Expanding the Applicability of an Innovative Laccase TTI in Intelligent Packaging by Adding an Enzyme Inhibitor to Change Its Coloration Kinetics.

Enzymatic time-temperature indicators (TTIs) usually suffer from instability and inefficiency in practical use as food quality indicator during storage. The aim of this study was to address the aforementioned problem by immobilizing laccase on electrospun chitosan fibers to increase the stability and minimize the usage of laccase. The addition of NaN3, as and enzyme inhibitor, was intended to extend this laccase TTI coloration rate and activation energy (Ea) range, so as to expand the application range of TTIs for evaluating changes in the quality of foods during storage. A two-component time-temperature indicator was prepared by immobilizing laccase on electrospun chitosan fibers as a TTI film, and by using guaiacol solution as a coloration substrate. The color difference of the innovative laccase TTI was discovered to be <3, and visually indistinguishable when OD500 reached 3.2; the response reaction time was regarded as the TTI's coloration endpoint. Enzyme immobilization and the addition of NaN3 increased coloration Km and reduced coloration Vmax. The coloration Vmax decreased to 64% when 0.1 mM NaN3 was added to the TTI, which exhibited noncompetitive inhibition and a slower coloration rate. Coloration hysteresis appeared in the TTI with NaN3, particularly at low temperatures. For TTI coloration, the Ea increased to 29.92-66.39 kJ/mol when 15-25 µg/cm2 of laccase was immobilized, and the endpoint increased to 11.0-199.5 h when 0-0.10 mM NaN3 was added. These modifications expanded the applicability of laccase TTIs in intelligent food packaging.

Enzymatic Time-Temperature Indicator Prototype Developed by Immobilizing Laccase on Electrospun Fibers to Predict Lactic Acid Bacterial Growth in Milk during Storage.

Laccase was immobilized on a chitosan/polyvinyl alcohol/tetraethylorthosilicate electrospun film (ceCPTL) and colored with guaiacol to obtain a laccase time-temperature indicator (TTI) prototype. The activation energy (Ea) of coloration of the prototype was 50.89-33.62 kJ/mol when 8-25 µg/cm2 laccase was immobilized on ceCPTL, and that of lactic acid bacteria (LAB) growth in milk was 73.32 kJ/mol. The Ea of coloration of the TTI prototype onto which 8-10 µg/cm2 laccase was immobilized was in the required range for predicting LAB growth in milk. The coloration endpoint of the TTI prototype onto which 10 µg/cm2 (0.01 U) laccase was immobilized could respond to the LAB count reaching 106 colony-forming units (CFU)/mL in milk during a static temperature response test, and the prediction error was discovered to be low. In dynamic temperature response experiments with intermittent temperature changes between 4 and 25 °C, the coloration rate of the laccase TTI prototype was consistent with LAB growth. The results of this study indicate that the laccase TTI prototype can be applied as a visual monitoring indicator to assist in evaluating milk quality in cold chains.

An Ensemble Classifier with Case-Based Reasoning System for Identifying Internet Addiction.

Internet usage has increased dramatically in recent decades. With this growing usage trend, the negative impacts of Internet usage have also increased significantly. One recurring concern involves users with Internet addiction, whose Internet usage has become excessive and disrupted their lives. In order to detect users with Internet addiction and disabuse their inappropriate behavior early, a secure Web service-based EMBAR (ensemble classifier with case-based reasoning) system is proposed in this study. The EMBAR system monitors users in the background and can be used for Internet usage monitoring in the future. Empirical results demonstrate that our proposed ensemble classifier with case-based reasoning (CBR) in the proposed EMBAR system for identifying users with potential Internet addiction offers better performance than other classifiers.

Hurdle Effect of Antimicrobial Activity Achieved by Time Differential Releasing of Nisin and Chitosan Hydrolysates from Bacterial Cellulose.

We investigated the combined antimicrobial effect of nisin and chitosan hydrolysates (CHs) by regulating the antimicrobial reaction order of substances due to differential releasing rate from hydroxypropylmethylcellulose-modified bacterial cellulose (HBC). The minimum inhibitory concentration of nisin against Staphylococcus aureus and that of CHs against Escherichia coli were 6 IU and 200 µg/mL, respectively. Hurdle and additive effects in antimicrobial tests were observed when nisin was used 6 h before CH treatment against S. aureus; similar effects were observed when CH was used before nisin treatment against E. coli. Simultaneously combined treatment of nisin and CHs exhibited the low antimicrobial effect. HBC was then selected as the carrier for the controlled release of nisin and CHs. A 90% inhibition in the growth of S. aureus and E. coli was achieved when 30 IU-nisin-containing HBC and 62.5 µg/mL-CH-containing HBC were used simultaneously. The controlled release of nisin and CHs by using HBC minimized the interaction between nisin and CHs as well as increased the number of microbial targets.

The bioactive composite film prepared from bacterial cellulose and modified by hydrolyzed gelatin peptide.

The hydrolyzed gelatin peptides, obtained from the hydrolysis of Tilapia nilotica skin gelatin with alcalase and pronase E, were fractionated using an ultrafiltration system into hydrolyzed gelatin peptides-a (10 kDa membrane), hydrolyzed gelatin peptides-b1, and hydrolyzed gelatin peptides-b2 (5 kDa membrane) fractions. The highest oxygen radical absorbance capacity was observed in hydrolyzed gelatin peptides-b2, which contained more nonpolar amino acids than the other hydrolyzed gelatin peptides. Hydrolyzed gelatin peptides-b2 at a concentration of 12.5 mg/ml exhibited the highest proliferation ability and increased the expression of Type I procollagen mRNA, which indicated an enhanced collagen synthesis. Hydrolyzed gelatin peptides protected Detroit 551 cells from 2,2'-azobis(2-amidinopropane) dihydrochloride-induced oxidative damage and increased cell viability. Hydroxylpropylmethyl cellulose-modified bacterial cellulose and dried fabricated biofilm were less eligible for Detroit 551 cell proliferation than bacterial cellulose. The release of hydrolyzed gelatin peptides in bacterial cellulose film was slower than that in hydroxylpropylmethyl cellulose-modified bacterial cellulose and dried fabricated biofilm; thus, bacterial cellulose film and hydroxylpropylmethyl cellulose-modified bacterial cellulose and dried fabricated biofilm are suitable candidates for applications in delayed release type and rapid release type biofilms, respectively.

Influence of alanine uptake on Staphylococcus aureus surface charge and its susceptibility to two cationic antibacterial agents, nisin and low molecular weight chitosan.

Low molecular weight chitosan (LMWC) and nisin, recognized as cationic antibacterial agents (CAAs), inhibit bacterial growth by interacting with the anionically charged cell wall. In this study, alanine uptake significantly reduced the anionic cell surface charge, as determined by the zeta potential (ZP) measurements, of Staphylococcus aureus, resulting from the incorporation of d-alanine into the cell wall. Minimum inhibitory concentration (MIC) tests and growth inhibition curves revealed that LMWC and nisin possessed inverse antibacterial activity against three strains of S. aureus, depending on the strains' net charge. A twofold reduction in the MIC value of nisin was obtained against S. aureus, inoculated in a 1.0% d- or l-alanine-augmented trypticase soy broth medium. A flocculation test demonstrated that neutralizing the anionic surface charge using d-alanine reduced the adsorption of S. aureus onto LMWC. Furthermore, the reduced surface net charge could enhance the colonization capacity of S. aureus on glass.

Isochaihulactone protects PC12 cell against H(2)O(2) induced oxidative stress and exerts the potent anti-aging effects in D-galactose aging mouse model.

AIM: to investigate the effect of isochaihulactone (also known as K8), a lignan compound of Bupleurum scorzonerifolium, on H(2)O(2)-induced cytotoxicity in neuronally differentiated PC12 cells (nPC12). METHODS: viability of neuronal PC12 cells was measured using MTT assay. Protein expression was determined by Western blot. Apoptotic cells was determined using TUNEL assay. D-galactose aging mice were used as a model system to study the anti-oxidant effects of isochaihulactone in vivo. RESULTS: pretreatment with isochaihulactone (5-10 micromol/L) increased cell viability and decreased membrane damage, generation of reactive oxygen species and degradation of poly (ADP-ribose) polymerase in H(2)O(2)-treated nPC12 cells and also decreased the expression of cyclooxygenase-2, via downregulation of NF-kappaB, resulting in a decrease in lipid peroxidation. The results suggest that isochaihulactone is a potential antioxidant agent. In a murine aging model, in which chronic systemic exposure to D-galactose (D-gal) causes the acceleration of senescence, administration of isochaihulactone (10 mgxkg(-1)xd(-1), sc) for 7 weeks concomitant with D-gal injection significantly increased superoxide dismutase and glutathione peroxidase activities and decreased the MDA level in plasma. Furthermore, H&E staining to quantify cell death within hippocampus showed that percentage of pyknotic nuclei in the D-gal-treated mice were much higher than in control. CONCLUSION: the results suggest that isochaihulactone exerts potent anti-aging effects against D-gal in mice possibly via antioxidative mechanisms.

Antimicrobial peptide of an anti-lipopolysaccharide factor modulates of the inflammatory response in RAW264.7 cells.

In this study, to clarify the protective mechanism of a peptide from shrimp anti-lipopolysaccharide (LPS) factor (SALF) against endotoxin shock, we evaluated the effects of the SALF and LPS on the production and release of tumor necrosis factor (TNF)-alphain vitro using the RAW264.7 murine macrophage cell line. Stimulation by LPS induced the production of inflammatory cytokines, and the SALF was able to modulate TNF-alpha production in LPS-stimulated RAW264.7 cells. Microarray studies revealed a transcriptional profile which was assessed in the presence or absence of the SALF by a quantitative real-time polymerase chain reaction. Pretreatment with the SALF significantly downregulated the expression of nuclear factor (NF)-kappaB in the presence of LPS. In contrast, pretreatment with the SALF significantly elevated the expressions of Anp32a, CLU, and SLPI, which are considered to be immune-related genes in the presence of LPS. Inhibitor studies suggested that the SALF's modulation of LPS-induced TNF-alpha production involved a complex mechanism with mitogen-activated protein kinase kinase, calcium, and protein kinase C. The data from this study, which imply that the SALF can suppress TNF-alpha production, suggest a role for the SALF in the defense mechanism which can potentially be applied to mammals for endotoxin treatment.

In situ modification of bacterial cellulose network structure by adding interfering substances during fermentation.

In an attempt to obtain bacterial cellulose (BC) with improved rehydration ability, Tween 80, urea, fluorescent brightener, hydroxypropylmethyl cellulose (HPMC) and carboxymethyl cellulose (CMC) were introduced into BC fermentation medium. Measurements of the mechanical strength of the resulting BCs (TBC, UBC, FBC, HBC and CBC) showed a decline except for UBC. SEM images showed that, although the cellulose bundle widths of FBC, HBC and CBC increase, the cellulose network void in FBC grew, while those in HBC and CBC shrank. X-ray diffraction and FT-IR analysis demonstrated that the addition of HPMC and CMC reduced the degree of crystallinity in their corresponding MBCs from 70.54% to 52.23% and 45.38%, respectively. HBC and CBC also exhibited the highest rehydration ability among all MBCs as well as the lowest crystallinity. The in situ modification with HPMC and CMC during fermentation can effectively improve rehydration ability of BC by altering its network structure.

Shrimp anti-lipopolysaccharide factor peptide enhances the antitumor activity of cisplatin in vitro and inhibits HeLa cells growth in nude mice.

The antitumor activity of the shrimp anti-lipopolysaccharide factor (SALF), an antimicrobial peptide, was not previously examined. In this study, a synthetic SALF was tested for antitumor activity using HeLa cells as the study model. We show that the SALF inhibited the proliferation of HeLa cells and reduced colony formation in a soft agar assay. An enhanced effect was observed when the SALF and cisplatin were used in combination, which caused significant inhibition of HeLa cells. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that the SALF altered the membrane structure similar to what a lytic peptide does. A flow cytometric analysis, qRT-PCR, and Western blotting showed that the SALF induced apoptosis, activated caspases-6, -7, and -9, and downregulated Bcl-2 and nuclear factor (NF)-kappaB suggesting that the SALF induces apoptosis through the death receptor/NF-kappaB signaling pathway. An in vivo analysis revealed that the SALF displayed significant tumor suppressive activity in mice with tumor xenografts. Overall, these results indicated that the SALF possesses the potential to be a novel therapeutic agent for treating cervical cancer.

An embedded mobile ECG reasoning system for elderly patients.

With the increase in the number senior citizens and chronic diseases, the number of elderly patients who need constant assistance has increased. One key point of all critical care for elderly patient is the continuous monitoring of their vital signs. Among these, the ECG signal is used for noninvasive diagnosis of cardiovascular diseases. Also, there is a pressing need to have a proper system in place for patient identification. Errors in patient identification, and hence improper administration of medication can lead to disastrous results. This paper proposes a novel embedded mobile ECG reasoning system that integrates ECG signal reasoning and RF identification together to monitor an elderly patient. As a result, our proposed method has a good accuracy in heart beat recognition, and enables continuous monitoring and identification of the elderly patient when alone. Moreover, in order to examine and validate our proposed system, we propose a managerial research model to test whether it can be implemented in a medical organization. The results prove that the mobility, usability, and performance of our proposed system have impacts on the user's attitude, and there is a significant positive relation between the user's attitude and the intent to use our proposed system.

Immune response and inhibition of bacterial growth by electrotransfer of plasmid DNA containing the antimicrobial peptide, epinecidin-1, into zebrafish muscle.

Bacterial infections represent serious diseases in aquaculture, rapidly leading to fish death by septicemia. We investigated whether the electrotransfer of green fluorescent protein gene fusion epinecidin-1 (CMV-gfp-epi) DNA into zebrafish muscle could regulate the fish immune response and inhibit bacterial growth. Electroporation parameters such as the number of pulses, voltage, and amount of plasmid DNA were analyzed, and results demonstrated the greatest mRNA expression level of gfp-epi relative to beta-actin was obtained with a pulse number of 4, a voltage strength of 100 V/cm, a concentration of DNA of 90 microg/fish, and electroporation for 96 h. In addition, the cytomegalovirus (CMV) promoter exhibited higher activity compared to the mylz promoter in muscle for electrotransfer in zebrafish. GFP fluorescence and gfp-epi mRNA expression in tissues after electroporation were also studied by a polymerase chain reaction, immunohistochemistry, and fluorescence microscopy. gfp-epi expression was significantly correlated with decreased bacterial numbers and immune-related gene expression. These data demonstrate that electroporation of epinecidin-1 might have provoked an inflammatory response that accounts for the improvement in bacterial clearance.

Design and synthesis of cationic antimicrobial peptides with improved activity and selectivity against Vibrio spp.

Extensive use of classical antibiotics has led to the growing emergence of many resistant strains of pathogenic bacteria. Evidence has suggested that cationic antimicrobial peptides (AMPs) are of greatest potential to represent a new class of antibiotics. The largest group of AMPs comprises peptides that fold into an amphipathic alpha-helical conformation when interacting with the target microorganism. In the current study, a series of cationic AMPs of 20 amino acids was designed and synthesised based on four structural parameters, including charge, polar angle, hydrophobicity and hydrophobic moment. The effect of these parameters on antimicrobial activity and selectivity was assessed by structural and biological analyses. Our results indicated that high hydrophobicity and amphipathicity (hydrophobic moment) were correlated with increased haemolytic activity, whilst antimicrobial activity was found to be less dependent on these factors. Three of the synthetic AMPs (GW-Q4, GW-Q6 and GW-H1) showed higher antimicrobial activity and selectivity against a broad spectrum of Gram-positive and Gram-negative bacteria compared with the naturally occurring AMPs magainin 2a and pleurocidin. This study also demonstrates that these rationally designed cationic and amphipathic helical AMPs exhibited high selectivity against several Vibrio spp. and are potential agents for future use in the treatment of these marine pathogens.