Isolation of three different sizes of exosomes in an Asian population with different retinal diseases before and after treatment: preliminary results.

Exosomes are membranous structures measuring between 40-120 nm that are secreted by various cells of the human body into the body fluid system. Exosomes contain proteins, mRNA, miRNA, and signaling molecules, and physiologically they assist in the intercellular transport of proteins and RNA molecules. In this study, we used an immunoaffinity filter paper platform combined with scanning electron microscopy and microfluidic systems to detect the size of exosomes within the aqueous humor. Eight aqueous humor samples showed three distinct sizes of exosomes that were significantly different on scanning electron microscopy(P < 0.01). We further used nanoparticle tracking analysis to assess the size distribution of exosomes within the aqueous humor. We found significantly different distributions of exosomes between patients with three different ocular diseases and patients with normal cataracts as controls. An obvious peak of exomeres(size around 35 nm)was found in the patients with central retinal vein occlusion and vitreous hemorrhage. Flare-ups of large exosomes(size 90-120 nm)were found in the patients with the inflammatory ocular disease pars planitis. No obvious peaks in exomeres or large exosomes were found in the control group. There was a high association between the distribution of exosomes and the pathogenesis of ocular diseases. After intravitreal anti-vascular endothelial growth factor treatment, the aqueous humor from the patients with neovascular diseases showed a significant reduction in exosomes in nanoparticle tracking analysis. These findings suggest that at least three distinct sizes of exosomes exist in the aqueous humor:(1)exomeres:<35 nm;(2)small exosomes:60-80 nm; and (3)large exosomes:90-120 nm. Different sizes of exosomes may have different implications in normal or diseased eyes.

Three different sized exosomes were identified in aqueous humor.The distribution of exosome size was significantly different between the patients with inflammatory and neovascularization retinal diseases.After intravitreal anti-vascular endothelial growth factor treatment, the aqueous humor from patients with neovascular diseases showed a significant reduction in exosomes in nanoparticle tracking analysis.

Polysaccharide of Ganoderma lucidum Ameliorates Cachectic Myopathy Induced by the Combination Cisplatin plus Docetaxel in Mice.

Cachexia is a lethal muscle-wasting syndrome associated with cancer and chemotherapy use. Mounting evidence suggests a correlation between cachexia and intestinal microbiota, but there is presently no effective treatment for cachexia. Whether the Ganoderma lucidum polysaccharide Liz-H exerts protective effects on cachexia and gut microbiota dysbiosis induced by the combination cisplatin plus docetaxel (cisplatin + docetaxel) was investigated. C57BL/6J mice were intraperitoneally injected with cisplatin + docetaxel, with or without oral administration of Liz-H. Body weight, food consumption, complete blood count, blood biochemistry, and muscle atrophy were measured. Next-generation sequencing was also performed to investigate changes to gut microbial ecology. Liz-H administration alleviated the cisplatin + docetaxel-induced weight loss, muscle atrophy, and neutropenia. Furthermore, upregulation of muscle protein degradation-related genes (MuRF-1 and Atrogin-1) and decline of myogenic factors (MyoD and myogenin) after treatment of cisplatin and docetaxel were prevented by Liz-H. Cisplatin and docetaxel treatment resulted in reducing comparative abundances of Ruminococcaceae and Bacteroides, but Liz-H treatment restored these to normal levels. This study indicates that Liz-H is a good chemoprotective reagent for cisplatin + docetaxel-induced cachexia. IMPORTANCE Cachexia is a multifactorial syndrome driven by metabolic dysregulation, anorexia, systemic inflammation, and insulin resistance. Approximately 80% of patients with advanced cancer have cachexia, and cachexia is the cause of death in 30% of cancer patients. Nutritional supplementation has not been shown to reverse cachexia progression. Thus, developing strategies to prevent and/or reverse cachexia is urgent. Polysaccharide is a major biologically active compound in the fungus Ganoderma lucidum. This study is the first to report that G. lucidum polysaccharides could alleviate chemotherapy-induced cachexia via reducing expression of genes that are known to drive muscle wasting, such as MuRF-1 and Atrogin-1. These results suggest that Liz-H is an effective treatment for cisplatin + docetaxel-induced cachexia.

Lumenal Galectin-9-Lamp2 interaction regulates lysosome and autophagy to prevent pathogenesis in the intestine and pancreas.

Intracellular galectins are carbohydrate-binding proteins capable of sensing and repairing damaged lysosomes. As in the physiological conditions glycosylated moieties are mostly in the lysosomal lumen but not cytosol, it is unclear whether galectins reside in lysosomes, bind to glycosylated proteins, and regulate lysosome functions. Here, we show in gut epithelial cells, galectin-9 is enriched in lysosomes and predominantly binds to lysosome-associated membrane protein 2 (Lamp2) in a Asn(N)-glycan dependent manner. At the steady state, galectin-9 binding to glycosylated Asn175 of Lamp2 is essential for functionality of lysosomes and autophagy. Loss of N-glycan-binding capability of galectin-9 causes its complete depletion from lysosomes and defective autophagy, leading to increased endoplasmic reticulum (ER) stress preferentially in autophagy-active Paneth cells and acinar cells. Unresolved ER stress consequently causes cell degeneration or apoptosis that associates with colitis and pancreatic disorders in mice. Therefore, lysosomal galectins maintain homeostatic function of lysosomes to prevent organ pathogenesis.

MiR-145 mediates zebrafish hepatic outgrowth through progranulin A signaling.

MicroRNAs (miRs) are mRNA-regulatory molecules that fine-tune gene expression and modulate both processes of development and tumorigenesis. Our previous studies identified progranulin A (GrnA) as a growth factor which induces zebrafish hepatic outgrowth through MET signaling. We also found that miR-145 is one of potential fine-tuning regulators of GrnA involved in embryonic hepatic outgrowth. The low level of miR-145 seen in hepatocarinogenesis has been shown to promote pathological liver growth. However, little is known about the regulatory mechanism of miR-145 in embryonic liver development. In this study, we demonstrate a significant decrease in miR-145 expression during hepatogenesis. We modulate miR-145 expression in zebrafish embryos by injection with a miR-145 mimic or a miR-145 hairpin inhibitor. Altered embryonic liver outgrowth is observed in response to miR-145 expression modulation. We also confirm a critical role of miR-145 in hepatic outgrowth by using whole-mount in situ hybridization. Loss of miR-145 expression in embryos results in hepatic cell proliferation, and vice versa. Furthermore, we demonstrate that GrnA is a target of miR-145 and GrnA-induced MET signaling is also regulated by miR-145 as determined by luciferase reporter assay and gene expression analysis, respectively. In addition, co-injection of GrnA mRNA with miR-145 mimic or MO-GrnA with miR-145 inhibitor restores the liver defects caused by dysregulation of miR-145 expression. In conclusion, our findings suggest an important role of miR-145 in regulating GrnA-dependent hepatic outgrowth in zebrafish embryonic development.

Benzo[a]pyrene-induced cell cycle progression occurs via ERK-induced Chk1 pathway activation in human lung cancer cells.

Benzo[a]pyrene (B[a]P) is a potent lung carcinogen derived from tobacco smoking and environmental contamination. This study aimed to investigate the signal transduction pathway responsible for B[a]P-induced non-small cell lung cancer (NSCLC) development. We exposed the human NSCLC cell lines Calu-1, CL3, H1299, CH27, H23, and H1355 to B[a]P and assessed cell cycle progression using flow cytometry. Expression of cell cycle mediators was measured using Western blot analyses and electrophoretic mobility shift assays (EMSAs). B[a]P exposure dramatically induced S-phase accumulation in H1355 cells. Phospho-p53 (Ser15 and Ser20), phospho-ERK, phospho-p38, and Bax were significantly increased in H1355 cells whereas phospho-Rb was decreased in these cells. In addition, B[a]P induced phosphorylation of checkpoint kinase-1 (Chk1) but not Chk2. EMSA experiments revealed a slower migrating band after c-Myc bound the E-box in response to B[a]P treatment, which was abolished upon the addition of the ERK inhibitor PD98059 in H1355 cells. Phospho-ERK inhibition and dominant negative mutant Chk1 expression reversed B[a]P-induced S phase accumulation and downregulated phospho-Chk1 and phospho-ERK expression. Taken together, these results suggest that activation of ERK and its downstream mediator Chk1 may contribute to B[a]P-induced S phase accumulation in H1355 cells. The results could help in the development of lung cancer treatments that target the Chk1 pathway through ERK.

RIG-I specifically mediates group II type I IFN activation in nervous necrosis virus infected zebrafish cells.

The type I interferon (IFN) response has been shown to be crucial for the survival of zebrafish larvae infected with nervous necrosis virus (NNV). Teleost type I IFNs can be divided into two groups, based on their cysteine content. While teleost group I IFNs have been extensively studied in terms of their regulation and anti-viral properties, the characteristics of teleost group II IFNs have been relatively unexplored. In this study, we describe the mechanism by which group II IFNs are activated in response to NNV infection in a zebrafish cell line, by focusing on the relationship between type I IFNs and pattern recognition receptors. Expression profile analysis of infected cells by microarray and qPCR revealed signaling activation of two pattern recognition receptors (PRRs): RIG-I like receptors (RLRs) and MyD88-dependent Toll-like receptors (TLRs). Knockdown of retinoic acid-inducible gene I (RIG-I) specifically repressed induction of group II IFNs (IFNϕ2, IFNϕ3) by NNV infection. Furthermore, Ingenuity Pathway Analysis (IPA) was used to demonstrate that RIG-I knockdown results in down-regulation of the inflammatory response in NNV-infected cells. Taken together, our results indicate that RIG-I plays an essential role in zebrafish group II type I IFN induction and the inflammatory response to NNV infection.

Progranulin regulates zebrafish muscle growth and regeneration through maintaining the pool of myogenic progenitor cells.

Myogenic progenitor cell (MPC) is responsible for postembryonic muscle growth and regeneration. Progranulin (PGRN) is a pluripotent growth factor that is correlated with neuromuscular disease, which is characterised by denervation, leading to muscle atrophy with an abnormal quantity and functional ability of MPC. However, the role of PGRN in MPC biology has yet to be elucidated. Here, we show that knockdown of zebrafish progranulin A (GrnA) resulted in a reduced number of MPC and impaired muscle growth. The decreased number of Pax7-positive MPCs could be restored by the ectopic expression of GrnA or MET. We further confirmed the requirement of GrnA in MPC activation during muscle regeneration by knockdown and transgenic line with muscle-specific overexpression of GrnA. In conclusion, we demonstrate a critical role for PGRN in the maintenance of MPC and suggest that muscle atrophy under PGRN loss may begin with MPC during postembryonic myogenesis.

Differentiation of pancreatic acinar cells to hepatocytes requires an intermediate cell type.

BACKGROUND & AIMS: The appearance of hepatic foci in pancreas has been well-documented in animal experiments and in patients with pancreatic cancer. We previously demonstrated that transdifferentiation of pancreatic exocrine cells to hepatocytes required members of the CCAAT enhancer binding protein family. Although the molecular basis of hepatic transdifferentiation is understood, the early cellular events remain to be defined. METHODS: Dexamethasone and oncostatin M were used to induce transdifferentiation of primary cultures of mouse acinar cells and exocrine cell lines into hepatocytes. Fluorescent-activated cell sorting was used to identify intermediate cell types and side-population characteristics. Cre-loxP-based lineage tracing was used to investigate whether acinar cells contribute directly to hepatocytes via intermediates that express adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2). RESULTS: Lineage tracing studies showed that hepatocytes were derived directly from pancreatic cells via ABCG2-expressing intermediates. Exposure of cells to insulin increased Akt phosphorylation, ABCG2 expression, and hepatic transdifferentiation. Inhibition of the phosphoinositide 3-kinase pathway, through addition of LY294002 or overexpression of a dominant-negative form of Akt, was sufficient to prevent transdifferentiation. When ABCG2-expressing cells were incubated with glucagon-like-peptide 1 or epidermal growth factor, the intermediate cells could differentiate into insulin-producing beta-like cells. CONCLUSIONS: The phosphoinositide 3-kinase pathway is important in the transdifferentiation of acinar cells to hepatocytes and those hepatocytes arise from acinar cells via ABCG2-expressing intermediates. Furthermore, ABCG2-expressing cells are multipotent and able to differentiate into hepatocytes and insulin-producing beta cells.