Sensitive analysis of miRNAs via primer exchange reaction integrated with hairpin catalytic reaction.

MicroRNAs (miRNAs) can serve as potential biological targets for early screening, targeted therapy, and prognosis in ovarian cancer (OC). However, sensitive and reliable quantification and identification of miRNA remain a huge challenge. Herein, we proposed a simple and reliable approach for the ultra-sensitive detection of miRNA by integrating endonuclease-III (Exo-III) assisted signal recycle, primer exchange reaction (PER), and hairpin catalytic reaction (HCR). In this method, target miRNA specifically binds with toehold sequence to form a blunt 3' terminus in the detection probe (dumbbell probe) that can be recognized by Exo-III, and to initiate subsequent signal amplifications. Based on this, the approach is successfully utilized in detecting OC related miRNAs with high sensitivity (limit of detection for miRNA-211 was 13 aM) and stability. By simply changing the toehold sequence in detection probe, the established approach can be easily extended to other miRNA detection. We believe that the platform is robust in detecting OS related biomarkers and is promising in renovating cancer diagnostic tools.

Effect of CaCl2 on 2 heat-induced whey protein concentrate fibrillation pathways: Spontaneous and nuclear induction.

Amyloid fibrils have many excellent functional properties that facilitate their applications in the food industry. There are 2 pathways for whey protein concentrate (WPC) to form amyloid fibril aggregates: spontaneous pathway and nuclear induction pathway. Low ionic strength is a necessary condition for the spontaneous pathway to proceed successfully. In this paper, the effect of salt ions on 2 WPC fibrillation pathways was investigated by adding CaCl2. The results demonstrated WPC fibrils were unable to form normally through spontaneous pathway as adding CaCl2; but still could form through nuclear induction pathway with 20 to 30 mM CaCl2, the nuclei accelerated the fibrillation process led to the resistance to the disordered aggregation brought by CaCl2. Moreover, divalent cations (Ca2+, Mg2+) had much stronger effects than monovalent cations (Na+) on fibril formation, and the results of X-ray photoelectron spectrum together with Fourier-transform infrared spectroscopy suggested that Ca2+ had a greater effect on the fibril formation than Cl-.

An aptamer-exonuclease III (Exo III)-assisted amplification-based lateral flow assay for sensitive detection of Escherichia coli O157:H7 in milk.

Escherichia coli O157:H7 (E. coli O157:H7), one of the most widespread foodborne pathogens, can cause a series of diseases and even lead to death. In this study, a highly sensitive method was developed by combining aptamer-exonuclease III (Exo III)-assisted amplification with lateral flow assay (LFA) based on gold nanoparticles (AuNP). The compound of single-stranded (ss) DNA-anti-E. coli O157:H7 aptamer (ssDNA-aptamer) was formed by hybridization between designed target ssDNA and aptamer. When E. coli O157:H7 was present, target bacteria were bound with the aptamer, and the free target ssDNA was hybridized with the probes of the designed hairpin (HP) structure. Exo III digests the 3' double-stranded blunt end of the complex and releases the enzyme product. Because the remaining sequence of the HP of the designed enzyme product was the same as the target ssDNA sequence, the target ssDNA could be amplified. Finally, the enhanced target ssDNA was combined with AuNP-LFA to achieve visual detection of E. coli O157:H7. The quantitative ability of this platform for E. coli O157:H7 was 7.6 × 101 cfu/mL in pure culture, and the detection limit in milk was 8.35 × 102 cfu/mL. This LFA was highly specific to E. coli O157:H7, and the time for detection of E. coli O157:H7 in milk was 4 h. Hence, this system has important application prospects in the detection of pathogenic bacteria in dairy products.

Comparative experiments of electrical conductivity from whey protein concentrates conventional film and nanofibril film.

We compared the electrical conductivity from two different aggregates of whey protein concentrates (WPC) film: conventional amorphous aggregation at natural pH (pH 6.5) and amyloid fibrils at a low pH (pH 2.0) far away from the isoelectric point. The two types of film fabricated by these solutions with different aggregate structures showed large variations in electrical conductivity and other properties. The WPC fibril film (pH 2.0) exhibited higher electrical conductivity than that of the conventional WPC film (pH 6.5), improved mechanical properties and oil resistance, due to varying morphology, higher surface hydrophobicity and more (absolute value) surface charge of film-forming solutions. The evidence from this study suggests that fibrilized WPC with high-ordered and ß-sheets-rich structures fabricated high electrical conductivity film, which broadens the potential application of fibrils as functional bio-nanomaterials.

Sensitive, Low-Background-Signal miRNA Analysis via the Self-Priming-Initiated Color Reaction Loaded on a Rolling Circle Amplification Product.

MicroRNAs (miRNAs) have been regarded as potential biomarkers in evaluating various diseases, such as pregnancy-induced hypertension and cancers. However, sensitive and reliable miRNA detection is still a challenge due to the low amplification efficiency and high background signal. Herein, we developed a colorimetric method for miRNA detection utilizing the self-priming-initiated color reaction loaded on a rolling circle amplification (RCA) product. In this method, a biotin-labeled RCA product is fixed on the surface of the streptavidin-coated wells, and the interfering components in samples are removed to avoid false reactions, thus reducing the background signals. Two signal amplification processes, including RCA and self-priming-initiated chain extension, endow the method with high sensitivity and a low limit of detection at the 10 fM level. In conclusion, our approach offers a promising perspective on sensitive and reliable miRNA detection and has the potential to be further utilized in biomedical research and early cancer detection.

Interactions between corn starch and lingonberry polyphenols and their effects on starch digestion and glucose transport.

This study investigated the interaction mechanism between corn starch (CS) and lingonberry polyphenols (LBP) during starch gelatinization, focusing on their effects on starch structure and physicochemical properties. Moreover, it explored the effect of this interaction on starch digestion and glucose transport. The results indicated that LBP interacted non-covalently with CS during starch gelatinization, disrupted the short-range ordered structure of starch, decreased gelatinization enthalpy of starch, and formed a dense network structure. Furthermore, the incorporation of LBP remarkably reduced the digestibility of CS. In particular, the addition of 10 % LBP decreased the terminal digestibility (C∞) from 77.87 % to 60.43 % and increased the amount of resistant starch (RS) by 21.63 %. LBP was found to inhibit α-amylase and α-glucosidase in a mixed manner. Additionally, LBP inhibited glucose transport in Caco-2 cells following starch digestion. When 10 % LBP was added, there was a 34.17 % decrease in glucose transport compared with starch digestion without LBP. This study helps establish the foundation for the development of LBP-containing starch or starch-based healthy foods and provides new insights into the mechanism by which LBP lowers blood glucose.

Soy Protein Isolate Interacted with Acrylamide to Reduce the Release of Acrylamide in the In Vitro Digestion Model.

Acrylamide (AA), a common carcinogen, has been found in many dietary products.. This study aimed to explore the interaction of soybean protein isolate (SPI) with AA and further research the different effects of SPI on the AA release due to interactions in the in vitro digestion model. Analysis of variance was used to analyze the data. The results suggested that AA could bind with SPI in vitro, leading to the variation in SPI structure. The intrinsic fluorescence of SPI was quenched by AA via static quenching. The non-covalent (van der Waals forces and hydrogen bonding) and covalent bonds were the main interaction forces between SPI and AA. Furthermore, the release of AA significantly decreased due to its interaction with SPI under simulated gastrointestinal conditions. SPI had different effects on the AA release rate after different treatments. The thermal (80, 85, 90, and 95 °C for either 10 or 20 min) and ultrasound (200, 300, and 400 W for either 15, 30, or 60 min) treatments of SPI were useful in reducing the release of AA. However, the high pressure-homogenized (30, 60, 90, and 120 MPa once, twice, or thrice) treatments of SPI were unfavorable for reducing the release of AA.

Covalent Interaction between High Hydrostatic Pressure-Pretreated Rice Bran Protein Hydrolysates and Ferulic Acid: Focus on Antioxidant Activities and Emulsifying Properties.

Rice bran protein hydrolysates (RBPH) pretreated with high hydrostatic pressure (HHP) covalently interacted with ferulic acid (FA) (0.5 to 2.5 mg/mL) under alkaline conditions. The structural and functional properties of the conjugates were investigated. The results revealed that the FA binding equivalent on RBPH increased from 6.03 to 207.64 nmol/mg. FTIR spectral analysis indicated that the content of α-helix increased, whereas the contents of ß-sheet, ß-turn, and random coil decreased. The surface hydrophobicity (H0) of RBPH increased, the fluorescence intensity decreased, and the tertiary structure changed because of covalent interactions between RBPH and FA. The emulsifying activity index of RBPH-FA (1.5 mg/mL) was 35.10% higher than that of the control, whereas FA concentrations higher than 1.5 mg/mL had a negative effect on emulsifying properties. RBPH-FA (2.5 mg/mL) exhibited the strongest antioxidant activity. This study provides a new reference for the application of RBPH-FA conjugates in food processing.

Investigation on the interaction of acrylamide with soy protein isolate: Exploring the binding mechanism in vitro.

Acrylamide (AA), which is a carcinogen in humans, has been a research focus in terms of food risk assessment. However, few published studies have explored protein strategies to reduce the health risks of AA. The objective of this study was to investigate the binding of AA with soy protein isolate (SPI) and elucidate the binding mechanism. The results showed that AA could bind with nontreated, heat-treated, high-pressure homogenization-treated, and ultrasound-treated SPI in vitro. Fourier-transform infrared spectroscopy suggested that secondary structure of SPI changed significantly after binding with AA in the nontreated and different treated groups. Moreover, fluorescence quenching experiments suggested that the quenching of SPI by AA was static quenching and hydrogen bonds, hydrophobic interactions, and van der Waals forces were involved in this process. PRACTICAL APPLICATION: The study of SPI and AA binding could provide a new perspective for reducing the bioaccessibility of AA in human body by using protein. The results showed that SPI could potentially be used as a novel health strategy to reduce the harm of AA in the human body.

MicroRNA­16 inhibits endometrial stromal cell migration and invasion through suppression of the inhibitor of nuclear factor­κB kinase subunit ß/nuclear factor­κB pathway.

Accumulating evidence has demonstrated that endometrial stromal cells (ESCs) are responsible for the pathogenesis of endometriosis (Ems), which is characterized by the presence of functional endometrial­like tissues outside the uterine cavity. Abnormal expression of microRNAs (miRNAs) in ESCs may be implicated in the etiology of Ems; however, the exact mechanisms have yet to be fully elucidated. The aim of the present study was to investigate the effects of miRNAs on ESCs and the underlying mechanisms. Using a microarray assay, microRNA­16 (miR­16) was found to be significantly downregulated in the ectopic endometrial tissues in patients with Ems, compared with that in eutopic endometrial tissues. Overexpression of miR­16 significantly suppressed the migration and invasion of ESCs, whereas miR­16 inhibition exerted the opposite effects. Furthermore, dual luciferase reporter assay demonstrated that miR­16 directly targeted the inhibitor of nuclear factor (NF)­κB kinase subunit ß (IKKß) and suppressed its translation. It was observed that the expression of IKKß was upregulated and inversely correlated with miR­16 levels in the ectopic endometrial tissues in patients with Ems. Additionally, knockdown of IKKß by si­IKKß mimicked the effects of miR­16 overexpression on ESCs, while the promoting effects of IKKß overexpression on the migration and invasion of ESCs were attenuated by miR­16 overexpression. Finally, miR­16 inhibited the activation of the NF­κB pathway by targeting IKKß. Collectively, these results demonstrated that miR­16 may suppress Ems by inhibiting the IKKß/NF­κB pathway, suggesting that miR­16 may be a useful target in the treatment of Ems.

Protective effect of seabuckthorn berry juice against acrylamide-induced oxidative damage in rats.

Acrylamide (AA), classified as a probable carcinogen, can be neurotoxic, genotoxic, and can damage DNA. This study explored the ability of seabuckthorn berries juice (SBJ) to alleviate AA-induced toxic injury in rats. Twenty-four adult male Sprague-Dawley (SD) rats were randomly divided into four groups: control group, AA group (40 mg/kg), AA + SBJ (40 mg/kg AA and 5 mL/kg SBJ), and AA + vitamin C (VC) group (positive control group, 40 mg/kg AA and 100 mg/kg VC). At the end of the experiment, rats in AA group showed a marked decrease in the rate of weight gain, hind extremity abduction, and ataxia. Obvious anomalies were seen in plasma biochemical parameters (P < 0.05), and different degrees of injury were observed upon histological examination of five tissues (hippocampus, cerebellum, liver, small intestine, and kidney). Compared to the control group, levels of superoxide dismutase, catalase, and glutathione were significantly decreased, while malondialdehyde was elevated (P < 0.05). SBJ treatment reduced the abnormal of behavior, hematological index, antioxidant enzyme, and tissue damage caused by AA in rats. PRACTICAL APPLICATION: Seabuckthorn berries are wild berries rich in vitamin C and polyphenols, which have good antioxidant properties. In this experiment, SBJ has a significant alleviating effect on AA-induced oxidative damage in rats. Therefore, we speculate that SBJ may relieve the oxidative damage caused by diet or other forms of AA exposure in the general population. At the same time, this experiment also provides new ideas for alleviating AA-induced in vivo toxicity.

A novel DNA vaccine for protective immunity against virulent Mycobacterium bovis in mice.

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB). The proteins Ag85B, MPB64, and ESAT-6 are the major immunogenic antigens of M. bovis; these proteins play important roles in inducing immune responses that confer resistance against infections. In the present study, we used pcDNA3.1(+) as a vector and constructed various DNA vaccines with the genes encoding the three antigens mentioned above. This procedure involved the following steps: fusion of two genes (pcDNA-MPB64-Ag85B, pcMA), fusion of three genes (pcDNA-MPB64-Ag85B-ESAT-6, pcMAE), bivalent combinations (pcDNA-Ag85B+pcDNA-MPB64, pcA+M), and trivalent combinations (pcDNA-Ag85B+pcDNA-MPB64+pcDNA-ESAT-6, pcA+M+E). The immune response to the DNA vaccines was evaluated based on serum antibody titers, lymphocyte proliferation assay, and titers of the cytokines interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). The protective efficacy following challenge with a virulent M. bovis strain, C68001, was evaluated based on survival rate, bacterial loads in lung tissue, and histopathologic changes. A significant 2-fold increase in serum antibody levels was observed in mice vaccinated with fusion DNA (two or three genes). Furthermore, the lymphocyte proliferation (SI) values and the levels of IFN-gamma and IL-2 were higher in mice vaccinated with fusion DNA (two or three genes) than in those immunized with polyvalent combination DNA vaccines (P<0.05). Additionally, the fusion DNA vaccines provided protection that was superior to that provided by the polyvalent combination DNA vaccines following challenge with M. bovis strain C68001. The protective efficacy of the fusion DNA vaccines in mice immunized three times was equivalent to the protective efficacy in mice immunized once with the Bacillus Calmette-Guerin (BCG) vaccine. This suggests that fusion DNA vaccine represent a promising approach for the prevention of bTB.

A novel fusion protein-based indirect enzyme-linked immunosorbent assay for the detection of bovine tuberculosis.

Enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine tuberculosis has been widely explored over the years. Three Mycobacterium bovis-specific antigen genes, namely, mpb70, mpb83, and esat-6 were recombined in tandem by spliced overlap extension technology and expressed in Escherichia coli to obtain the fusion protein (rM70-83-E6). Western blot analysis showed that rM70-83-E6 can specifically react with bovine tuberculosis-positive sera but not those from cattle infected with other bovine diseases such as bovine paratuberculosis. An indirect ELISA (iELISA) method was established with rM70-83-E6 as the diagnostic antigen. The diagnostic criteria were determined using 150 serum samples from healthy cattle. Analyses of 85 serum samples from cattle with bovine tuberculosis and 100 serum samples from healthy cattle demonstrated that the sensitivity of the iELISA was 69.4% (59/85) and the specificity was 96.0% (96/100). Moreover, 46 out of 67 purified protein derivative (PPD) skin test-positive samples were also positive by iELISA, giving a positive coincidence of 68.7%, while all 50 PPD skin test-negative samples were negative by iELISA, giving a negative coincidence of 100%. The total coincidence between iELISA and the PPD skin test was 82.1%. This study demonstrated that iELISA using rM70-83-E6 antigen is simple, sensitive and easy to perform and can be used to analysis of a large number of samples for serodiagnosis of bovine tubercuiosis.

Safety evaluation of soybean protein isolate oxidized by a hydroxyl radical-generating system.

The oxidative modification of soybean protein isolate (SPI) induced by a hydroxyl radical-generating system (HRGS) has a broad range of applications. However, few toxicology studies exist on this material. The safety of HRGS-oxidized SPI was assessed using subchronic and genotoxicity studies. A 30-day subchronic study (250, 500 and 1000 mg/kg∙BW) in rats showed no significant adverse effects on food consumption, body weight (BW), mortality, hematology, biochemistry, necropsy, organ weight or histopathology. The result of an Ames test showed that HRGS-oxidized SPI was not mutagenic to the test strains. The results of a bone marrow micronucleus test and mouse sperm abnormality test showed HRGS-oxidized SPI (417.5, 835.0 and 1670.0 mg/kg⋅BW) did not produce any aberrant effects on bone marrow cells or mouse sperm. Therefore, HRGS-oxidized SPI showed no genotoxicity in vivo or in vitro. In conclusion, these results support the safe use of HRGS-oxidized SPI as a food and dietary supplement.

Silymarin protects against acrylamide-induced neurotoxicity via Nrf2 signalling in PC12 cells.

Silymarin (SM) is a well-known antioxidant, anti-inflammatory and anti-cancer compound extracted from the milk thistle. Here, we investigated the protective effect of SM against acrylamide (AA)-induced neurotoxicity, mainly caused by oxidative stress, via activation of the nuclear transcription factor E2-related factor 2 (Nrf2) signalling pathway in PC12 cells. The MTT reduction assay was used to measure cell viability in various drug-treated groups and demonstrated that SM could increase cell viability in AA-treated PC12 cells. We then measured the reactive oxygen species (ROS) levels by the peroxide-sensitive fluorescent probe DCFH-DA and intracellular glutathione (GSH) and malondialdehyde (MDA) levels by absorption spectrophotometry. Our data revealed that SM could reduce ROS and MDA levels and increase GSH levels in AA-induced PC12 cells. To identify a potential mechanism for SM-induced protection, we measured the mRNA and protein expression levels of Nrf2 and its downstream target antioxidants glutathione peroxidase (Gpx), glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM) by quantitative real-time PCR and Western blot, respectively. The results suggested that SM could activate Nrf2 signalling and increase the expression of Nrf2, Gpx, GCLC and GCLM in AA-treated PC12 cells. In conclusion, SM can effectively alleviate AA-induced neurotoxicity in PC12 cells.

Key role of peptidoglycan on acrylamide binding by lactic acid bacteria.

The primary purpose of this study was to analyze the ability of four peptidoglycan (PGN) from different lactic acid bacteria to bind acrylamide (AA) and to identify the binding mechanism. In this study, to clarify the possible binding interactions among AA and components of PGN, chemical components, surface structure, amino acids component, and functional groups of peptidoglycans were studied. It was found that PGN from Lactobacillus plantarum 1.0065 had the highest ability to bind AA with 87%. Furthermore, a significant positive relation was found between the carbohydrate content of PGN and percentage of bind AA, and the content of four specific amino acids of PGN and AA binding ability were also positive correlated. Thereinto, alanine of PGN had a significant impact on AA binding among four amino acids. Additionally, the C-O (carboxyl, polysaccharides, and arene), C=O amide, and N-H amines groups of PGN were involved in AA binding.

The protective immune response induced by B cell epitope of classical swine fever virus glycoprotein E2.

Classical swine fever virus (CSFV) envelope glycoprotein E2 is a major protective immunogen responsible for eliciting neutralizing antibodies and conferring protective immunity against the virus. Based on the core sequence (TAVSPTTLR, 829-837 aa) of the B cell linear epitope of the CSFV E2 protein identified by Lin et al., two oligonucleotides MF and MR were synthesized and used to construct by PCR a gene cassette encoding a 15 amino acid polypeptide M (CTAVSPTTLRTEVVK), which spans 828-842 amino acids of E2. The gene cassette was fused in-frame to 3' terminal of glutathione S transferase gene (GST) of the prokaryotic expression vector pGEX-6p-1, resulting in the recombinant plasmid pGEX-M. After transformation into Escherichia coli BL21 a soluble fusion protein GST-M with expected size of 28 kDa was expressed after inducing with isopropyl-beta-d-thiogalactoside (IPTG). Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the purified GST-M had good reactivity with swine anti-CSFV serum and rabbit anti-CSFV E2 serum. Further vaccination trials showed that the fusion protein GST-M could elicit effectively immune response protecting rabbits and pigs from virulent challenge. This study showed a possibility for developing epitope-based vaccines against CSFV.

Paenibacillin A, a new 2(1H)-pyrazinone ring-containing natural product from the endophytic bacterium Paenibacillus sp. Xy-2.

A new 2(1H)-pyrazinone ring-containing natural product, paenibacillin A (1), together with five known diketopiperazine derivatives 2-6 and two known isoflavones 7-8, was isolated from the culture of an endophytic bacterium Paenibacillus sp. Xy-2. The structure of compound 1 was elucidated by extensive spectral methods, including UV, IR, HR-ESI-MS, 1D and 2D NMR and ECD experiments. Compound 1 exhibited moderate cytotoxicity against HL-60 cell line with IC50 value of 50.48 µM.

Butenolide derivatives from the plant endophytic fungus Aspergillus terreus.

Three new butenolides containing 5-hydroxyfuran-2(5H)-one core, asperteretal A (1), asperteretal B (2), and asperteretal C (3), together with seven known butenolides (4-10), were obtained from an endophytic fungus Aspergillus terreus PR-P-2 isolated from the plant Camellia sinensis var. assamica. The structures of compounds 1-3 were elucidated on the basis of detailed spectroscopic analysis including UV, IR, HRESIMS, 1D and 2D NMR, and ECD spectra. Compounds 1, 3, 5 and 6-8 showed potent inhibitory effects on NO production in RAW 264.7 lipopolysaccharide-induced macrophages, and compounds 5 and 8 also exhibited moderate cytotoxicity against HL-60 cell line.

Evaluation of multicomponent recombinant vaccines against Actinobacillus pleuropneumoniae in mice.

BACKGROUND: Porcine contagious pleuropneumonia (PCP) is a highly contagious disease that is caused by Actinobacillus pleuropneumoniae (APP) and characterized by severe fibrinous necrotizing hemorrhagic pleuropneumonia, which is a severe threat to the swine industry. In addition to APP RTX-toxins I (ApxI), APP RTX-toxin II (ApxII), APP RTX-toxin III (ApxIII) and Outer membrane protein (OMP), there may be other useful antigens that can contribute to protection. In the development of an efficacious vaccine against APP, the immunogenicities of multicomponent recombinant subunit vaccines were evaluated. METHODS: Six major virulent factor genes of APP, i.e., apxI, apxII, apxIII, APP RTX-toxins IV (apxIV), omp and type 4 fimbrial structural (apfa) were expressed. BALB/c mice were immunized with recombinant ApxI ( rApxI), recombinant ApxII (rApxII), recombinant ApxIII (rApxIII) and recombinant OMP (rOMP) (Group I); rApxI, rApxII, rApxIII, recombinant ApxIV (rApxIV), recombinant Apfa (rApfa) and rOMP (Group II); APP serotype 1 (APP1) inactivated vaccine (Group III); or phosphate-buffered saline (PBS) (Control group), respectively. After the first immunization, mice were subjected to two booster immunizations at 2-week intervals, followed by challenge with APP1 Shope 4074 and APP2 S1536. RESULTS: The efficacy of the multicomponent recombinant subunit vaccines was evaluated on the basis of antibody titers, survival rates, lung lesions and indirect immunofluorescence (IIF) detection of APP. The antibody level of Group I was significantly higher than those of the other three groups (P < 0.05). The survival rate of Group I was higher than that of Groups II and III (P < 0.05) and the control (P < 0.01). Compared with the other three groups, the lungs of Group I did not exhibit obvious hemorrhage or necrosis, and only showed weak and scattered fluorescent dots by IIF detection. CONCLUSION: The result indicates that the multicomponent recombinant subunit vaccine composed of rApxI, rApxII, rApxIII and rOMP can provide effective cross-protection against homologous and heterologous APP challenge.