Establishment of microneurovascular units in alginate hydrogel microspheres to reveal the anti-hypoxic effect of salidroside.

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Evaluation of aconitine cardiotoxicity with a heart-on-a-particle prepared by a microfluidic device.

A heart-on-a-particle model based on multicompartmental microgel is proposed to simulate the heart microenvironment and study the cardiotoxicity of drugs. The relevant microgel was fabricated by a biocompatible microfluidic-based approach, where heart function-related HL-1 and HUVEC cells were arranged in separate compartments. Finally, the mechanism of aconitine-induced heart toxicity was elucidated using mass spectrometry and molecular biotechnology.

Gravity-Oriented Microfluidic Device for Biocompatible End-to-End Fabrication of Cell-Laden Microgels.

Droplet microfluidics are extensively utilized to generate monodisperse cell-laden microgels in biomedical applications. However, maintaining cell viability is still challenging due to overexposure to harsh conditions in subsequent procedures that recover the microgels from the oil phase. Here, a gravity-oriented microfluidic device for end-to-end fabrication of cell-laden microgels is reported, which integrates dispersion, gelation, and extraction into a continuous workflow. This innovative on-chip extraction, driven by native buoyancy and kinetically facilitated by pseudosurfactant, exhibits 100% retrieval efficiency for microgels with a wide range of sizes and stiffnesses. The viability of encapsulated cells is perfectly maintained at ≈98% with minimal variations within and between batches. The end-to-end fabrication remarkably enhances the biocompatibility and practicality of microfluidics-based cell encapsulation and is promising to be compatible with various applications ranging from single-cell analysis to clinical therapy.

Recent advancements in single-cell metabolic analysis for pharmacological research.

Cellular heterogeneity is crucial for understanding tissue biology and disease pathophysiology. Pharmacological research is being advanced by single-cell metabolic analysis, which offers a technique to identify variations in RNA, proteins, metabolites, and drug molecules in cells. In this review, the recent advancement of single-cell metabolic analysis techniques and their applications in drug metabolism and drug response are summarized. High-precision and controlled single-cell isolation and manipulation are provided by microfluidics-based methods, such as droplet microfluidics, microchamber, open microfluidic probe, and digital microfluidics. They are used in tandem with variety of detection techniques, including optical imaging, Raman spectroscopy, electrochemical detection, RNA sequencing, and mass spectrometry, to evaluate single-cell metabolic changes in response to drug administration. The advantages and disadvantages of different techniques are discussed along with the challenges and future directions for single-cell analysis. These techniques are employed in pharmaceutical analysis for studying drug response and resistance pathway, therapeutic targets discovery, and in vitro disease model evaluation.

Stability of the Arginine-Ornithine-Citrulline Cycle Maintained by Tumor-Stroma Interactions in Cell Coculture Hydrogel Microspheres.

Tumor-stroma interaction is the core process of tumor metastasis. Therefore, building a model of tumor-stromal cell communication is crucial for understanding the tumor metastasis process and curing cancer completely. In this research, a controllable three-dimensional (3D) tumor-stroma coculture microsphere model based on droplet microfluidic technology was developed to culture human lung cancer cells (A549 cell) and fibroblast cells (NIH-3T3 cell) using core-shell hydrogel microspheres to partition different kinds of cells. In our coculture model, tumor cells show a trend of epithelial-mesenchymal transition (EMT): a decrease in the number of surface E-cadherin and an increase in the number of N-cadherin. At the same time, fibroblasts are activated into cancer-associated fibroblasts (CAFs) as the level of interleukin-6 (IL-6) released is increased. In addition, an interesting phenomenon was discovered; in the absence of fibroblasts, the metabolism of the tumor cell culture alone leads to arginine depletion and citrulline accumulation, whereas a coculture can maintain the arginine-ornithine-citrulline cycle to reach equilibrium after 72 h, and the balance increases the stress resistance of tumor cells. This discovery may provide a new direction for understanding tumor resistance.

Effect of Tryptophan Metabolites on Cell Damage Revealed by Bacteria-Cell Interactions in Hydrogel Microspheres.

This work presented an alternative approach for studying bacteria-cell interactions in three-dimensional (3D) hydrogel microspheres formed by the cross-linking reaction of alginate and calcium-ethylenediaminetetraacetic acid (EDTA-Ca) produced in a microfluidic chip. During the co-culture process of hepatocytes (HepG2) and Escherichia coli (E. coli) 25922, we concluded that the content change of tryptophan metabolites detected via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was related to the cell damage level and the change of interleukin (IL-22) detected by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was related to the ways of co-cultivation. Compared to the two-dimensional (2D) adherent cell culture process in a Petri dish (2D), the co-culture process of HepG2 and E. coli 25922 in hydrogel microspheres indicated more information about metabolism such as the appearance of indole-3-propionic acid (IPA) and possibly IL-22. The method provides a new perspective to investigate the bacteria-cell interaction and it could be a promising tool in the study of gut microbiota and human health.

Microfluidic Probe for In-Situ Extraction of Adherent Cancer Cells to Detect Heterogeneity Difference by Electrospray Ionization Mass Spectrometry.

The pathological studies of cancer tissues and cell molecules could provide an early diagnosis for the treatment of cancer. In this work, we have designed a microfluidic surface extractor (MSE). The MSE has been coupled with electrospray mass spectrometry (extraction reagent, methanol; optimum flow rate, 0.5 mL/h) to analyze the phospholipid content of different tumor cells. Three types of cancer cell lines, including A549 cells, HepG2 cells, and U87 cells, were investigated, and the principle component analysis (PCA: linear discriminant analysis (LDA), PC1 97.2%; PC2, 2.8%) was carried out to analyze the difference in the lipid contents. The LDA revealed heterogeneity among the cancer cells. The designed MSE could have potential applications in the clinical analysis of cancer tissues.

Therapeutic inhibition of breast cancer bone metastasis progression and lung colonization: breaking the vicious cycle by targeting α5ß1 integrin.

At diagnosis, 10 % of breast cancer patients already have locally advanced or metastatic disease; moreover, metastasis eventually develops in at least 40 % of early breast cancer patients. Osteolytic bone colonization occurs in 80-85 % of metastatic breast cancer patients and is thought to be an early step in metastatic progression. Thus, breast cancer displays a strong preference for metastasis to bone, and most metastatic breast cancer patients will experience its complications. Our prior research has shown that the α5ß1 integrin fibronectin receptor mediates both metastatic and angiogenic invasion. We invented a targeted peptide inhibitor of activated α5ß1, Ac-PHSCN-NH2 (PHSCN), as a validated lead compound to impede both metastatic invasion and neovascularization. Systemic PHSCN monotherapy prevented disease progression for up to 14 months in Phase I clinical trial. Here, we report that the next-generation construct, Ac-PhScN-NH2 (PhScN), which contains D-isomers of histidine (h) and cysteine (c), is greater than 100,000-fold more potent than PHSCN at blocking basement membrane invasion. Moreover, PhScN is also up to 10,000-fold more potent than PHSCN at inhibiting lung extravasation and colonization in athymic mice for both MDA-MB-231 metastatic and SUM149PT inflammatory breast cancer cells. Furthermore, we show that systemic treatment with 50 mg/kg PhScN monotherapy reduces established intratibial MDA-MB-231 bone colony progression by 80 %. Thus, PhScN is a highly potent, well-tolerated inhibitor of both lung colonization and bone colony progression.

A D-amino acid containing peptide as a potent, noncovalent inhibitor of α5ß1 integrin in human prostate cancer invasion and lung colonization.

Primary tumors often give rise to disseminated tumor cells (DTC's), which acquire full malignancy after invading distant site(s). Thus, DTC's may be a productive target for preventing prostate cancer metastasis progression. Our prior research showed that PHSCN peptide (Ac-PHSCN-NH2) targets activated α5ß1 integrin to prevent invasion and metastasis in preclinical adenocarcinoma models, and disease progression in Phase I clinical trial. Here, we report that D-stereoisomer replacement of histidine and cysteine in PHSCN produces a highly potent derivative, Ac-PhScN-NH2 (PhScN). PhScN was 27,000- to 150,000-fold more potent as an inhibitor of basement membrane invasion by DU 145 and PC-3 prostate cancer cells. A large increase in invasion-inhibitory potency occurred after covalent modification of the sulfhydryl group in PHSCN to prevent disulfide bond formation; while the potency of covalently modified PhScN was not significantly increased. Thus PhScN and PHSCN invasion inhibition occurs by a noncovalent mechanism. These peptides also displayed similar cell surface binding dissociation constants (Kd), and competed for the same site. Consistent with its increased invasion-inhibitory potency, PhScN was also a highly potent inhibitor of lung extravasation and colonization in athymic nude mice: it was several hundred- or several thousand-fold more potent than PHSCN at blocking extravasation by PC-3 or DU 145 cells, and 111,000- or 379,000-fold more potent at inhibiting lung colonization, respectively. Furthermore, systemic 5 mg/kg PhScN monotherapy was sufficient to cause complete regression of established, intramuscular DU 145 tumors. PhScN thus represents a potent new family of therapeutic agents targeting metastasis by DTC's to prevent parallel progression in prostate cancer.

Role of α(5)ß(1) Integrin Up-regulation in Radiation-Induced Invasion by Human Pancreatic Cancer Cells.

RADIOTHERAPY IS USED IN THE MANAGEMENT OF PANCREATIC CANCER BECAUSE OF ITS HIGH PROPENSITY FOR LOCOREGIONAL RELAPSE: one third of patients succumb to localized disease. Thus, strategies to improve the efficacy of radiotherapy in pancreatic cancer are important to pursue. We used naturally serum-free, selectively permeable basement membranes and confocal microscopy of fluorescent antibody-stained human Panc-1, MiaPaCa-2, and BxPC-3 pancreatic cancer cell lines to investigate the effects of ionizing radiation on α(5)ß(1) integrin fibronectin receptor expression and on α(5)ß(1)-mediated invasion. We report that radiation rapidly induces pancreatic cancer cell invasion, and that radiation-induced invasion is caused by up-regulation of α(5)ß(1) integrin fibronectin receptors by transcriptional and/or postendocytic recycling mechanisms. We also report that radiation causes α(5)ß(1) up-regulation in Panc-1, MiaPaCa-2, and BxPC-3 tumor xenografts and that upregulated α(5)ß(1) colocalizes with upregulated early or late endosomes in Panc-1 or BxPC-3 tumors, respectively, although it may colocalize significantly with both endosome types in MiaPaCa-2 tumors. Our results suggest that systemic inhibition of α(5)ß(1)-mediated invasion might be an effective way to reduce radiation-induced pancreatic cancer cell invasion, thereby improving the efficacy of radiotherapy.

The PHSCN dendrimer as a more potent inhibitor of human breast cancer cell invasion, extravasation, and lung colony formation.

The α5ß1 integrin fibronectin receptor is an attractive therapeutic target in breast cancer because it plays key roles in invasion and metastasis. While its inactive form is widely expressed, activated α5ß1 occurs only on tumor cells and their associated vasculature. The PHSCN peptide has been shown to bind activated α5ß1 preferentially, thereby blocking invasion in vitro, and inhibiting growth, metastasis and tumor recurrence in preclinical models. Moreover in a recent Phase I clinical trial, systemic PHSCN monotherapy was well tolerated, and metastatic disease failed to progress for 4-14 months in 38% of patients receiving it. A significantly more potent PHSCN derivative, the PHSCN-polylysine dendrimer (Ac-PHSCNGGK-MAP) has recently been developed. We report that it is 1280- to 6700-fold more potent than the PHSCN peptide at blocking α5ß1 mediated SUM-149 PT and MDA-MB-231 human breast cancer cell invasion of naturally occurring basement membranes in vitro. Chou-Talalay analysis of these data suggested that invasion inhibition by the PHSCN dendrimer was highly synergistic. We also report that, consistent with its enhanced invasion-inhibitory potency, the PHSCN dendrimer is 700- to 1100-fold more effective than the PHSCN peptide at preventing SUM-149 PT and MDA-MB-231 extravasation in the lungs of athymic, nude mice. Our results also show that many extravasated SUM-149 PT and MDA-MB-231 cells go on to develop into metastatic colonies, and that pretreatment with the PHSCN dendrimer is more than 100-fold more effective at reducing lung colony formation. Since many patients newly diagnosed with breast cancer already have locally advanced or metastatic disease, the availability of a well-tolerated, nontoxic systemic therapy that can prevent metastatic progression by blocking invasion could be very beneficial.

Increased potency of the PHSCN dendrimer as an inhibitor of human prostate cancer cell invasion, extravasation, and lung colony formation.

Activated alpha5beta1 integrin occurs specifically on tumor cells and on endothelial cells of tumor-associated vasculature, and plays a key role in invasion and metastasis. The PHSCN peptide (Ac-PHSCN-NH(2)) preferentially binds activated alpha5beta1, to block invasion in vitro, and inhibit growth, metastasis and tumor recurrence in preclinical models of prostate cancer. In Phase I clinical trial, systemic Ac-PHSCN-NH(2) monotherapy was well tolerated, and metastatic disease progression was prevented for 4-14 months in one-third of treated patients. We have developed a significantly more potent derivative, the PHSCN-polylysine dendrimer (Ac-PHSCNGGK-MAP). Using in vitro invasion assays with naturally serum-free basement membranes, we observed that the PHSCN dendrimer was 130- to 1900-fold more potent than the PHSCN peptide at blocking alpha5beta1-mediated invasion by DU 145 and PC-3 human prostate cancer cells, whether invasion was induced by serum, or by the Ac-PHSRN-NH(2) peptide, under serum-free conditions. The PHSCN dendrimer was also approximately 800 times more effective than PHSCN peptide at preventing DU 145 and PC-3 extravasation in the lungs of athymic mice. Chou-Talalay analysis suggested that inhibition of both invasion in vitro and extravasation in vivo by the PHSCN dendrimer are highly synergistic. We found that many extravasated DU 145 and PC-3 cells go onto develop into metastatic colonies, and that a single pretreatment with the PHSCN dendrimer was 100-fold more affective than the PHSCN peptide at reducing lung colony formation. Since many patients newly diagnosed with prostate cancer already have locally advanced or metastatic disease, the availability of a well-tolerated, nontoxic systemic therapy, like the PHSCN dendrimer, which prevents metastatic progression by inhibiting invasion, could be very beneficial.

alpha(5)beta(1) Integrin Ligand PHSRN Induces Invasion and alpha(5) mRNA in Endothelial Cells to Stimulate Angiogenesis.

Angiogenesis requires endothelial cell invasion and is crucial for wound healing and for tumor growth and metastasis. Invasion of native collagen is mediated by the alpha(5)beta(1) integrin fibronectin receptor. Thus, alpha(5)beta(1) up-regulation on the surfaces of endothelial cells may induce endothelial cell invasion to stimulate angiogenesis. We report that the interaction of alpha(5)beta(1) with its PHSRN peptide ligand induces human microvascular endothelial cell invasion and that PHSRN-induced endothelial cell invasion is regulated by alpha(4)beta(1) integrin and requires matrix metalloproteinase 1 (MMP-1). Moreover, our results show that exposure to PHSRN causes rapid, specific up-regulation of surface levels of alpha(5)beta(1) integrin and significantly increases alpha(5) integrin mRNA in microvascular endothelial cells. Consistent with these results, alpha(5) small interfering RNA abrogates PHSRN-induced surface alpha(5) and MMP-1 up-regulation, as well as blocking invasion induction. We also observed dose-dependent, PHSRN-induced alpha(5)beta(1) integrin up-regulation on endothelial cells in vivo in Matrigel plugs. We further report that the PHSCN peptide, an alpha(5)beta(1)-targeted invasion inhibitor, blocks PHSRN-induced invasion, alpha(5)beta(1) up-regulation, alpha(5) mRNA induction, and MMP-1 secretion in microvascular endothelial cells and that systemic PHSCN administration prevents PHSRN-induced alpha(5)beta(1) up-regulation and angiogenesis in Matrigel plugs. These results demonstrate a critical role for alpha(5)beta(1) integrin and MMP-1 in mediating the endothelial cell invasion and angiogenesis and suggest that PHSRN-induced alpha(5) transcription and alpha(5)beta(1) up-regulation may form an important feed-forward mechanism for stimulating angiogenesis.

Spatial organization of neurons in the dorsal motor nucleus of the vagus synapsing with intragastric cholinergic and nitric oxide/VIP neurons in the rat.

The dorsal motor nucleus of the vagus (DMV) contains preganglionic neurons that control gastric motility and secretion. Stimulation of different parts of the DMV results in a decrease or an increase in gastric motor activities, suggesting a spatial organization of vagal preganglionic neurons in the DMV. Little is known about how these preganglionic neurons in the DMV synapse with different groups of intragastric motor neurons to mediate contraction or relaxation of the stomach. We used pharmacological and immunohistochemical methods to characterize intragastric neural pathways involved in mediating gastric contraction and relaxation in rats. Microinjections of L-glutamate (L-Glu) into the rostral or caudal DMV produced gastric contraction and relaxation, respectively, in a dose-related manner. Intravenous infusion of hexamethonium blocked these actions, suggesting mediation via preganglionic cholinergic pathways. Atropine inhibited gastric contraction by 85.5 +/- 4.5%. Gastric relaxation was reduced by intravenous administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 52.5 +/- 11.9%) or VIP antagonist (56.3 +/- 14.9%). Combined administration of L-NAME and VIP antagonist inhibited gastric relaxation evoked by L-Glu (87.8 +/- 4.3%). Immunohistochemical studies demonstrated choline acetyltransferase immunoreactivity in response to L-Glu microinjection into the rostral DMV in 88% of c-Fos-positive intragastric myenteric neurons. Microinjection of L-Glu into the caudal DMV evoked expression of nitric oxide (NO) synthase and VIP immunoreactivity in 81 and 39%, respectively, of all c-Fos-positive intragastric myenteric neurons. These data indicate spatial organization of the DMV. Depending on the location, microinjection of L-Glu into the DMV may stimulate intragastric myenteric cholinergic neurons or NO/VIP neurons to mediate gastric contraction and relaxation.

Computationally analyzing the possible biological function of YJL103C--an ORF potentially involved in the regulation of energy process in yeast.

Although the complete genomes of a number of organisms have been sequenced, the biological functions of many genes are still not known. Because experimentally studying the functions of those genes one by one requires tremendous time, it is vital to use published resources like microarray gene expression data for computational analysis of gene functions. One example is YJL103C, a yeast gene of unknown function in the Saccharomyces Genome Database (SGD). It is possible to quickly infer its biological function by computational analysis. In this study, we present an efficient model to explore the biological function of a novel gene using microarray data. We showed that the expression pattern of YJL103C is most similar to the genes in the energy group and respiratory chain subgroup. We further found that YJL103C contains a HAP2,3,4 box in its promoter region and a cytochrome C heme-binding signature in its protein sequence. Our findings define a potential role for YJL103C in the regulation of energy metabolism, specifically in the process of oxidative phosphorylation. Similar bioinformatics methods can be applied to infer the biological functions of other novel genes in organisms for which microarray data are available. In this work, we selected a single gene of unknown function as a case study. By focusing on the power of computer analysis and bioinformatics on the available microarray data, we have determined the likely biological function of YJL103C. Our study provides a method by which to explore the potential function of other genes currently annotated as having an unknown function in any organism for which global gene expression data are available.

Cholecystokinin synthesizes and secretes leptin in isolated canine gastric chief cells.

It is well recognized that a product of obese (ob) locus and body weight control hormone, leptin, acts on both short-term satiety for meal-induced termination of food intake (gastric phase) and long-term satiety for energy expenditure via the hypothalamus. The considerable sources of leptin are chief cells for gastric phase and adipocytes for the long-term satiety. The objective of this study was to demonstrate if CCK enhances leptin synthesis and secretion in isolated canine gastric chief cells. Confocal immunofluorescence studies showed that the CCK-A receptor and leptin were colocalized in the endoplasm. Western blotting demonstrated that canine chief cells expressed the leptin peptide and its protein level was enhanced by CCK treatment. An ELISA further showed that CCK dose-dependently secreted leptin from isolated canine chief cells. This was reproduced by the high-affinity CCK-A receptor agonist, CCK-OPE. These results indicate that canine chief cells synthesize and secrete leptin in response to CCK via the high-affinity state of the CCK-A receptor.