Hen Egg White Lysozyme (HEWL) Confers Resistance to Verticillium Wilt in Cotton by Inhibiting the Spread of Fungus and Generating ROS Burst.

Verticillium wilt is a soil-borne vascular disease caused by the fungal pathogen Verticillium dahliae. It causes great harm to upland cotton (Gossypium hirsutum) yield and quality. A previous study has shown that Hen egg white lysozyme (HEWL) exerts strong inhibitory activity against V. dahliae in vitro. In the current study, we introduced the HEWL gene into cotton through the Agrobacterium-mediated transformation, and the exogenous HEWL protein was successfully expressed in cotton. Our study revealed that HEWL was able to significantly inhibit the proliferation of V. dahlia in cotton. Consequently, the overexpression of HEWL effectively improved the resistance to Verticillium wilt in transgenic cotton. In addition, ROS accumulation and NO content increased rapidly after the V. dahliae inoculation of plant leaves overexpressing HEWL. In addition, the expression of the PR genes was significantly up-regulated. Taken together, our results suggest that HEWL significantly improves resistance to Verticillium wilt by inhibiting the growth of pathogenic fungus, triggering ROS burst, and activating PR genes expression in cotton.

Metabolic Engineering of Model Microorganisms for the Production of Xanthophyll.

Xanthophyll is an oxidated version of carotenoid. It presents significant value to the pharmaceutical, food, and cosmetic industries due to its specific antioxidant activity and variety of colors. Chemical processing and conventional extraction from natural organisms are still the main sources of xanthophyll. However, the current industrial production model can no longer meet the demand for human health care, reducing petrochemical energy consumption and green sustainable development. With the swift development of genetic metabolic engineering, xanthophyll synthesis by the metabolic engineering of model microorganisms shows great application potential. At present, compared to carotenes such as lycopene and ß-carotene, xanthophyll has a relatively low production in engineering microorganisms due to its stronger inherent antioxidation, relatively high polarity, and longer metabolic pathway. This review comprehensively summarized the progress in xanthophyll synthesis by the metabolic engineering of model microorganisms, described strategies to improve xanthophyll production in detail, and proposed the current challenges and future efforts needed to build commercialized xanthophyll-producing microorganisms.

Comparison of Activity and Safety of DSPAα1 and Its N-Glycosylation Mutants.

DSPAα1 is a potent rude thrombolytic protein with high medicative value. DSPAα1 has two natural N-glycan sites (N153Q-S154-S155, N398Q-K399-T400) that may lead to immune responses when administered in vivo. We aimed to study the effect of its N-glycosylation sites on DSPAα1 in vitro and in vivo by mutating these N-glycosylation sites. In this experiment, four single mutants and one double mutant were predicted and expressed in Pichia pastoris. When the N398Q-K399-T400 site was mutated, the fibrinolytic activity of the mutant was reduced by 75%. When the N153Q-S154-S155 sites were inactivated as described above, the plasminogen activating activity of its mutant was reduced by 40%, and fibrin selectivity was significantly reduced by 21-fold. The introduction of N-glycosylation on N184-G185-A186T and K368N-S369-S370 also considerably reduced the activity and fibrin selectivity of DSPAα1. The pH tolerance and thermotolerance of all mutants did not change significantly. In vivo experiments also confirmed that N-glycosylation mutations can reduce the safety of DSPAα1, lead to prolonged bleeding time, non-physiological reduction of coagulation factor (α2-AP, PAI) concentration, and increase the risk of irregular bleeding. This study ultimately demonstrated the effect of N-glycosylation mutations on the activity and safety of DSPAα1.

Influence of the COVID-19 Pandemic on the Prevalence Pattern of Allergens.

INTRODUCTION: The effect of the COVID-19 pandemic on allergic diseases is not certain, as people's living habits and the environment have been affected by the pandemic. The present study described the influence of the COVID-19 pandemic on the allergen sensitization rate in patients with allergic diseases in central China. The results provide reliable epidemiological data for the prevention and control of allergic diseases during the COVID-19 epidemic. METHODS: Data were collected from a total of 6,915 patients with symptoms of allergic diseases who visited the Third Xiangya Hospital of Central South University in China for allergen testing from January 1, 2018, to December 31, 2021. Patients were divided into a children group (<14 years old), youth group (15∼44 years old), middle-aged group (45∼59 years old), and elderly group (>60 years old). Immunoblotting was used to detect 20 serum allergen-specific IgE (sIgE) antibodies in patient serum samples. We compared the positive rates of various allergens in different age and sex groups before and during the COVID-19 epidemic, and the prevalence data of sIgE sensitization were analysed. RESULTS: Among the 6,915 patients with symptoms of allergic diseases, 2,838 (41.04%) patients were positive for at least one of the allergens. The top three positive rates of inhaled allergens were Dermatophagoides farinae (1,764 cases, 25.51%), Dermatophagoides pteronyssinus (1,616 cases, 23.37%), and house dust (645 cases, 9.33%). The top three positive rates of food allergens were eggs (686 cases, 9.92%), milk (509 cases, 7.36%), and crabs (192 cases, 2.78%). The total positive rate of allergens was higher in men (46.99%) than in women (37.30%). Compared to 2 years before the COVID-19 epidemic, the rate of sensitization to indoor inhalant allergens increased, but outdoor inhalant allergens showed no significant change. The positive rates of milk and eggs peaked during the outbreak of COVID-19 (2020) then declined in 2021. The total positive rate of allergens was higher in males than females before and during the COVID-19 epidemic, but more allergens were different between males and females during the pandemic. Compared to middle-aged and older adults, the children and youth groups were more susceptible to allergic diseases, and they exhibited an increasing positive rate for most common allergens, especially indoor inhalant allergens, during the COVID-19 epidemic than before the pandemic. CONCLUSION: D. pteronyssinus and D. farinae are the most common allergens in South China. Under the background of normalization of epidemic prevention, indoor inhaled allergens should be first in the prevention and control of allergic diseases, and a combination of various indoor cleaning measures should be used to improve the efficiency of interventions.

Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2.

Bat plasminogen activators α2 (DSPAα2) has extremely high medicinal value as a powerful natural thrombolytic protein. However, wild-type DSPAα2 has two N-glycosylation sites (N185 and N398) and its non-human classes of high-mannose-type N-glycans may cause immune responses in vivo. By mutating the N-glycosylation sites, we aimed to study the effect of its N-glycan chain on plasminogen activation, fibrin sensitivity, and to observe the physicochemical properties of DSPAα2. A logical structure design was performed in this study. Four single mutants and one double mutant were constructed and expressed in Pichia pastoris. When the N398 site was eliminated, the plasminogen activator in the mutants had their activities reduced to ~40%. When the N185 site was inactivated, there was a weak decrease in the plasminogen activation of its mutant, while the fibrin sensitivity significantly decreased by ~10-fold. Neither N-glycosylation nor deglycosylation mutations changed the pH resistance or heat resistance of DSPAα2. This study confirms that N-glycosylation affects the biochemical function of DSPAα2, which provides a reference for subsequent applications of DSPAα2.

The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris.

BACKGROUND: N-glycosylation is one of the most important post-translational modifications. Many studies have shown that N-glycosylation has a significant effect on the secretion level of heterologous glycoproteins in yeast cells. However, there have been few studies reporting a clear and unified explanation for the intracellular mechanism that N-glycosylation affect the secretion of heterologous glycoproteins so far. Pichia pastoris is an important microbial cell factory producing heterologous protein. It is of great significance to study the effect of N-glycosylation on the secretion level of heterologous protein. Camel chymosin is a glycoprotein with higher application potential in cheese manufacturing industry. We have expressed camel prochymosin in P. pastoris GS115, but the lower secretion level limits its industrial application. This study attempts to increase the secretion level of prochymosin through N-glycosylation, and explore the molecular mechanism of N-glycosylation affecting secretion. RESULTS: Adding an N-glycosylation site at the 34th amino acid of the propeptide of prochymosin significantly increased its secretion in P. pastoris. N-glycosylation improved the thermostability of prochymosin without affecting the enzymatic activity. Immunoprecipitation coupled to mass spectrometry (IP-MS) analysis showed that compared with the wild prochymosin (chy), the number of proteins interacting with N-glycosylated mutant (chy34) decreased, and all differential interacting proteins (DIPs) were down-regulated in chy34-GS115 cell. The DIPs in endoplasmic reticulum were mainly concentrated in the misfolded protein pathway. Among the five DIPs in this pathway, overexpression of BiP significantly increased the secretion of chy. The knockout of the possible misfolded protein recognition elements, UDP-glycose:glycoprotein glucosyltransferase 1 and 2 (UGGT1/2) had no effect on the growth of yeast cells and the secretion of prochymosin. CONCLUSIONS: In conclusion, N-glycosylation increased the secretion of prochymosin in P. pastoris trough the adjustment of intracellular interacted proteins. The results of our study may help to elucidate the molecular mechanism of N-glycosylation affecting secretion and provide a new research method to improve the secretion of heterologous glycoprotein in P. pastoris.

Distinctions Between Fecal and Intestinal Mucosal Microbiota in Subgroups of Irritable Bowel Syndrome.

BACKGROUND AND AIMS: Recent studies have shown that changes in the intestinal microbiota contribute to the pathogenesis of irritable bowel syndrome (IBS). This study aimed to investigate the characteristics of the fecal and intestinal mucosal microbiota in IBS patients, and the correlation between microbiota and clinical manifestations. METHODS: Fecal and intestinal mucosal samples were collected from 14 constipation-predominant IBS (IBS-C) patients, 20 diarrhea-predominant IBS (IBS-D) patients, and 20 healthy controls (HCs). 16S rRNA gene sequencing and fluorescence in situ hybridization were used for the analysis of samples. RESULTS: Community richness and diversity of the fecal microbiota in IBS patients were significantly reduced compared with the HCs. The mucosal samples in IBS patients showed decreased Bifidobacterium and increased Bacteroides caccae compared with HCs; Eubacterium and Roseburia were decreased in IBS-C patients and increased in IBS-D patients. A comparison of the fecal and mucosal microbiota in IBS patients showed significantly increased Bifidobacterium in fecal samples and a decrease in mucosal samples in IBS-C patients; Bacteroides caccae and Roseburia were significantly reduced in fecal samples and increased in mucosal samples of IBS patients. A correlation between microbiota and clinical manifestations in IBS patients showed that Bacteroides caccae and Roseburia in fecal samples and Bifidobacterium and Eubacterium in mucosal samples were associated with abdominal pain and distention. CONCLUSIONS: Distinct differences exist between the fecal and intestinal mucosal microbiota in IBS patients, with the changes in the latter appearing more consistent with the pathophysiology of IBS. Changes in intestinal microbiota were associated with the clinical manifestations in IBS.

Dysregulated behaviour of hair follicle stem cells triggers alopecia and provides potential therapeutic targets.

Due to a steady increase in the number of individuals suffering from alopecia, this condition has recently received increasing attention. Alopecia can be caused by various pathological, environmental or psychological factors, eventually resulting in abnormalities in hair follicle (HF) structures or HF regeneration disorders, especially dysregulated hair follicle stem cell (HFSC) behaviour. HFSC behaviour includes activation, proliferation and differentiation. Appropriate HFSC behaviour sustains a persistent hair cycle (HC). HFSC behaviour is mainly influenced by HFSC metabolism, ageing and the microenvironment. In this review, we summarize recent findings on how HFSC metabolism, ageing and the microenvironment give rise to hair growth disorders, as well as related genes and signalling pathways. Recent research on the application of stem cell-based hair tissue engineering and regenerative medicine to treat alopecia is also summarized. Determining how dysregulated HFSC behaviour underlies alopecia would be helpful in identifying potential therapeutic targets.

Non-invasive neuromodulation effects on painful diabetic peripheral neuropathy: a systematic review and meta-analysis.

Diabetic Peripheral Neuropathy (DPN) typically is accompanied by painful symptoms. Several therapeutic agents have been tried for symptomatic relief, but with varying results. The use of non-invasive neuromodulation (NINM) is a potential treatment option for DPN. The objective of our study is to evaluate NINM effects on pain rating and nerve conduction velocity in DPN patients. The search was carried out in seven databases until Aug 30th, 2019. Finally, twenty studies met the inclusion criteria. We found a significant reduction of pain scores by central NINMs (effect size [ES] = - 0.75, 95% CI = - 1.35 to - 0.14), but not by the overall peripheral techniques (electrical and electromagnetic) (ES = - 0.58, 95% CI = - 1.23 to 0.07). However, the subgroup of peripheral electrical NINMs reported a significant higher effect (ES = - 0.84, 95% CI = - 1.57 to - 0.11) compared to electromagnetic techniques (ES = 0.21; 95% CI = - 1.00 to 1.42, I2 = 95.3%) . Other subgroup analysis results show that NINMs effects are higher with intensive protocols and in populations with resistant symptoms or intolerance to analgesic medications. Besides, NINMs can increase motor nerves velocity (ES = 1.82; 95% CI = 1.47 to 2.17), and there were no effects on sensory nerves velocity (ES = 0.01, 95% CI = - 0.79 to 0.80). The results suggest that central and peripheral electrical NINMs could reduce neuropathic pain among DPN patients, without reported adverse events. Well-powered studies are needed to confirm that NINM techniques as an alternative effective and safe treatment option.

Inhibiting HMGB1-RAGE axis prevents pro-inflammatory macrophages/microglia polarization and affords neuroprotection after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) favors a persistent pro-inflammatory macrophages/microglia-mediated response with only a transient appearance of anti-inflammatory phenotype of immune cells. However, the mechanisms controlling this special sterile inflammation after SCI are still not fully elucidated. It is known that damage-associated molecular patterns (DAMPs) released from necrotic cells after injury can trigger severe inflammation. High mobility group box 1(HMGB1), a ubiquitously expressed DNA binding protein, is an identified DAMP, and our previous study demonstrated that reactive astrocytes could undergo necroptosis and release HMGB1 after SCI in mice. The present study aimed to explore the effects and the possible mechanism of HMGB1on macrophages/microglia polarization, as well as the neuroprotective effects by HMGB1 inhibition after SCI. METHODS: In this study, the expression and the concentration of HMGB1 was determined by qRT-PCR, ELISA, and immunohistochemistry. Glycyrrhizin was applied to inhibit HMGB1, while FPS-ZM1 to suppress receptor for advanced glycation end products (RAGE). The polarization of macrophages/microglia in vitro and in vivo was detected by qRT-PCR, immunostaining, and western blot. The lesion area was detected by GFAP staining, while neuronal survival was examined by Nissl staining. Luxol fast blue (LFB) staining, DAB staining, and western blot were adopted to evaluate the myelin loss. Basso-Beattie-Bresnahan (BBB) scoring and rump-height Index (RHI) assay was applied to evaluate locomotor functional recovery. RESULTS: Our data showed that HMGB1 can be elevated and released from necroptotic astrocytes and HMGB1 could induce pro-inflammatory microglia through the RAGE-nuclear factor-kappa B (NF-κB) pathway. We further demonstrated that inhibiting HMGB1 or RAGE effectively decreased the numbers of detrimental pro-inflammatory macrophages/microglia while increased anti-inflammatory cells after SCI. Furthermore, our data showed that inhibiting HMGB1 or RAGE significantly decreased neuronal loss and demyelination, and improved functional recovery after SCI. CONCLUSIONS: The data implicated that HMGB1-RAGE axis contributed to the dominant pro-inflammatory macrophages/microglia-mediated pro-inflammatory response, and inhibiting this pathway afforded neuroprotection for SCI. Thus, therapies designed to modulate immune microenvironment based on this cascade might be a prospective treatment for SCI.

Non-invasive prediction model for high-risk esophageal varices in the Chinese population.

BACKGROUND: There are two types of esophageal varices (EVs): high-risk EVs (HEVs) and low-risk EVs, and HEVs pose a greater threat to patient life than low-risk EVs. The diagnosis of EVs is mainly conducted by gastroscopy, which can cause discomfort to patients, or by non-invasive prediction models. A number of non-invasive models for predicting EVs have been reported; however, those that are based on the formula for calculation of liver and spleen volume in HEVs have not been reported. AIM: To establish a non-invasive prediction model based on the formula for liver and spleen volume for predicting HEVs in patients with viral cirrhosis. METHODS: Data from 86 EV patients with viral cirrhosis were collected. Actual liver and spleen volumes of the patients were determined by computed tomography, and their calculated liver and spleen volumes were calculated by standard formulas. Other imaging and biochemical data were determined. The impact of each parameter on HEVs was analyzed by univariate and multivariate analyses, the data from which were employed to establish a non-invasive prediction model. Then the established prediction model was compared with other previous prediction models. Finally, the discriminating ability, calibration ability, and clinical efficacy of the new model was verified in both the modeling group and the external validation group. RESULTS: Data from univariate and multivariate analyses indicated that the liver-spleen volume ratio, spleen volume change rate, and aspartate aminotransferase were correlated with HEVs. These indexes were successfully used to establish the non-invasive prediction model. The comparison of the models showed that the established model could better predict HEVs compared with previous models. The discriminating ability, calibration ability, and clinical efficacy of the new model were affirmed. CONCLUSION: The non-invasive prediction model for predicting HEVs in patients with viral cirrhosis was successfully established. The new model is reliable for predicting HEVs and has clinical applicability.

MicroRNA-383 inhibits proliferation, migration, and invasion in hepatocellular carcinoma cells by targeting PHF8.

BACKGROUND: To study the effect of microRNA-383 (miR-383) on cell proliferation, migration, and invasion of hepatocellular carcinoma (HCC) cells, and explore its mechanism. METHODS: The expressions of miR-383 and plant homology domain that refers to protein 8 (PHF8) were detected in tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot respectively. The miR-383 group (transfected miR-383 mimics), miR-con group (transfected miR-con), si-con group (transfected si-con), si-PHF8 group (transfected si-PHF8), miR-383 + ctrl group (cotransfected miR-383 mimics and pcDNA-3.1), miR-383 + PHF8 group (cotransfected miR-383 mimics and pcDNA-3.1-PHF8) were transfected into HepG2 cells by liposome method. Cell proliferation, migration and invasion were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) or trans-well assays respectively. The luciferase activity of each group was detected by dual luciferase reporter gene assay. RESULTS: Compared with normal adjacent tissues, the expression of miR-383 was significantly down-regulated and the expression of PHF8 was significantly up-regulated (p < .05). Compared with normal hepatocellular cell LO2, the expression of miR-383 was significantly reduced (p < .05) in HCC cells. Moreover, overexpression of miR-383 or silencing of PHF8 significantly inhibited the proliferation, migration, and invasion of HCC cells. In addition, PHF8 was targeted by miR-383 and its restoration rescued the inhibitory effect of miR-383 on cell proliferation, migration, and invasion of HCC cells. CONCLUSION: miR-383 could inhibit the proliferation, migration, and invasion of HCC cells by targeting PHF8, which will provide a basis for miR-383 targeted therapy for HCC.

Ozone therapy promotes the differentiation of basal keratinocytes via increasing Tp63-mediated transcription of KRT10 to improve psoriasis.

Psoriasis is a chronic immune-mediated inflammatory dermatosis. Recently, ozone therapy has been applicated to psoriasis treatment; however, the mechanism by which ozone therapy improves psoriasis remains unclear. The excessive proliferation and the differentiation of basal keratinocytes have been considered critical issues during pathological psoriasis process, in which keratin 6 (KRT6) and KRT10 might be involved. In the present study, KRT6, IL-17 and IL-22 protein within psoriasis lesions was decreased, while KRT10 and Tp63 protein in psoriasis lesions was increased by ozone treatment in both patient and IMQ mice psoriatic tissues. In the meantime, ozone treatment down-regulated KRT6 mRNA and protein expression while up-regulated KRT10 mRNA and protein expression within IL-22 treated primary KCs; the cell viability of KCs was suppressed by ozone treatment. Moreover, Tp63 bound to KRT10 promoter region to activate its transcription in basal keratinocytes; the promotive effects of ozone on Tp63 and KRT10 were significantly reversed by Tp63 silence. Both TP63 and KRT10 mRNA expression were significantly increased by ozone treatment in psoriasis lesions; there was a positive correlation between Tp63 and KRT10 expression within tissue samples, suggesting that ozone induces the expression of Tp63 to enhance the expression of KRT10 and the differentiation of keratinocytes, therefore improving the psoriasis. In conclusion, the application of ozonated oil could be an efficient and safe treatment for psoriasis; ozone promotes the differentiation of keratinocytes via increasing Tp63-mediated transcription of KRT10, therefore improving psoriasis.

A Na+/H+ antiporter, K2-NhaD, improves salt and drought tolerance in cotton (Gossypium hirsutum L.).

KEY MESSAGE: Overexpression of K2-NhaD in transgenic cotton resulted in phenotypes with strong salinity and drought tolerance in greenhouse and field experiments, increased expression of stress-related genes, and improved regulation of metabolic pathways, such as the SOS pathway. Drought and salinity are major abiotic stressors which negatively impact cotton yield under field conditions. Here, a plasma membrane Na+/H+ antiporter gene, K2-NhaD, was introduced into upland cotton R15 using an Agrobacterium tumefaciens-mediated transformation system. Homozygous transgenic lines K9, K17, and K22 were identified by PCR and glyphosate-resistance. TAIL-PCR confirmed that T-DNA carrying the K2-NhaD gene in transgenic lines K9, K17 and K22 was inserted into chromosome 3, 19 and 12 of the cotton genome, respectively. Overexpression of K2-NhaD in transgenic cotton plants grown in greenhouse conditions and subjected to drought and salinity stress resulted in significantly higher relative water content, chlorophyll, soluble sugar, proline levels, and SOD, CAT, and POD activity, relative to non-transgenic plants. The expression of stress-related genes was significantly upregulated, and this resulted in improved regulation of metabolic pathways, such as the salt overly sensitive pathway. K2-NhaD transgenic plants growing under field conditions displayed strong salinity and drought tolerance, especially at high levels of soil salinity and drought. Seed cotton yields in transgenic line were significantly higher than in wild-type plants. In conclusion, the data indicate that K2-NhaD transgenic lines have great potential for the production of stress-tolerant cotton under field conditions.

Long-term Outcomes of Autologous Peripheral Blood Stem Cell Transplantation in Patients With Cirrhosis.

BACKGROUND & AIMS: Peripheral blood stem cells (PBSCs) mobilized with colony-stimulating factor can promote liver regeneration and increase liver function in patients with liver diseases. However, the long-term effects of stem cell treatments on survival and risk of hepatocellular carcinoma (HCC) in patients with cirrhosis have not been determined. We investigated the long-term effects of autologous stem cell transplantation and risk of HCC in patients with cirrhosis. METHODS: We performed a retrospective analysis of 2 cohorts of patients with decompensated cirrhosis who received transplantations of autologous PBSCs (n = 282) or standard medical treatment (SMT, n = 286) in China from January 1, 2006, through December 31, 2016. Patients were followed up until death or liver transplantation. Mortality data were obtained by case records and confirmed by telephone calls. Survival time was calculated and HCC was confirmed by computed tomography or ultrasound. We used propensity score matching to adjust the differences between the 2 groups. Survival and incidence of HCC were analyzed and Cox proportional hazard regression was used to determine the prognostic factors. RESULTS: After propensity score matching, time of survival was significantly higher in the PBSC group than the SMT group (P = .001). The adjusted rate of 5-year survival was 71.2% in the PBSC group and 52.1% in the SMT group. The overall incidence of HCC did not differ significantly between the PBSC and SMT groups (21.1% vs 20.4%; P = .999). Significant improvement of liver functions was observed at 1 year, 2 years, 3 years, and 5 years after PBSC transplantation compared with the SMT group. CONCLUSIONS: In a long-term analysis of patients with decompensated cirrhosis, autologous transplants of PBSCs significantly improved long-term survival compared with a control group. PBSC transplant did not appear to increase the risk of HCC.

[Bactericidal effect of ozonated camellia oil on Staphylococcus aureus in vitro].

OBJECTIVE: To explore a new method for detecting the bactericidal effect of oiling agent in vitro, and to determine the disinfectant effecacy of ozonated camellia oil on Staphylococcus aureus.
 Methods: Suspension of Staphylococcus aureus was prepared and innoculated on the LB plate by plate scribing method. After culture overnight, 21 bacterial monoclones with the same diameter were selected and divided into 3 groups: A negative control group, a baseoil (camellia oil) group and an ozonated camellia oil group. We used a ring to isolate the single clone and added oil inside the ring, cultured the whole plate over night, picked out each single clone (with gel) to 5 mL LB medium and cultured it for 12 h. The final concentration of the LB medium was detected by plate count method and turbidimetry.
 Results: According to the plate count method and turbidimetry, the bacterial concentration in the ozonated camellia oil group was lower than that in the negative control group and base oil group (P<0.001).
 Conclusion: Bacterial monoclone culture method shows that ozonated camellia oil can significantly kill Staphylococcus aureus, and this method is an effective method for evaluating the bactericidal function of the oiling agent in vitro.

Establishment and Assessment of Mice Models of Type 2 Diabetes Mellitus.

Objective To establish type 2 diabetes mellitus(T2DM)KM mouse models via the combined use of high-calorie diet and multiple administration of low-dose streptozotocin(STZ). Methods Based on the randomized number table,30 KM mice were equally and randomly divided into 2 groups:modeling group and control group. Mice in the modeling group were given foods with high calories for one month and injected with 30 mg/kg STZ via the left lower abdominal cavity for 2-4 consecutive days,while mice in the control group were fed with standard maintenance foods and the same dose of citrate buffer solution. The general conditions including food and water intake and mice weight were recorded. Blood glucose level was measured 1,2,4,5,12,and 21 weeks after STZ injection. When the glucose level became stabilized,the serum insulin and blood lipids [including total cholesterol(TC),triacylglycerol(TG),high-density lipoprotein(HDL) and low-density lipoprotein(LDL)],and hemoglobin a1c (HbA1c)were measured,and oral glucose tolerance test were performed. Results The modeling group had a 100% survival rate. After STZ injection,the body weight of mice in the modeling group reached the peak in the forth week,and later the growth rate decreased,still significantly lower than that of control group mice till the 21st week(t=3.160,P=0.006). Their blood glucose level was significantly higher than that of mice before STZ injection and in the control group(all P<0.05);as time went on,it was also rising,and it remained high till the 21st week [(26.38±1.34)mmol/L]. In the 4th week,the fasting blood glucose of mice in the modeling group was(11.86±3.33)mmol/L,which was significantly higher than that of mice in the control group [(6.37±1.27)mmol/L](t=-3.830,P=0.002). Fasting serum insulin of mice in the modeling group showed no significant difference compared with control group [(5.73±0.24)mU/L vs.(5.48±0.32)mU/L;t=-0.863,P=0.416]. Insulin sensitivity index was 0.0145±0.0039,which was significantly lower than that(0.0267±0.0039)in control group(t=4.414,P=0.003). In the 6th week,the blood glucose levels of mice in the modeling group were(15.35±1.82),(26.45±1.07),(25.58±1.46),and(26.15±1.00)mmol/L 0,30,60,and 120 min after oral gavage of D-glucose,which were all significantly higher than those in the control group [(6.88±1.75)(t=-8.203,P=0.000),(17.65±2.94)(t=-6.884,P=0.000),(13.18±2.04)(t=-12.110,P=0.000),and(7.37±3.40)mmol/L(t=-12.969,P=0.000)]. In the 8th week,serum TC and TG levels of mice in the modeling group were(3.83±0.06)and(2.20±0.20)mmol/L,which were significantly higher than those in the control group [(3.10±0.10)(t=11.000,P=0.000)and(0.90±0.10)mmol/L(t=10.070,P=0.000)]. HDL level of mice in the modeling group was(2.03±0.06)mmol/L,which was significantly lower than that in the control group [(2.48±0.02)mmol/L;t=11.662,P=0.000]. LDL level was increased but showed no significant difference [(0.34±0.08)mmol/L vs.(0.26±0.02)mmol/L](t=1.680,P=0.168). HbA1c content of mice in the modeling group was(7.30±0.31)%,which was significantly higher than that(4.40±0.32)% in the control group(t=-11.587,P=0.000). Conclusion KM mice models of T2DM were successfully established after high-calorie diet and multiple administration of low-dose STZ.

Enzymatic modification of chitosan by cinnamic acids: Antibacterial activity against Ralstonia solanacearum.

This study aimed to identify chitosan polymers that have antibacterial activity against the bacterial wilt pathogen. The chitosan polymers were enzymatically synthesized using chitosan and five cinnamic acids (CADs): caffeic acid (CA), ferulic acid (FA), cinnamic acid (CIA), p-coumaric acid (COA) and chlorogenic acid (CHA), using laccase from Pleurotus ostreatus as a catalyst. The reaction was performed in a phosphate buffered solution under heterogenous reaction conditions. The chitosan derivatives (CTS-g-CADs) were characterized by FT-IR, XRD, TGA and SEM. FT-IR demonstrated that the reaction products bound covalently to the free amino groups or hydroxyl groups of chitosan via band of amide I or ester band. XRD showed a reduced packing density for grafted chitosan comparing to original chitosan. TGA demonstrated that CTS-g-CADs have a higher thermostability than chitosan. Additionally, chitosan and its derivatives showed similar antibacterial activity. However, the IC50 value of the chitosan-caffeic acid derivative (CTS-g-CA) against the mulberry bacterial wilt pathogen RS-5 was 0.23mg/mL, which was two-fifths of the IC50 value of chitosan. Therefore, the enzymatically synthesized chitosan polymers can be used to control plant diseases in biotechnological domains.

The Quality Changes of Postharvest Mulberry Fruit Treated by Chitosan-g-Caffeic Acid During Cold Storage.

This study aimed to characterize the effects of chitosan-g-caffeic acid (CTS-g-CA) on improving the quality and extending the shelf life of postharvest mulberry fruit during storage at 4 °C for 18 d. CTS-g-CA was enzymatically synthesized using laccase from Pleurotus ostreatus as a catalyst. The synergistic effects of CTS-g-CA treatment on mulberry fruit were evaluated using a co-toxicity factor (cf). The results showed that the rotting rate of CTS-g-CA-treated fruit was 37.67% (compared with that of the control at 97.67%) on day 18. The weight loss and malondialdehyde (MDA) contents of the CTS-g-CA-treated mulberry fruit were significantly lower (P < 0.05) than those of the control, CA, CTS, and CA+CTS treatments. Moreover, the DPPH and ABTS radical scavenging activities of the CTS-g-CA treatment were both higher than those of the control. Furthermore, the CTS-g-CA treatment also maintained higher levels of main active substances, such as anthocyanins, ascorbic acid, polyphenols and flavones, in mulberry fruit than the other treatments. Therefore, CTS-g-CA could be used to improve the quality and extend the shelf life of postharvest mulberry fruit during cold storage.

An effective biphase system accelerates hesperidinase-catalyzed conversion of rutin to isoquercitrin.

Isoquercitrin is a rare, natural ingredient with several biological activities that is a key precursor for the synthesis of enzymatically modified isoquercitrin (EMIQ). The enzymatic production of isoquercitrin from rutin catalyzed by hesperidinase is feasible; however, the bioprocess is hindered by low substrate concentration and a long reaction time. Thus, a novel biphase system consisting of [Bmim][BF4]:glycine-sodium hydroxide (pH 9) (10:90, v/v) and glyceryl triacetate (1:1, v/v) was initially established for isoquercitrin production. The biotransformation product was identified using liquid chromatography-mass spectrometry, and the bonding mechanism of the enzyme and substrate was inferred using circular dichroism spectra and kinetic parameters. The highest rutin conversion of 99.5% and isoquercitrin yield of 93.9% were obtained after 3 h. The reaction route is environmentally benign and mild, and the biphase system could be reused. The substrate concentration was increased 2.6-fold, the reaction time was reduced to three tenths the original time. The three-dimensional structure of hesperidinase was changed in the biphase system, which α-helix and random content were reduced and ß-sheet content was increased. Thus, the developed biphase system can effectively strengthen the hesperidinase-catalyzed synthesis of isoquercitrin with high yield.