Protocol for embedded 3D printing of heart tissues using thiol-norbornene collagen.

Here, we present a protocol for 3D printing heart tissues using thiol-norbornene photoclick collagen (NorCol). We describe steps for synthesizing NorCol, preparing bioink and the support bath, and cell-laden printing. We then detail procedures for the loading of C2C12 cells into NorCol, ensuring structural integrity and cell viability after printing. This protocol is adaptable to various cell lines and allows for the printing of diverse complex structures, which can be used in drug screening and disease modeling.

Quantifying the effect of non-ionic surfactant alkylphenol ethoxylates on the persistence of thiabendazole on fresh produce surface.

BACKGROUND: Understanding the role of adjuvants in pesticide persistence is crucial to develop effective pesticide formulations and manage pesticide residues in fresh produce. This study investigated the impact of a commercial non-ionic surfactant product containing alkylphenol ethoxylates (APEOs) on the persistence of thiabendazole on apple and spinach surfaces against the 30 kg m-3 baking soda (sodium bicarbonate, NaHCO3 ) soaking, which was used to remove the active ingredient (AI) in the cuticular wax layer of fresh produce through alkaline hydrolysis. Surface-enhanced Raman scattering (SERS) mapping method was used to quantify the residue levels on fresh produce surfaces at different experimental scenarios. Four standard curves were established to quantify surface thiabendazole in the absence and presence of APEOs, on apple and spinach leaf surfaces, respectively. RESULTS: Overall, the result showed that APEOs enhanced the persistence of thiabendazole over time. After 3 days of exposure, APEOs increased thiabendazole surface residue against NaHCO3 hydrolysis on apple and spinach surfaces by 5.39% and 10.47%, respectively. CONCLUSION: The study suggests that APEOs led to more pesticide residues on fresh produce and greater difficulty in washing them off from the surfaces using baking soda, posing food safety concerns. © 2023 Society of Chemical Industry.

Accurate age-grading of field-aged mosquitoes reared under ambient conditions using surface-enhanced Raman spectroscopy and artificial neural networks.

Age-grading mosquitoes are significant because only older mosquitoes are competent to transmit pathogens to humans. However, we lack effective tools to do so, especially at the critical point where mosquitoes become a risk to humans. In this study, we demonstrated the capability of using surface-enhanced Raman spectroscopy and artificial neural networks to accurately age-grade field-aged low-generation (F2) female Aedes aegypti mosquitoes held under ambient conditions (error was 1.9 chronological days, in the range 0-22 days). When degree days were used for model calibration, the accuracy was further improved to 20.8 degree days (approximately equal to 1.4 chronological days), which indicates the impact of temperature fluctuation on prediction accuracy. This performance is a significant advancement over binary classification. The great accuracy of this method outperforms traditional age-grading methods and will facilitate effective epidemiological studies, risk assessment, vector intervention monitoring, and evaluation.

Understanding the impact of a non-ionic surfactant alkylphenol ethoxylate on surface-enhanced Raman spectroscopic analysis of pesticides on apple surfaces.

Pesticide active ingredients (AIs) are often applied with adjuvants to facilitate the stability and functionality of AIs in agricultural practice. The objective of this study is to investigate the role of a common non-ionic surfactant, alkylphenol ethoxylate (APEO), on the surface-enhanced Raman spectroscopic (SERS) analysis of pesticides as well as its impact on pesticide persistence on apple surfaces, as a model fresh produce surface. The wetted areas of two AIs (thiabendazole and phosmet) mixed with APEO were determined respectively to correct the unit concentration applied on apple surfaces for a fair comparison. SERS with gold nanoparticle (AuNP) mirror substrates was applied to measure the signal intensity of AIs with and without APEO on apple surfaces after a short-term (45 min) and a long-term (5 days) exposure. The limit of detection (LOD) of thiabendazole and phosmet using this SERS-based method were 0.861 ppm and 2.883 ppm, respectively. The result showed that APEO decreased the SERS signal for non-systemic phosmet, while increased SERS intensity of systemic thiabendazole on apple surfaces after 45 min pesticide exposure. After 5 days, the SERS intensity of thiabendazole with APEO was higher than thiabendazole alone, and there was no significant difference between phosmet with and without APEO. Possible mechanisms were discussed. Furthermore, a 1% sodium bicarbonate (NaHCO3) washing method was applied to test the impact of APEO on the persistence of the residues on apple surfaces after short-term and long-term exposures. The results indicated that APEO significantly enhanced the persistence of thiabendazole on plant surfaces after a 5-day exposure, while there was no significant impact on phosmet. The information obtained facilitates a better understanding of the impact of the non-ionic surfactant on SERS analysis of pesticide behavior on and in plants and helps further develop the SERS method for studying complex pesticide formulations in plant systems.

Microcarriers promote the through interface movement of mouse trophoblast stem cells by regulating stiffness.

Mechanical force is crucial in the whole process of embryonic development. However, the role of trophoblast mechanics during embryo implantation has rarely been studied. In this study, we constructed a model to explore the effect of stiffness changes in mouse trophoblast stem cells (mTSCs) on implantation: microcarrier was prepared by sodium alginate using a droplet microfluidics system, and mTSCs were attached to the microcarrier surface with laminin modifications, called T(micro). Compared with the spheroid, formed by the self-assembly of mTSCs (T(sph)), we could regulate the stiffness of the microcarrier, making the Young's modulus of mTSCs (367.70 ± 79.81 Pa) similar to that of the blastocyst trophoblast ectoderm (432.49 ± 151.90 Pa). Moreover, T(micro) contributes to improve the adhesion rate, expansion area and invasion depth of mTSCs. Further, T(micro) was highly expressed in tissue migration-related genes due to the activation of the Rho-associated coiled-coil containing protein kinase (ROCK) pathway at relatively similar modulus of trophoblast. Overall, our study explores the embryo implantation process with a new perspective, and provides theoretical support for understanding the effect of mechanics on embryo implantation.

A Uterus-Inspired Niche Drives Blastocyst Development to the Early Organogenesis.

The fundamental physical features such as the mechanical properties and microstructures of the uterus need to be considered when building in vitro culture platforms to mimic the uterus for embryo implantation and further development but have long been neglected. Here, a uterus-inspired niche (UN) constructed by grafting collagen gels onto polydimethylsiloxane based on a systematic investigation of a series of parameters (varying concentrations and thicknesses of collagen gel) is established to intrinsically specify and simulate the mechanics and microstructures of the mouse uterus. This brand-new and unique system is robust in supporting embryo invasion, as evidenced by the special interaction between the embryos and the UN system and successfully promoting E3.5 embryo development into the early organogenesis stage. This platform serves as a powerful tool for developmental biology and tissue engineering.

3D printed controllable microporous scaffolds support embryonic development in vitro.

Little is known about the complex molecular and cellular events occurring during implantation, which represents a critical step for pregnancy. The conventional 2D culture could not support postimplantation embryos' normal development, and 3D conditions shed light into the "black box". 3D printing technology has been widely used in recapitulating the structure and function of native tissues in vitro. Here, we 3D printed anisotropic microporous scaffolds to culture embryos by manipulating the advancing angle between printed layers, which affected embryo development. The 30° and 60° scaffolds promote embryo development with moderate embryo-scaffold attachments. T-positive cells and FOXA2-positive cells were observed to appear in the posterior region of the embryo and migrated to the anterior region of the embryo on day 7. These findings demonstrate a 3D printed stand that supports embryonic development in vitro and the critical role of 3D architecture for embryo implantation, in which additive manufacturing is a versatile tool.

Gut Microbiota-Derived Resveratrol Metabolites, Dihydroresveratrol and Lunularin, Significantly Contribute to the Biological Activities of Resveratrol.

Although resveratrol (RES) is barely detectable in the plasma and tissues upon oral consumption, collective evidence reveals that RES presents various bioactivities in vivo, including anti-inflammation and anti-cancer. This paradox necessitates further research on profiling and characterizing the biotransformation of RES, as its metabolites may contribute profound biological effects. After 4-week oral administration, 11 metabolites of RES were identified and quantified in mice by HPLC-MS/MS, including dihydro-resveratrol (DHR), lunularin (LUN), and conjugates (sulfates and glucuronides) of RES, DHR and LUN. Importantly, DHR, LUN, and their conjugates were much more abundantly distributed in tissues, gastrointestinal tract (GIT), and biological fluids compared to RES and its conjugates. Moreover, we established that DHR and LUN were gut bacteria-derived metabolites of RES, as indicated by their depletion in antibiotic-treated mice. Furthermore, the biological activities of RES, DHR, and LUN were determined at physiologically relevant levels. DHR and LUN exhibited stronger anti-inflammatory and anti-cancer effects than RES at the concentrations found in mouse tissues. In summary, our study profiled the tissue distribution of the metabolites of RES after its oral administration in mice and uncovered the important role of gut microbial metabolites of RES in the biological activities of RES in vivo.

Microcarriers in application for cartilage tissue engineering: Recent progress and challenges.

Successful regeneration of cartilage tissue at a clinical scale has been a tremendous challenge in the past decades. Microcarriers (MCs), usually used for cell and drug delivery, have been studied broadly across a wide range of medical fields, especially the cartilage tissue engineering (TE). Notably, microcarrier systems provide an attractive method for regulating cell phenotype and microtissue maturations, they also serve as powerful injectable carriers and are combined with new technologies for cartilage regeneration. In this review, we introduced the typical methods to fabricate various types of microcarriers and discussed the appropriate materials for microcarriers. Furthermore, we highlighted recent progress of applications and general design principle for microcarriers. Finally, we summarized the current challenges and promising prospects of microcarrier-based systems for medical applications. Overall, this review provides comprehensive and systematic guidelines for the rational design and applications of microcarriers in cartilage TE.

Gastrointestinal biotransformation and tissue distribution of pterostilbene after long-term dietary administration in mice.

The metabolic fate of dietary compounds is closely related to their biological functions. Pterostilbene (PT) is a methylated stilbene found in many plant foods. Herein, we investigated gastrointestinal biotransformation and tissue distribution of PT in mice fed with 0.05% PT (w/w) for 5 weeks. PT and its major metabolites i.e. PT sulfate (PT-S), pinostilbene, pinostilbene sulfate, hydroxylated PT and hydroxylated PT sulfate were identified and quantified in the mucosa and content of the digestive tissues, blood, urine and vital organs. The results showed PT underwent demethylation, hydroxylation and conjugation in the small intestine, while the conjugated metabolites were largely deconjugated in the colon. Anaerobic fermentation with mouse cecal bacteria demonstrated the microbiota mediated deconjugation and demethylation of PT-S and PT, respectively. In conclusion, oral consumption of PT led to extensive biotransformation in mouse gastrointestinal tract and the metabolites of PT might play important roles in the bioactivity of PT.

Protective effects of non-extractable phenolics from strawberry against inflammation and colon cancer in vitro.

Strawberry is a rich source of phenolics. However, most studies focused on extractable phenolics (EP) while neglecting non-extractable phenolics (NEP). The aim of this study was to characterize EP and NEP from strawberry (Fragaria × ananassa) and determine their anti-inflammatory and anti-colon cancer potentials in cell culture models. NEP contained flavonols, flavanols and phenolic acids that were released through alkaline hydrolysis. NEP dose-dependently inhibited lipopolysaccharides -induced NO production in RAW 264.7 macrophage. Western blotting showed that NEP reduced the expression levels of pro-inflammatory proteins such as iNOS and c-FOS, but increased the expression level of antioxidative protein, such as HO-1. Moreover, NEP markedly suppressed proliferation of human colon cancer HCT116 cells via inducing G2/M phase cell cycle arrest and apoptosis. Collectively, these findings illustrated preventive effects of strawberry NEP against inflammation and colon cancer, shedding light on potential contribution of NEP from strawberry as a health-promoting agent.

Selective Targeting of Class I Histone Deacetylases in a Model of Human Osteosarcoma.

Dysregulation of histone deacetylases (HDACs) is associated with the pathogenesis of human osteosarcoma, which may present an epigenetic vulnerability as well as a therapeutic target. Domatinostat (4SC-202) is a next-generation class I HDAC inhibitor that is currently being used in clinical research for certain cancers, but its impact on human osteosarcoma has yet to be explored. In this study, we report that 4SC-202 inhibits osteosarcoma cell growth in vitro and in vivo. By analyzing cell function in vitro, we show that the anti-tumor effect of 4SC-202 involves the combined induction of cell-cycle arrest at the G2/M phase and apoptotic program, as well as a reduction in cell invasion and migration capabilities. We also found that 4SC-202 has little capacity to promote osteogenic differentiation. Remarkably, 4SC-202 revised the global transcriptome and induced distinct signatures of gene expression in vitro. Moreover, 4SC-202 decreased tumor growth of established human tumor xenografts in immunodeficient mice in vivo. We further reveal key targets regulated by 4SC-202 that contribute to tumor cell growth and survival, and canonical signaling pathways associated with progression and metastasis of osteosarcoma. Our study suggests that 4SC-202 may be exploited as a valuable drug to promote more effective treatment of patients with osteosarcoma and provide molecular insights into the mechanism of action of class I HDAC inhibitors.

In-vivo biotransformation of citrus functional components and their effects on health.

Citrus, one of the most popular fruits worldwide, contains various functional components, including flavonoids, dietary fibers (DFs), essential oils (EOs), synephrines, limonoids, and carotenoids. The functional components of citrus attract special attention due to their health-promoting effects. Food components undergo complex biotransformation by host itself and the gut microbiota after oral intake, which alters their bioaccessibility, bioavailability, and bioactivity in the host body. To better understand the health effects of citrus fruits, it is important to understand the in-vivo biotransformation of citrus functional components. We reviewed the biotransformation of citrus functional components (flavonoids, DFs, EOs, synephrines, limonoids, and carotenoids) in the body from their intake to excretion. In addition, we described the importance of biotransformation in terms of health effects. This review would facilitate mechanistic understanding of the health-promoting effect of citrus and its functional components, and also provide guidance for the development of health-promoting foods based on citrus and its functional components.

Research Progress on the Anti-Cancer Molecular Mechanisms of Huaier.

Huaier (Trametes robiniophila Murr), a Chinese traditional herb of medicine, has demonstrated promising curative effects in clinical treatment for various tumors. There are documented experiments showing the biological functions of Huaier with its antineoplastic molecular mechanisms: restraining proliferation and metastasis, arresting cell cycle, inducing apoptosis, pyrosis, and autophagy, anti-intratumoral angiogenesis, attenuating characteristics of tumor stem-like cells, interfering with the function of the tumor-related immune system, reversing drug resistance, and enhancing the sensitivity to chemotherapeutic drugs, etc. In addition, studies suggest that non-coding RNA (ncRNA) acts a pivotal part in cancer occurrence and development, and demonstrates that Huaier adjusts the performance of certain lncRNA (long non-coding RNA) and proceeds to affect the microRNA and its target genes, rendering an anti-tumor effect. Huaier also modulates the expression of lncRNA to attenuate the activity of ncRNA-sponged microRNA and then inhibits the expression of downstream target genes. We summarize and illustrate the experimentally confirmed anti-cancer molecular mechanisms of Huaier, to inspire new ideas for researchers in relevant fields.

Identification of Xanthomicrol as a Major Metabolite of 5-Demethyltangeretin in Mouse Gastrointestinal Tract and Its Inhibitory Effects on Colon Cancer Cells.

5-Demethyltangeretin (5DT) is a unique polymethoxyflavone mainly found in the peel of citrus, and has shown potent suppressive effects on multiple human cancer cells. Biotransformation plays a critical role in the biological activities of dietary bioactive components because their metabolites may exert significant bioactivities. In the present study, the metabolic fate of 5DT in mouse gastrointestinal (GI) tract after long-term oral intake and the anti-cancer effects of its major metabolite were determined. It was found that 5DT underwent extensive biotransformation after oral ingestion in mice. A major demethylated metabolite was produced via phase I metabolism, while conjugates (glucuronide and sulfate) were generated via phase II metabolism. Specifically, 4'-position on the B ring of 5DT was the major site for demethylation reaction, which led to the production of xanthomicrol (XAN) as a major metabolite. More importantly, the level of XAN in the colon was significantly higher than that of 5DT in 5DT-fed mice. Thus, we further determined the suppressive effects of XAN on human colon cancer HCT116 cells. We found that XAN effectively inhibited the proliferation of HCT116 cells by arresting cell cycle and inducing cellular apoptosis, which was further evidenced by upregulated p53 and p21 and downregulated cyclin D and CDK4/6 level. In conclusion, this study identified XAN as a major metabolite of 5DT in mouse GI tract, and demonstrated its suppressive effects on HCT116 colon cancer cells.

Effect of alkaline extraction pH on structure properties, solubility, and beany flavor of yellow pea protein isolate.

In the current study, the impact of alkaline extraction pH (8.5, 9.0, and 9.5) on chemical composition, molecular structure, solubility and aromatic profile of PPI was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the quantification of free sulfhydryl group and disulfide bond contents, size exclusion chromatography with multi-angle static light scattering and refractive index (SEC-MALS-RI), circular dichroism (CD) spectroscopy, and headspace solid phase micro extraction gas chromatography-mass spectroscopy (HS-SPME-GC-MS). We found that protein recovery yield increased from 49.20% to 57.56% as the alkaline extraction pH increased from 8.5 to 9.5. However, increasing the extraction pH promoted the formation of protein aggregates which decreased the percent protein solubility although there was no influence on protein secondary structure. PPI extracted at pH 9.0 possessed the lowest beany flavor as revealed by the selected six beany flavor markers including alcohols, aldehydes, ketones and pyrazine. The lowest lipoxygenase activity at pH 9.0 may contribute to the least beany flavor in PPI. Therefore, pH 9.0 was found to be the optimal condition for preparing premium PPI in terms of yield, functionality, and aromatic profile using alkaline extraction-isoelectric precipitation process. The findings could have fundamental implications for the preparation and utilization of pea proteins in food applications.

A Facile Method to Fabricate Anisotropic Extracellular Matrix with 3D Printing Topological Microfibers.

Natural tissues and organs have different requirements regarding the mechanical characteristics of response. It is still a challenge to achieve biomaterials with anisotropic mechanical properties using an extracellular matrix with biological activity. We have improved the ductility and modulus of the gelatin matrix using 3D printed gelatin microfibers with different concentrations and topologies and, at the same, time achieved anisotropic mechanical properties. We successfully printed flat microfibers using partially cross-linked gelatin. We modified the 10% (w/v) gelatin matrix with microfibers consisting of a gelatin concentration of 14% (w/v), increasing the modulus to about three times and the elongation at break by 39% in parallel with the fiber direction. At the same time, it is found that the microfiber topology can effectively change the matrix ductility, and changing the modulus of the gelatin used in the microfiber can effectively change the matrix modulus. These findings provide a simple method for obtaining active biological materials that are closer to a physiological environment.

Non-extractable polyphenols from cranberries: potential anti-inflammation and anti-colon-cancer agents.

Cranberries (Vaccinium macrocarpon) are full of polyphenols, which display various health benefits. Most studies have focused on extractable polyphenols (EPs) rather than non-extractable polyphenols (NEPs) but NEPs may possess important biological functions. The objective of this work was to characterize EP and NEP fractions from whole cranberries and determine their potential as anti-inflammation and anti-colon-cancer agents. Our results showed that of the identified polyphenols, anthocyanins were the major ones in the cranberry EP fraction, while phenolic acids were most abundant in the NEP fraction. The oxygen radical absorbance capacity (ORAC) of the NEPs was significantly higher than that of the EPs. Both the EPs and NEPs showed anti-inflammatory effects in inhibiting LPS-induced production of nitric oxide in macrophages. At the concentrations tested, the NEPs showed significantly higher inhibition of the production of nitric oxide in macrophages than the EPs, which was accompanied by decreased expression of inducible nitric oxide synthase (iNOS) and increased expression of HO-1. EP and NEP samples showed anti-cancer capacities in HCT116 cells. And the NEPs showed stronger inhibitory effects on the viability and colony formation capacity of human colon cancer HCT116 cells than the EPs. In a flow cytometry analysis, the NEPs caused cell cycle arrest at the G0/G1 phase and induced significant cellular apoptosis in colon cancer cells. Overall, our results suggested that both the EP and NEP fractions from cranberries were bioactive, and importantly, the NEP fraction showed promising anti-inflammation and anti-colon-cancer potential.

Identification of a new benzophenone from Psidium guajava L. leaves and its antineoplastic effects on human colon cancer cells.

Psidium guajava L. leaves have a long history of being consumed as herbal teas in many countries. The aim of this study was to identify compounds with anticancer potentials from Psidium guajava L. leaves. Utilizing various extraction and chromatographical techniques, we have isolated one new (2) and two known compounds (1, 3). Structural analyses by the spectroscopic methods of TOF-MS, 1H NMR, 13C NMR, HSQC, and HMBC identified these three compounds as guavinoside E (1), 3,5-dihydroxy-2,4-dimethyl-1-O-(6'-O-galloyl-ß-d-glucopyranosyl)-benzophenone (2), and guavinoside B (3). Cell viability assays showed that compounds 2 and 3 inhibited the growth of HCT116 human colon cancer cells in a dose-dependent manner, where compound 2 was more potent than compound 3. Based on flow cytometry analysis, compound 2 showed stronger activity in inducing cellular apoptosis in cancer cells than compound 3. Furthermore, compounds 2 and 3 modulated expression levels of key proteins involved in cell proliferation and apoptotic signaling. Specifically, compound 2 increased the levels of p53, p-ERK1/2, p-JNK, and cleaved caspases 8 and 9, and compound 3 increased the levels of p53 and cleaved caspase 8. Overall, this study provided identities of three bioactive compounds from P. guajava L. leaves and their anti-cancer effects against human colon cancer cells, which could facilitate the utilization of these compounds and P. guajava L. leaves as potential chemoprevention agents against colon carcinogenesis.

Inhibitory effects of 7,7'-bromo-curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation.

This study aimed to determine the capacity of 7,7'-bromo-curcumin (CUR-Br), a curcumin analogue with higher chemical stability than curcumin (CUR), in the suppression of mouse ear edema. Male CD-1 mice were topically pre-treated with either CUR or CUR-Br for 30 min prior to an application of 12-O-tetradecanoylphorbol-13-acetate. After 6 h, mice were killed, and ear punches were measured for their weight and thickness as a marker of edema and inflammation. CUR-Br demonstrated a higher anti-inflammatory efficacy compared to CUR. CUR and CUR-Br at 1.0 µmol suppressed the TPA-induced increase in the ear weight by 26.0% and 57.2%, and decreased TPA-induced increase in the ear thickness by 22.2% and 84.7%, respectively. The inhibitory effects of Cur-Br were associated with decreased levels of inflammatory cytokines (IL-1ß, IL-2, IL-6, KC/GRO, IL-10, IL-17, and IL-23). In addition, CUR-Br significantly downregulated expression of pro-inflammatory signaling proteins such as p-STAT3, STAT3, PI3K, AKT, p-p65, and COX-2. Overall, our results demonstrated that the curcumin analogue, CUR-Br, showed stronger anti-inflammatory properties than CUR in inhibiting TPA-induced inflammatory response in mouse skin.