Expression of the umami taste receptor T1R1/T1R3 in porcine testis of: Function in regulating testosterone synthesis and autophagy in Leydig cells.

Testosterone is a vital male hormone responsible for male sexual characteristics. The taste receptor family 1 subunit 3 (T1R3) regulates testosterone synthesis and autophagy in non-taste cells, and the links with the taste receptor family 1 subunit 1 (T1R1) for umami perception. However, little is known about these mechanisms. Thus, we aimed to determine the relationship between the umami taste receptor (T1R1/T1R3) and testosterone synthesis or autophagy in testicular Leydig cells of the Xiang pig. There was a certain proportion of spermatogenic tubular dysplasia in the Xiang pig at puberty, in which autophagy was enhanced, and the testosterone level was increased with a weak expression of T1R3. Silenced T1R3 decreased testosterone level and intracellular cyclic adenosine monophosphate (cAMP) content and inhibited the messenger RNA (mRNA) expression levels of testosterone synthesis enzyme genes [steroidogenic acute regulatory protein (StAR), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (3ß-HSD1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and hydroxysteroid 17-beta dehydrogenase 3 (17ß-HSD3)]. In addition, T1R3 increased the number of acidic autophagy bubbles and upregulated the expression levels of autophagy markers [Microtubule-associated protein 1 A/1B-light chain 3 (LC3) and Beclin-1] in testicular Leydig cells of the Xiang pig. Using an umami tasting agonist (10 mM L-glutamate for 6 h), the activation of T1R1/T1R3 enhanced the testosterone synthesis ability by increasing the intracellular cAMP level and upregulated the expression levels of StAR, 3ß-HSD1, CYP17A1 and 17ß-HSD3 in Leydig cells. Furthermore, the number of acidic autophagy bubbles decreased in the T1R1/T1R3-activated group with the downregulation of the expression levels of the autophagy markers, including LC3 and Beclin-1. These data suggest that the function of T1R1/T1R3 expressed in testicular Leydig cells of the Xiang pig is related to testosterone synthesis and autophagy.

Exploring characteristics of placental transcriptome and cord serum metabolome associated with low birth weight in Kele pigs.

The neonate with low birth weight (LBW) resulted from intrauterine growth retardation (IUGR) exists a substantial risk of postpartum death. Placental insufficiency is responsible for inadequate fetal growth; however, the pathological mechanisms of placental dysfunction-induced IUGR in pigs remain unclear. In this study, the characteristics of placental morphology, placental transcriptome, and cord serum metabolome were explored between the Kele piglets with LBW and the ones with normal birth weight (NBW). Results showed that LBW was a common occurrence in Kele piglets. The LBW placentas showed inferior villus development and lower villi density compared to NBW placentas. There were 1024 differentially expressed genes (DEGs) identified by transcriptome analysis between the LBW and NBW placentas, of which 218 and 806 genes were up- and down-regulated in the LBW placentas, respectively. PPI network analysis showed that ITGB2, CD4, IL6, ITGB3, LCK, RAC2, CD8A, JAK3, TYROBP, and CXCR4 were hub genes in all DEGs. From GO and KEGG enrichment analysis, DEGs were primarily enriched in immunological response, cell adhesion, immune response, cytokine-cytokine receptor interaction, and PI3K-Akt signaling pathway. By using metabolomic analysis, a total of 115 differential metabolites in the cord serum of LBW and NBW piglets were found, mostly linked to amino acid metabolism and sphingolipid metabolism. In comparison to NBW piglets, LBW piglets had lower levels of arginine, isoleucine, and aspartic acid in the cord. Taken together, these data revealed dysplasia of the placental villus, insufficient supply of nutrients, and abnormal immune function of the placenta may be associated with the occurrence and development of LBW in Kele pigs.

RACK1 may participate in placental development at mid-gestation via regulating trophoblast cell proliferation and migration in pigs.

Intrauterine growth restriction (IUGR) is a severe complication in swine production. Placental insufficiency is responsible for inadequate fetal growth, but the specific etiology of placental dysfunction-induced IUGR in pigs remains poorly understood. In this work, placenta samples supplying the lightest weight (LW) and mean weight (MW) pig fetuses in the litter at Day 65 (D65) of gestation were collected, and the relationship between fetal growth and placental morphologies and functions was investigated using histomorphological analysis, RNA sequencing, quantitative polymerase chain reaction, and in vitro experiment in LW and MW placentas. Results showed that the folded structure of the epithelial bilayer of LW placentas followed a poor and incomplete development compared with that of MW placentas. A total of 654 differentially expressed genes (DEGs) were screened out between the LW and MW placentas, and the gene encodes receptor for activated C kinase 1 (RACK1) was found to be downregulated in LW placentas. The DEGs were mainly enriched in translation, ribosome, protein synthesis, and mammalian target of rapamycin (mTOR) signaling pathway according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In vitro experiments indicated that the decreased RACK1 in LW placentas may be involved in abnormal development of placental folds (PFs) by inhibiting the proliferation and migration of porcine trophoblast cells. Taken together, these results revealed that RACK1 may be a vital regulator in the development of PFs via regulating trophoblast cell proliferation and migration in pigs.

Acoustofluidic assembly of primary tumor-derived organotypic cell clusters for rapid evaluation of cancer immunotherapy.

Cancer immunotherapy shows promising potential for treating breast cancer. While patients may have heterogeneous treatment responses for adjuvant therapy, it is challenging to predict an individual patient's response to cancer immunotherapy. Here, we report primary tumor-derived organotypic cell clusters (POCCs) for rapid and reliable evaluation of cancer immunotherapy. By using a label-free, contactless, and highly biocompatible acoustofluidic method, hundreds of cell clusters could be assembled from patient primary breast tumor dissociation within 2 min. Through the incorporation of time-lapse living cell imaging, the POCCs could faithfully recapitulate the cancer-immune interaction dynamics as well as their response to checkpoint inhibitors. Superior to current tumor organoids that usually take more than two weeks to develop, the POCCs can be established and used for evaluation of cancer immunotherapy within 12 h. The POCCs can preserve the cell components from the primary tumor due to the short culture time. Moreover, the POCCs can be assembled with uniform fabricate size and cell composition and served as an open platform for manipulating cell composition and ratio under controlled treatment conditions with a short turnaround time. Thus, we provide a new method to identify potentially immunogenic breast tumors and test immunotherapy, promoting personalized cancer therapy.

Microfluidics guided by deep learning for cancer immunotherapy screening.

Immunocyte infiltration and cytotoxicity play critical roles in both inflammation and immunotherapy. However, current cancer immunotherapy screening methods overlook the capacity of the T cells to penetrate the tumor stroma, thereby significantly limiting the development of effective treatments for solid tumors. Here, we present an automated high-throughput microfluidic platform for simultaneous tracking of the dynamics of T cell infiltration and cytotoxicity within the 3D tumor cultures with a tunable stromal makeup. By recourse to a clinical tumor-infiltrating lymphocyte (TIL) score analyzer, which is based on a clinical data-driven deep learning method, our platform can evaluate the efficacy of each treatment based on the scoring of T cell infiltration patterns. By screening a drug library using this technology, we identified an epigenetic drug (lysine-specific histone demethylase 1 inhibitor, LSD1i) that effectively promoted T cell tumor infiltration and enhanced treatment efficacy in combination with an immune checkpoint inhibitor (anti-PD1) in vivo. We demonstrated an automated system and strategy for screening immunocyte-solid tumor interactions, enabling the discovery of immuno- and combination therapies.

Understanding Immune-Driven Brain Aging by Human Brain Organoid Microphysiological Analysis Platform.

The aging of the immune system drives systemic aging and the pathogenesis of age-related diseases. However, a significant knowledge gap remains in understanding immune-driven aging, especially in brain aging, due to the limited current in vitro models of neuroimmune interaction. Here, the authors report the development of a human brain organoid microphysiological analysis platform (MAP) to discover the dynamic process of immune-driven brain aging. The organoid MAP is created by 3D printing that confines organoid growth and facilitates cell and nutrition perfusion, promoting organoid maturation and their committment to forebrain identity. Dynamic rocking flow is incorporated into the platform that allows to perfuse primary monocytes from young (20 to 30-year-old) and aged (>60-year-old) donors and culture human cortical organoids to model neuroimmune interaction. The authors find that the aged monocytes increase infiltration and promote the expression of aging-related markers (e.g., higher expression of p16) within the human cortical organoids, indicating that aged monocytes may drive brain aging. The authors believe that the organoid MAP may provide promising solutions for basic research and translational applications in aging, neural immunological diseases, autoimmune disorders, and cancer.

Evaluation of cancer immunotherapy using mini-tumor chips.

Rationale: Predicting tumor responses to adjuvant therapies can potentially help guide treatment decisions and improve patient survival. Currently, tumor pathology, histology, and molecular profiles are being integrated into personalized profiles to guide therapeutic decisions. However, it remains a grand challenge to evaluate tumor responses to immunotherapy for personalized medicine. Methods: We present a microfluidics-based mini-tumor chip approach to predict tumor responses to cancer immunotherapy in a preclinical model. By uniformly infusing dissociated tumor cells into isolated microfluidic well-arrays, 960 mini-tumors could be uniformly generated on-chip, with each well representing the ex vivo tumor niche that preserves the original tumor cell composition and dynamic cell-cell interactions and autocrine/paracrine cytokines. Results: By incorporating time-lapse live-cell imaging, our mini-tumor chip allows the investigation of dynamic immune-tumor interactions as well as their responses to cancer immunotherapy (e.g., anti-PD1 treatment) in parallel within 36 hours. Additionally, by establishing orthotopic breast tumor models with constitutive differential PD-L1 expression levels, we showed that the on-chip interrogation of the primary tumor's responses to anti-PD1 as early as 10 days post tumor inoculation could predict the in vivo tumors' responses to anti-PD1 at the endpoint of day 24. We also demonstrated the application of this mini-tumor chip to interrogate on-chip responses of primary tumor cells isolated from primary human breast and renal tumor tissues. Conclusions: Our approach provides a simple, quick-turnaround solution to measure tumor responses to cancer immunotherapy.

Rapid Profiling of Tumor-Immune Interaction Using Acoustically Assembled Patient-Derived Cell Clusters.

Tumor microenvironment crosstalk, in particular interactions between cancer cells, T cells, and myeloid-derived suppressor cells (MDSCs), mediates tumor initiation, progression, and response to treatment. However, current patient-derived models such as tumor organoids and 2D cultures lack some essential niche cell types (e.g., MDSCs) and fail to model complex tumor-immune interactions. Here, the authors present the novel acoustically assembled patient-derived cell clusters (APCCs) that can preserve original tumor/immune cell compositions, model their interactions in 3D microenvironments, and test the treatment responses of primary tumors in a rapid, scalable, and user-friendly manner. By incorporating a large array of 3D acoustic trappings within the extracellular matrix, hundreds of APCCs can be assembled within a petri dish within 2 min. Moreover, the APCCs can preserve sensitive and short-lived (≈1 to 2-day lifespan in vivo) tumor-induced MDSCs and model their dynamic suppression of T cell tumor toxicity for up to 24 h. Finally, using the APCCs, the authors succesully model the combinational therapeutic effect of a multi-kinase inhibitor targeting MDSCs (cabozantinib) and an anti-PD-1 immune checkpoint inhibitor (pembrolizumab). The novel APCCs may hold promising potential in predicting treatment response for personalized cancer adjuvant therapy as well as screening novel cancer immunotherapy and combinational therapy.

Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats.

Cathepsin D (CTSD), the major lysosomal aspartic protease that is widely expressed in different tissues, potentially regulates the biological behaviors of various cells. Follicular granulosa cells are responsive to the increase of ovulation number, hence indirectly influencing litter size. However, the mechanism underlying the effect of CTSD on the behaviors of goat granulosa cells has not been fully elucidated. This study used immunohistochemistry to analyze CTSD localization in goat ovarian tissues. Moreover, western blotting was applied to examine the differential expression of CTSD in the ovarian tissues of monotocous and polytocous goats. Subsequently, the effects of CTSD knockdown on cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific traits, including bone morphogenetic protein receptor IB (BMPR-IB), follicle-stimulating hormone (FSHR), and inhibin α (INHA), were determined in granulosa cells. Results showed that CTSD was expressed in corpus luteum, follicle, and granulosa cells. Notably, CTSD expression in the monotocous group was significantly higher than that in the polytocous group. In addition, CTSD knockdown could improve granulosa cell proliferation, inhibit cell apoptosis, and significantly elevate the expression of proliferating cell nuclear antigen (PCNA) and B cell lymphoma 2 (Bcl-2), but it lowered the expression of Bcl-2-associated X (Bax) and caspase-3. Furthermore, CTSD knockdown significantly reduced the ratios of cells in the G0/G1 and G2/M phases but substantially increased the ratio of cells in the S phase. The expression levels of cyclin D2 and cyclin E were elevated followed by the obvious decline of cyclin A1 expression. However, the expression levels of BMPR-IB, FSHR, and INHA clearly increased as a result of CTSD knockdown. Hence, our findings demonstrate that CTSD is an important factor affecting the litter size trait in goats by regulating the granulosa cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific trait.

An organoid-based screen for epigenetic inhibitors that stimulate antigen presentation and potentiate T-cell-mediated cytotoxicity.

In breast cancer, genetic heterogeneity, the lack of actionable targets and immune evasion all contribute to the limited clinical response rates to immune checkpoint blockade therapy. Here, we report a high-throughput screen based on the functional interaction of mouse- or patient-derived breast tumour organoids and tumour-specific cytotoxic T cells for the identification of epigenetic inhibitors that promote antigen presentation and potentiate T-cell-mediated cytotoxicity. We show that the epigenetic inhibitors GSK-LSD1, CUDC-101 and BML-210, identified by the screen, display antitumour activities in orthotopic mammary tumours in mice, that they upregulate antigen presentation mediated by the major histocompatibility complex class I on breast tumour cells and that treatment with BML-210 substantially sensitized breast tumours to the inhibitor of the checkpoint programmed death-1. Standardized measurements of tumour-cell killing activity facilitated by tumour-organoid-T-cell screens may help with the identification of candidate immunotherapeutics for a range of cancers.

Metabolomic analysis of exosomal-markers in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a worldwide malignancy with high mortality rates and poor prognosis due to the lack of effective biomarkers for early detection. Exosomes have been extensively explored as attractive biomarkers for cancer diagnosis and treatment. However, little is known about exosome metabolomics and their roles in ESCC. Here, we performed a targeted metabolomic analysis of plasma exosomes and identified 196 metabolites, mainly including lipid fatty acids, benzene, amino acids, organic acids, carbohydrates and fatty acyls. We systematically compared metabolome patterns of exosomes via machine learning from patients with recrudescence and patients without recrudescence and demonstrated a marker set consisting of 3'-UMP, palmitoleic acid, palmitaldehyde, and isobutyl decanoate for predicting ESCC recurrence with an AUC of 98%. These metabolome signatures of exosomes retained a high absolute fold change value at all ESCC stages and were very likely associated with cancer metabolism, which could be potentially applied as novel biomarkers for diagnosis and prognosis of ESCC.

Effects of N-Acetylcysteine on the reproductive performance, oxidative stress and RNA sequencing of Nubian goats.

N-acetylcysteine (NAC) has been found to enhance the protective ability of cells to counter balance oxidative stress and inflammation. To investigate the effects of dietary NAC supplementation on the reproductive performance of goats, the reproductive performance and endometrial transcriptome of goats fed with diets with NAC (NAC group) and without NAC supplementation (control group) were compared. Results showed that the goats fed with 0.03% and 0.05% NAC had similar litter size, birth weight, nitric oxide (NO), sex hormones and amino acids levels compared with the goats of the control group. However, feeding with 0.07% NAC supplementation from day 0 to day 30 of gestation remarkably increased the litter size of goats. The goats of the 0.07% NAC group presented increased levels of NO relative to the control group, but their sex hormones and amino acids showed no differences. Comparative transcriptome analysis identified 207 differentially expressed genes (DEGs) in the endometrium between the control and the 0.07% NAC groups. These DEGs included 146 upregulated genes and 61 downregulated genes in the 0.07% NAC group. They were primarily involved in the cellular response to toxic substances, oxidoreductase activity, immune receptor activity, signalling receptor binding, cytokine-cytokine receptor interactions, PI3K-Akt signalling pathway and PPAR signalling pathway. In conclusion, results showed that dietary 0.07% NAC supplementation exerted a beneficial effect on the survival of goat embryos at the early pregnancy stage. Such positive outcome might be due to the increased NO production and affected expression of genes involved in the anti-inflammation pathways of the endometrium.

miR-31-5p Regulates 14-3-3 ɛ to Inhibit Prostate Cancer 22RV1 Cell Survival and Proliferation via PI3K/AKT/Bcl-2 Signaling Pathway.

INTRODUCTION: Prostate cancer (PCa) is one of the most common malignancies, and almost all patients with advanced PCa will develop castration-resistant prostate cancer (CRPC) after receiving endocrine therapy. Effective treatment for patients with CRPC has not been established. Novel approaches are needed to identify therapeutic targets for CRPC. PURPOSE: Recent research studies have found that members of the 14-3-3 family play an important role in the development and progression of PCa. Previous results have shown that 14-3-3 ɛ is significantly upregulated in several cancers. This study aimed to identify novel miRNAs that regulate 14-3-3 ɛ expression and therapeutic targets for CRPC. METHODS: In this study, we used computation and experimental approaches for the prediction and verification of the miRNAs targeting 14-3-3 ɛ, and investigated the potential roles of 14-3-3 ɛ in the survival and proliferation of 22RV1 cells. RESULTS: We confirm that mir-31-5p is downregulated in 22RV1 cells and acts as a tumor suppressor by regulating 14-3-3 ɛ. Ectopic expression of miR-31-5p or 14-3-3 ɛ interference significantly inhibits cell proliferation, invasion, and migration in 22RV1 cells, as well as promotes cell apoptosis via the PI3K/AKT/Bcl-2 signaling pathway. Moreover, 14-3-3 ɛ is required for the miR-31-5p-mediated upregulation of the PI3K/AKT/Bcl-2 signaling pathway. CONCLUSION: Our findings provide information on the underlying mechanisms of miR-31-5p/14-3-3 ɛ in 22RV1 cell proliferation and apoptosis through the PI3K/AKT/Bcl-2 signaling pathway. These results suggest that miR-31-5p and 14-3-3 ɛ may potentially be utilized as novel prognostic markers and therapeutic targets for PCa treatment.

Trapping cell spheroids and organoids using digital acoustofluidics.

The precise positioning and arrangement of cell spheroids and organoids are critical to reconstructing complex tissue architecture for tissue engineering and regenerative medicine. Here, we present a digital acoustofluidic method to manipulate cell spheroids and organoids with unprecedented dexterity. By introducing localized vibrations via a C-shaped integrated digital transducer (IDT), we can generate a trapping node to immobilize cell spheroids with a diameters ranging from 20µm to 300µm. Moreover, we digitally trapped multiple cell spheroids atop the C-shaped IDTs within a closed or open microfluidic chamber. By programming the trapping nodes within a 3 × 3 C-shaped IDT array, we can precisely position cell spheroids into designed patterns. We also demonstrated that our digital acoustofluidic device can accurately control the interaction of spheroid cells and organoids. Along with a simple fabrication procedure and setup, our digital acoustofluidic method can provide precisely manipulate and position various cell spheroids or organoids in a contactless, label-free, and highly biocompatible manner. We believe this technology can be widely used for tissue engineering, regenerative medicine, and fundamental cell biology research.

One-Stop Microfluidic Assembly of Human Brain Organoids To Model Prenatal Cannabis Exposure.

Prenatal cannabis exposure (PCE) influences human brain development, but it is challenging to model PCE using animals and current cell culture techniques. Here, we developed a one-stop microfluidic platform to assemble and culture human cerebral organoids from human embryonic stem cells (hESC) to investigate the effect of PCE on early human brain development. By incorporating perfusable culture chambers, air-liquid interface, and one-stop protocol, this microfluidic platform can simplify the fabrication procedure and produce a large number of organoids (169 organoids per 3.5 cm × 3.5 cm device area) without fusion, as compared with conventional fabrication methods. These one-stop microfluidic assembled cerebral organoids not only recapitulate early human brain structure, biology, and electrophysiology but also have minimal size variation and hypoxia. Under on-chip exposure to the psychoactive cannabinoid, Δ-9-tetrahydrocannabinol (THC), cerebral organoids exhibited reduced neuronal maturation, downregulation of cannabinoid receptor type 1 (CB1) receptors, and impaired neurite outgrowth. Moreover, transient on-chip THC treatment also decreased spontaneous firing in these organoids. This one-stop microfluidic technique enables a simple, scalable, and repeatable organoid culture method that can be used not only for human brain organoids but also for many other human organoids including liver, kidney, retina, and tumor organoids. This technology could be widely used in modeling brain and other organ development, developmental disorders, developmental pharmacology and toxicology, and drug screening.

Rapid Microfluidic Formation of Uniform Patient-Derived Breast Tumor Spheroids.

Breast cancer is a highly complex, heterogeneous, and multifactorial disease that poses challenges for rapid and efficient treatment and development of personalized therapy. Here, we describe a rapid and reliable method to generate three-dimensional (3D) tumor spheroids in vitro that recapitulate an individual patient's tumor for testing treatments. By employing droplet microfluidics and scaffold materials, tumor cells were encapsulated into a large number of Matrigel-in-oil droplets with precise control over cell numbers and components per droplet. After removal of the oil, large numbers of uniform tumor spheroids were formed within a few hours via Matrigel-supported cell self-assembly. Our microfluidic technique produces uniform-sized tumor spheroids in less than 1 day. This method was used to reproducibly and rapidly generate uniform-sized tumor spheroids derived from patients' breast tumor tissues. As a proof-of-concept application, this method was used to quickly evaluate cancer treatments. We demonstrated that our microfluidic patient-derived tumor cultures not only preserve the genetic characteristics of the original tumor tissue but also provide heterogeneous responses to targeted therapies within 2 days. We believe this method will enable a timely and reliable 3D in vitro culture model, which may be applicable to personalized treatment prediction, drug discovery, and toxicity testing.

Profiling Cell-Matrix Adhesion Using Digitalized Acoustic Streaming.

Profiling the kinetics of cell-matrix adhesion is of great importance to understand many physiological and pathological processes such as morphogenesis, tissue homeostasis, wound healing, and tumorigenesis. Here, we developed a novel digital acoustofluidic device for parallel profiling cell-matrix adhesion at single-cell level. By introduction of localized and uniform acoustic streaming into an open chamber microfluidic device, the adherent cells within the open chamber can be detached by the streaming-induced Stokes drag force. By digital regulation of pulsed acoustic power from a low level to high levels, the hundreds of adherent cells can be ruptured from the fibronectin-coated substrate accordingly, and their adhesive forces (from several pN to several nN) and kinetics can be determined by the applied power and cell incubation time. As a proof-of-concept application for studying cancer metastasis, we applied this technique to measure the adhesion strength and kinetics of human breast cancer cells to extracellular matrix such as fibronectin and compared their metastatic potentials by measuring the rupture force of cancer cells representing malignant (MCF-7 cells and MDA-MB-231 cells) and nonmalignant (MCF-10A cells) states. Our acoustofluidic device is simple, easy to operate, and capable of measuring, in parallel, hundreds of individual cells' adhesion forces with a resolution at the pN level. Thus, we expect this device could be widely used for both fundamental cell biology research as well as development of cancer diagnostics and tissue engineering technologies.

Quantitative ultra-high-performance liquid chromatography-tandem mass spectrometry for determination of dexmedetomidine in pediatric plasma samples: Correlation with genetic polymorphisms.

Dexmedetomidine is an important sedative agent administered as premedication to achieve procedural sedation in children. To describe the correlation between the genetic state and the concentration of dexmedetomidine, it is necessary to develop a specific, time-saving and economical method for detection of dexmedetomidine in plasma samples. In this work, an ultra-high-performance liquid chromatography (UHPLC)-tandem mass spectrometry method has been established and validated for detection of dexmedetomidine in plasma from pediatric population. After a simple liquid-liquid extraction with an organic solution, the analytes were separated on an ACQUITY BEH C18 column (2.1 mm × 50 mm, 1.7 µm particle size) by gradient elution with the mobile phase of acetonitrile and 1‰ aqueous formic acid (flow rate 0.3 mL min-1 ). Mass spectrometry measurements were performed under the positive selected reaction monitoring and the mass transitions monitored were m/z 201.3 → 95.1, 204.2 → 98.0 for dexmedetomidine and deuterated medetomidine (internal standard), respectively. Validation of the method based on China Food and Drug Administration guidelines showed acceptable selectivity. The UHPLC method employed a stable isotope-labeled internal standard, showed good specificity and was successfully used to detect dexmedetomidine in plasma samples from 260 pediatric patients. A subsequent application of this method to a pharmacogenetic study was also described. Importantly, this is the first study to report the correlation between CYP2A6 rs835309 activity and concentration of dexmedetomidine.

High-throughput acoustofluidic fabrication of tumor spheroids.

Three-dimensional (3D) culture of multicellular spheroids, offering a desirable biomimetic microenvironment, is appropriate for recapitulating tissue cellular adhesive complexity and revealing a more realistic drug response. However, current 3D culture methods are suffering from low-throughput, poor controllability, intensive-labor, and variation in spheroid size, thus not ready for many high-throughput screening applications including drug discovery and toxicity testing. Herein, we developed a high-throughput multicellular spheroid fabrication method using acoustofluidics. By acoustically-assembling cancer cells with low-cost and disposable devices, our method can produce more than 12 000 multicellular aggregates within several minutes and allow us to transfer these aggregates into ultra-low attachment dishes for long-term culture. This method can generate more than 6000 tumor spheroids per operation, and reduce tumor spheroid formation time to one day. Our platform has advantages in forming spheroids with high throughput, short time, and long-term effectiveness, and is easy-to-operation. This acoustofluidic spheroid assembly method provides a simple and efficient way to produce large numbers of uniform-sized spheroids for biomedical applications in translational medicine, pharmaceutical industry and basic life science research.

Acoustic disruption of tumor endothelium and on-demand drug delivery for cancer chemotherapy.

Chemotherapy has been the most widely used treatment against cancer, however, it is limited by its systemic toxicity as well as resistance developed by tumors' physical barriers. Herein, we propose a novel acoustically-mediated treatment regime to on-demand release therapeutics and disrupt tumor structures. By programming a high intensity focused ultrasound transducer, we can locally and digitally release gemcitabine (GEM) as well as open the local blood-tumor barrier or even tumor stroma to enhance intratumor drug delivery via acoustically-oscillating bubbles and liposomes. In our experiments, we modeled tumor endothelium by culturing a monolayer of murine endothelial cells (2H11) on transwell membrane. We locally disrupted the cultured endothelium to enhance drug penetration by using perfluorocarbon liquid droplets as breaking probes and protoporphyrin IX hybridized liposomes as drug carriers. We also demonstrated an on-demand release of GEM by digitally triggering the break of drug carriers. Moreover, we validated the acoustic tumor endothelium disruption in vivo by monitoring penetration of dye (Evans blue) in solid tumors. Therefore, we present an acoustically-mediated delivery method that both releases drug on-demand locally and opens the blood-tumor barrier to enhance drug penetration. This sets the ground for further clinical cancer therapy to improve many systemic cancer treatments.