Biocatalytic cascade to polysaccharide amination.

BACKGROUND: Chitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from Fusarium graminearum for oxidation, this study compared the performance of CvATA (from Chromobacterium violaceum) and SpATA (from Silicibacter pomeroyi) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated. RESULTS: Herein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure. CONCLUSIONS: Using oxidase-amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme.

Utilising standard samples instead of randomly collected food waste in composting: Implementation strategy and feasibility evaluation.

Randomly collected food waste results in inaccurate experimental data with poor reproducibility for composting. This study investigated standard food waste samples as replacements for randomly collected food waste. A response surface methodology was utilised to analyse data from a 28-day compost process optimisation experiment using collected food waste, and the optimal combination of composting parameters was derived. Experiments using different standard food waste samples (high oil and salt, high oil and sugar, balanced diet, and vegetarian) were conducted for 28 days under optimal conditions. The ranking of differences between the standard samples and collected food waste was vegetarian > balanced diet > high oil and sugar > high oil and salt. Statistical analysis indicated t-tests for increased oil and salt samples and collected food waste were not significant, and Cohen's d effect values were minimal. High oil and salt samples can be used as replacements for collected food waste in composting experiments.

Effects of Voltage and Treatment Time of Pulsed Electric Field on Electroporation in Rhizoctonia solani.

The pulsed electric field (PEF) of µs duration can induce electroporation by causing permanent damage to the membrane, leading to cell death. The microbe was treated by a homemade PEF generator instrument. The sterilization effect of PEF on the Rhizoctonia solani was observed by scanning electron microscope (SEM) and transmission electron microscope (TEM), and the leakage of the intracellular contents was measured with a conductometer and an ultraviolet spectrophotometer. The increases in the electrical conductivity and the optical density (OD) value indicated that the cell membrane was damaged, and the intracellular contents overflowed. As a result, according to our experimental conditions, the optimum condition was the high-pulsed electric voltage of 26 kV, and the treatment time was 4 min. It could be concluded that the PEF could damage the cell membrane, and the ratio of electroporation reached 100%, which provides a new method of killing R. solani efficiently.

Prediction of cooling effect of constant temperature community bin based on BP neural network.

In order to explore the influence of outdoor microclimate on the cooling effect of constant temperature community bin, the temperature prediction model was predicted. The temperature and microclimate data sets of the community bin were collected in summer from May 2021 to September 2021. The climatic characteristics included cloudy and sunny conditions, and the environmental factors included outdoor temperature, air speed, air relative humidity, and solar radiation intensity. Stepwise regression method was used to test the significance of environmental factors, and the corresponding regression equation was obtained. BP neural network was used to establish temperature prediction models under cloudy and sunny conditions, respectively. The results showed that the coefficient of determination (R2) of the two models was above 0.8, and the environmental factors with significant influence were screened out. The root mean square error (RMSE) between the training value and the actual value established by BP neural network was 0.83 °C, and the determination coefficient (R2) was 0.968. Under sunny conditions, the root mean square error (RMSE) of predicted value and measured value was 0.65 °C, and the determination coefficient (R2) was 0.982. According to the analysis of the sample data, it showed that the BP neural network was more accurate than stepwise regression, and could be used to predict the temperature of community bin, which provided model basis for the practical application of intelligent temperature control community bin in summer.

Custom-Made Antibiotic Cement-Coated Nail for the Treatment of Infected Bone Defect.

BACKGROUND: Longbone infected bone defect remains a great challenge due to multiple surgeries, long-term treatment duration, and uncertain prognosis. Treatment principles include eradication/debridement, stabilization, and antibiotic administration. An antibiotic cement-coated nail has shown great prospects due to both local antibiotic elution and stabilization of bone defects. However, the current fabrication technique remains to be improved. METHODS: For the first time, we described a new method for custom-made cement-coated nail fabrication based on a 3D printing technique. A retrospective study of 19 consecutive patients with long bone infected bone defects from one medical center was conducted who met the inclusion and exclusion criteria from November 2016 to May 2020. The treatment involved thorough debridement, custom-made antibiotic cement-coated nail filling, and culture-specific systemic antibiotic treatment guided by a multidisciplinary team. Clinical and radiographic examinations (X-ray and CT scans) were used to evaluate bony union. Clinical and laboratory examinations were used to evaluate the infection control. The SF-36 score was used to evaluate patients' quality of life pre- and postoperatively. RESULTS: The mean follow-up was 98.8 weeks (ranging from 40 to 192). All cases achieved infection control, 3 cases achieved bone healing after one-stage operation, and 12 cases achieved bone healing after a two-stage bone graft procedure. At the last follow-up, none of the 19 patients had infection recurrence or 1 case had failure of the protective plate. The pre- and postoperative SF-36 score showed that there were statistical differences in all the 9 aspects. CONCLUSIONS: The precise custom-made antibiotic cement-coated intramedullary nail through the 3D printing technique used in this study is an effective strategy for the treatment of infected bone defects of long bone. This technique may help to increase the infection control rate and promote bone healing.

Effects of Pulsed Electric Field on the Cell Wall and Infection Activity of Rhizoctonia solani.

This paper adopts the Design-Expert software to design an orthogonal experiment with a pulse voltage amplitude of 30 kV, processing time of three minutes, and a pulse width of 45 µs as the center points, in order to study the effects of the pulsed electric field on the cell wall and infection activity of Rhizoctonia solani. High-voltage pulse power was used to treat the bacteria solution with the pulsed electric field. Untreated Rhizoctonia solani were used as the control group. Transmission electron microscope images were used to analyze the cell wall damage. ANOVA was performed on the experimental results and the fitting degree of the model was good (F>>1). Response surface analysis was used to optimize the parameters based on chitin content and polygalacturonase activity. The optimal treatment conditions were obtained as a pulse voltage amplitude of 25 kV, processing time of 2.54 min, and a pulse width of 34.35 µs. On this basis, experiments were designed to verify the optimized conditions. The results demonstrated that, under the optimal processing conditions, the damage index of the cell wall of Rhizoctonia solani was 9.59% lower in chitin content and 83.05% lower in polygalacturonase activity compared with those of the control group. All indexes were significantly different (P < 0.001), which is consistent with the parameter optimization results. The results provide a theoretical basis for the pulsed electric field assisted sterilization and reference for the design of plant protection machinery in the latter stage.

Analysis of Factors Influencing the Transmembrane Voltage Induced in Filamentous Fungi by Pulsed Electric Fields.

This article studies the sterilization effects of high-voltage pulsed electric field (PEF) of technology on filamentous fungi. A cell dielectric model was proposed based on the physical structure of filamentous fungi. Basic theories of the electromagnetic field were comprehensively applied, and the multiphysics field simulation software COMSOL Multiphysics was used for more detailed study. The effects of PEF treatment parameters and microbial characteristic parameters on the resulting cell membrane and nuclear membrane changes were simulated and analyzed. The results showed significant effects on the transmembrane voltage of the cell membrane and nuclear membrane from the electric field intensity, pulse duration, cell membrane thickness, superposition effect of the pulses. However, the amount of hyphae had little effect, and the number of cell nuclei and the thickness of the cell walls had almost no effect on the transmembrane voltage of the cell membranes and the nuclear membranes. The results provide theoretical support for applying high-voltage PEFs to kill fungi in practical applications.

[Enhanced expressions of Wnt5a/JNK signaling pathway-related molecules in the lung tissues of asthmatic rats].

OBJECTIVE: To observe the expression of Wnt5a/c-Jun N-terminal kinase (JNK) signaling pathway in the lung of asthmatic rats. METHODS: The experimental rats were randomly divided into two groups, the model group and the control group. After the rat models of asthma were established, airway pathological changes were observed by HE staining; the expression of Wnt5a mRNA in the lung tissues and peripheral blood cells was examined by real-time quantitative PCR; and the levels of phospho-c-Jun (p-c-Jun) and phospho-c-Jun N-terminal kinase (p-JNK) proteins in the lung tissues were measured by Western blotting. RESULTS: Compared with the control group, asthmatic rats presented with thickened bronchial mucosa with more obvious chrysanthemum fold, narrower airway, and more inflammatory cells infiltrated around trachea and pulmonary vessels. The inner airway wall and smooth muscle layer were much thicker in rats of asthma group than those of control group. The levels of Wnt5a mRNA, p-c-Jun and p-JNK proteins in the lung and blood of asthmatic rats increased as compared with the control group. CONCLUSION: The expressions of Wnt5a mRNA, p-JNK and p-c-Jun proteins were elevated in asthmatic rats.

[An increased of Th17 cells and IL-23 and IL-17 in the lymph and blood of rats with bronchial asthma].

OBJECTIVE: To investigate whether immune response could change the number of CD4(+);IL-17(+);Th17 cells and the concentrations of IL-17 and IL-23 in the serum and lymph of asthmatic rats. METHODS: Both serum and lymph CD4(+);IL-17(+);Th17 cell numbers were measured by flow cytometry. The concentrations of IL-17 and IL-23 in the serum and lymph were assayed by ELISA. The expression of IL-23 p19 mRNA was examined by quantitative real-time PCR. RESULTS: Mononuclear cells (MNC) were collected from the blood and lymph of rats 0, 24 and 48 hours after final ovalbumin stimulation and were subsequently cultured for 18 hours. The serum and lymph of the asthma group produced higher amounts of CD4(+);IL-17(+);Th17 cells, and higher levels of IL-23p19 mRNA, than those of the healthy controls (P < 0.05). In addition, compared to lymph, serum had significantly lower amounts of CD4(+);IL-17(+);Th17 cells, decreased expression of IL-23p19 mRNA, and reduced IL-23 and IL-17 concentratons (P < 0.05). Cytokines and cell numbers of Th17 cells from the asthma group significantly increased compared with that of control group. CONCLUSION: These data suggested that asthma-driven CD4(+);IL-17(+);Th17-cell proliferation and release of IL-17 and IL-23 may be associated with different immune activities between the serum and lymph.

[Influence of anti-ICOS antibody on quantity and function of CD4(+);CD25(+);Foxp3(+);Treg from lymph and blood of rats with bronchial asthma].

AIM: To investigate the influences of anti-ICOS antibody (anti-ICOSAb) on quantity and function of CD4(+);CD25(+);Foxp3(+);Treg cells from lymph and peripheral blood of rats with bronchial asthma. METHODS: The mononuclear cells (MNC) from lymph and blood were co-cultured with anti-ICOSAb, and then the percentage of CD4(+);CD25(+);Foxp3(+);Treg cells were analyzed by flow cytometer (FCM) and the levels of IL-10 and TGF-ß1 in supernatants were determined by ELISA. RESULTS: The MNC were collected from lymph and blood at 0, 24 and 48 h after the last challenge, respectively, and the cells were cultured for 96 h in vitro. The percentage of CD4(+);CD25(+);Foxp3(+);Treg cells in the MNC from lymph was significantly higher than that from blood in each group (P<0.05); The percentage of CD4(+);CD25(+);Foxp3(+);Treg cells in the MNC from lymph and blood in asthma group was significantly lower compare with the normal control group (P<0.05); The percentage of CD4(+);CD25(+);Foxp3(+);Treg cells in the MNC from lymph and blood in the anti-ICOSAb group obviously decreased compare with the asthma group (P<0.05). At 0 h after the last challenge, the level of IL-10 in the supernatant of MNC from lymph and blood in the anti-ICOSAb group were significantly lower than that of the control and asthma groups (P<0.05), while there were no significant differences of TGF-ß1 expression in the supernatant of MNC from lymph and blood in each group at different time points. CONCLUSION: Blocking the ICOS/ICOSL signaling pathway by anti-ICOSAb could exacerbate the deficiency of CD4(+);CD25(+);Foxp3(+);Treg cells from lymph and blood in bronchial asthmatic rat, meanwhile inhibit the CD4(+);CD25(+);Foxp3(+);Treg cells secreting IL-10 at 0 h after the last challenge, but have no significant effect on the secretion of TGF-ß1.

[Changes and the clinical significance of CD4⁺ CD25⁺ regulatory T cells and Th17 cells in peripheral blood of infants with respiratory syncytial virus bronchiolitis].

AIM: To observe the percentages of CD4(+);CD25(+); regulatory T cells (Tregs) and Th17 cells and the levels of IL-10, TGF-ß and IL-17 in peripheral blood of infants with respiratory syncytial virus (RSV) bronchiolitis. The relationship between above cells, cytokines and RSV bronchiolitis was determined. METHODS: Thirty-three infants with RSV bronchiolitis, twenty-eight infants with non-RSV pneumonia and twenty-six healthy infants were enrolled. The percentages of Tregs and Th17 cells in peripheral blood were detected by flow cytometer (FCM), and the levels of IL-10, TGF-ß and IL-17 in plasma were determined by ELISA. RESULTS: The percentage of Tregs and the levels of IL-10 and TGF-ß in infants with RSV bronchiolitis were significantly lower than those in infants with non-RSV pneumonia and healthy infants (P<0.05), while the percentage of Th17 cells and the level of IL-17 in infants with RSV bronchiolitis were significantly higher than those in infants with non-RSV pneumonia and healthy infants (P<0.05). CONCLUSION: The imbalance between Tregs and Th17 cells in peripheral blood of infants with RSV bronchiolitis may be one of the pathogenesis of RSV bronchiolitis.

[Expression of RhoA in the lung tissue of acute lung injury rats and the influence of RhoA on the expression of IL-8 and IL-10].

AIM: To investigate the expression of RhoA in the lung tissue of acute lung injury (ALI) rats induced by oleic acid (OA) and explore the influence of RhoA on the level of IL-8 and IL-10 in serum. Discussion RhoA and acute lung injury relationship. METHODS: Establish rat oleic acid acute lung injury model, Wet/Dry weights (W/D) were detected. Pathological changes of the lung tissue were examined. The levels of IL-8 and IL-10 in serum were detected by ELISA and the expression of RhoA in homogenate of lung tissue was determined by PCR. RESULTS: W/D and ALI score in ALI group at 6, 12, 24 and 48 h were higher than those in control group (P<0.01) and intervention group at the same time point (P<0.05). The level of IL-10(ng/L) in serum in ALI group at 2, 6, 12, 24 and 48 h was remarkably higher than that in control group (P<0.01), but was lower compared with intervention group at the same time point (P<0.01). The level of IL-8(pg/L) in serum at 2, 6, 12, 24 and 48 h in ALI group were higher than those in control group(P<0.01) and intervention group at the same time point(P<0.01).Compared with that of control group, the expression of RhoA in homogenate of lung tissue in ALI group at 2, 6, 12, 24 and 48 h was higher(P<0.01), but there was no difference between ALI group and intervention group at the same time point(P>0.05). CONCLUSION: The expression of RhoA in lung tissue of ALI rats induced by OA is increased. RhoA expression increase that can cause lung damage degree increase in rats. RhoA can aggravate the pathology changes of ALI rats, which might be related to up regulating expression of IL-8 and down regulating expression of IL-10 by RhoA.

Rosmarinic acid inhibits proliferation and induces apoptosis of hepatic stellate cells.

Hepatic stellate cells (HSCs), activated during liver injury, are defined as the most important target in the therapy of hepatic fibrosis. In the present study, we evaluated the effect of Rosmarinic acid (RosA) on the proliferation and apoptosis in activated hepatic stellate cells (HSC-T6), which is useful to decrease this cell population. The proliferation of HSC-T6 was significantly inhibited after treated with various concentrations of RosA for different times. Flow cytometric analyses and transmission electron microscope (TEM) observations revealed that HSC-T6 treated with RosA underwent apoptosis in a time dependent manner and displayed typical apoptotic features in the cells. The phosphorylation in signal transducer and activator of transcription protein-3 (STAT3), which regulates cell survival, proliferation and differentiation in a variety of tissues, was markedly decreased as the result of Western blot assay and correlated with downregulation of CyclinD1 and B cell lymphoma/leukemia-2 (Bcl-2). In conclusion, these results suggested that RosA was able to inhibit proliferation and induce apoptosis in HSC-T6, partly due to the inhibition of phosphorylation in STAT3, which contributed to the reversal of hepatic fibrosis.

[Study about outcomes of non-pharmacological conservative treatment for lumbar spinal stenosis].

OBJECTIVE: To describe the short-term outcomes of a non-pharmacological conservative approach to patients with LSS. METHODS: This is a prospective consecutive case series with short-term follow-up of 21 consecutive patients who were diagnosed with LSS. Patients recruited from the outpatients of orthopaedic department and rehabilitation department in the Peking University People's Hospital from March 2010 to March 2011. Patients had baseline interviews with follow-up questionnaires in the end of the first and the third month. MAIN OUTCOME MEASURES: pain intensity was measured using the Numerical Rating Scale (NRS) and disability was measured using the Roland Morris Disability Questionnaire (RMDQ), as well as the 36-item Short Form Health Surrey (SF-36) and efficacy assessment for evaluation. RESULTS: All of 21 eligible consenting patients initially enrolling completed the follow-up. Pain at worst, functional status, quality of life improved significantly in the end of the first month. These were considered to be clinically meaningful in the end of the third month. No patients went on to require surgery. No major complications of treatment were noted. CONCLUSIONS: A non-pharmacological conservative treatment may be useful and safe in bringing about clinically meaningful improvement in pain and disability in patients with LSS. Before surgical management, a non-surgical approach should be taken into account at first.

[The comparison of CD4(+) CD25(+) Treg, IL-10 and TGF-beta from lymph and blood in bronchial asthmatic rat and the effect of dexamethasone on it].

AIM: To observe the expression of CD4(+) CD25(+) Foxp3(+) Treg, IL-10 and TGF-beta in lymph and blood, as well as the effect of dexamethasone on it and investigate the relationship between them. METHODS: Lymph and blood samples of 0 h, 24 h, 48 h after the last challenge were collected from the rat model of asthma. The percentage of CD4(+) CD25(+) Foxp3(+) Treg were detected by flow cytometer (FCM), while the levels of IL-10 and TGF-beta were determined by ELISA. RESULTS: The percentage of CD4(+) CD25(+) Foxp3(+) Treg in the blood and the levels of IL-10 and TGF-beta in the plasma in cases of asthma group were significantly lower than that of the control and therapy group at different time points (P<0.05); The percentage of CD4(+) CD25(+) Foxp3(+) Treg and the level of IL-10 in lymph were significantly higher than that of blood (P<0.05), however, the level of TGF-beta in lymph were significantly lower than that of blood (P<0.05); There were no significant differences which the percentage of CD4(+) CD25(+) Foxp3(+) Treg in the blood and the levels of IL-10 and TGF-beta in the plasma between control group and therapy group (P>0.05). CONCLUSION: There is a disbalance both quantity and function of CD4(+) CD25(+) Foxp3(+) in the lymph of bronchial asthmatic rat, and the percentage of CD4(+) CD25(+) Foxp3(+) Treg in lymph are significantly higher than that of blood, which dexamethasone may effectively increase the percentage of CD4(+) CD25(+) Foxp3(+) Treg in bronchial asthmatic rat.