Insights into peptide profiling of sturgeon myofibrillar proteins with low temperature vacuum heating.

BACKGROUND: Protein oxidation during food processing causes changes in the balance of protein-molecular interactions and protein-water interactions, ultimately leading to protein denaturation, which results in the loss of a range of functional properties. Therefore, how to control the oxidative modification of proteins during processing has been the focus of research. RESULTS: In the present study, the intrinsic fluorescence value of the myofibrillar proteins (MP) decreased and the surface hydrophobicity value increased, indicating that the heat treatment caused a significant change in the conformation of the MP. With an increase in heating temperature, protein carbonyl content increased, total sulfhydryl content decreased, and protein secondary structure changed from α-helix to ß-sheet, indicating that protein oxidation and aggregation occurred. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that heat treatment can lead to the degradation of proteins, especially myosin heavy chain, although actin had a certain thermal stability. In total, 733 proteins were identified by proteomics, and the protein oxidation caused by low temperature vacuum heating (LTVH) was determined to be mild oxidation dominated by malondialdehyde and 4-hydroxynonenal by oxidation site division. CONCLUSION: The present study has revealed the effect of LTVH treatment on the protein oxidation modification behavior of sturgeon meat, and explored the effect mechanism of LTVH treatment on the processing quality of sturgeon meat from the perspective of protein oxidation. The results may provide a theoretical basis for the precise processing of aquatic products. © 2023 Society of Chemical Industry.

Characteristics and antioxidant properties of Harpadon nehereus protein hydrolysate-xylose conjugates obtained from the Maillard reaction by ultrasound-assisted wet heating in a natural deep eutectic solvents system.

BACKGROUND: Harpadon nehereus is a high-protein marine fish. A valuable way to add value to H. nehereus is to convert it into protein hydrolysate. The Maillard reaction is an effective way to improve the functional properties of peptides and proteins, which are affected by many factors such as reactant concentration, water activity, pH, temperature, and heating time. However, the traditional Maillard reaction method is inefficient. The purpose of this study was therefore to explore the effect of the ultrasound-assisted wet heating method on the Maillard reaction of H. nehereus protein hydrolysate (HNPH) in a new-type green solvent - a natural hypereutectic solvent (NADES). RESULTS: Harpadon nehereus protein hydrolysate-xylose (Xy) conjugates were prepared via a Maillard reaction in a NADES system using an ultrasound-assisted wet heating method. The effects of different treatment conditions on the Maillard reaction were studied. The optimized glycation degree (DG) of HNPH-Xy conjugates was obtained with a water content of 10%, a reaction temperature of 80 °C, a reaction time of 35 min, and an ultrasonic power level of 300 W. Compared with HNPH, the structure of HNPH-Xy conjugates were significantly changed. Moreover, the functional properties and antioxidant activity of HNPH-Xy were all superior to the HNPH. CONCLUSIONS: An ultrasound-assisted wet-heating Maillard reaction between HNPH and Xy in the NADES system could be a promising way to improve the functional properties of HNPH. © 2023 Society of Chemical Industry.

Inhibitory effects of ultrasonic and rosmarinic acid on lipid oxidation and lipoxygenase in large yellow croaker during cold storage.

Lipid oxidation will lead to the deterioration of flavor, color and texture of aquatic products with high fatty acid content. The mechanism of ultrasound (US) combined with rosmarinic acid (RA) on lipid oxidation and endogenous enzyme activities of large yellow croaker during cold-storage (4 ℃) was investigated. The result showed that the US and RA have synergistic effects in delaying lipid oxidation and inhibiting endogenous lipase and lipoxygenase (LOX) activities related to oxidation. The inhibition of LOX activity by RA was dose-dependent, and US showed a negative effect on the inhibition of enzyme activity in the presence of low concentration RA. Moreover, RA changes the enzyme structure through static fluorescence quenching and interaction with enzyme molecules. Hydrogen bonding and hydrophobic interaction are the main interaction forces between RA and LOX. This study could provide basic mechanism of US treatment cooperating with polyphenols to inhibit lipid oxidation during food preservation.

Effect of tea polyphenols on sturgeon myofibrillar protein structure in the in vitro anti-glycation model mediated by low temperature vacuum heating.

The binding mechanism between tea polyphenols and sturgeon myofibrillar protein (SMP) in the early stage (0, 2, 4 min), middle stage (6, 10 min) and late stage (15 min) of low temperature vacuum heating (LTVH) in an in vitro anti-glycation model was investigated. The result indicated that the protein cross-linking during LTVH treatment were mainly induced by tea polyphenols. The loss rate of free arginine (Arg) and free lysine (Lys) of SMP at the late stage of LTVH treatment (15 min) was 73.95 % and 83.16 %, respectively. The hydrophobic force and disulfide bond were the main force between tea polyphenols and SMP in the middle and late stage of LTVH treatment. The benzene ring and phenolic hydroxyl group of tea polyphenols can interact with the amino acid residues of SMP, which was exothermic and entropy-increasing. This study provides new insights in the interaction mechanisms between tea polyphenols-protein during heat treatment process.

Fabrication of glycated yeast cell protein via Maillard reaction for delivery of curcumin: improved environmental stability, antioxidant activity, and bioaccessibility.

BACKGROUND: The application of curcumin (CUR) in the food industry is limited by its instability, hydrophobicity and low bioavailability. Yeast cell protein (YCP) is a by-product of spent brewer's yeast, which has the potential to deliver bioactive substances. However, the environmental stresses such as pH, salt and heat treatment has restricted its application in the food industry. Maillard reaction as a non-enzymatic browning reaction can improve protein stability under environmental stress. RESULTS: The CUR was successfully encapsulated into the hydrophobic core of YCP/glycated YCP (GYCP) and enhanced by hydrogen bonding, resulting in static fluorescence quenching of YCP/GYCP. The average diameter and dispersibility of GYPC-CUR nanocomplex were significantly improved after glucose glycation (121.40 nm versus 139.70 nm). Moreover, the encapsulation capacity of CUR was not influenced by glucose glycation. The oxidative stability and bioaccessibility of CUR in nanocomplexes were increased compared with free CUR, especially complexed with GYCP conjugates. CONCLUSION: Steric hindrance provided by glucose conjugation improved the enviriomental stability, oxidative activity and bioaccessibility of CUR in nanocomplexes. Thus, glucose-glycated YCP has potential application as a delivery carrier for hydrophobic compounds in functional foods. © 2022 Society of Chemical Industry.

Cutting and Bonding Parafilm® to Fast Prototyping Flexible Hanging Drop Chips for 3D Spheroid Cultures.

A fast and low-cost fabrication process of flexible hanging drop chips for 3D spheroid cultures was proposed by cutting and bonding Parafilm®, a cohesive thermoplastic. The Parafilm® Hanging Drop Chip (PHDC) was assembled by two-layer of Parafilm® sheet with different sizes of holes. The hole on the upper layer of the Parafilm® is smaller than the hole on the bottom layer. The impact of hole size and sample volume on hanging drop formation and 3D spheroid formations in the hanging drop were investigated. The results showed that 20 µL solution on PHDC with a 3 mm hole could form stabile drop and facilitate spheroid formation. The initial cell number determinates the size of the formed spheroids. Exchanging liquid from the upper hole of the PHDC enables the co-culture of two types of cells in one spheroid and drug efficacy testing in hanging drops. The relative expression of cell adhesion and hypoxia-related genes from spheroids in hanging drop and conventional culture plate suggested the relevance of 3D spheroids and in vivo tumor tissue. The economical hanging drop chip can be fabricated without wet chemistry or expensive fabrication equipment, strengthening its application potential in conventional biological laboratories.

A smartphone-supported portable micro-spectroscopy/imaging system to characterize morphology and spectra of samples at the microscale.

A smartphone-based analysis system is favored for point-of-care testing applications. The present work proposes a novel micro-spectroscopy/imaging system comprising a portable spectrometer as an optical sensor and a compact homemade microscope to acquire the image and spectra of micron-scale regions. Protein concentration quantification based on the bicinchoninic acid method was demonstrated with the proposed micro-spectroscopy/imaging system to analyse the spectrometer signals. Morphologies of onion endothelial and human breast cancer cells, used as biological sample models, were characterized to demonstrate the microscopic imaging capacity of the device. The ability to simultaneously obtain morphological and spectral information using the proposed portable device was demonstrated by examining the 10 µm sub-pixels of a smartphone screen. These results highlight the potential for adopting a smartphone-based micro-spectroscopy/imaging system for point-of-care testing.

The use of amphiphilic copolymer in the solid dispersion formulation of nimodipine to inhibit drug crystallization in the release media: Combining nano-drug delivery system with solid preparations.

Solid dispersion is a widely used method to improve the dissolution and oral bioavailability of water-insoluble drugs. However, due to the strong hydrophobicity, the drug crystallization in the release media after drug dissolution and the resulted decreased drug absorption retards the use of solid dispersions. It is widely known that the amphiphilic copolymer can encapsulate the hydrophobic compounds and help form stable nano-dispersions in water. Inspired by this, we tried to formulate the solid dispersion of nimodipine by using amphipathic copolymer as one of the carriers. Concerning the solid dispersions, there are many important points involved in these formulations, such as the miscibility between the drug and the carriers, the storage stability of solid dispersions, the dissolution enhancement and so on. In this study, a systemic method is proposed. In details, the supersaturation test and the glass transition temperature (Tg) measurement to predict the crystallization inhibition, the ratios of different components and the storage stability, the interactions among the components were investigated in detail by nuclear magnetic resonance (1H NMR) and isothermal titration calorimetry (ITC) and, the final dissolution and oral bioavailability enhancement. It was found that the amphiphilic copolymer used in the solid dispersion encouraged the formation the drug loading micelles in the release media and, finally, the problem of drug crystallization in the dissolution process was successfully solved.

On-chip RT-LAMP and colorimetric detection of the prostate cancer 3 biomarker with an integrated thermal and imaging box.

To achieve low-cost, compact, and portable nucleic acid testing, an integrated device containing a three-dimensional printing fabricated reverse transcription loop-mediated isothermal amplification (RT-LAMP) chip, a thermal module, and an imaging module was developed. Samples and RT-LAMP reagents were loaded on a sponge-like polyvinyl alcohol pad on a chip, whereas the colorimetric detection zone was a dry paper pre-loaded with Calcine. The sealed chip was incubated on the integrated thermal module, and the RT-LAMP products were pressed into the Calcine pre-loaded dry paper by a stick. Colorimetric changes could be visually observed by the naked eye or imaged with a smartphone camera through the imaging module. For detection of the prostate cancer antigen 3 (PCA3) biomarker, LAMP primers were designed and verified. The specificity of Calcine pre-loaded dry paper based on colorimetric detection of positive LAMP products was investigated. The reaction conditions for on-chip RT-LAMP such as amplification time, temperature, and volume were optimized. Finally, a detection limit of 0.34 fg/µL RNA was achieved with the proposed on-chip RT-LAMP and colorimetric detection method for PCA3. Since the thermal plate is powered by a 12-V battery and the color change can be imaged with a smartphone, the integrated platform can be operated on-site, highlighting its potential in point-of-care testing applications.

Contrast-enhanced ultrasound features of mediastinal lymphomas and thymic epithelial tumors.

OBJECTIVES: To summarize the contrast-enhanced ultrasound (CEUS) features of mediastinal lymphomas and thymic epithelial tumors (including thymomas and thymic carcinomas) and to explore the value of CEUS in the differential diagnosis of lymphomas and thymic epithelial tumors. METHODS: Sixty-nine patients with 69 mediastinal lesions who underwent CEUS and had disease confirmed by histopathology were enrolled in the study. There were 33 cases of lymphoma, 19 cases of thymic carcinoma, and 17 cases of thymoma. CEUS features, including the enhancement pattern, enhancement distribution, enhancement time, inner necrosis status, wash out pattern, and vascular morphology, were evaluated in each group. RESULTS: Thymomas often presented with homogeneous (88.2%, 15/17) and late (88.2%, 15/17) enhancement and a low rate of inner necrosis (17.6%, 3/17). Late (73.7%, 14/19), heterogeneous (68.4%, 13/19), and centripetal (63.2%, 12/19) enhancement were more often observed in thymic carcinoma, as was a high rate of inner necrosis (78.9%, 15/19). Lymphomas showed a homogeneous enhancement rate of 57.6% (19/33) and a late enhancement rate of 54.5% (18/33). The rate of inner necrosis for lymphomas was 45.5% (15/33). The diagnostic accuracy of this finding for distinguishing thymic epithelial tumors from lymphomas was 63.8%, the sensitivity was 80.6%, and the specificity was 45.5%. Enlarged blood vessels were a feature specific to lymphomas, while small vessels arranged in a comb shape was a feature specific to thymic epithelial tumors. CONCLUSION: This study describes the CEUS features of common mediastinal tumors and may stimulate further studies in this field.

Spontaneous formation of tumor spheroid on a hydrophilic filter paper for cancer stem cell enrichment.

Emerging evidence has demonstrated that cancer stem cells (CSCs) play critical roles in tumor invasion, metastasis and recurrence. The specific targeting capability on CSCs is of high importance for the development of effective anti-tumor therapeutics. However, isolation, enrichment and cultivation of these special and rare groups of tumor cells for in vitro analyses is a nontrivial job and requires particular culture medium and environmental control. Herein, we established a low-cost and efficient method for CSC enrichment by culturing prostate cancer cells on a hydrophilic filter paper. We found that tumor spheroids could form spontaneously on a pristine filter paper solely with regular cell culture medium. The paper-grown cells had elevated expression of putative CSC markers, indicating increased stemness of the cancer cells. Moreover, increased resistance of the chemotherapeutic drug doxorubicin was observed on the formed CSC spheroids compared to regular culture. The properties of the filter paper were characterized to investigate the underlying mechanism behind the promoted tumor spheroid formation. The obtained results suggested that the excellent hydrophilicity of the cellulose fibers retarded the hydrophobic interaction-mediated cell anchoring on the cellulose fibers, while the limited space/niche between fibers promoted the aggregation of cells. In addition, biocompatible paper-based materials are able to realize convenient assembly of tissue-like structures for developing in vitro disease models or organs-on-paper applications. Therefore, hydrophilic filter papers could be a low-cost material for construction of various assay platforms for isolating and enriching CSCs, screening anti-tumor drugs, and constructing tumor models in vitro.

Separation and Characterization of Prostate Cancer Cell Subtype according to Their Motility Using a Multi-Layer CiGiP Culture.

Cancer cell metastasis has been recognized as one hallmark of malignant tumor progression; thus, measuring the motility of cells, especially tumor cell migration, is important for evaluating the therapeutic effects of anti-tumor drugs. Here, we used a paper-based cell migration platform to separate and isolate cells according to their distinct motility. A multi-layer cells-in-gels-in-paper (CiGiP) stack was assembled. Only a small portion of DU 145 prostate cancer cells seeded in the middle layer could successfully migrate into the top and bottom layers of the stack, showing heterogeneous motility. The cells with distinct migration were isolated for further analysis. Quantitative PCR assay results demonstrated that cells with higher migration potential had increased expression of the ALDH1A1, SRY (sex-determining region Y)-box 2, NANOG, and octamer-binding transcription 4. Increased doxorubicin tolerance was also observed in cells that migrated through the CiGiP layers. In summary, the separation and characterization of prostate cancer cell subtype can be achieved by using the multi-layer CiGiP cell migration platform.

Protective and Heat Retention Effects of Thermo-sensitive Basement Membrane Extract (Matrigel) in Hepatic Radiofrequency Ablation in an Experimental Animal Study.

PURPOSE: To evaluate the protective effect of using thermo-sensitive basement membrane extract (Matrigel) for hydrodissection to minimize thermal injury to nearby structures and to evaluate its heat sink effect on the ablation zone in radiofrequency ablation (RFA) of the liver. MATERIALS AND METHODS: First, the viscosity profile and heat sink effect of Matrigel were assessed during RFA in vitro and ex vivo. Fresh pig liver tissue was used, and the temperature changes in Matrigel and in 5% dextrose in water (D5W) during RFA were recorded. Then, the size of the ablation zone in the peripheral liver after RFA was measured. Second, in an in vivo study, 45 Sprague-Dawley rats were divided into three groups of 15 rats each (Matrigel, D5W and control). In the experimental groups, artificial ascites with 10 ml of Matrigel or D5W were injected using ultrasound guidance prior to RFA. The frequency of thermal injury to the nearby organs was compared among the three groups, with assessments of several locations: near the diaphragm, the abdominal wall and the gastrointestinal (GI) tract. Finally, the biological degradation of Matrigel by ultrasound was evaluated over 60 days. RESULTS: First, Matrigel produced a greater heat retention (less heat sink) effect than D5W during ex vivo ablation (63 ± 9 vs. 26 ± 6 °C at 1 min on the surface of the liver, P < 0.001). Hepatic ablation zone volume did not differ between the two groups. Second, thermal injury to the nearby structures was found in 14 of 15 cases (93.3%) in the control group, 8 of 15 cases (53.3%) in the D5W group, and 1 of 15 cases (6.7%) in the Matrigel group. Significant differences in the thermal injury rates for nearby structures were detected among the three groups (P < 0.001). The most significant difference in the thermal injury rate was found in locations near the GI tract (P = 0.003). Finally, Matrigel that was injected in vivo was gradually degraded during the following 60 days. CONCLUSIONS: Using thermo-sensitive Matrigel as a hydrodissection material might help reduce the frequency of collateral thermal injury to nearby structures, especially in locations close to the GI tract, compared to conventional D5W. Additionally, Matrigel did not increase the heat sink effect on the ablation zone during ablation and was degraded over time in vivo.

Microflow imaging of contrast-enhanced ultrasound for evaluation of neovascularization in peripheral lung cancer.

The aim of this study was to investigate the role of microflow imaging (MFI) of contrast-enhanced ultrasound (CEUS) for evaluating microvascular architecture of different types of peripheral lung cancer (PLC) and to explore the correlated pathological basis.Ninety-five patients with PLC were enrolled in this study. Two radiologists independently evaluated the microvascular architecture of PLC with MFI. The interobserver agreement was measured with Kappa test. The diagnosis value of MFI was calculated. With pathological analysis, the correlation between MFI and microvascular density (MVD)/microvascular diameter (MD) was evaluated.Of the 95 PLCs, MFI were mainly classified "dead wood" (27.4%, 25.3%), "vascular" (47.4%, 49.5%), and "cotton" (20.0%, 20.0%) patterns by the 2 readers. Kappa test showed a good agreement between the 2 readers (Kappa = 0.758). The "dead wood" can be regarded as a specific diagnostic factor for squamous carcinoma; the sensitivity, specificity, and accuracy was 62.9%, 93.3%, and 82.1%, respectively. The "vascular" and "cotton" patterns correlated well with adenocarcinoma and SCLC (small cell lung cancer); diagnostic sensitivity, specificity, and accuracy were 86.7%, 65.7%, and 78.9%, respectively. MVD of "dead wood" was lower than "vascular" and "cotton," while MD was bigger than the other 2 patterns (P < 0.05). There was a good correlation between MFI and histopathological types of PLC as well as between MFI and MVD/MD (P < 0.05).MFI has the advantage to display the microvascular architecture of PLCs and might become a promising diagnostic method of histopathological types of PLC. MFI features also correlated well with its pathological basis, including MVD and MD.

Role of Arrival Time Difference Between Lesions and Lung Tissue on Contrast-Enhanced Sonography in the Differential Diagnosis of Subpleural Pulmonary Lesions.

OBJECTIVES: The purpose of this study was to explore the diagnostic value of the arrival time difference between lesions and surrounding lung tissue on contrast-enhanced sonography of subpleural pulmonary lesions. METHODS: A total of 110 patients with subpleural pulmonary lesions who underwent both conventional and contrast-enhanced sonography and had a definite diagnosis were enrolled. After contrast agent injection, the arrival times in the lesion, lung, and chest wall were recorded. The arrival time differences between various tissues were also calculated. RESULTS: Statistical analysis showed a significant difference in the lesion arrival time, the arrival time difference between the lesion and lung, and the arrival time difference between the chest wall and lesion (all P < .001) for benign and malignant lesions. Receiver operating characteristic curve analysis revealed that the optimal diagnostic criterion was the arrival time difference between the lesion and lung, and that the best cutoff point was 2.5 seconds (later arrival signified malignancy). This new diagnostic criterion showed superior diagnostic accuracy (97.1%) compared to conventional diagnostic criteria. CONCLUSIONS: The individualized diagnostic method based on an arrival time comparison using contrast-enhanced sonography had high diagnostic accuracy (97.1%) with good feasibility and could provide useful diagnostic information for subpleural pulmonary lesions.

BF02, a recombinant TNFR2 fusion protein, alleviates adjuvant arthritis by regulating T lymphocytes in rats.

AIM: To investigate the therapeutic effects of BF02 on adjuvant arthritis (AA) in rats and the regulatory effects of BF02 on T lymphocyte function. METHODS: SD rats received a single intradermal injection of Freund's complete adjuvant emulsion into the right hind metatarsal footpad. After the onset of AA, the rats were injected BF02 (1, 3, or 9 mg/kg, sc) every 3 d for a total of 15 d. Intragastric administration of methotrexate (MTX, 0.5 mg/kg, every 3 d for a total of 15 d) was taken as the positive control drug. Arthritis index, swollen joint count, ankle joint histopathology, spleen histopathology and the paw radiography were used for evaluating the drug effects on AA rats. T lymphocyte function was assessed by measuring T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression levels, and percentage of T lymphocyte subsets. RESULTS: In the AA rats, remarkable secondary inflammatory responses exhibited, accompanied by significantly higher levels of IL-1, IL-6, TNF-α, IL-17, LTα, RANKL, and MMP-13. The expression of IL17 and TNF-α mRNAs was also substantially higher than in normal rats. The percentages of CD3(+)CD4(+) and CD4(+)CD25(+) T lymphocytes were increased, whereas the percentages of CD4(+)CD62L(+) and CD4(+)CD25(+)FoxP3(+) T lymphocytes were decreased. Treatment of the AA rats with BF02 (9 mg/kg) or MTX significantly decreased the arthritis index, swollen joint count and arthritis global assessment. Moreover, both BF02 (9 mg/kg) and MTX significantly inhibited T lymphocyte proliferation, and blocked the above mentioned aberrance in T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression, and percentages of T lymphocyte subsets. CONCLUSION: BF02 exerts therapeutic effects on AA rats via the regulation of T lymphocytes.

Inhibitory effect of p53 upregulated modulator of apoptosis targeting siRNA on hypoxia/reoxygenation-induced cardiomyocyte apoptosis in rats.

OBJECTIVES: The effect of p53 upregulated modulator of apoptosis (PUMA) in hypoxia/reoxygenation-induced cardiomyocyte injuries in rats was investigated. METHODS: PUMA-targeting (si-PUMA) and scramble siRNAs were designed and transfected into primarily rat cardiomyocytes in vitro. RESULTS: RT-PCR and Western blot analysis showed that 50 nmol/l of si-PUMA can specifically inhibit PUMA expression. MTT assay and lactate dehydrogenase activity detection showed that the cell survival rate in the si-PUMA group was enhanced and that the lactate dehydrogenase enzymatic activity dramatically decreased compared with the control group (p < 0.01). Spectrophotometry, as well as annexin V and propidium iodide staining, combined with flow cytometry, revealed that caspase-3 activity in the si-PUMA group was downregulated and the apoptotic rate was decreased (p < 0.01). RT-PCR also showed that Bax expression was downregulated and Bcl-2 expression was upregulated in the si-PUMA group, compared with the control group (p < 0.05). si-PUMA protects cardiomyocytes from apoptosis. CONCLUSION: PUMA mediates hypoxia/reoxygenation-induced cardiomyocyte apoptosis, which can be a potential target of gene therapy for ischemia/reperfusion cardiomyocyte injuries.