Modeling angiogenesis in the human brain in a tissue-engineered post-capillary venule.

Angiogenesis plays an essential role in embryonic development, organ remodeling, wound healing, and is also associated with many human diseases. The process of angiogenesis in the brain during development is well characterized in animal models, but little is known about the process in the mature brain. Here, we use a tissue-engineered post-capillary venule (PCV) model incorporating stem cell derived induced brain microvascular endothelial-like cells (iBMECs) and pericyte-like cells (iPCs) to visualize the dynamics of angiogenesis. We compare angiogenesis under two conditions: in response to perfusion of growth factors and in the presence of an external concentration gradient. We show that both iBMECs and iPCs can serve as tip cells leading angiogenic sprouts. More importantly, the growth rate for iPC-led sprouts is about twofold higher than for iBMEC-led sprouts. Under a concentration gradient, angiogenic sprouts show a small directional bias toward the high growth factor concentration. Overall, pericytes exhibited a broad range of behavior, including maintaining quiescence, co-migrating with endothelial cells in sprouts, or leading sprout growth as tip cells.

Engineering the human blood-brain barrier at the capillary scale using a double-templating technique.

In vitro blood-brain barrier (BBB) models have played an important role in studying processes such as immune cell trafficking and drug delivery, as well as contributing to the understanding of mechanisms of disease progression. Many biological and pathological processes in the cerebrovasculature occur in capillaries and hence the lack of robust hierarchical models at the capillary scale is a major roadblock in BBB research. Here we report on a double-templating technique for engineering hierarchical BBB models with physiological barrier function at the capillary scale. We first demonstrate the formation of hierarchical vascular networks using human umbilical vein endothelial cells. We then characterize barrier function in a BBB model using brain microvascular endothelial-like cells (iBMECs) differentiated from induced pluripotent stem cells (iPSCs). Finally, we characterize immune cell adhesion and transmigration in response to perfusion with the inflammatory cytokine tumor necrosis factor-alpha, and show that we can recapitulate capillary-scale effects, such as leukocyte plugging, observed in mouse models. Our double-templated hierarchical model enables the study of a wide range of biological and pathological processes related to the human BBB.

Thermo-Sensitive PLGA-PEG-PLGA Tri-Block Copolymer Hydrogel as Three-Dimensional Cell Culture Matrix for Ovarian Cancer Cells.

Thermo-sensitive hydrogels which could encapsulate cells and provide a three dimensional (3D) microenvironment have great potential in building new cell culture models in vitro. In this study, a thermal responsive hydrogel based on PLGA-PEG-PLGA tri-block copolymers was developed as matrix for 3D ovarian cancer cell culturing. The gelation of PLGA-PEG-PLGA tri-block copolymer was concentration-dependent. SEM images showed the pores were suitable for the formation of 3D cell structures. Cell morphological results showed that large aggregates of ovarian cancer cells (HO8910) were formed after cultured for 10 days. Therefore, hydrogel based on PLGA-PEG-PLGA tri-block copolymers hold potential as in vitro cell culture matrix for ovarian cancer cells.

Effect of Ultrasonic Treatment on the Physicochemical Properties of Bovine Plasma Protein-Carboxymethyl Cellulose Composite Gel.

In order to improve the stability of bovine plasma protein-carboxymethyl cellulose composite gels and to expand the utilization of animal by-product resources, this study investigated the impact of different ultrasound powers (300, 400, 500, 600, and 700 W) and ultrasound times (0, 10, 20, 30, and 40 min) on the functional properties, secondary structure and intermolecular forces of bovine plasma protein-carboxymethyl cellulose composite gel. The results showed that moderate ultrasonication resulted in the enhancement of gel strength, water holding capacity and thermal stability of the composite gels, the disruption of hydrogen bonding and hydrophobic interactions between gel molecules, the alteration and unfolding of the internal structure of the gels, and the stabilization of the dispersion state by electrostatic repulsive forces between the protein particles. The content of α-helices, ß-turns, and ß-sheets increased and the content of random curls decreased after sonication (p < 0.05). In summary, appropriate ultrasound power and time can significantly improve the functional and structural properties of composite gels. It was found that controlling the thermal aggregation behavior of composite gels by adjusting the ultrasonic power and time is an effective strategy to enable the optimization of composite gel texture and water retention properties.

Effects of oxidative stress and protein S-nitrosylation interactions on mitochondrial pathway apoptosis and tenderness of yak meat during postmortem aging.

To reveal the interaction of oxidative stress and protein S-nitrosylation on mitochondrial pathway apoptosis and tenderness development in postmortem yak meat. Herein, we selected yak longissimus dorsi muscle as the research object and treated hydrogen peroxide (H2O2) with S-nitrosoglutathione agent (GSNO) as well as Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) in mixed injections with 0.9 % saline as a control group, followed by incubation at 4 °C for 12, 24, 72, 120 and 168 h. Results showed that this interaction significantly increased mitochondrial ROS and NO content (P < 0.05) while weakening the antioxidant capacity of GSH and TRX redox response systems or accelerating the Ca2+ release process, leading to mitochondrial functional impairment and increased apoptosis rate. Notably, the H2O2 + L-NAME group showed more pronounced apoptosis. Hence, we suggest that the interaction between oxidative stress and protein S-nitrosylation could positively regulate yak meat tenderization.

Discovery of a botanical compound as a broad-spectrum inhibitor against gut microbial ß-glucuronidases from the Tibetan medicine Rhodiola crenulata.

Gut microbial ß-glucuronidases (gmß-GUS) played crucial roles in regulating a variety of endogenous substances and xenobiotics on the circulating level, thus had been recognized as key modulators of drug toxicity and human diseases. Inhibition or inactivation of gmß-GUS enzymes has become a promising therapeutic strategy to alleviate drug-induced intestinal toxicity. Herein, the Rhodiola crenulata extract (RCE) was found with potent and broad-spectrum inhibition on multiple gmß-GUS enzymes. Subsequently, the anti-gmß-GUS activities of the major constituents in RCE were tested and the results showed that 1,2,3,4,6-penta-O-galloyl-ß-d-glucopyranose (PGG) acted as a strong and broad-spectrum inhibitor on multiple gmß-GUS (including EcGUS, CpGUS, SaGUS, and EeGUS). Inhibition kinetic assays demonstrated that PGG effectively inhibited four gmß-GUS in a non-competitive manner, with the Ki values ranging from 0.12 µM to 1.29 µM. Docking simulations showed that PGG could tightly bound to the non-catalytic sites of various gmß-GUS, mainly via hydrogen bonding and aromatic interactions. It was also found that PGG could strongly inhibit the total gmß-GUS activity in mice feces, with the IC50 value of 1.24 µM. Collectively, our findings revealed that RCE and its constituent PGG could strongly inhibit multiple gmß-GUS enzymes, suggesting that RCE and PGG could be used for alleviating gmß-GUS associated enterotoxicity.

Enzyme-Based Biosensors and Their Applications.

Enzymes constitute an extremely important class of biomacromolecules with diverse catalytic functions, which have been validated as key mediators for regulating cellular metabolism and maintaining homeostasis in living organisms [...].

An ex Vivo Model Enables Systematic Investigation of the Intestinal Absorption and Transcytosis of Oral Particulate Nanocarriers.

Nanoparticulate formulations are being developed toward enhancing the bioavailability of orally administrated biologics. However, the processes mediating particulate carriers' intestinal uptake and transport remains to be fully elucidated. Herein, an optical clearing-based whole tissue mount/imaging strategy was developed to enable high quality microscopic imaging of intestinal specimens. It enabled the distribution of nanoparticles within intestinal villi to be quantitatively analyzed at a cellular level. Two-hundred and fifty nm fluorescent polystyrene nanoparticles were modified with polyethylene glycol (PEG), Concanavalin A (ConA), and pectin to yield mucopenetrating, enterocyte targeting, and mucoadhesive model nanocarriers, respectively. Introducing ConA on the PEGylated nanoparticles significantly increased their uptake in the intestinal epithelium (∼4.16 fold for 200 nm nanoparticle and ∼2.88 fold for 50 nm nanoparticles at 2 h). Moreover, enterocyte targeting mediated the trans-epithelial translocation of 50 nm nanoparticles more efficiently than that of the 200 nm nanoparticles. This new approach provides an efficient methodology to obtain detailed insight into the transcytotic activity of enterocytes as well as the barrier function of the constitutive intestinal mucus. It can be applied to guide the rational design of particulate formulations for more efficient oral biologics delivery.

Magnetic microspheres mimicking certain functions of macrophages: Towards precise antibacterial potency for bone defect healing.

A variety of novel biomaterials have recently been developed to promote bone regeneration. However, the current biomaterials cannot accurately and effectively resist bacterial invasion. In this study, we constructed microspheres that mimic certain functions of macrophages as additives to bone repair materials, which can be manipulated as demanded to resist bacteria effectively and protect bone defect healing. Firstly, we prepared gelatin microspheres (GMSs) by an emulsion-crosslinking method, which were subsequently coated with polydopamine (PDA). Then, amino antibacterial nanoparticles obtained by a nanoprecipitation-self-assembly method and commercial amino magnetic nanoparticles were modified onto these PDA-coated GMSs to construct the functionalized microspheres (FMSs). The results showed that the FMSs possessed a rough topography and could be manipulated by a 100-400 â€‹mT static magnetic field to migrate directionally in unsolidified hydrogels. Moreover, in vitro experiments with near-infrared (NIR) showed that the FMSs had a sensitive and recyclable photothermal performance and could capture and kill Porphyromonas gingivalis by releasing reactive oxygen species. Finally, the FMSs were mixed with osteogenic hydrogel precursor, injected into the Sprague-Dawley rat periodontal bone defect of maxillary first molar (M1), and subsequently driven by magnetism to the cervical surface of M1 and the outer surface of the gel system for targeted sterilization under NIR, thus protecting the bone defect healing. In conclusion, the FMSs had excellent manipulation and antimicrobial performances. This provided us with a promising strategy to construct light-magnetism-responsive antibacterial materials to build a beneficial environment for bone defect healing.

LF-NMR determination of water distribution and its relationship with protein- related properties of yak and cattle during postmortem aging.

The water distribution have a profound influence on meat quality, and proteins play a critical role in water distribution. The water distribution detected with proton NMR and its relationship with protein related properties were investigated. Three populations of water were detected: bound water (T21, P21), immobilized water (T22, P22), and free water (T23, P23). The decreased T22 and T23 indicated an increase in water-holding capacity in both muscles from 3 days of aging. The P22 in cattle was higher than that in yak and the P23 in cattle was lower than that in yak, suggesting that cattle exhibited a greater water-holding capacity compared to yak. Moreover, postmortem aging affected muscle protein oxidation, denaturation, and degradation. Correlation analysis suggested that protein oxidation and denaturation caused muscle water loss and protein degradation could allow the muscle to retain water. It provides a basis for the optimization of quality of meat and products.

Preparation of Complementary Food for Infants and Young Children with Beef Liver: Process Optimization and Storage Quality.

In this study, fuzzy mathematics and response surface modeling were applied to optimize the preparation process of beef liver paste and characterize the proximate composition, sensory and physicochemical qualities, and in vitro simulated digestive properties while refrigerated at 0-4 °C (0, 3, 7, 15, 30, 45, and 60 days). The results showed that the optimal preparation process was 4.8% potato starch, 99.4% water, 10.2% olive oil, and a 3:2 ratio of chicken breast and beef liver. The beef liver paste prepared contained essential amino acids for infants and children, with a protein content of 10.29 g/100 g. During storage, the pH of the beef liver paste decreased significantly (p < 0.05) on day 7, texture and rheological properties decreased significantly after 30 days, a* values increased, L* and b* values gradually decreased, and TVB-N and TBARS values increased significantly (p < 0.05) on day 7 but were below the limit values during the storage period (TVB-N value ≤ 15 mg/100 g, TBARS value ≤ 1 mg/Kg). In vitro simulated digestion tests showed better digestibility and digestive characteristics in the first 15 days. The results of this study provide a reference for the development of beef liver products for infant and child supplementation.

Effect of Cooking Method and Doneness Degree on Volatile Compounds and Taste Substance of Pingliang Red Beef.

This study used gas chromatography-ion mobility spectrometry (GC-IMS) and high-performance liquid chromatography (HPLC) methods to examine the impact of cooking methods and doneness on volatile aroma compounds and non-volatile substances (fatty acids, nucleotides, and amino acids) in Pingliang red beef. The flavor substances' topographic fingerprints were established, and 45 compounds were traced to 71 distinct signal peaks. Pingliang red beef's fruity flavor was enhanced thanks to the increased concentration of hexanal, styrene, and 2-butanone that resulted from instant boiling. The levels of 3-methylbutanal, which contributes to the characteristic caramel-chocolate-cheese aroma, peaked at 90 min of boiling and 40 min of roasting. The FFA content was reduced by 28.34% and 27.42%, respectively, after the beef was roasted for 40 min and instantly boiled for 10 s (p > 0.05). The most distinctive feature after 30 min of boiling was the umami, as the highest levels of glutamate (Glu) (p < 0.05) and the highest equivalent umami concentration (EUC) values were obtained through this cooking method. Additionally, adenosine-5'-monophosphate (AMP) and inosine-5'-monophosphate (IMP) decreased with increasing doneness compared to higher doneness, indicating that lower doneness was favorable in enhancing the umami of the beef. In summary, different cooking methods and doneness levels can affect the flavor and taste of Pingliang red beef, but it is not suitable for high-doneness cooking.

Preparation of Bovine Hides Gelatin by Ultra-High Pressure Technique and the Effect of Its Replacement Fat on the Quality and In Vitro Digestion of Beef Patties.

Beef skin gelatin can be used as a good substitute for animal fat in meat patties. In this paper, the effect of different parameters on low-fat beef patties with cowhide gelatin substituted for beef fat (0, 25%, 50%, 75%, 100%) prepared by ultra-high pressure assisted technology was investigated by texture, cooking loss, and sensory scores. The beef patties were also stored at 0-4 °C for 0, 7, 14, 21, and 28 d. The differences and changing rules of fatty acid and amino acid compositions and contents of beef patties with different fat contents were investigated by simulating gastrointestinal digestion in vitro. The optimal process formulation of low-fat beef patties with cowhide gelatin was determined by experimental optimization as follows: ultra-high pressure 360 MPa, ultra-high of pressure time of 21 min, NaCl addition of 1.5%, compound phosphate addition of 0.3%. The addition of cowhide gelatin significantly increased monounsaturated fatty acids, polyunsaturated fatty acids, amino acid content, and protein digestibility of beef patties (p < 0.05). Moreover, with the extension of storage time, the content of saturated fatty acids was significantly higher (p < 0.05), the content of monounsaturated and polyunsaturated fatty acids was significantly lower (p < 0.05), the content of amino acids was significantly lower (p < 0.05), and protein digestibility was significantly lower (p < 0.05) under all substitution ratios. Overall, beef patties with 75% and 100% substitution ratios had better digestibility characteristics. The results of this study provide a theoretical basis for gelatin's potential as a fat substitute for beef patties and for improving the quality of low-fat meat products.

The influence of physiological and pathological perturbations on blood-brain barrier function.

The blood-brain barrier (BBB) is located at the interface between the vascular system and the brain parenchyma, and is responsible for communication with systemic circulation and peripheral tissues. During life, the BBB can be subjected to a wide range of perturbations or stresses that may be endogenous or exogenous, pathological or therapeutic, or intended or unintended. The risk factors for many diseases of the brain are multifactorial and involve perturbations that may occur simultaneously (e.g., two-hit model for Alzheimer's disease) and result in different outcomes. Therefore, it is important to understand the influence of individual perturbations on BBB function in isolation. Here we review the effects of eight perturbations: mechanical forces, temperature, electromagnetic radiation, hypoxia, endogenous factors, exogenous factors, chemical factors, and pathogens. While some perturbations may result in acute or chronic BBB disruption, many are also exploited for diagnostic or therapeutic purposes. The resultant outcome on BBB function depends on the dose (or magnitude) and duration of the perturbation. Homeostasis may be restored by self-repair, for example, via processes such as proliferation of affected cells or angiogenesis to create new vasculature. Transient or sustained BBB dysfunction may result in acute or pathological symptoms, for example, microhemorrhages or hypoperfusion. In more extreme cases, perturbations may lead to cytotoxicity and cell death, for example, through exposure to cytotoxic plaques.

A tissue-engineered model of the blood-tumor barrier during metastatic breast cancer.

Metastatic brain cancer has poor prognosis due to challenges in both detection and treatment. One contributor to poor prognosis is the blood-brain barrier (BBB), which severely limits the transport of therapeutic agents to intracranial tumors. During the development of brain metastases from primary breast cancer, the BBB is modified and is termed the 'blood-tumor barrier' (BTB). A better understanding of the differences between the BBB and BTB across cancer types and stages may assist in identifying new therapeutic targets. Here, we utilize a tissue-engineered microvessel model with induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (iBMECs) and surrounded by human breast metastatic cancer spheroids with brain tropism. We directly compare BBB and BTB in vitro microvessels to unravel both physical and chemical interactions occurring during perivascular cancer growth. We determine the dynamics of vascular co-option by cancer cells, modes of vascular degeneration, and quantify the endothelial barrier to antibody transport. Additionally, using bulk RNA sequencing, ELISA of microvessel perfusates, and related functional assays, we probe early brain endothelial changes in the presence of cancer cells. We find that immune cell adhesion and endothelial turnover are elevated within the metastatic BTB, and that macrophages exert a unique influence on BTB identity. Our model provides a novel three-dimensional system to study mechanisms of cancer-vascular-immune interactions and drug delivery occurring within the BTB.

Effects of Salt Soaking Treatment on the Deodorization of Beef Liver and the Flavor Formation of Beef Liver Steak.

In this study, based on the evaluation of fishy value and sensory evaluation, this study determined that soaking in a 1% salt solution for 60 min had a significant impact on the deodorization of beef liver (p < 0.05). The results showed that salt infiltration promoted the release of fishy substances, improving the edible and processing performance of beef liver. The identification of flavor compounds in raw and roasted beef liver via GC-IMS implies that (E)-2-octenal-M, (E)-3-penten-2-one-M, ethyl acetate-M, ethyl acetate-D, and methanethiol are closely related to improving the flavor of beef liver; among them, (E)-2-octenal-M, (E)-3-penten-2-one-M, and methanethiol can cause beef liver odor, while nonanal-M, octanal-M, benzene acetaldehyde, n-hexanol-D, butyl propanoate-M, heptanal-D, heptanal-M, and 3-methylthiopropanal-M had significant effects on the flavor formation of beef liver steak. The determination of reducing sugars revealed that salt soaking had no significant effect on the reducing sugar content of beef liver, and the beef liver steak was significantly reduced (p < 0.05), proving that reducing sugars promoted the formation of beef liver steak flavor under roasting conditions. Fatty acid determination revealed that salt soaking significantly reduced the content of polyunsaturated fatty acids in beef liver (p < 0.05), promoting the process of fat degradation and volatile flavor production in the beef liver steak. Salt plays a prominent role in salting-out and osmosis during deodorization and flavor improvement. Through controlling important biochemical and enzymatic reactions, the release of flavor substances in a food matrix was increased, and a good deodorization effect was achieved, which lays a foundation for further research on the deodorization of beef liver and the flavor of beef liver steak.

Corrigendum: The influence of physiological and pathological perturbations on blood-brain barrier function.

[This corrects the article DOI: 10.3389/fnins.2023.1289894.].

Discovery of 4'-trifluoromethylchalcones as novel, potent and selective hCYP1B1 inhibitors without concomitant AhR activation.

Human cytochrome P450 1B1 (hCYP1B1), an extrahepatic cytochrome P450 enzyme over-expressed in various tumors, has been validated as a promising target for preventing and treating cancers. Herein, two series of chalcone derivatives were synthesized to discover potent hCYP1B1 inhibitors without AhR agonist effect. Structure-activity relationship (SAR) studies demonstrated that 4'-trifluoromethyl on the B-ring strongly enhanced the anti-hCYP1B1 effects, identifying A9 as a promising lead compound. Further SAR analysis on A9 derivatives (modified A-ring of 4'-trifluoromethylchalcone) showed that introducing 2-methoxyl improved the anti-hCYP1B1 effect and selectivity, while introducing a methoxyl at the C-4 site was beneficial for avoiding AhR activation. Ultimately, five 4'-trifluoromethyl chalcones were identified as potent hCYP1B1 inhibitors (IC50 < 10 nM), while B18 exhibits the most potent anti-hCYP1B1 effect (IC50 = 3.6 nM), suitable metabolic stability and good cell-permeability. B18 also acted as an AhR antagonist and could down-regulate hCYP1B1 in living systems. Mechanistic studies showed that B18 potently inhibited hCYP1B1 in a competitive inhibition manner (Ki = 3.92 nM), while docking simulations revealed that B18 could tightly bind to the catalytic cavity of hCYP1B1 mainly via hydrophobic and hydrogen-bonding interactions. Furthermore, B18 could potently inhibit hCYP1B1 in living cells and showed remarkable anti-migration ability on MFC-7 cells. Taken together, this study deciphered the SARs of chalcones as hCYP1B1 inhibitors and provided several potent hCYP1B1 inhibitors as promising candidates for the development of more efficacious anti-migration agents.

Mechano-biomimetic hydrogel 3D cell cultivation as a strategy to improve mammalian cell protein expression.

Eukaryotic expression systems are frequently employed for the production of recombinant proteins as therapeutics as well as research tools. Among which mammalian cell protein expression approach is the most powerful one, which can express complex proteins or genetic engineered biological drugs, such as PD-1. However, the high expense, which partially derives from its low protein yielding efficiency, limited the further application of such approach in large scale production of target proteins. To address this issue, we proposed a novel technique to promote the protein production efficiency of mammal cells without using conventional genetic engineered approaches. By placing 293T cells in a hydrogel 3D cell culture platform and adjusting the stress relaxation of the matrix hydrogel, cells formed multicellular spheroids by self-organization. In particular, the multicellular spheroids have a significantly enhanced ability to transiently express multiple proteins (SHH-N, PD-1 and PDL-1). We also examined in detail the mechanism underlying this phenomenon, and found that the reorganization of cytoskeleton during spheroids formation enhances the translation process of protein by recruiting ribosomes. Overall, this finding provides a novel approach for subsequent improvement of large-scale mammalian protein expression cell systems.

Brain microvascular endothelial cell dysfunction in an isogenic juvenile iPSC model of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of cytosine-adenine-guanine (CAG) repeats in the huntingtin gene, which leads to neuronal loss and decline in cognitive and motor function. Increasing evidence suggests that blood-brain barrier (BBB) dysfunction may contribute to progression of the disease. Studies in animal models, in vitro models, and post-mortem tissue find that disease progression is associated with increased microvascular density, altered cerebral blood flow, and loss of paracellular and transcellular barrier function. Here, we report on changes in BBB phenotype due to expansion of CAG repeats using an isogenic pair of induced pluripotent stem cells (iPSCs) differentiated into brain microvascular endothelial-like cells (iBMECs). We show that CAG expansion associated with juvenile HD alters the trajectory of iBMEC differentiation, producing cells with ~ two-fold lower percentage of adherent endothelial cells. CAG expansion is associated with diminished transendothelial electrical resistance and reduced tight junction protein expression, but no significant changes in paracellular permeability. While mutant huntingtin protein (mHTT) aggregates were not observed in HD iBMECs, widespread transcriptional dysregulation was observed in iBMECs compared to iPSCs. In addition, CAG expansion in iBMECs results in distinct responses to pathological and therapeutic perturbations including angiogenic factors, oxidative stress, and osmotic stress. In a tissue-engineered BBB model, iBMECs show subtle changes in phenotype, including differences in cell turnover and immune cell adhesion. Our results further support that CAG expansion in BMECs contributes to BBB dysfunction during HD.