Recent advances in the fabrication of pH-sensitive indicators films and their application for food quality evaluation.

Over a few decades, anthocyanin (ACN)-based colorimetric indicators in intelligent packaging systems have been widely used to monitor the freshness or spoilage of perishable food products. Most of the perishable food products are highly susceptible to enzymatic/microbial spoilage and produce several volatile or nonvolatile organic acid and nitrogenous compounds. As a result, the natural pH of fresh foods significantly changes. Fabrication of CAN-based colorimetric indicators in intelligent packaging systems is an advanced technique that monitors the freshness or spoilage of perishable foods based on the display of color variations at varying pH values. This study focuses on the advancement of pH-sensitive indicators and extraction of colorimetric indicators from commercially available natural sources. Moreover, the fabrication techniques and widespread industrial applications of such indicators have also been discussed. In addition, readers will get information about the color-changing and antioxidant mechanisms of ACN-based indicator films in food packaging.

Pectin mediates the mechanism of host blood glucose regulation through intestinal flora.

Pectin is a complex polysaccharide found in plant cell walls and interlayers. As a food component, pectin is benefit for regulating intestinal flora. Metabolites of intestinal flora, including short-chain fatty acids (SCFAs), bile acids (BAs) and lipopolysaccharides (LPS), are involved in blood glucose regulation. SCFAs promote insulin synthesis through the intestine-GPCRs-derived pathway and hepatic adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway to promote hepatic glycogen synthesis. On the one hand, BAs stimulate intestinal L cells and pancreatic α cells to secrete Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) through receptors G protein-coupled receptor (TGR5) and farnesoid X receptor (FXR). On the other hand, BAs promote hepatic glycogen synthesis through AMPK pathway. LPS inhibits the release of inflammatory cytokines through Toll-like receptors (TLRs)-myeloid differentiation factor 88 (MYD88) pathway and mitogen-activated protein kinase (MAPK) pathway, thereby alleviating insulin resistance (IR). In brief, both SCFAs and BAs promote GLP-1 secretion through different pathways, employing strategies of increasing glucose consumption and decreasing glucose production to maintain normal glucose levels. Notably, pectin can also directly inhibit the release of inflammatory cytokines through the -TLRs-MYD88 pathway. These data provide valuable information for further elucidating the relationship between pectin-intestinal flora-glucose metabolism.

Separation and Purification of Hydroxyl-α-Sanshool from Zanthoxylum armatum DC. by Silica Gel Column Chromatography.

Hydroxyl-α-sanshool is the main alkylamide produced by Zanthoxylum armatum DC., and it is responsible for numbness after consuming Z. armatum-flavored dishes or food products. The present study deals with the isolation, enrichment, and purification of hydroxyl-α-sanshool. The results indicated that the powder of Z. armatum was extracted with 70% ethanol and then filtrated; the supernatant was concentrated to get pasty residue. Petroleum ether (60-90 °C) and ethyl acetate at a 3:2 ratio, with an Rf value of 0.23, were chosen as the eluent. Petroleum ether extract (PEE) and ethyl acetate-petroleum ether extract (E-PEE) were used as the suitable enriched method. Afterward, the PEE and E-PEE were loaded onto silica gel for silica gel column chromatography. Preliminary identification was carried out by TLC and UV. The fractions containing mainly hydroxyl-α-sanshool were pooled and dried by rotary evaporation. Lastly, all of the samples were determined by HPLC. The yield and recovery rates of hydroxyl-α-sanshool in the p-E-PEE were 12.42% and 121.65%, respectively, and the purity was 98.34%. Additionally, compared with E-PEE, the purity of hydroxyl-α-sanshool in the purification of E-PEE (p-E-PEE) increased by 88.30%. In summary, this study provides a simple, rapid, economical, and effective approach to the separation of high-purity hydroxyl-α-sanshool.

Nucleic Acid Hybridization Enhanced Luminescence for Rapid and Sensitive RNA and DNA Based Diagnostics.

Long-lived emissive nucleic acid probes are widely used in biochemical analysis due to their programmable structures, high signal-to-background ratio, and high sensitivity. Homogeneous detection based on long-lived emissive nucleic acid probes is often achieved through Förster resonance energy transfer (FRET), which suffers from the limitation of a narrow effective distance range. Herein, a new strategy of accessing nucleic acid hybridization-responsive luminescent probes is presented. The photoluminescence (PL) of a Lumi4-Tb complex internally modified with DNA is switched on by nucleic acid hybridization, after which the PL is increased up to 20 times. PL lifetime analysis revealed a possible mechanism of luminescence enhancement. Due to the flexibility of single-stranded nucleic acid chains, the bases and phosphate groups can coordinate with the Tb(III), which reduces the stability of the Tb complex and results in weak PL. After hybridization, the rigid double helix structure suppresses the coordination between Tb(III) and the bases or phosphate groups, causing luminescence enhancement. As the DNA sequence can be freely designed, an array of probes for different DNA or RNA targets can be created with the same Tb complex. Moreover, the novel probe design can afford pM detection limits of DNA or RNA without any nucleic acid amplification and exhibits great potential for nucleic acid detection in clinical diagnosis.

Potential implications of polyphenols on aging considering oxidative stress, inflammation, autophagy, and gut microbiota.

Naturally occurring compounds polyphenols are secondary metabolites of plants, comprised several categories, namely, flavonoids, phenolic acids, lignans and stilbenes. The biological aging process is driven by a series of interrelated mechanisms, including oxidative stress, inflammation status, and autophagy function, through diverse signaling pathways. Moreover, the crucial role of gut microbiota in regulating aging and health status was widely demonstrated. In recent years, the potential anti-aging benefits of polyphenols have been gaining increasing scientific interest due to their capability to modulate oxidative damage, inflammation, autophagy, and gut microbiota. This review highlights the influence of polyphenols in preventing aging disorders and augmenting lifespan based on the influence of oxidative stress, inflammation, autophagy, and gut microbiota, and encourages research on novel polyphenol-based strategies and clinical trials to develop a nutrition-oriented holistic anti-aging therapy.

Novel liquid fermentation medium of Cordyceps militaris and optimization of hydrothermal reflux extraction of cordycepin.

In this study, we developed a novel liquid fermentation medium of Cordyceps militaris using pupa powder and wheat bran as nitrogen resources instead of the traditionally used peptone. This process not only reduced the cost by approximately 50%, but increased production by over 30%. Then, we explored a method to extract and purify cordycepin by combining hydrothermal reflux extraction with macroporous resin adsorption, which is inexpensive and suitable for the industrial production. The optimum conditions for hydrothermal reflux were extracting three times at 95 °C with 1:10 sample-to-water ratio, and the cordycepin purity with macroporous resin HPD-100 reached 95.23%.[Formula: see text].

Portable and Field-Ready Detection of Circulating MicroRNAs with Paper-Based Bioluminescent Sensing and Isothermal Amplification.

We present a paper-based system that integrates bioluminescence resonance energy transfer (BRET) and isothermal amplification for the analysis of tumor-associated circulating microRNAs (miRNAs) in clinical serum samples. The analysis procedure could be easily accomplished with two pieces of functionalized paper and a low-cost smartphone-based device, which enables sequence-specific quantification of femtomolar miRNAs, without the need for tedious handling of aqueous reactions and operation of sophisticated equipment. Furthermore, the analytical performance of the proposed paper-based system was highly stable at room temperature, demonstrating its capability for cold-chain-free and remote deployment. These qualities highlight the practical utility of our method for the portable and field-ready miRNA diagnostic tests in resource-limited settings.

The Marriage of Protein and Lanthanide: Unveiling a Time-Resolved Fluorescence Sensor Array Regulated by pH toward High-Throughput Assay of Metal Ions in Biofluids.

A protein/lanthanide complex (BSA/Tb3+)-based sensor array in two different pH buffers has been designed for high-throughput recognition and time-resolved fluorescence (TRF) detection of metal ions in biofluids. BSA, which acted as an antenna ligand, can sensitize the fluorescence of Tb3+ (i.e., antenna effect), while the presence of metal ions would lead to the corresponding conformational change of BSA for altering the antenna effect accompanied by a substantial TRF performance of Tb3+. This principle has also been fully proved by both experimental characterizations and coarse-grained molecular dynamics (CG-MD) studies. By using Tris-HCl buffer with different pHs (at 7.4 and 8.5), 17 metal ions have been well-distinguished by using our proposed BSA/Tb3+ sensor array. Moreover, the sensor array has the potential to discriminate different concentrations of the same metal ions and a mixture of metal ions. Remarkably, the detection of metal ions in biofluids can be realized by utilizing the presented sensor array, verifying its practical applications. The platform avoids the synthesis of multiplex sensing receptors, providing a new method for the construction of convenient and feasible lanthanide complex-based TRF sensing arrays.

Molecular Tweezers-like Calix[4]arene Based Alkaline Earth Metal Cation (Ca2+, Sr2+, and Ba2+) Chemosensor and Its Imaging in Living Cells and Zebrafish.

Although alkaline earth metal cations play an important role in our daily life, little attention has been paid to the field of fast quantitative analysis of their content due to a lack of satisfactory precision and a fast and convenient means of detection. In this study, we have designed a set of molecular tweezers based on the calix[4]arene chemosensor L, which was found to exhibit high selectivity and sensitivity toward Ca2+, Sr2+, and Ba2+ (by UV-vis and fluorescence methods) with low detection limits of the order of 10-7 to 10-8 M and high association constants (of the order of 106). More significantly, sensor L not only can recognize Ca2+, Sr2+, and Ba2+ but also can further discriminate between these three cations via the differing red shifts in their UV-vis spectra (560 nm for L·Ca2+, 570 nm for L·Sr2+, and 580 nm for L·Ba2+ complex) which is attributed to their different atomic radii. A rare synergistic effect for the recognition mechanism has been demonstrated by 1H NMR spectroscopic titration. Sensor L constructed a high shielding field by the cooperation of Tris with alkaline earth metal ion after complex. Additionally, the presence of acetoxymethyl group in sensor L results in enhancement of cell permeability, and as a consequence, sensor L exhibited excellent sensing and imaging (in vivo) in living cells and in zebrafish.

Discovery and identification of antimicrobial peptides in Sichuan pepper (Zanthoxylum bungeanum Maxim) seeds by peptidomics and bioinformatics.

Antimicrobial peptides (AMPs) have generated growing attention because of the increasing bacterial resistance. However, the discovery and identification of AMPs have proven to be challenging due to the complex purification procedure associated with conventional methods. For the reasons given above, it is necessary to explore more efficient ways to obtain AMPs. We established a new method for discovery and identification of novel AMPs by proteomics and bioinformatics from Zanthoxylum bungeanum Maxim seeds protein hydrolysate directly. This process was initially achieved by employing ultra-performance liquid chromatography-electrospray ionization-mass spectrometry/mass (UPLC-ESI-MS/MS) spectrometry to identify peptides derived from Z. bungeanum Maxim seed protein hydrolysates. Three online servers were introduced to predict potential AMPs. Sixteen potential AMPs ranging from 1.5 to 2.7 kDa were predicted and chemically synthesized, one of which, designated NP-6, inhibited activity against all the tested strains according to antimicrobial assay. Time-killing assay indicated that NP-6 could quickly kill almost all the Escherichia coli within 180 min and Staphylococcus aureus at 360 min. Moreover, the simulation 3D structure of NP-6 was consisted of α-helix and random coil, and this was verified by circular dichroism (CD) spectra. At last, the scanning electron microscope (SEM) images of E. coli and S. aureus treated by NP-6 demonstrated that NP-6 had a significant effect on bacteria cell morphology. Our findings provide an efficient approach for discovery of AMPs, and Z. bungeanum Maxim seeds may be a nature resource to extract antimicrobial agents.

Mechanism of antimicrobial peptide NP-6 from Sichuan pepper seeds against E. coli and effects of different environmental factors on its activity.

A novel antimicrobial peptide named NP-6 was identified in our previous work. Here, the mechanisms of the peptide against Escherichia coli (E. coli) were further investigated, as well as the peptide's resistance to temperature, pH, salinity, and enzymes. The transmission electron microscopy (TEM), confocal laser scanning microcopy (CLSM), and flow cytometric (FCM) analysis, combined with measurement of released K+, were performed to evaluate the effect of NP-6 E. coli cell membrane. The influence of NP-6 on bacterial DNA/RNA and enzyme was also investigated. The leakage of K+ demonstrated that NP-6 could increase the permeability of E. coli cell membrane. The ATP leakage, FCM, and CLSM assays suggested that NP-6 caused the disintegration of bacterial cell membrane. The TEM observation indicated that NP-6 could cause the formation of empty cells and debris. Besides, the DNA-binding assay indicated that NP-6 could bind with bacterial genomic DNA in a way that ethidium bromide (EB) did, and suppress the migration of DNA/RNA in gel retardation. Additionally, NP-6 could also affect the activity of ß-galactosidase. Finally, the effect of different surroundings such as heating, pH, ions, and protease on the antimicrobial activity of NP-6 against E. coli was also investigated. Results showed that the peptide was heat stable in the range of 60~100 °C and performed well at pH 6.0~8.0. However, the antimicrobial activity of NP-6 decreased significantly in the presence of Mg2+/Ca2+, and after incubation with trypsin/proteinase K. The results will provide a theoretical support in the further application of NP-6.

Manganese(II)-doped zinc/germanium oxide nanoparticles as a viable fluorescent probe for visual and time-resolved fluorometric determination of ascorbic acid and its oxidase.

A method is described for the determination of ascorbic acid (AA) in complex biological fluids. It based on maganese(II)-doped zinc/germanium oxide nanoparticles (Mn@ZnGe NPs) with appealing time-resolved phosphorescence (TRP). TRP can provide a background-free reporter signal in analytical methods. The absorption of AA overlaps the excitation band of Mn@ZnGe NPs at 254 nm. This reduces the intensity of fluorescence via an inner filter effect (IFE) with increasing concentration of AA. Typical experimental conditions include an emission peak at 536 nm, a delay time of 50 µs and a counting time of 2 ms. This method can detect AA in a range of 5-500 µM with a 0.13 µM limit of detection. If AA is oxidized by the enzyme AA oxidase (AAOx), dehydroascorbic acid will be formed which doesn't absorb at 254 nm. Hence, the IFE cannot occur and fluorescence is not reduced. The strategy can be used to quantify AAOx in the activity range of 1-4 U·mL-1. By using a handheld UV lamp and a smart phone with a color-scanning feature, the feasibility for visual detection and real-time/onsite quantitative scanometric monitoring of AA and AAOx is demonstrated. Graphical abstract Schematic presentation of a fluorometric method for determination of ascorbic acid (AA) and ascorbic oxidase and a scanometric visual assay. It based on the use of maganese(II)-doped zinc/germanium oxide nanoparticles (Mn@ZnGe NPs) with appealing time-resolved phosphorescence (TRP) and the inner-filter effect (IFE) between AA and Mn@ZnGe NPs.

A rapid, ratiometric, enzyme-free, and sensitive single-step miRNA detection using three-way junction based FRET probes.

MicroRNAs (miRNAs) are single stranded endogenous molecules composed of only 18-24 nucleotides which are critical for gene expression regulating the translation of messenger RNAs. Conventional methods based on enzyme-assisted nucleic acid amplification techniques have many problems, such as easy contamination, high cost, susceptibility to false amplification, and tendency to have sequence mismatches. Here we report a rapid, ratiometric, enzyme-free, sensitive, and highly selective single-step miRNA detection using three-way junction assembled (or self-assembled) FRET probes. The developed strategy can be operated within the linear range from subnanomolar to hundred nanomolar concentrations of miRNAs. In comparison with the traditional approaches, our method showed high sensitivity for the miRNA detection and extreme selectivity for the efficient discrimination of single-base mismatches. The results reveal that the strategy paved a new avenue for the design of novel highly specific probes applicable in diagnostics and potentially in microscopic imaging of miRNAs in real biological environments.

Electrochemistry Study of Permselectivity and Interfacial Electron Transfers of a Branch-Tailed Fluorosurfactant Self-Assembled Monolayer on Gold.

We investigated the permselectivity and interfacial electron transfers of an amphiphilic branch-tailed fluorosurfactant self-assembled monolayer (FS-SAM) on a gold electrode by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The FS-SAM was prepared by a self-assembly technique and a "click" reaction. The barrier property and interfacial electron transfers of the FS-SAM were also evaluated using various probes with different features. The FS-SAM allowed a higher degree of permeation by small hydrophilic (Cl- and F-) electrolyte ions than large hydrophobic (ClO4- and PF6-) ones. Meanwhile, the redox reaction of the Fe(CN)63- couple was nearly completely blocked by the FS-SAM, whereas the electron transfer of Ru(NH3)63+ was easier than that of Fe(CN)63-, which may be due to the underlying tunneling mechanism. For hydrophobic dopamine, the hydrophobic bonding between the FS-SAM exterior fluoroalkyl moieties and the hydrophobic probes, as well as the hydration resistance from the interior hydration shell around the oligo (ethylene glycol) moieties, hindered the transport of hydrophobic probes into the FS-SAM. These results may have profound implications for understanding the permselectivity and electron transfers of amphiphilic surfaces consisting of molecules containing aromatic groups and branch-tailed fluorosurfactants in their structures.

Chemical composition and antimicrobial activities of volatile oil extracted from Chrysanthemum morifolium Ramat.

Volatile oil in Chrysanthemum morifolium Ramat (C. morifolium) was extracted by the method of water vapor distillation and its chemical components was identified by gas-chromatography coupled with mass spectrometry (GC-MS). The volatile oil are evaluated for antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella enteritids, Pseudomonas aeruginosa and Bacillus subtilis. Effects of surfactant, temperature, pH and ultraviolet light on antibacterial activity stability of volatile oil were analyzed too. Total 56 compounds were identified in C. morifolium volatile oil. The main constituents in C. morifolium volatile oil were monoterpenes and sesquiterpenes compounds, including hydrocarbons, esters, aldehydes, ketones, phenols and organic acids. α-curcumene was the most abundant volatile component (12.55%). The volatile oil showed promising antibacterial activity against 5 selected strains. The inhibitory effect on P. aeruginosa exhibited maximum inhibition zone diameter 20.43 mm, and E. coli showed 12.29 mm. The volatile oil treated with surfactant Tween 20 showed the strongest antibacterial activity, followed by Tween 80 and the SDS lowest, which showed the lowest. pH also had different effect on antibacterial activity stability of the C. morifolium volatile oil. No significant difference effect on antibacterial activity stability of volatile oil was observed with temperature and UV treatment.

[Inhibition of WIP1 in A549 cells enhances the sensitivity of cells to arsenic treatment].

OBJECTIVE: To explore if inhibiting the expression of wild-type p53-induced phosphatase 1( WIP1) could enhance the sensitivity of A549 cells to arsenic. METHODS: To inhibit expression of WIP1, WIP1 siRNA was transferred into A549 cells by using Lipofectamine 2000. Then the protein expression levels of P53 phosphorylation proteins and their downstream effectors were detected by western-blot analysis. Cell apoptosis were assessed by Annexin V-FITC stain assay. The sensitivity of transferred cells to arsenic was detected by using MTT assay. RESULTS: The mRNA and protein expression level of WIP1 were all decreased by 70 % in A549 cells transferred with WIP1 siRNA. Western-blot analysis indicated that P53 phosphorylation process was much accelerated in WIP1-inhibited cells after arsenic treatment. For example, compared to control cells, an significant decrease in P53 ser15 expression and an increase in P53 ser46 expression was found in WIP1-inhibited cells when treated with As_2O_3( 5-40 µmol/L). In addition, compared to control group, the expression of P21 decreased whereas PUMA increased in WIP1-inhibited cells when treated with As_2O_3( 10-40 µmol/L). Cell viability of WIP1-inhibited cells after As_2O_3 treatment( 5-40 µmol/L) was significantly higher than that of the control group, which may be due to a high apoptosis rate in WIP1-inhibited cells. CONCLUSION: WIP1 could be used as a new target in arsenic-base anticancer therapies.

[Improving the expression of TP53INP1 in A549 cells enhances the sensitivity of cells to arsenic treatment].

OBJECTIVE: To explore if improving the expression of TP53INP1 could enhance the sensitivity of A549 cells to arsenic. METHODS: The eukaryotic express vector containing TP53INP1 gene was transferred into A549 cells by using lentivirus vector. Cell apoptosis and cell viability after arsenic treatment were assessed by flow cytometry and MTT assay, respectively. RESULTS: The protein expression level of TP53INP1 was increased in A549 cells transferred with eukaryotic express vector containing TP53INP1 gene, which led to an increase in apoptosis and a decrease in cell viability. Compared with A549 cells, significant increase in apoptosis was found in A549-TP53INP1 cells when treated with As_2O_3( 5- 40 µmol/L). In addiation, the IC50 of As_2O_3 in A549-TP53INP1(( 44. 64 ± 6. 84) µmol/L) cells was significantly lower than that of the A549 group(( 54. 25 ± 6. 13) µmol/L)( P < 0. 05). CONCLUSION: Enhancement of TP53INP1 can significantly improve apoptosis response and enhance sensitivity of A549 cells to arsenic. It is suggested that TP53INP1 could be used as a new target in arsenic-based cancer treatment.

[4-amino-1,8-naphthalimide on the Sensitive effect of arsenic trioxide in hepatocellular carcinoma cells].

OBJECTIVE: To study the effect and mechanism of 4-amino-1, 8-naphthalimide (4-AN) on the sensitive effect of arsenic trioxide (ATO) in hepatocellular carcinoma cells. METHODS: Hepatocellular carcinoma HepG2 cells were divided into two groups according to whether they were treated with 4-AN or not. Cell viability was evaluated by MTT assay, population doubling experiment and colony formation assay; genic mechanism was explored by 8-OH-dG assay, single cell gel electrophoresis (comet assay) and microriucleus test. RESULTS: At 2-10 micromol/L concentration of ATO, the cell viability and colony formation efficiency of the combinatio group (4-AN+ATO) were significantly lower than that of the ATO group (P<0.05); moreover, the tail-length (L-Tail) and olive tail moment (OTM) in comet assay were notablely higher than that of the ATO group (P<0.05). At 2-20 micromol/L concentration of ATO, the population doubling time and 8-OH-dG in combination group were significantly higher than that of ATO group (P<0.05). Results from DNA damage repair assay showed that the efficiency of DNA damage repair in combination group was remarkably lower than that of ATO group (P<0.05). At 5-20 micromol/L concentration of ATO, the frequency of micronucleated cells in combination group was significantly higher than that of ATO group (P<0.05). CONCLUSION: 4-AN can significantly increase the sensitivity of ATO in treatment with hepatocellular carcinoma cells and prevent DNA damage repair may be a primary mechanism for this effect.

Rapid in situ detection of ultratrace 2,4-dinitrotoluene solids by a sandwiched paper-like electrochemical sensor.

This work reported the rapid in situ detection of ultratrace 2,4-dinitrotoluene (DNT) solids on various substrates by a sandwiched paper-like electrochemical sensor. The sensor, prepared by a simple electroless deposition method without using special instruments, possessed a unique thin-film structure of an insulated polyvinylidene fluoride (PVDF) membrane in between two gold (Au) conducting layers. The resulting gold-PVDF sandwich (GPVDFS) array exhibited excellent flexibility, porosity and electrochemical performance as a highly integrated dual-electrode sensor platform. The infiltration of nonvolatile ionic liquid (IL) electrolytes containing ferrocene (Fc) into the GPVDFS array produced a paper-like electrochemical sensor, which can directly detect ultratrace DNT solids on various substrate surfaces (e.g., plant leaves, gloves and metal knives) with detection limit as low as 0.33 ng/mm(2). The critical role of Fc in the detection of DNT at this dual-electrode sensor was explored. The compensating electrochemical oxidation of Fc at the counter/reference electrode was found to be essential to the reduction of DNT at the working electrode when IL electrolytes were employed. The present work thus demonstrated the promising applications of paper-based porous electrode arrays in developing IL-based electrochemical sensors for the in situ detection of analyte solids in complicated environments.

Nanomechanical analysis of yeast cells in CdSe quantum dot biosynthesis.

QD biosynthesis affects the mechanical strength of yeast cells. The intracellular synthesis of CdSe QD in yeast cells incubated with Na2 SeO3 and subsequently with CdCl2 increases the glucan content of their cell walls, resulting in their enhanced mechanical strength.