Impact of microorganisms on key processes of organic acid metabolism during the occurrence and disappearance of paocai pellicle.

In vegetable fermentation, pellicle is a common quality deterioration phenomenon. This study investigates the characteristics of glucose, organic acids, amino acids, and biogenic amines during the pellicle occurrence and disappearance of paocai. The results revealed a slight increase in pH of the fermentation system after pellicle occurred, and glucose was the main carbohydrate that microbial activity primary relied on. The microorganisms responsible for pellicle formation consumed organic acids in brine, but the lactic acid in paocai gradually increased and exceeded 25 mg/g. The appearance of pellicle caused a decrease in total free amino acids from 200.390 mg/100 g to 172.079 when pellicle occurred, whereas its impact on biogenic amines was not apparent. Through Kyoto Encyclopedia of Genes and Genomes pathway enrichment of metagenomics sequencing data, screening, and sorting of the key enzymes involved in organic acid metabolism, it was observed that the composition and species of the key microorganisms capable of metabolizing organic acids were more abundant before the appearance of pellicle. When pellicle occurred, lactic acid may be metabolized by Lactobacillus plantarum; in contrast, Lactobacillus and Pichia were associated with citric acid metabolism, and Lactobacillus, Pichia, Saccharomycodes, and Kazachstania were linked to malic acid metabolism. Moreover, Prevotella, Kazachstania, Lactobacillus, Vibrio, and Siphonobacter were implicated in succinic acid metabolism. Additionally, the production of tartaric acid and oxalic acid in paocai and brine resulted from abiotic effects. This knowledge offers a theoretical basis for precise control of paocai fermentation process. PRACTICAL APPLICATION: Our study revealed the specific situation of the metabolites produced by the microorganisms during the pollution and recovery process of pellicle in paocai fermentation, especially the effect of pellicle on the key process of organic acid metabolism. These research results provided theoretical basis for precise control of paocai fermentation.

Antibiofilm Activity and Mechanism of Linalool against Food Spoilage Bacillus amyloliquefaciens.

Pellicle biofilm-forming bacteria Bacillus amyloliquefaciens are the major spoilage microorganisms of soy products. Due to their inherent resistance to antibiotics and disinfectants, pellicle biofilms formed are difficult to eliminate and represent a threat to food safety. Here, we assessed linalool's ability to prevent the pellicle of two spoilage B. amyloliquefaciens strains. The minimum biofilm inhibitory concentration (MBIC) of linalool against B. amyloliquefaciens DY1a and DY1b was 4 µL/mL and 8 µL/mL, respectively. The MBIC of linalool had a considerable eradication rate of 77.15% and 83.21% on the biofilm of the two strains, respectively. Scanning electron microscopy observations revealed that less wrinkly and thinner pellicle biofilms formed on a medium supplemented with 1/2 MBIC and 1/4 MBIC linalool. Also, linalool inhibited cell motility and the production of extracellular polysaccharides and proteins of the biofilm matrix. Furthermore, linalool exposure reduced the cell surface hydrophobicity, zeta potential, and cell auto-aggregation of B. amyloliquefaciens. Molecular docking analysis demonstrated that linalool interacted strongly with quorum-sensing ComP receptor and biofilm matrix assembly TasA through intermolecular hydrogen bonds, hydrophobic contacts, and van der Waals forces interacting with site residues. Overall, our findings suggest that linalool may be employed as a potential antibiofilm agent to control food spoilage B. amyloliquefaciens.

Peptide profiles and allergy-reactivity of extensive hydrolysates of milk protein.

Milk protein concentrate (MPC) is one of the major allergens in food. This study aimed to analyze the peptide profiles and potential allergenicity of the extensive hydrolysates of MPC (EMPHs) using the peptidomics approach. Results demonstrated that when the hydrolysis time was 4 h, the degree of hydrolysis of the four EMPHs (AX, Alcalase-Protamex), (AD, Alcalase-Protease A 2SD), (AE, Alcalase-Flavourzyme) and (AH, Alcalase-ProteAXH) were 12.45 %, 18.48 %, 18.87 % and 16.77 %, respectively. The results of size exclusion chromatography showed no significant difference, when the hydrolysis time exceeded 3 h. A total of 16 allergic peptides were identified in the EMPHs by LC-MS/MS. The peptide profiles and the coverage of master protein of the four EMPHs were different. The results of the enzyme-linked immunoassay and KU812 cell model showed that the allergenicity of the EMPHs samples was significantly reduced. This study provided strong support for the application of EMPHs in hypoallergenic formula foods.

Identification of pellicle formation related microorganisms in traditional Sichuan paocai through metagenomic sequence and the effects of Baijiu/Salt on pellicle and volatile components.

The occurrence of pellicle on the surface of paocai brine is a common undesirable phenomenon during the multi-rounds of paocai fermentation, which is mainly caused by the growth of microorganisms related to pellicle formation. But the detailed information on these microorganisms and volatile components produced by them, as well as the changes of the microorganisms during the process of paocai recovery, are still rare in the literature. Therefore, the purpose of this study was (1) to analyze the pellicle formation related microorganisms by comparing the differential microorganisms in initial brine and the brine when pellicle occurred through metagenomic sequencing technology, (2) to explore the changes of microorganisms in the fermentation system after addition Baijiu and/or salt, and (3) to further detect the VOCs in paocai samples by gas chromatography-mass spectrometry (GC-MS). The relationship between VOCs and the selected marker microorganisms was also determined. The results showed that the diversity of fungi was increased when pellicle formed, the pellicle formation related microorganisms mainly belonged to six genus, including Kazachstania, Lactobacillus, Pichia, Candida, Lachancea, and Saccharomyces. Apart from the unknown function and basic life activities of microorganisms, the metabolic activities of these pellicle formation related microorganisms were mainly carbohydrate transport and metabolism, and amino acid transport and metabolism. The growth of pellicle formation related microorganisms could be inhibited by adding Baijiu (1.5% v/v), but the addition of salt (7% salt (w/v) did not promote this inhibitory effect. Through PCA analysis, it was found that the VOCs of paocai were significantly affected by adding Baijiu and Baijiu and salt. The undesirable smell at the beginning of pellicle formation may be related to Propanoic acid, hexyl ester, 1,3-Dimethyl-1-cyclohexene, Oxime-, methoxy-phenyl- and Phenylethyl Alcohol.

PtrHAT22, as a higher hierarchy regulator, coordinately regulates secondary cell wall component biosynthesis in Populus trichocarpa.

Homeodomain-leucine zipper (HD-Zip) II transcription factors (TFs) have been reported to play vital roles in diverse biological processes of plants. However, it remains unclear whether HD-Zip II TFs regulate secondary cell wall (SCW) in woody plants. In this study, we performed the functional characterization of a Populus trichocarpa HD-Zip II TF, PtrHAT22, which encodes a nuclear localized transcription repressor predominantly expressing in secondary developing tissues. Overexpression of PtrHAT22 showed arrested growths, including reduced heights and diameters above the ground, small leaves, and decreased biomass. Meanwhile, the contents of lignin, cellulose, and thickness of SCW significantly decreased, whilst the content of hemicellulose obviously increased in PtrHAT22 transgenic poplar. The expressions of some wood-associated TFs and structural genes significantly changed accordingly with the alternations of SCW characteristics in PtrHAT22 transgenic poplar. Furthermore, PtrHAT22 directly repressed the promoter activities of PtrMYB20, PtrMYB28, and PtrCOMT2, and bind two cis-acting elements that were specifically enriched in their promoter regions. Taken together, our results suggested that PtrHAT22, as a higher hierarchy TF like PtrWNDs, exerted coordination regulation of poplar SCW component biosynthesis through directly and indirectly regulating structural genes and different hierarchy TFs of SCW formation network.

A label-free electrochemical aptasensor based on AuNPs-loaded zeolitic imidazolate framework-8 for sensitive determination of aflatoxin B1.

In this study, an electrochemical aptasensor for sensitive detection of aflatoxin B1 (AFB1) was fabricated by electrodepositing gold nanoparticles (AuNPs) on the glass carbon electrode (GCE) modified with zeolitic imidazolate framework-8 (ZIF-8). The high specific surface area of AuNPs/ZIF-8 nanocomposite increased the aptamers loading on the electrode surface. When compared to other previously reported sensors, the developed aptasensor exhibited a wider linear range of 10.0 to 1.0 × 105 pg/mL with a lower limit of detection (LOD) of 1.82 pg/mL under the optimized conditions. Furthermore, the obtained results revealed that the constructed aptasensor exhibited outstanding selectivity, reproducibility and stability. Moreover, the aptasensor was successfully employed to detect the AFB1 in corn oil and peanut oil samples, with recoveries ranging from 93.49% to 106.9%, demonstrating the potential application value of this methodology.

Incorporation of Lipids into Wheat Bran Cellulose/Wheat Gluten Composite Film Improves Its Water Resistance Properties.

This work evaluated the improvement effects of lipids incorporation on water resistance of composite biodegradable film prepared with wheat bran cellulose/wheat gluten (WBC/WG) using an alkaline-ethanol film forming system. Four types of lipids, paraffin wax (PW), beeswax (BW), paraffin oil (PO), and oleic acid (OA), were tested. We found that PW, BW, and PO incorporation at 5-20% improved water vapor permeability (WVP) and surface hydrophobicity of prepared films. Particularly, incorporation of 15% BW could best improve the water resistance properties of the film, with the lowest WVP of 0.76 × 10-12 g/cm·s·Pa and largest water contact angle (WCA) of 86.18°. Incorporation of OA led to the decline in moisture barrier properties. SEM images revealed that different lipids incorporation changed the morphology and of the composite film, and cross-sectional morphology indicated BW-incorporated film obtained more uniform and compact structures compared to other films. Moreover, Fourier transform infrared spectra indicated that the incorporation of PW or BW enhanced the molecular interactions between the film components, confirmed by the chemical shift of characteristic peaks at 3277 and 1026 cm-1. Differential scanning calorimetry results revealed that incorporation of PW, BW, and PO increased films' melting point, decomposition temperatures, and enthalpy values. Furthermore, the presence of most lipids decreased tensile strength and elongation at the break of the film. Overall, the composite film containing 15% BW obtained the most promising water resistance performance and acceptable mechanical properties, and it thus most suitable as a hydrophobic biodegradable material for food packaging.

Role of miR-92a-3p/PTEN axis in regulation of pancreatic cancer cell proliferation and metastasis. / 微小RNA-92a-3p靶向PTENè°ƒæŽ§èƒ°è ºç™Œç»†èƒžå¢žæ®–å’Œè½¬ç§».

OBJECTIVES: To explore the effect of miR-92a-3p on the proliferation and metastasis of pancreatic cancer cells via targeting phosphatase and tension homolog deleted on chromosome ten (PTEN). METHODS: MiR-92a-3p expression and PTEN protein levels were quantified in a normal pancreatic cells (HPDE6-C7) and 5 pancreatic cancer cell lines (Panc-1, BxPC-3, AsPC-1,MIA Paca-2, and Capan-2) by real-time PCR and Western blotting, respectively. BxPC-3 and Panc-1 cells were selected for further experiment. After transfection of normal control (NC) mimics (NC mimics group), miR-92a-3p mimics (miR-92a-3p mimics group), NC inhibitor or miR-92a-3p inhibitor (NC inhibitor group or miR-92a-3p inhibitor group), the proliferation of BxPC-3 and Panc-1 cells was measured by cell counting kit-8 (CCK-8), and the migration of them was measured by Transwell assay, and the levels of PTEN protein were measured by Western blotting. In addition, wild-type PTEN 3'-UTR (wt-PTEN 3'UTR) and mutant-type PTEN 3'-UTR (mut-PTEN 3'UTR) luciferase reporter vectors were constructed and co-transfected with NC mimics, miR-92a-3p mimic, NC inhibitor or miR-92a-3p inhibitor into 293T tool cells, and then the dual luciferase reporter assay was performed to examine the regulative correlation between miR-92a-3p and PTEN. The BxPC-3 cells were divided into 4 groups: a NC inhibitor+si-NC group, a miR-92a-3p inhibitor+si-NC group, a NC inhibitor+si-PTEN group, and a miR-92a-3p inhibitor+si-PTEN group. The Panc-1 cells were also assigned into 4 groups: a NC mimics+NC group, a miR-92a-3p mimics+si-NC group, a NC mimics+ PTEN group, and a miR-92a-3p mimics+PTEN group. The proliferation of Panc-1 cells was measured by CCK-8; the cell migration was measured by Transwell assay, and the levels of PTEN protein were measured by Western blotting. RESULTS: The miR-92a-3p was highly expressed in pancreatic cancer cell lines (allP<0.01), while the PTEN protein levels were lower in pancreatic cancer cell lines (allP<0.05)compared with that in the HPDE6-C7 cells. Compared with the NC mimics group, the cell viability of BxPC-3 and Panc-1 cells were both increased in the miR-92a-3p mimics group (bothP<0.01); compared with the inhibitor group, the cell viability of BxPC-3 and Panc-1 cells were both decreased in the miR-92a-3p inhibitor group (bothP<0.01). Compared with the NC mimics group, the cell number of BxPC-3 and Panc-1 cells through micropores were increased in the miR-92a-3p mimics group (bothP<0.01); compared with the inhibitor group, the cell number of BxPC-3 and Panc-1 cells through micropores were decreased in the miR-92a-3p inhibitor group (bothP<0.01). Compared with NC mimics group, the activity of dual luciferaseof wt-PTEN3'-UTR was inhibited in the miR-92a-3p mimics group (P<0.01); compared with the NC inhibitor group, the activity of dual luciferase of wt-PTEN3'-UTR was promoted in the miR-92a-3p inhibitor group (P<0.01). Compared with the miR-92a-3p inhibitor+si-NC group, the suppressive effects of miR-92a-3p on the proliferation and metastasis of BxPC-3 cells was restored in the miR-92a-3p inhibitor+si-PTEN group; while compared with the miR-92a-3p mimics+NC group, the positive effects of miR-92a-3p overexpression on the proliferation and metastasis of Panc-1 cells was restored in the miR-92a-3p mimics+PTEN group. CONCLUSIONS: The highly expressed miR-92a-3p in pancreatic cancer cells can decrease the protein levels of PTEN, thereby enhancing the proliferation and metastasis activity of pancreatic cancer cells.

Extraction Optimization and Evaluation of the Antioxidant and α-Glucosidase Inhibitory Activity of Polysaccharides from Chrysanthemum morifolium cv. Hangju.

In order to evaluate the antioxidant and α-glucosidase activities of polysaccharides from Chrysanthemum morifolium cv. Hangju (CMPs), the response surface methodology was applied to optimize the parameters for extraction progress of CMPs by ultrasound, with heat reflex extraction (HRE) performed as the control. The difference in the physicochemical properties of polysaccharides obtained by the two methods were also investigated. The maximum yields (8.29 ± 0.18%) of polysaccharides extracted by ultrasonic assisted extraction (UAE) were obtained under the optimized conditions of ultrasonic power 501 W, extraction time 19 min, and ratio of liquid-to-raw material 41 mL/g. Polysaccharides extracted by UAE possessed lower protein contents (2.56%) and higher uronic acids contents (7.08%) and low molecular weight fractions than that by HRE. No significant differences were found in monosaccharide composition and Fourier transform infrared (FT-IR) spectra of polysaccharides extracted by UAE and HRE, while polysaccharides by UAE possessed stronger antioxidant and α-glucosidase inhibitory activities. Therefore, UAE was an efficient way to obtain CMPs.

Clinical observation of end-to-end neuroanastomosis in the treatment of complete injury of the unilateral recurrent laryngeal nerve.

BACKGROUND: Complete injury of the recurrent laryngeal nerve (RLN) is a serious complication of thyroid surgery. Increasingly researches focus on the voice recovery of patients with RLN injury. This paper studied the effect of immediate end-to-end neuroanastomosis after complete injury of the unilateral RLN during thyroid surgery on postoperative vocal function. METHODS: Thirteen patients who underwent end-to-end neuroanastomosis for the treatment of complete injury of the unilateral RLN caused by thyroid surgery in Hunan Provincial People's Hospital between October 2009 and January 2020 were selected. The basic information, cause of RLN injury, postoperative voice recovery, recovery time, and subjective assessment of voice by auditory perception results (Grade, Roughness, Breathiness, Asthenia, and Strain, the GRBAS score) of the patients were recorded. RESULTS: Among the 13 cases with RLN injury, the cause of RLN injury in 10 cases was transection by sharp instruments, and the voice was recovered one day after the operation. The cause of RLN injury in one case was suture of the RLN branch, and the voice was recovered one day after the operation. The cause of RLN injury in two cases was thermal injury, and the times for voice recovery after end-to-end neuroanastomosis were 3 and 4 months. The patients with a GRBAS score of 0 or 1 recovered their voice one day after the operation. The GRBAS score reached 1 in the two thermal injury cases, with voice recovery at 3 and 4 months after surgery. By 6 months, the voice recovery rate of the patients was 100%. CONCLUSIONS: After complete injury of the unilateral RLN, immediate end-to-end anastomosis of the RLN can maximally preserve the postoperative vocal function of patients.

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].

Preparation and characterization of indicator films from carboxymethyl-cellulose/starch and purple sweet potato (Ipomoea batatas (L.) lam) anthocyanins for monitoring fish freshness.

A novel indicator film from carboxymethyl-cellulose (CMC)/starch (S) and purple sweet potato anthocyanins (PSPA) was prepared and characterized. The Fourier transform infrared spectroscopic (FT-IR) analysis indicated that new interaction were generated between the components of the film, and the scanning electron microscopic (SEM) and X-ray diffraction spectra (XRD) showed that PSPA was well dispersed in CMC and starch-based film. The results from differential scanning calorimetric (DSC) analysis revealed that the novel film had increased melting temperature. The addition of PSPA into the film resulted in the increase of tensile strength but the decrease of elongation at break and moisture content. The analysis of color change demonstrated that the film color was changed from red to blue and green when exposed to different pH or ammonia. In their application trial, the SCA film was used as labels to monitor the freshness of fish stored at 20 °C, and the results showed that the color of the film was changed from red to blue when the fish became spoiled. The observation was consistent with the TVB-N value, which was higher than the set limit (20 mg/100 g). Hence, the novel indicator film could be used as intelligent food packaging to monitor fish freshness.

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.

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.

Properties comparison between free and immobilized wheat esterase using glass fiber film.

In this paper, the catalytic performance of non-purified esterase from wheat bran immobilized on glass fibre membrane carrier is established, the immobilization conditions observed were enzyme 1 mL, phosphate buffer 3 mL (pH 7.0), immobilization time 1 h, immobilization temperature 29 °C. After carrier functionalization some characteristics of immobilized enzyme were studied, the results showed that immobilized enzyme presenting improved characteristic than that of free enzyme. The optimum pH for free and immobilized enzymes were found to be 8 and 7, respectively. As for optimum temperature for free and immobilized enzymes were observed to be 30 °C and 40 °C, respectively. When the enzyme was immobilized on glass fibre membranes, its Km increased about 7 times. In addition, storage and thermal stability of the free wheat esterase were increased by as a result of membrane immobilization, after 12 days of storage, the immobilized enzyme still retained about 91.10% of its original activity at 4 °C, indicating a great potential in industrial application.

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.

The Size Flexibility of Ferritin Nanocage Opens a New Way to Prepare Nanomaterials.

Ferritins are ubiquitous iron storage proteins where Fe(II) sequestration prevents not only its spontaneous oxidation to Fe(III) but also production of toxic free radicals. Recently, scientists have subverted these nature functions and used ferritin cage structures of nanometer dimensions for encapsulation of guest molecules such as anti-cancer drugs or bioactive nutrients based on pH induced ferritin disassembly and reassembly property. However, prior to this study, ferritin nanocage was required to disassemble only under harsh pH conditions (≤2.0 or ≥11.0), followed by reassembly at near neutral pH. Such harsh conditions can cause protein or guest molecules damage to a great extent during this pH-induced unfolding-refolding process. Here, we provide evidence demonstrating that the apoferritin shell is flexible rather than rigid. Indeed, we found that two large complex molecules, uranyl acetate dihydrate and phosphotungstic acid, can reach the cavity of both plant and animal apoferritin followed by mineralization. Moreover, large organic compound such as curcumin and doxorubicin can also be encapsulated within ferritin cavity by its mixing with protein. This strategy will increase the use of ferritin in nanotechnology, and could be also applicable to other shell-like proteins as templates to prepare nanomaterials.

Immunolocalization of Jian Carp (Cyprinus Carpio Var. Jian) Cathepsin B: Cloning, Expression, Characterization, and Antibody Preparation.

Cathepsin B (CatB) cDNA of 759 bp from Jian carp (Cyprinus carpio var. Jian) with amino acid similarity of 99.6% to common carp was cloned. The mature CatB was expressed in Escherichia coli BL21 transferred with vector CatB-pET-30a. It was purified and identified as a single band (29 kDa) on SDS-PAGE. Optimum CatB activity was observed at 40 °C and pH 5.5. Mouse anti-CatB polyclonal antibody with a high titer of 1:256000 was prepared successfully and shown to specifically recognize the antigen both in prokaryotic cells and in the tissues of Jian carp according to western blotting and immunohistochemistry results. Immunolocation analysis showed that CatB distribution at protein level varied among the tested tissues. The results presented in this study may provide a significant reference for future research on the inherent relationship between CatB and the quality of fish or fish products at both the gene and protein levels.

A novel calcium supplement prepared by phytoferritin nanocages protects against absorption inhibitors through a unique pathway.

The consumption of milk is declining in industrialized countries, leading to inadequate calcium intake. Therefore, it is important to explore a new class of Ca-enriched nutrient for the fortification of food. In this work, we prepared a novel class of soluble and edible Ca-protein complexes where approximately 140 calcium ions were encapsulated within a phytoferritin nanocage. As an alternative to other organic and/or inorganic carriers, protein nanocages were found to provide a unique vehicle of biological origin for the intracellular delivery of calcium ions for supplementation. Such encapsulation can protect calcium ions within protein cages against dietary factors such as tannic acid (TA), oxalic acid (OA), and other divalent metal ions in foodstuffs. We demonstrated that the calcium-containing ferritin composites can be absorbed by Caco-2 cells through a process where a TfR1 receptor is involved, whereas the uptake of free calcium ions has been known to be associated with another receptor, DMT1, indicating that the calcium ions encapsulated in supramolecular protein cages can be internalized by the Caco-2 cells through a different pathway from its free analogs for calcium supplementation.

Stability and iron oxidation properties of a novel homopolymeric plant ferritin from adzuki bean seeds: a comparative analysis with recombinant soybean seed H-1 chain ferritin.

BACKGROUND: All reported plant ferritins are heteropolymers comprising two different H-type subunits. Whether or not homopolymeric plant ferritin occurs in nature is an open question. METHODS: A homopolymeric phytoferritin from adzuki bean seeds (ASF) was obtained by various protein purification techniques for the first time, which shares the highest identity (89.6%) with soybean seed H-1 ferritin (rH-1). Therefore, we compared iron oxidation activity and protein stability of ASF with those of rH-1 by stopped-flow combined with light scattering or UV/Vis spectrophotography, SDS- and native- PAGE analyses. Additionally, a new rH-1 variant (S68E) was prepared by site-directed mutagenesis approach to elucidate their difference in protein stability. RESULTS: At high iron loading of protein, the extension peptide (EP) of plant ferritin was involved in iron oxidation, and the EP of ASF exhibited a much stronger iron oxidative activity than that of rH-1. Besides, ASF is more stable than rH-1 during storage, which is ascribed to one amino acid residue, Ser68. CONCLUSIONS: ASF exhibits a different mechanism in iron oxidation from rH-1 at high iron loading of protein, and a higher stability than rH-1. These differences are mainly stemmed from their different EP sequences. GENERAL SIGNIFICANCE: This work demonstrates that plant cells have evolved the EP of phytoferritin to control iron chemistry and protein stability by exerting a fine tuning of its amino acid sequence.