A novel starch-active lytic polysaccharide monooxygenase discovered with bioinformatics screening and its application in textile desizing.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative cleavage of different types of polysaccharides have potential to be used in various industries. However, AA13 family LPMOs which specifically catalyze starch substrates have relatively less members than AA9 and AA10 families to limit their application range. Amylase has been used in enzymatic desizing treatment of cotton fabric for semicentury which urgently need for new assistant enzymes to improve reaction efficiency and reduce cost so as to promote their application in the textile industry. RESULTS: A total of 380 unannotated new genes which probably encode AA13 family LPMOs were discovered by the Hidden Markov model scanning in this study. Ten of them have been successfully heterologous overexpressed. AlLPMO13 with the highest activity has been purified and determined its optimum pH and temperature as pH 5.0 and 50 °C. It also showed various oxidative activities on different substrates (modified corn starch > amylose > amylopectin > corn starch). The results of enzymatic textile desizing application showed that the best combination of amylase (5 g/L), AlLPMO13 (5 mg/L), and H2O2 (3 g/L) made the desizing level and the capillary effects increased by 3 grades and more than 20%, respectively, compared with the results treated by only amylase. CONCLUSION: The Hidden Markov model constructed basing on 34 AA13 family LPMOs was proved to be a valid bioinformatics tool for discovering novel starch-active LPMOs. The novel enzyme AlLPMO13 has strong development potential in the enzymatic textile industry both concerning on economy and on application effect.

Enhanced compatibility between poly(lactic acid) and poly (butylene adipate-co-terephthalate) by incorporation of N-halamine epoxy precursor.

In this work, a new N-halamine precursor with two epoxy groups, 1,3-bis(2,3-epoxypropyl)-s-triazine-2,4,6-trione (BETT), was synthesized and used to enhance the compatibility between poly(lactic acid) (PLA) and poly (butylene adipate-co-terephthalate) (PBAT). The rheological analysis and GPC indicated that chain extension between PLA and PBAT occurred during the melt-blending in the presence of BETT. The PLA/PBAT chain extensions improved the compatibility between PLA and PBAT and hindered the crystallization of PLA. SEM images showed that PLA/PBAT blend gradually changed from the typical sea-island phase without BETT to a co-continuous structure with increase in amount of BETT. This showed that the interfacial compatibility between PLA and PBAT improved significantly on addition of BETT. Moreover, compared to PLA/PBAT blend, the mechanical properties of PLA/PBAT/BETT blends showed great improvement. Furthermore, the chlorinated PLA/PBAT/BETT sheets displayed excellent antibacterial activities against E. coli (CMCC 44103) and S. aureus (ATCC 6538) cultures, wherein the sheets with 17.5 ± 0.8 µg/cm2 of the active chlorine could kill all inoculated bacteria within 30 min.

[Whole blood leukocyte phagocytosis assay for Candida albicans based on flow cytometry].

OBJECTIVE: To establish a whole blood leukocyte phagocytosis assay for Candida albicans (C.albicans) based on flow cytometry (FCM). METHODS: C.albicans of mid-logarithmic growth phase was labeled by fluorescence probe carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), and then added into CD45-PC5 pre-stained human whole blood cells at a 10:1 multiplicity of infection (MOI) in 37DegreesCelsius. The cells were incubated for 10, 30 and 60 minutes. Phagocytosis rate of C.albicans by the CD45 positive cells in the blood was determined by FCM. RESULTS: In yeast extract peptone dextrose medium (YPD) and under the conditions of 37DegreesCelsius and 50 mL/L CO2, the logarithmic growth phase of C.albicans SC5314 was from the 5th to 11th hour. C.albicans were well stained by 10 mmol/L CFDA-SE after 30-minute incubation. After 10-, 30- and 60-minute incubation with SC5314 C.albicans with CD45⁺ cells, the phagocytosis rates measured by FCM were (80.1 ± 6.1)%, (83.8 ± 7.7)% and (92.3 ± 11.2)% for the neutrophils, (11.2 ± 3.6)%, (15.8 ± 4.4)% and (27.7 ± 6.8)% for the monocytes and (0.9 ± 0.3)%, (0.8 ± 0.4)% and (5.2 ± 1.6)% for the lymphocytes. CONCLUSION: The method for measuring whole blood leukocyte phagocytosis of C.albicans based on FCM is successfully established, and 30 minutes are the proper incubation time for the phagocytosis assay.

Suppression of NF-κB pathway by crocetin contributes to attenuation of lipopolysaccharide-induced acute lung injury in mice.

Crocetin, a carotenoid compound, has been shown to reduce expression of inflammation and inhibit the production of reactive oxygen species. In the present study, the effect of crocetin on acute lung injury induced by lipopolysaccharide (LPS) was investigated in vivo. In the mouse model, pretreatment with crocetin at dosages of 50 and 100 mg/kg reduced the LPS-induced lung oedema and histological changes, increased LPS-impaired superoxide dismutase (SOD) activity, and decreased lung myeloperoxidase (MPO) activity. Furthermore, treatment with crocetin significantly attenuated LPS-induced mRNA and the protein expressions of interleukin-6 (IL-6), macrophage chemoattractant protein-1 (MCP-1), and tumour necrosis factor-α (TNF-α) in lung tissue. In addition, crocetin at different dosages reduced phospho-IκB expression and NF-κB activity in LPS-induced lung tissue alteration. These results indicate that crocetin can provide protection against LPS-induced acute lung injury in mice.

Inhibitory effect on protein kinase Ctheta by Crocetin attenuates palmitate-induced insulin insensitivity in 3T3-L1 adipocytes.

Epidemiologic and experimental studies have pointed to an etiologic role of elevated plasma free fatty acids in insulin resistance, which is frequently associated with a state of low-grade inflammation. In this study, we investigated the effects of Crocetin, a unique carotenoid, on insulin resistance induced by palmitate in 3T3-L1 adipocytes. Exposure of palmitate led to an increase in insulin receptor substrate-1 (IRS-1) serine(307) phosphorylation as well as activation of c-Jun NH(2)-terminal kinase (JNK) and inhibitor kappaB kinase beta (IKKbeta), concomitantly with reductions of IRS-1 function and glucose metabolism. Interestingly, pretreatment with Crocetin almost reversed all of these abnormalities in a dose-dependent manner. IRS-1 serine(307) phosphorylation was significantly reduced by JNK or IKKbeta inhibitor, especially by combination of these two inhibitors. Moreover, palmitate treatment induced activation of protein kinase Ctheta (PKCtheta) while blocking PKCtheta significantly inhibited JNK and IKKbeta activation induced by palmitate or phorbol 12-myristate 13-acetate (PKC activator, PMA), and attenuated the palmitate-induced defects in insulin action. Crocetin demonstrated an impressive suppression in the activation of PKCtheta induced not only by palmitate but also by PMA in a dose-dependent manner. Taken together, Crocetin inhibited JNK and IKKbeta activation via suppression of PKCtheta phosphorylation, attenuating insulin insensitivity induced by palmitate in 3T3-L1 adipocytes.

Effect of crocetin on blood pressure restoration and synthesis of inflammatory mediators in heart after hemorrhagic shock in anesthetized rats.

Crocetin, a constituent of saffron, has been shown not only to prevent reactive oxygen species-induced hepatotoxicity and genotoxicity but also to increase whole-body oxygen consumption and survival. The present study was to determine whether crocetin has beneficial effects on cardiac injury caused by hemorrhagic shock and resuscitation in rats. Anesthetized rats were bled to reduce mean arterial pressure (MAP) to 35 +/- 5 mmHg for 60 min and then resuscitated with their withdrawn shed blood and isotonic sodium chloride solution. Crocetin was administered via the duodenum at 50 mg/kg 40 min after bleeding. We investigated MAP, serum creatine kinase activity, the activity of nuclear factor-kappaB, iNOS, and total superoxide dismutase (T-SOD), as well as levels of NO, malondialdehyde, TNF-alpha, and IL-6 in the heart at 2 h postresuscitation. Compared with control group, crocetin significantly increased MAP from 10 min after administration to the end of the protocol except the period between 75 and 90 min after initial bleeding, whereas serum creatine kinase activity was dramatically decreased at 2 h postresuscitation. Myocardial nuclear factor-kappaB activity, iNOS activity, NO, malondialdehyde, TNF-alpha, and IL-6 were significantly elevated, whereas T-SOD activity was suppressed in the control group if compared with those of sham animals. These parameters tended to be normalized in rats administered crocetin. These results suggest that crocetin blocks inflammatory cascades by inhibiting reactive oxygen species production and preserving T-SOD activity to ameliorate the cardiac injury caused by hemorrhage/resuscitation.