Ultrasound Viscosity Imaging in Breast Lesions: A Multicenter Prospective Study.

RATIONALE AND OBJECTIVES: To explore and validate the clinical value of ultrasound (US) viscosity imaging in differentiating breast lesions by combining with BI-RADS, and then comparing the diagnostic performances with BI-RADS alone. MATERIALS AND METHODS: This multicenter, prospective study enrolled participants with breast lesions from June 2021 to November 2022. A development cohort (DC) and validation cohort (VC) were established. Using histological results as reference standard, the viscosity-related parameter with the highest area under the receiver operating curve (AUC) was selected as the optimal one. Then the original BI-RADS would upgrade or not based on the value of this parameter. Finally, the results were validated in the VC and total cohorts. In the DC, VC and total cohorts, all breast lesions were divided into the large lesion, small lesion and overall groups respectively. RESULTS: A total of 639 participants (mean age, 46 years ± 14) with 639 breast lesions (372 benign and 267 malignant lesions) were finally enrolled in this study including 392 participants in the DC and 247 in the VC. In the DC, the optimal viscosity-related parameter in differentiating breast lesions was calculated to be A'-S2-Vmax, with the AUC of 0.88 (95% CI: 0.84, 0.91). Using > 9.97 Pa.s as the cutoff value, the BI-RADS was then modified. The AUC of modified BI-RADS significantly increased from 0.85 (95% CI: 0.81, 0.88) to 0.91 (95% CI: 0.87, 0.93), 0.85 (95% CI: 0.80, 0.89) to 0.90 (95% CI: 0.85, 0.93) and 0.85 (95% CI: 0.82, 0.87) to 0.90 (95% CI: 0.88, 0.92) in the DC, VC and total cohorts respectively (P < .05 for all). CONCLUSION: The quantitative viscous parameters evaluated by US viscosity imaging contribute to breast cancer diagnosis when combined with BI-RADS.

Novel Peptide Derived from Gadus morhua Stimulates Osteoblastic Differentiation and Mineralization through Wnt/ß-Catenin and BMP Signaling Pathways.

Marine biodiversity offers a wide array of active ingredient resources. Gadus morhua peptides (GMPs) showed excellent osteoprotective effects in ovariectomized mice. However, the potential osteogenesis mechanisms of key osteogenic peptides in GMP were seldom reported. In this study, a novel osteogenic peptide (GETNPADSKPGSIR, P-GM-2) was screened from GMP. P-GM-2 has a high stability coefficient and a strong interaction with epidermal growth factor receptor. Cell culture experiments showed that P-GM-2 stimulated the expression of osteogenic differentiation markers to promote osteoblast proliferation, differentiation, and mineralization. Additionally, P-GM-2 phosphorylates GSK-3ß, leading to the stabilization of ß-catenin and its translocation to the nucleus, thus initiating the activation of the Wnt/ß-catenin signaling pathway. Meanwhile, P-GM-2 could also regulate the osteogenic differentiation of preosteoblasts by triggering the BMP/Smad and mitogen-activated protein kinase signaling pathways. Further validation with specific inhibitors (ICG001 and Noggin) demonstrated that the osteogenic activity of P-GM-2 was revealed by the activation of the BMP and Wnt/ß-catenin pathways. In summary, these results provide theoretical and practical insights into P-GM-2 as an effective antiosteoporosis active ingredient.

Molecularly imprinted ratiometric fluorescence sensor for visual detection of 17ß-estradiol in milk: A generalized strategy toward imprinted ratiometric fluorescence construction.

17ß-Estradiol (E2) is the typical endocrine disruptor of steroidal estrogens and is widely used in animal husbandry and dairy processing. In the environment, even lower concentrations of E2 can cause endocrine dysfunction in organisms. Herein, we have developed a novel molecularly imprinted ratiometric fluorescent sensor based on SiO2-coated CdTe quantum dots (CdTe@SiO2) and 7-hydroxycoumarin with a post-imprint mixing strategy. The sensor selectively detected E2 in aqueous environments due to its two fluorescent signals with a self-correction function. The sensor has been successfully used for spiking a wide range of real water and milk samples. The results showed that the sensor exhibited good linearity over the concentration range 0.011-50 µg/L, obtaining satisfactory recoveries of 92.4-110.6% with precisions (RSD) < 2.5%. Moreover, this sensor obtained an ultra-low detection limit of 3.3 ng/L and a higher imprinting factor of 13.66. By using estriol (E3), as a supporting model, it was confirmed that a simple and economical ratiometric fluorescent construction strategy was provided for other hydrophobic substances.

Nutrition and Cardiovascular Disease: The Potential Role of Marine Bioactive Proteins and Peptides in Thrombosis Prevention.

Thrombus and cardiovascular diseases pose a significant health threat, and dietary interventions have shown promising potential in reducing the incidence of these diseases. Marine bioactive proteins and peptides have been extensively studied for their antithrombotic properties. They can inhibit platelet activation and aggregation by binding to key receptors on the platelet surface. Additionally, they can competitively anchor to critical enzyme sites, leading to the inhibition of coagulation factors. Marine microorganisms also offer alternative sources for the development of novel fibrinolytic proteins, which can help dissolve blood clots. The advancements in technologies, such as targeted hydrolysis, specific purification, and encapsulation, have provided a solid foundation for the industrialization of bioactive peptides. These techniques enable precise control over the production and delivery of bioactive peptides, enhancing their efficacy and safety. However, it is important to note that further research and clinical studies are needed to fully understand the mechanisms of action and therapeutic potential of marine bioactive proteins and peptides in mitigating thrombotic events. The challenges and future application perspectives of these bioactive peptides also need to be explored.

Absorption and transport properties of a codfish-derived peptide and its protective effect on bone loss in ovariectomized mice.

A potential osteogenic tetradecapeptide with the amino acid sequence GETNPADSKPGSIR (P-GM-2) was identified from Gadus morhua. The present study aimed to elucidate its absorption and transport properties using Caco-2/HT29-MTX co-culture monolayers and to evaluate its osteogenic activity using an ovariectomized mouse model. The results showed that P-GM-2 could cross Caco-2/HT29-MTX co-culture barriers intactly with an apparent permeability coefficient of 4.02 × 10-6 cm s-1via the TJ-mediated passive paracellular pathway. Pharmacokinetic results revealed that P-GM-2 was detectable in the blood of mice within 5 min of oral administration and reached its maximum concentration at 30 min. Furthermore, the oral administration of P-GM-2 for a duration of three months has been found to effectively regulate the secretion of key markers of bone turnover, thereby protecting against bone microstructure degeneration and bone loss in ovariectomized mice. Importantly, no toxicity related to the treatment was observed. Taken together, these findings offer valuable insights into the absorption and transport mechanisms of P-GM-2, highlighting its potential as a safe and effective active ingredient for preventing osteoporosis.

Oxidation-resistant nanoliposomes loaded with osteogenic peptides: Characteristics, stability and bioaccessibility.

Phosphatidylcholine (PC) oxidation leads to the fusion of nanoliposomes and leakage of containment compounds during the storage period. This study aims to improve the oxidation resistance by partially substituting PC in the osteogenic peptides (OPs) loaded nanoliposomes with hydrogenated phosphatidylcholine (HPC). The investigation assessed the characteristics, stability, and bioaccessibility of these novel nanoliposomes. By altering the PC/HPC mass ratio from 1:0 to 0:1, an increase in the encapsulation efficiency (EE), loading capacity (LC), polydispersity index (PDI), and bioaccessibility of OPs-loaded nanoliposomes was observed. Additionally, there was a decrease in thiobarbituric acid reactive substances (TBARS), peroxide value (POV), non-volatile aldehyde, and ketone. The stability of salt decreased when using HPC alone (0:1). The performance of OPs-loaded nanoliposomes with a PC/HPC mass ratio of 1:3 was found to be satisfactory in terms of storage and pH stability. Fluorescence spectroscopy, Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared spectroscopy (FTIR) revealed a tighter lipid aggregation, enhanced intermolecular van der Waals forces, and hydrogen bond formation in membranes of nanoliposomes containing HPC. The addition of HPC to the nanoliposomes delayed the release of peptides in simulated without affecting osteogenic activity. These results provide guidance for the development of oxidation-resistant nanoliposomes loaded with OPs products.

Absorption characteristics and the effect on vascular endothelial cell permeability of an anticoagulant peptide.

In the former report, the casein peptide TKLTEEEKNR (PfCN) exhibits strong thrombin inhibitory activity in vitro. Its absorption capabilities, however, are unclear. Therefore, we studied its absorption characteristics both in vivo and in vitro. PfCN was carried by cells from the apical chamber to the basolateral chamber via active translocation in Caco-2 cells. Meanwhile, it can also be transported by HUVECs. We found that PfCN can be taken up by HUVECs using confocal laser imaging. PfCN has been proven to have good absorption properties in in vivo experiments. After five minutes of oral treatment, PfCN was identified in the blood, peaking at 82.75 ± 36.52 ng/mL in 30 min. And PfCN vanished from the blood circulation after 120 min. According to in vivo experiments, excessive concentrations of PfCN will alter the permeability of HUVECs. As a result, there is a foundation for PfCN application in the food sector. Meanwhile, we also hope this article can give an idea to the researchers who studying the absorption of functional peptides.

Identification and characterisation of taste-enhancing peptides from oysters (Crassostrea gigas) via the Maillard reaction.

Oysters, which are flavourful edible marine products, have been utilised to produce Maillard reaction products (MRPs), which contribute to saltiness enhancement. Here, the molecular weight distribution, free amino acids, and taste characteristics of MRPs were analysed, while ultraviolet light was used to observe the Maillard reaction. Both thermal degradation and cross-linking reactions occur during the Maillard reaction. When the Maillard reaction time was 90 min, the saltiness, umami, and richness of the MRPs peaked, however bitterness reached its lowest value. Moreover, at an MRP concentration of 1.5 mg/mL, salts were reduced by 35.71% in a 3 mg/mL sodium chloride solution without reducing saltiness, based on sensory evaluation. Glycation sites of the MRPs, which are crucial for saltiness enhancement and derived from a variety of protein sources, were determined using nano-HPLC-MS/MS analysis. Our study establishes the foundation for preparing salt-enhancing peptides, accelerating the popularisation of oyster-derived flavouring agents.

Myosin heavy chain-derived peptide of Gadus morhua promotes proliferation and differentiation in osteoblasts and bone formation and maintains bone homeostasis in ovariectomized mice.

Gadus morhua is an important commercial fish rich in nutrients required for daily metabolism. However, the regulation of G. morhua peptides (GMP) on osteoblast growth remains unclear. In order to clarify the regulatory effects of GMP on osteoblasts, the effects of GMP on the growth of MC3T3-E1 cells were investigated, and the osteogenic peptides were identified and screened. The results showed that GMP promoted the proliferation and differentiation of osteoblasts by regulating the BMP/WNT signaling pathway at concentrations of 1-100 µg mL-1. Molecular docking studies showed that a decapeptide, MNKKREAEFQ (P-GM-1), had a high affinity for integrins 3VI4 and 1L5G (-CDOCKER interaction energy: 161.30, 212.27 kcal mol-1). Additionally, the proliferation rate of MC3T3-E1 cells was increased by 27%, and ALP activity was significantly increased under P-GM-1 treatment (100 µg mL-1). Moreover, P-GM-1 promotes bone formation, maintains bone homeostasis, and prevents osteoporosis in ovariectomized mice by regulating the BMP/Smad signaling pathway. This study confirmed the potential of GMP in the regulation of bone mineral density and provided a certain theoretical basis for the development of anti-osteoporosis active factors from GMP.

Crassostrea gigas-Based Bioactive Peptide Protected Thrombin-Treated Endothelial Cells against Thrombosis and Cell Barrier Dysfunction.

The activation of thrombin-treated endothelial cells resulted in disruption of the vascular tissues. A novel oyster-derived bioactive dodecapeptide (IEELEELEAER, P-2-CG) was reported to protect the human umbilical vein endothelial cells and their barrier function via the decrease of VE-cadherin disruption and the restoration of the F-actin arrangement. The promotion of the extrinsic pathway in this case triggers the release of tissue factors that occurs on the surface of the endothelial cells, thus changing the antithrombotic to prothrombotic. P-2-CG induced accordingly a prolongation of plasma clotting time and thrombin generation time, following the alteration of the antithrombotic phenotype. Furthermore, the antithrombotic activity of P-2-CG was also supported by the reduction of FXa and the inhibition of other factors release, for instance, inflammation factors, ROS, etc. In addition to its antithrombogenic role, P-2-CG displayed anti-inflammatory and antioxidant properties via the mitogen-activated protein kinase cascades and central signaling pathways as shown in an in vitro model of endothelial dysfunction.

Tyrosinase inhibitory effects of the peptides from fish scale with the metal copper ions chelating ability.

Tyrosinase inhibitors have important applications in the cosmetics, medical and food industries due to they can effectively inhibit the synthesis of melanin. In this study, tilapia scale polypeptides were used as raw materials, and high-purity polypeptides with metal copper ions chelating ability were obtained by enzymatic hydrolysis, column chromatography, and EDTA elution. In vitro cell model analysis showed that the fish squamous peptides could strongly inhibit the activity of tyrosinase. When the sample concentration was 5 mg·mL-1, its inhibition rate of tyrosinase reached to 59.73%, which had a better inhibition of enzyme activity compared with the positive control of the same concentration. The comprehensive results showed that the fish scale polypeptide with metal copper ions chelating ability could be a strong tyrosinase inhibitor, and might be used to prevent food browning in food-related fields, and could also be used for skin whitening in the fields of medicine and cosmetics.

Inhibitory effects of Atlantic cod (Gadus morhua) peptides on RANKL-induced osteoclastogenesis in vitro and osteoporosis in ovariectomized mice.

Atlantic cod (Gadus morhua) is one of the most important fishes in the world with high nutritional value and economic value. However, the impact and underlying mechanism of the G. morhua peptides (GMPs) on osteoclastogenesis and bone mineral density (BMD) regulation remain unclear. The purpose of this study was to investigate the effects of GMPs on osteoclast formation and anti-osteoporosis activity in vitro and in vivo. The results showed that GMPs significantly reduced receptor activator of nuclear factor (RANKL) induced tartrate-resistant acid phosphatase (TRAP) activity, and decreased the expression of osteoclast regulatory factors c-Fos and NFATc1 by inhibiting the activation of MAPK and NF-κB pathways, and thereby inhibiting osteoclast formation and bone resorption. In vivo, GMP protects mice against ovariectomy-induced bone loss by regulating the balance of major factors released in bone formation and resorption. Taken together, GMP could be a potential candidate or dietary supplement for the prevention of osteoporosis.

A novel anticoagulant peptide discovered from Crassostrea gigas by combining bioinformatics with the enzymolysis strategy: inhibitory kinetics and mechanisms.

A novel anticoagulant peptide (IEELEEELEAER) derived from oyster (Crassostrea gigas) was discovered by combining the emerging bioinformatics with the classical enzymolysis approach. The anticoagulant peptide drastically reduced the extrinsic clotting activity (49% residual PT activity) and impaired the intrinsic clotting activity (77% residual PT activity). Consistent with the clotting data, the thrombin peak height reduced to 88.7 from 123.4 nM, and the thrombin generation time delayed to 5.32 from 4.42 min when an extrinsic trigger was applied. The inhibitory kinetics of FXIa, FIXa, FXa, FIIa, and APC in a purified component system rationally explained the reduction of the extrinsic clotting activity and impairment of thrombin generation. Besides the inhibition of FXa and FIIa activity, the activation processes of FX and FII by an intrinsic/extrinsic tenase complex and prothrombinase were also damaged. The anticoagulant activity in the plasma system was the result of comprehensive inhibition of various factors. The research provided a frame for anticoagulant evaluation and inhibitory mechanism of bioactive peptides from food products.

Identification and analysis of transepithelial transport properties of casein peptides with anticoagulant and ACE inhibitory activities.

Casein is an excellent source for producing anticoagulant and angiotensin I-converting enzyme inhibitory (ACEI) peptides. Here, the anticoagulant and ACEI activities of the casein hydrolysate produced by in vitro simulated gastrointestinal (GI) digestion were evaluated. The casein hydrolysate showed potent anticoagulant activity by prolonging the thrombin time (TT) and activated partial thromboplastin time (APTT), and also presenting great ACEI activity, with an IC50 value of 0.52 mg mL-1. Subsequently, the transepithelial transport properties of the casein hydrolysate were analyzed by using the Caco-2 cell monolayer model. The peptides profile of the casein hydrolysate before and after it passed across the Caco-2 cell monolayer were identified by NanoLC-Q-TOF-MS/MS. The results showed that a total of 121 and 184 peptides were identified before and after casein hydrolysate moved through the Caco-2 cell monolayer, respectively. Eighty peptides were presented at both time points of the transport study. Among the 80 peptides, 26 of them were screened with a high possibility of exerting physiological roles after they were absorbed into the blood by in silico methods, and the physicochemical characteristics, e.g., hydrophobicity, net charge, and toxicity of the peptides also be evaluated. Our results provided a new prospect and approach for producing bioactive peptides from casein with anticoagulant and ACEI activities.

Pharmacokinetics and Transport of an Osteogenic Dodecapeptide.

A dodecapeptide with the amino acid sequence of IEELEEELEAER (PIE), identified from Mytilus edulis proteolysis hydrolysates, has shown good bone-forming activity in previous studies. The pharmacokinetics and transport of the PIE peptide in vivo or in vitro were investigated in this study. The results showed that the PIE peptide can be transported into monolayer Caco-2 cells, and the PIE peptide was identified in the serum after the mice reached the highest value of 173.60 ± 60.30 ng/mL, in which it was quantified by an optimized mass spectrometry method. In addition, the PIE peptide has a promoting effect on the bone morphogenetic protein pathway at the gene and protein levels. According to the distribution of PIE-FITC in ovariectomized mice after orally administrated PIE-FITC, it was confirmed that it can enter the gastrointestinal tract and serum, and reach the bones. Taken together, the PIE peptide can be absorbed well both in vitro and in vivo, and it could promote pre-osteoblast differentiation factors.

A bovine lactoferrin-derived peptide induced osteogenesis via regulation of osteoblast proliferation and differentiation.

Osteoporosis is a disease of aging, characterized by a decrease in bone quality and a reduction in bone strength. Promoting the activity of osteoblasts is a useful strategy for combating the progression of osteoporosis. As a novel bone growth factor, lactoferrin plays a role in the anabolic activity in bone by inducing the proliferation and differentiation of osteoblasts and inhibiting the formation of osteoclasts. However, potential peptides with osteogenic activity from lactoferrin have not been identified. In the present study, a peptide with osteogenic activity-LFP-C, fragment residues 624 to 632, derived from lactoferrin hydrolysates-was identified using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and screened using molecular docking analysis. The LFP-C peptide significantly increased the proliferation of mouse cell line MC3T3-E1 and had a promoting effect on alkaline phosphatase activity and calcium deposition. Moreover, LFP-C increased the proportion of osteoblasts in the G2 and M phases. The osteogenic mechanism of LFP-C was also studied by molecular docking. We found that LFP-C could bind to the key domain (Lys13-Thr15-Gln16-Leu17-Gly18-Asp22) of epidermal growth factor receptor, a vital receptor tyrosine kinase that leads to the activation of the mitogen-activated protein kinase pathway. The main interaction forces were interpolated charge, hydrophobicity, and hydrogen bonding. Results indicated that LFP-C may play an osteogenic role in a similar way to lactoferrin, by promoting the proliferation and differentiation of osteoblasts. The findings of this in vitro experiment also demonstrated that the molecular docking method could play a role in the screening process; this in silico approach allowed for faster and cheaper identification of a promising bioactive component.

Novel Anticoagulant Peptide from Lactoferrin Binding Thrombin at the Active Site and Exosite-I.

Thrombin is currently one of the important targets for the treatment and prevention of thrombosis. At present, there are few reports on the application of lactoferrin peptides in anticoagulation. In this study, a peptide with the amino acid sequence of LRPVAAEIY (LF-LR) derived from lactoferrin was shown to possess antithrombotic activity. LF-LR (5 mM) significantly prolonged activated partial thromboplastin time, prothrombin time, and thrombin time for 13.4, 1.7, and 5.1 s, respectively. It prolonged the coagulation time of fibrinogen from 15.3 ± 0.4 to 20.2 ± 0.5 s by affecting the conformation of thrombin. Using circular dichroism analysis, LF-LR can increase the α-helix content of thrombin from 25.6 to 56.7% and made the ß-sheet disappear. In addition, LF-LR also quenched fluorescence of thrombin at about 346 nm (λEx = 280 nm). By means of molecular docking, it was found that LF-LR could bind to both the active site and the exosite-I of thrombin, and the combined LYS60F, TRP60D, ASP189, LYS36, and ARG77A are typical amino acids in the two domains, respectively.

Anticoagulant Decapeptide Interacts with Thrombin at the Active Site and Exosite-I.

Thrombin can be used as a target for its inhibitors to prevent blood coagulation. A novel peptide (TKLTEEEKNR, PfCN) identified from αS2-casein (fragments 211-220) with high anticoagulant activity was screened and prepared. The activated partial thromboplastin time, prothrombin time, and thrombin time, at the concentration of 4 mM, prolonged about 19, 2.5 and 5.5 s, respectively. At the same concentration, the fibrinogen clotting time prolonged from 25.5 ± 0.7 to 38.3 ± 1.3 s. The thrombin inhibitory efficiency in vitro (IC50 value of 29.27 mM) and antithrombosis effect in vivo were determined. The secondary structure of thrombin, which was influenced by PfCN, indicates that PfCN can bind to thrombin. Isothermal titration calorimetry and the chromogenic substrate test showed that PfCN belongs to the bivalent thrombin inhibitor like bivalirudin. Although the effect was not as good as bivalirudin, in the animal experiment, bleeding occurred in the bivalirudin group but not in the PfCN group. Moreover, molecular docking illustrates the mechanism for the antithrombin activity of PfCN. These results indicated that PfCN could be used as an effective thrombin inhibitor with broad potential for the prevention of thrombotic acute pulmonary embolism and other thrombotic events.

Bone formation activity of an osteogenic dodecapeptide from blue mussels (Mytilus edulis).

A novel osteogenic dodecapeptide peptide (PIE), IEELEEELEAER, was purified from the protein hydrolysate of blue mussels (Mytilus edulis). PIE was identified using a capillary electrophoresis electrospray ionization-quadrupole-time of flight mass spectrometer. PIE showed a good reduction in the bone loss in ovariectomized mice, and it also increased the bone mineral density of the ovariectomized mice. PIE has a high affinity with integrins (PDB: , ). There are 8 and 12 amino acid residues from PIE that interact with integrins and , respectively. PIE accelerates the transformation of G0/G1 phase cells into G2 M phase cells, which promotes the growth of osteoblasts. PIE (100 µg mL-1) can enhance alkaline phosphatase (ALP) activity by 26.48% compared with the control, and it also inhibits the growth of osteoclasts and tartrate resistant acid phosphatase (TRAP) activity. Therefore, PIE may contribute to preventing osteoporosis both in vitro and in vivo.