestion with endo-ß-N-acetylglucosaminidase F2 (Endo F2), an experimental method was developed that can quickly release Fab glycans. Qualitative and glycan ratio analyses were carried out by ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). The test was divided into two steps: in the first step, a non-denaturing (native state) glycosidase excision test was performed on the CHO-cetuximab drug substance. The drug substance was diluted to 1.0 mg/mL by adding ultrapure water, following which 1.0 µL of Endo F2 was directly added to 100 µL of the drug substance for enzyme digestion at 37 ℃. Through HRMS, the data were deconvoluted to obtain the accurate mass of the drug substance. The results showed that when the digestion time of Endo F2 was 5 min, the glycans in the Fab segment could be completely removed, whereas those in the Fc segment were not affected. Rapid enzyme cutting of the Fab glycans was realized; simultaneously, it was concluded that this method was also very specific for the removal of Fab glycans. In the second step, an accurate ratio analysis test was performed on Fab glycans excised from CHO-cetuximab. The released Fab glycans were precipitated with ice ethanol, the supernatant was centrifuged and spin-dried, and then labeled with para-aminobenzyl (2-AB). 2-AB labeling could make glycans have fluorescent detectable signals, and after reconstitution in 70% acetonitrile aqueous solution, was detected by UPLC coupled with a fluorescence detector (FLR). Good chromatographic peak separation was obtained using a hydrophilic interaction chromatography (HILIC) column. Thus, the test enabled stable glycan ratio analysis. The molecular weight results for three independent Endo F2 digestion cycles for 5 min showed that the masses after digestion were similar; subsequently, glycan ratio analysis was performed based on HILIC. The results of three independent glycan ratio analysis experiments were also similar, indicating that the rapid enzyme digestion of Endo F2 followed by glycan ratio analysis after 5 min of digestion yielded good stability and reliability. Data obtained by measuring the samples produced using two different processes employed by our company showed that there were distinct differences in the glycan profiles of the two processes, especially in terms of the sialic acid glycoforms. These results prove that the method developed in this study can accurately analyze the ratio of glycans. Monitoring the antibody production process is important and meaningful for the evaluation of the process.

IAP-1 promoted cisplatin resistance in nasopharyngeal carcinoma via inhibition of caspase-3-mediated apoptosis.

Recurrent/metastatic nasopharyngeal carcinoma (NPC) is known for having a poor prognosis due to its unfavorable response to chemoradiotherapy. However, the specific processes involved remain poorly understood. This study focused on the cisplatin-resistance mechanism in NPC to help understand the occurrence of advanced NPC and aims to explore the potential therapeutic target for cisplatin-resistant NPC. Two cisplatin-resistant NPC cell lines, HNE-1/DDP and CNE-2/DDP, were established and the differentially expressed genes (DEGs) between parental and cisplatin-resistance cell lines, filtering from high-throughput sequencing results, were analyzed. Next, the effects of IAP-1 on cisplatin-resistant nasopharyngeal cancer cell proliferation, apoptosis, drug resistance and associated cell signaling were evaluated in vitro and in vitro. From our bioinformatic results, more than 15,000 differentially expressed genes (DEGs) were found between parental and resistant cell lines. Nine related DEGs were found in the classic platinum resistance pathway, three of which (ATM, IAP-1, and IAP-2) also appeared in the top five differentially expressed pathways, with elevated IAP-1 showing the highest fold change. Further studies revealed that high IAP-1 expression can lead to an increased cisplatin inhibitory concentration and apoptosis inhibition. IAP-1 silencing can induce upregulation of the caspase-3 and enhance the antiproliferation and proapoptotic effects of cisplatin. Clinical data also showed that IAP-1 overexpression was associated with a worse survival status. In summary, in vitro and in vivo experiments demonstrated that IAP-1 plays a vital role in cisplatin resistance by regulating caspase induced apoptosis and serve as a potential novel therapeutic target and a prognostic indicator for advanced NPC.

A Simulation Paradigm for Evaluation of Subtle Liver Lesions at Pediatric CT: Performance and Confidence.

Purpose: To create and validate a systematic observer performance platform for evaluation of simulated liver lesions at pediatric CT and to test this paradigm to measure the effect of radiation dose reduction on detection performance and reader confidence. Materials and Methods: Thirty normal pediatric (from patients aged 0-10 years) contrast material-enhanced, de-identified abdominal CT scans obtained from July 1, 2012, through July 1, 2016, were retrospectively collected from the clinical database. The study was exempt from institutional review board approval. Zero to three simulated, low-contrast liver lesions (≤6 mm) were digitally inserted by using software, and noise was added to simulate reductions in volume CT dose index (representing radiation dose estimation) of 25% and 50%. Pediatric, abdominal, and resident radiologists (three of each) reviewed 90 data sets in three sessions using an online interface, marking each lesion location and rating confidence (scale, 0-100). Statistical analysis was performed by using software. Results: Mixed-effects models revealed a significant decrease in detection sensitivity as radiation dose decreased (P < .001). The mean confidence of the full-dose and 25% dose reduction examinations was significantly higher than that of the 50% dose reduction examinations (P = .011 and .012, respectively) but not different from one another (P = .866). Dose was not a significant predictor of time to complete each case, and subspecialty was not a significant predictor of sensitivity or false-positive results. Conclusion: Sensitivity for lesion detection significantly decreased as dose decreased; however, confidence did not change between the full-dose and 25% reduced-dose scans. This suggests that readers are unaware of this decrease in performance, which should be accounted for in clinical dose reduction efforts.Keywords: Abdomen/GI, CT, Liver, Observer Performance, Pediatrics, Perception Image© RSNA, 2019.

[Screening of dissolved oxygen induced promoters in Corynebacterium crenatum and functional verification].

OBJECTIVE: Purpose of this work was to screen promoters which were induced by dissolved oxygen in Corynebacterium crenatum SYPA5-5. METHODS: Based on the genomic information of Corynebacterium, we identified target proteins which were the highest expression level of one copy of 27 known proteins from our earlier study. Based on the upstream sequence of the coding gene sequences, we amplified two promoters, named P-tkt and P-fum. The tac promoter of recombinant pDXW-8-cat and pDXW-8-gfp were replaced by the new promoters, and the recombinant plasmids were transformed into E. coli JM109 and C. crenatum SYPA5-5 respectively. At different dissolved oxygen level, we compared the function of promoters by the expression of chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP). RESULTS: Results show that we identified 2 target proteins, which were transketolase and fumarate hydratase. At the high dissolved oxygen level the CAT activity of E. coli JM109/pDXW-Ptkt-cat and C. crenatum SYPA5-5/pDXW-Ptkt-cat were 3.032 U/mg and 1.987 U/mg, which were 2.8-fold and 3.2-fold than that in the low dissolved oxygen, respectively. We got similar results by using a 5-L fermentor. CONCLUSION: The P-tkt promoter induced by high dissolved oxygen was suitable for fermentation to produce amino acids and beneficial to improve ability of synthesis and metabolism of amino acids at high dissolved oxygen level.