Hepatic glucuronyl C5-epimerase combats obesity by stabilising GDF15.

BACKGROUND & AIMS: Disturbed hepatic metabolism frequently results in excessive lipid accumulation in the adipose tissue. However, the specific role of the liver-adipose axis in maintaining lipid homeostasis, as well as the underlying mechanism, has not yet been fully elucidated. In this study, we investigated the role of hepatic glucuronyl C5-epimerase (Glce) in the progression of obesity. METHODS: We determined the association between the expression of hepatic Glce and body mass index (BMI) in obese patients. Obesity models were established in hepatic Glce-knockout and wild-type mice fed a high-fat diet (HFD) to understand the effect of Glce on obesity development. The role of Glce in the progression of disrupted hepatokine secretion was examined via secretome analysis. RESULTS: Hepatic Glce expression was inversely correlated with BMI in obese patients. Moreover, Glce level was found to be decreased in the liver of a HFD murine model. Hepatic Glce deficiency led to impaired thermogenesis in adipose tissue and exacerbated HFD-induced obesity. Interestingly, decreased level of growth differentiation factor 15 (GDF15) was observed in the culture medium of Glce-knockout mouse hepatocytes. Treatment with recombinant GDF15 obstructed obesity progression derived from the absence of hepatic Glce, similar to the effect of Glce or its inactive mutant overexpressed both in vitro and in vivo. Furthermore, liver Glce deficiency led to diminished production and increased degradation of mature GDF15, resulting in reduced hepatic GDF15 secretion. CONCLUSIONS: Hepatic Glce deficiency facilitated obesity development, and decreased Glce expression further reduced hepatic secretion of GDF15, thereby perturbing lipid homeostasis in vivo. Therefore, the novel Glce-GDF15 axis plays an important role in maintaining energy balance and may act as a potential target for combating obesity. IMPACT AND IMPLICATIONS: Evidence suggests that GDF15 plays a key role in hepatic metabolism; however, the molecular mechanism for regulating its expression and secretion is largely unknown. Our work observes that hepatic Glce, as a key Golgi-localised epimerase, may work on the maturation and post-translational regulation of GDF15. Hepatic Glce deficiency reduces the production of mature GDF15 protein and facilitates its ubiquitination, resulting in the aggravation of obesity development. This study sheds light on the new function and mechanism of the Glce-GDF15 axis in lipid metabolism and provides a potential therapeutic target against obesity.

Constant-rate perfused array chip for high-throughput screening of drug permeability through brain endothelium.

The development of an in vitro model for predicting drug permeability through the human blood-brain barrier (BBB) will greatly accelerate the development of neural therapy. Previously reported platforms for BBB model construction cannot meet the requirements of constant-rate and high-throughput flow, as well as compatibility with the commercial meter for real-time transendothelial electrical resistance (TEER) measurement. Herein, a constant-rate perfused array chip (cPAC) was developed to establish a brain endothelium model for screening drug permeability. The cPAC consisted of 24 units with four layers. Three reservoirs on the top had a 0.5 mm center-to-center spacing, enabling real-time detection of the TEER with the commercial volt-ohm meter. With the optimized chip design, the constant-rate and high-throughput flow by gravity was achieved. Compared with the static culture of the Transwell, the brain endothelium model on the cPAC exhibited superior performance in barrier function, efflux functionality of the transporters, and reversible osmotic opening of the brain endothelium. More importantly, the permeability of model drugs on the cPAC matched the in vivo results with the correlation coefficient reaching 0.994. Finally, the brain endothelium model was cocultured with 3D tumor cells for simultaneous evaluation of drug permeability and brain tumor therapy. The drug efficacy at the target cells on the coculture model was also consistent with clinical findings. These results demonstrated that this platform provides a promising tool for brain endothelium model establishment to predict drug permeability and brain therapy. We anticipate the cPAC to be widely accepted for establishing various barrier models.

Establishment and large-scale validation of a three-dimensional tumor model on an array chip for anticancer drug evaluation.

Two-dimensional (2D) tumor model has always poorly predicted drug response of animal model due to the lack of recapitulation of tumor microenvironment. Establishing a biomimetic, controllable, and cost-effective three-dimensional (3D) model and large-scale validation of its in vivo predictivity has shown promise in bridging the gap between the 2D tumor model and animal model. Here, we established a matrigel-based 3D micro-tumor model on an array chip for large-scale anticancer drug evaluation. Compared with the 2D tumor model, the 3D tumor model on the chip showed spheroid morphology, slower proliferation kinetics, and comparable reproducibility. Next, the results of the chemotherapeutic evaluation from 18 drugs against 27 cancer cell lines showed 17.6% of drug resistance on the 3D tumor model. Moreover, the evaluation results of targeted drugs showed expected sensitivity and higher specificity on the 3D tumor model compared with the 2D model. Finally, the evaluation results on the 3D tumor model were more consistent with the in vivo cell-derived xenograft model, and excluded 95% false-positive results from the 2D model. Overall, the matrigel-based 3D micro-tumor model on the array chip provides a promising tool to accelerate anticancer drug discovery.

A Novel Pectin-Like Glycopeptide Isolated from the Fruit of Fructus Mori Impedes Aggregation and Production of Aß42.

The fruit of Fructus Mori is food and medicine, which has been demonstrated to have a significant neuroprotective effect. However, the effective constituent remains unknown. We speculate that the glycopeptide in the extract of the fruit has similar activity. To address this hypothesis, we isolated a novel pectin-like glycopeptide (FMP-6-S4) with a molecular weight of 11.23 kDa from the fruit. It contains about 20% of peptide comprising 17 amino acids and 80% glycan consisting of L-rhamnose (L-Rha), D-galactose (D-Gal), D-galacturonic acid (D-GalA), L-arabinose (L-Ara) and d-glucose (D-Glc) in molar ratios of 7.25:4.62:77.66:5.62:4.85. The backbone of the glycan part consisted of 1,4-linked α-D-GalpA and 1, 2-linked α-L-Rhap, while the branches were composed of hexenuronic acid (HexA) substituted at the C-3 position of partial galacturonic acid, and traces of galactose, glucose, and arabinose were substituted at the C-4 position of rhamnose. The in vitro experiments revealed that FMP-6-S4 might inhibit Aß42 (ß-amyloid peptides 42) aggregation and decrease Aß42 production by modulating APP (amyloid precursor protein) processing.

An integrated biomimetic array chip for establishment of collagen-based 3D primary human hepatocyte model for prediction of clinical drug-induced liver injury.

Drug-induced liver injury (DILI) is a leading cause of therapy failure in the clinic and also contributes much to acute liver failure cases. Investigations of predictive sensitivity in animal models have limitations due to interspecies differences. Previously reported in vitro models of liver injury based on primary human hepatocytes (PHHs) cannot meet the requirements of high physiological fidelity, low cost, simple operation, and high throughput with improved sensitivity. Herein, we developed an integrated biomimetic array chip (iBAC) for establishing extracellular matrix (ECM)-based models. A collagen-based 3D PHH model was constructed on the iBAC as a case for the prediction of clinical DILI at throughput. The iBAC has a three-layer structure with a core component of 3D implanting holes. At an initial cell seeding numbers of 5000-10,000, the collagen-based 3D PHH model was optimized with improved and stabilized liver functionality, including cell viability, albumin, and urea production. Moreover, basal activities of most metabolic enzymes on the iBAC were maintained for at least 12 days. Next, a small-scale hepatotoxicity screening indicated that the 3D PHH model on the iBAC was more sensitive for predicting hepatotoxicity than the 2D PHH model on the plate. Finally, a large-scale screening of liver toxicity using 122 clinical drugs further demonstrated that the collagen-based 3D PHH model on the iBAC had superior predictive sensitivity compared to all previously reported in vitro models. These results indicated the importance of 3D collagen for liver physiological functionality and hepatotoxicity prediction. We anticipant it being a promising tool for risk assessment of drug-induced hepatotoxicity with a widespread acceptance in drug industry.

Survival outcomes in prostate cancer patients with a prior cancer.

BACKGROUND: To shed light on the survival outcomes of prostate cancer (PCa) patients diagnosed after a prior cancer and identify prognostic factors for overall survival (OS) and cancer-specific survival (CSS) in PCa patients. METHODS: In the primary group, a total of 1,778 PCa patients with a prior cancer were identified in the Surveillance, Epidemiology, and End Results (SEER) database from 2005 to 2015, retrospectively. Baseline characteristics and causes of death (COD) of these patients were collected and compared. In the second group, a total of 10,296 PCa patients [5,148 patients with PCa as the only malignancy and 5,148 patients with PCa as their second primary malignancy (SPM)] diagnosed between 2010 and 2011 were extracted to investigate the impact of prior cancers on survival outcomes. RESULTS: In PCa patients with a prior cancer, the most common type of prior cancer was from gastrointestinal system (29.92%), followed by urinary system (21.37%). Patients were more likely to die of the prior caner, and those with prior cancer from respiratory system had the worst survival outcomes. Moreover, the overall ratios in patients with stage (PCa) I-II and III-IV diseases were 0.21 and 1.65, indicating that patients with higher stage diseases were more likely to die of PCa. In the second group, patients with PCa as the SPM had worse OS than those with PCa as the first primary cancer. Lastly, prognostic factors for OS and CSS in PCa patients were explored. CONCLUSIONS: PCa remains to be an important COD for patients with a prior malignancy, especially for those with high-stage diseases. PCa patients with a prior cancer had worse survival outcomes than those without.

Long non-coding RNA GClnc1 knockdown suppresses progression of epithelial ovarian cancer by recruiting FOXC2 to disrupt the NOTCH1/NF-κB/Snail pathway.

PURPOSE: Epithelial ovarian cancer (EOC) is a highly fatal gynecological cancer. A long noncoding RNA (lncRNA) gastric cancer-associated lncRNA1 (GClnc1) has been revealed to play critical roles in metastasis. Therefore, the present study aims to explore the correlation between GClnc1 and the metastasis and progression of EOC. METHODS: First, 57 paired EOC and paracancerous tissues were collected to detect GClnc1 expression by RT-qPCR. Subsequently, OVC1 and SKOV3 cells with GClnc1 silencing/overexpression were developed to detect changes in cell activity, apoptosis, migration and invasion abilities. Then, the subcellular localization of GClnc1 was detected by nuclear/cytoplasmic fractionation, ISH and FISH assays. The binding relationships between GClnc1 and forkhead box protein C2 (FOXC2), and between FOXC2 and NOTCH1 were predicted and verified. RESULTS: GClnc1 was significantly overexpressed in EOC tissues, and knockdown of GClnc1 inhibited cell viability and promoted apoptosis. Moreover, GClnc1 in the nucleus bound to the transcription factor FOXC2, thereby activating the transcription of NOTCH1. NOTCH1 overexpression enhanced the proliferation and epithelial-mesenchymal transition of SKOV3 and OVC1 cells. Moreover, NOTCH1 activated the NF-κB/Snail signaling. Finally, in vivo experiments demonstrated that GClnc1 knockdown suppressed the growth and metastasis of SKOV3 and OVC1 cells in vivo. CONCLUSIONS: GClnc1 promoted NOTCH1 transcription by recruiting FOXC2, thereby activating the NF-κB/Snail signaling and promoting EOC cell growth and metastasis.

Generation of Gradients on a Microfluidic Device: Toward a High-Throughput Investigation of Spermatozoa Chemotaxis.

Various research tools have been used for in vitro detection of sperm chemotaxis. However, they are typically poor in maintenance of gradient stability, not to mention their low efficiency. Microfluidic device offers a new experimental platform for better control over chemical concentration gradient than traditional ones. In the present study, an easy-handle diffusion-based microfluidic chip was established. This device allowed for conduction of three parallel experiments on the same chip, and improved the performance of sperm chemotaxis research. In such a chip, there were six channels surrounding a hexagonal pool. The channels are connected to the hexagon by microchannels. Firstly, the fluid flow in the system was characterized; secondly, fluorescein solution was used to calibrate gradient profiles formed in the central hexagon; thirdly, sperm behavior was observed under two concentration gradients of progesterone (100 pM and 1 mM, respectively) as a validation of the device. Significant differences in chemotactic parameters were recognized between experimental and control groups (p < 0.05). Compared with control group, sperm motility was greatly enhanced in 1 mM group (p < 0.05), but no significant difference was found in 100 pM group. In conclusion, we proposed a microfluidic device for the study of sperm chemotaxis that was capable of generating multi-channel gradients on a chip and would help reduce experimental errors and save time in experiment.

Quantifying biased response of axon to chemical gradient steepness in a microfluidic device.

Axons are very sensitive to molecular gradients and can discriminate extremely small differences in gradient steepness. Microfluidic devices capable of generating chemical gradients and adjusting their steepness could be used to quantify the sensitivity of axonal response. Here, we present a versatile and robust microfluidic device that can generate substrate-bound molecular gradients with evenly varying steepness on a single chip to precisely quantify axonal response. In this device, two solutions are perfused into a central channel via two inlets while partially flowing into two peripheral channels through interconnecting grooves, which gradually decrease the fluid velocity along the central channel. Molecular gradients with evenly and gradually decreased steepness can therefore be generated with a high resolution that is less than 0.05%/mm. In addition, the overall distribution range and resolution of the gradient steepness can be highly and flexibly controlled by adjusting various parameters of the device. Using this device, we quantified the hippocampal axonal response to substrate-bound laminin and ephrin-A5 gradients with varying steepnesses. Our results provided more detailed information on how and to what extent different steepnesses guide hippocampal neuron development during the initial outgrowth. Furthermore, our results show that axons can sensitively respond to very shallow laminin and ephrin-A5 gradients, which could effectively initiate biased differentiation of hippocampal neurons in the steepness range investigated in this study.

Nanoelectrode for amperometric monitoring of individual vesicular exocytosis inside single synapses.

Chemical neurotransmission occurs at chemical synapses and endocrine glands, but up to now there was no means for direct monitoring of neurotransmitter exocytosis fluxes and their precise kinetics from inside an individual synapse. The fabrication of a novel finite conical nanoelectrode is reported perfectly suited in size and electrochemical properties for probing amperometrically inside what appears to be single synapses and monitoring individual vesicular exocytotic events in real time. This allowed obtaining direct and important physiological evidences which may yield important and new insights into the nature of synaptic communications.

Simultaneous generation of gradients with gradually changed slope in a microfluidic device for quantifying axon response.

Over the past decades, various microfluidic devices have been developed to investigate the role of the molecular gradient in axonal development; however, there are very few devices providing quantitative information about the response of axons to molecular gradients with different slopes. Here, we propose a novel laminar-based microfluidic device enabling simultaneous generation of multiple gradients with gradually changed slope on a single chip. This device, with two asymmetrically designed peripheral channels and opposite flow direction, could generate gradients with gradually changed slope in the center channel, enabling us to investigate simultaneously the response of axons to multiple slope gradients with the same batch of neurons. We quantitatively investigated the response of axon growth rate and growth direction to substrate-bound laminin gradients with different slopes using this single-layer chip. Furthermore, we compartmented this gradient generation chip and a cell culture chip by a porous membrane to investigate quantitatively the response of axon growth rate to the gradient of soluble factor netrin-1. The results suggested that contacting with a molecular gradient would effectively accelerate neurites growth and enhance axonal formation, and the axon guidance ratio obviously increased with the increase of gradient slope in a proper range. The capability of generating a molecular gradient with continuously variable slopes on a single chip would open up opportunities for obtaining quantitative information about the sensitivity of axons and other types of cells in response to gradients of various proteins.

Non-neutral nonsynonymous single nucleotide polymorphisms in human ABC transporters: the first comparison of six prediction methods.

Nonsynonymous single nucleotide polymorphisms (nsSNPs) in coding regions that can lead to amino acid changes may cause alteration of protein function and account for susceptibility to disease and altered drug/xenobiotic response. Abundant nsSNPs have been found in genes coding for human ATP-binding cassette (ABC) transporters, but there is little known about the relationship between the genotype and phenotype of nsSNPs in these membrane proteins. In addition, it is unknown which prediction method is better suited for the prediction of non-neutral nsSNPs of ABC transporters. We have identified 2,172 validated nsSNPs in 49 human ABC transporter genes from the Ensembl genome database and the NCBI SNP database. Using six different algorithms, 41 to 52% of nsSNPs in ABC transporter genes were predicted to have functional impacts on protein function. Predictions largely agreed with the available experimental annotations. Overall, 78.5% of non-neutral nsSNPs were predicted correctly as damaging by SNAP, which together with SIFT and PolyPhen, was superior to the prediction methods Pmut, PhD-SNP, and Panther. This study also identified any amino acids that were likely to be functionally critical but have not yet been studied experimentally. There was significant concordance between the predicted results of SIFT and PolyPhen. Evolutionarily non-neutral (destabilizing) amino acid substitutions are predicted to be the basis for the pathogenic alteration of ABC transporter activity that is associated with disease susceptibility and altered drug/xenobiotic response.