A novel 2-aminophenalenone-based fluorescent probe designed for monitoring H2O2 for in vitro and in vivo bioimaging.

A significant compound in living organisms, hydrogen peroxide (H2O2) plays a dual role as a signalling molecule in cellular communication and as a pivotal biomarker in assessing disease and oxidative stress. Thus, the detection of abnormal changes in H2O2 levels is essential to understanding its function and involvement in biological systems. The growing demand to meet the specific needs for applications, particularly in biological systems, has sharpened focus on highly sensitive, highly selective molecular sensors and, in turn, heightened interest in these diagnostic tools with innovative designs. In our study, 2-aminophenalenone (2-AP) was used for the first time as a fluorophore in a fluorescent probe. The 2-APB molecule obtained from the reaction of 2-AP with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzyl chloroformate exhibited a highly selective and sensitive (i.e. 62 nM) detection profile for H2O2 compared with the other reactive oxygen species, anions, and metal cations. Moreover, offering naked-eye detection in aqueous solutions, 2-APB demonstrated excellent sensing performance, detection and real-time monitoring in relation to exogenous H2O2 in cells and endogenous H2O2 in zebrafish embryos.

A zebrafish model for studying the mechanisms of newborn hyperbilirubinemia and bilirubin-induced neurological damage.

Unresolved neonatal hyperbilirubinemia may lead to the accumulation of excess bilirubin in the body, and bilirubin in neural tissues may induce toxicity. Bilirubin-induced neurological damage (BIND) can result in acute or chronic bilirubin encephalopathy, causing temporary or lasting neurological dysfunction or severe damage resulting in infant death. Although serum bilirubin levels are used as an indication of severity, known and unknown individual differences affect the severity of the symptoms. The mechanisms of BIND are not yet fully understood. Here, a zebrafish newborn hyperbilirubinemia model is developed and characterized. Direct exposure to excess bilirubin induced dose- and time-dependent toxicity linked to the accumulation of bilirubin in the body and brain. Introduced bilirubin was processed by the liver, which increased the tolerance of larvae. BIND in larvae was demonstrated by morphometric measurements, histopathological analyses and functional tests. The larvae that survived hyperbilirubinemia displayed mild or severe morphologies associated with defects in eye movements, body posture and swimming problems. Interestingly, a plethora of mild to severe clinical symptoms were reproduced in the zebrafish model.

Dopamine-Conjugated Bovine Serum Albumin Nanoparticles Containing pH-Responsive Catechol-V(III) Coordination for In Vitro and In Vivo Drug Delivery.

V(III) instead of commonly used Fe(III) provided a rich tris-catechol-metal coordination at pH 7.4, which is important for slow drug release at physiological pH. Bovine serum albumin (BSA) functionalized with catechol-containing dopamine (D) and cross-linked using tris-catechol-V(III) coordination yielded pH-responsive compact D-BSA NPs (253 nm). However, conversion to bis- and/or mono-catechol-V(III) complexes in an acidic medium resulted in degradation of NPs and rapid release of doxorubicin (DOX). It was shown that D-BSA NPs entered cancerous MCF-7 cells (66%) more efficiently than non-cancerous HEK293T (33%) in 3 h. Also, DOX-loaded NPs reduced cell viability of MCF-7 by 75% and induced apoptosis in a majority of cells after 24 h. Biodegradability and lack of hemolytic activity were shown in vitro, whereas a lack of toxicity was shown in histological sections of zebrafish. Furthermore, 30% of circulating tumor cells in vasculature in 24 h were killed by DOX-loaded NPs shown with the zebrafish CTC xenograft model.

Effect of non-repetitive linker on in vitro and in vivo properties of an anti-VEGF scFv.

Single chain antibody fragments (scFvs) are favored in diagnostic and therapeutic fields thanks to their small size and the availability of various engineering approaches. Linker between variable heavy (VH) and light (VL) chains of scFv covalently links these domains and it can affect scFv's bio-physical/chemical properties and in vivo activity. Thus, scFv linker design is important for a successful scFv construction, and flexible linkers are preferred for a proper pairing of VH-VL. The flexibility of the linker is determined by length and sequence content and glycine-serine (GS) linkers are commonly preferred for scFvs based on their highly flexible profiles. Despite the advantage of this provided flexibility, GS linkers carry repeated sequences which can cause problems for PCR-based engineering approaches and immunogenicity. Here, two different linkers, a repetitive GS linker and an alternative non-repetitive linker with similar flexibility but lower immunogenicity are employed to generate anti-Vascular Endothelial Growth Factor scFvs derived from bevacizumab. Our findings highlight a better in vitro profile of the non-repetitive linker such as a higher monomer ratio, higher thermal stability while there was no significant difference in in vivo efficacy in a zebrafish embryonic angiogenesis model. This is the first study to compare in vivo efficacy of scFvs with different linkers in a zebrafish model.

Transcriptomics Profiling Identifies Cisplatin-Inducible Death Receptor 5 Antisense Long Non-coding RNA as a Modulator of Proliferation and Metastasis in HeLa Cells.

Cisplatin is a well-known cancer chemotherapeutic agent but how extensively long non-coding RNA (lncRNA) expression is modulated by cisplatin is unknown. It is imperative to employ a comprehensive approach to obtain a better account of cisplatin-mediated changes in the expression of lncRNAs. In this study, we used a transcriptomics approach to profile lncRNAs in cisplatin-treated HeLa cells, which resulted in identification of 10,214 differentially expressed lncRNAs, of which 2,500 were antisense lncRNAs. For functional analyses, we knocked down one of the cisplatin inducible lncRNAs, death receptor 5 antisense (DR5-AS) lncRNA, which resulted in a morphological change in HeLa cell shape without inducing any cell death. A second round of transcriptomics-based profiling revealed differential expression of genes associated with immune system, motility and cell cycle in DR5-AS knockdown HeLa cells. Cellular analyses showed that DR5-AS reduced cell proliferation and caused a cell cycle arrest at S and G2/M phases. Moreover, DR5-AS knockdown reduced the invasive capacity of HeLa cells in zebrafish xenograft model. These results suggest that cisplatin-mediated pleiotropic effects, such as reduction in cell proliferation, metastasis and cell cycle arrest, may be mediated by lncRNAs.

TAp73ß Can Promote Hepatocellular Carcinoma Dedifferentiation.

Hepatocyte dedifferentiation is a major source of hepatocellular carcinoma (HCC), but its mechanisms are unknown. We explored the p73 expression in HCC tumors and studied the effects of transcriptionally active p73ß (TAp73ß) in HCC cells. Expression profiles of p73 and patient clinical data were collected from the Genomic Data Commons (GDC) data portal and the TSVdb database, respectively. Global gene expression profiles were determined by pan-genomic 54K microarrays. The Gene Set Enrichment Analysis method was used to identify TAp73ß-regulated gene sets. The effects of TAp73 isoforms were analyzed in monolayer cell culture, 3D-cell culture and xenograft models in zebrafish using western blot, flow cytometry, fluorescence imaging, real-time polymerase chain reaction (RT-PCR), immunohistochemistry and morphological examination. TAp73 isoforms were significantly upregulated in HCC, and high p73 expression correlated with poor patient survival. The induced expression of TAp73ß caused landscape expression changes in genes involved in growth signaling, cell cycle, stress response, immunity, metabolism and development. Hep3B cells overexpressing TAp73ß had lost hepatocyte lineage biomarkers including ALB, CYP3A4, AFP, HNF4α. In contrast, TAp73ß upregulated genes promoting cholangiocyte lineage such as YAP, JAG1 and ZO-1, accompanied with an increase in metastatic ability. Our findings suggest that TAp73ß may promote malignant dedifferentiation of HCC cells.

lncRNA HOTAIR overexpression induced downregulation of c-Met signaling promotes hybrid epithelial/mesenchymal phenotype in hepatocellular carcinoma cells.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) are both reversible processes, and regulation of phenotypical transition is very important for progression of several cancers including hepatocellular carcinoma (HCC). Recently, it is defined that cancer cells can attain a hybrid epithelial/mesenchymal (hybrid E/M) phenotype. Cells with hybrid E/M phenotype comprise mixed epithelial and mesenchymal properties, they can be more resistant to therapeutics and also more capable of initiating metastatic lesions. However, the mechanisms regulating hybrid E/M in HCC are not well described yet. In this study, we investigated the role of the potential crosstalk between lncRNA HOTAIR and c-Met receptor tyrosine kinase, which are two essential regulators of EMT and MET, in acquiring of hybrid E/M phenotype in HCC. METHODS: Expression of c-Met and lncRNA HOTAIR were defined in HCC cell lines and patient tissues through HCC progression. lncRNA HOTAIR was overexpressed in SNU-449 cells and its effects on c-Met signaling were analyzed. c-Met was overexpressed in SNU-398 cells and its effect on HOTAIR expression was analyzed. Biological significance of HOTAIR/c-Met interplay was defined in means of adhesion, proliferation, motility behavior, invasion, spheroid formation and metastatic ability. Effect of ectopic lncRNA HOTAIR expression on phenotype was defined with investigation of molecular epithelial and mesenchymal traits. RESULTS: In vitro and in vivo experiments verified the pivotal role of lncRNA HOTAIR in acquisition of hybrid E/M phenotype through modulating expression and activation of c-Met and its membrane co-localizing partner Caveolin-1, and membrane organization to cope with the rate limiting steps of metastasis such as survival in adhesion independent microenvironment, escaping from anoikis and resisting to fluidic shear stress (FSS) in HCC. CONCLUSIONS: Our work provides the first evidence suggesting a role for lncRNA HOTAIR in the modulation of c-Met to promote hybrid E/M phenotype. The balance between lncRNA HOTAIR and c-Met might be critical for cell fate decision and metastatic potential of HCC cells. Video Abstract.

Thioredoxin interacting protein promotes invasion in hepatocellular carcinoma.

BACKGROUND: Considerable evidence suggests that oxidative stress plays an essential role in the progression of hepatocellular carcinoma (HCC). While acquired resistance to oxidative stress is the main driver of aggressive cell phenotype, the underlying mechanisms remain unknown. Here, we tested the hypothesis that elevated expression of Thioredoxin-interacting protein (TXNIP) is a main regulator of the aggressive phenotype in HCC. MATERIALS AND METHODS: To test this hypothesis, we measured TXNIP expression levels in 11 HCC cell lines by qPCR and western blotting. In addition, 80 pairs of HCC tissues and matched liver tissues of 73 cases, as well as 11 normal liver tissue samples were examined by immunohistochemistry. Besides, TXNIP expression levels were analyzed by Oncomine Platform in seven independent microarray datasets. Finally, the functional role of TXNIP in HCC was investigated in vitro and in vivo by silencing and overexpression studies. RESULTS: Our results show that TXNIP expression is significantly increased in HCC compared to non-tumor counterparts (p < 0.0001) as well as to normal (p < 0.0001) and cirrhotic (p < 0.0001) liver tissues. Moreover, stable overexpression of TXNIP in HCC cells (i) significantly increases ROS levels, (ii) induces EMT phenotype, (iii) increases motility, invasion and 3D branching tubulogenesis, (iv) decreases apoptosis, and (v) elevates in vivo metastasis in zebrafish embryos. Finally, we identify sinusoidal/stromal and cytoplasmic TXNIP staining patterns as risk factors for intrahepatic vascular invasion (p:0.0400). CONCLUSION: Our results strongly suggest that overexpression of TXNIP has a pivotal role in HCC progression by inducing cell survival, invasion, and metastasis.

THADA Regulates the Organismal Balance between Energy Storage and Heat Production.

Human susceptibility to obesity is mainly genetic, yet the underlying evolutionary drivers causing variation from person to person are not clear. One theory rationalizes that populations that have adapted to warmer climates have reduced their metabolic rates, thereby increasing their propensity to store energy. We uncover here the function of a gene that supports this theory. THADA is one of the genes most strongly selected during evolution as humans settled in different climates. We report here that THADA knockout flies are obese, hyperphagic, have reduced energy production, and are sensitive to the cold. THADA binds the sarco/ER Ca2+ ATPase (SERCA) and acts on it as an uncoupler. Reducing SERCA activity in THADA mutant flies rescues their obesity, pinpointing SERCA as a key effector of THADA function. In sum, this identifies THADA as a regulator of the balance between energy consumption and energy storage, which was selected during human evolution.

Tissue-specific coupling between insulin/IGF and TORC1 signaling via PRAS40 in Drosophila.

PRAS40 has recently been identified as a protein that couples insulin/IGF signaling (IIS) to TORC1 activation in cell culture; however, the physiological function of PRAS40 is not known. In this study, we investigate flies lacking PRAS40. Surprisingly, we find both biochemically and genetically that PRAS40 couples IIS to TORC1 activation in a tissue-specific manner, regulating TORC1 activity in ovaries but not in other tissues of the animal. PRAS40 thereby regulates fertility but not growth of the fly, allowing distinct physiological functions of TORC1 to be uncoupled. We also show that the main function of PRAS40 in vivo is to regulate TORC1 activity, and not to act as a downstream target and effector of TORC1. Finally, this work sheds some light on the question of whether TORC1 activity is coupled to IIS in vivo.

Dynamic Fgf signaling couples morphogenesis and migration in the zebrafish lateral line primordium.

The collective migration of cells in the form of cohesive tissues is a hallmark of both morphogenesis and repair. The extrinsic cues that direct these complex migrations usually act by regulating the dynamics of a specific subset of cells, those at the leading edge. Given that normally the function of tissue migration is to lay down multicellular structures, such as branched epithelial networks or sensory organs, it is surprising how little is known about the mechanisms that organize cells behind the leading edge. Cells of the zebrafish lateral line primordium switch from mesenchyme-like leader cells to epithelial rosettes that develop into mechanosensory organs. Here, we show that this transition is regulated by an Fgf signaling circuit that is active within the migrating primordium. Point sources of Fgf ligand drive surrounding cells towards a ;non-leader' fate by increasing their epithelial character, a prerequisite for rosette formation. We demonstrate that the dynamic expression of Fgf ligands determines the spatiotemporal pattern of epithelialization underlying sensory organ formation in the lateral line. Furthermore, this work uncovers a surprising link between internal tissue organization and collective migration.