Anti-PD-1 therapy achieves favorable outcomes in HBV-positive non-liver cancer.

Anti-PD-1 therapy has shown promising outcomes in the treatment of different types of cancer. It is of fundamental interest to analyze the efficacy of anti-PD-1 therapy in cancer patients infected with hepatitis B virus (HBV) since the comorbidity of HBV and cancer is widely documented. We designed a multicenter retrospective study to evaluate the efficacy of anti-PD-1 therapy on non-liver cancer patients infected with HBV. We found anti-PD-1 therapy achieved much better outcomes in HBV+ non-liver cancer patients than their HBV- counterparts. We performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) from esophageal squamous cell carcinoma (ESCC) patients. We found both cytotoxicity score of T cells and MHC score of B cells significantly increased after anti-PD-1 therapy in HBV+ ESCC patients. We also identified CX3CR1high TEFF, a subset of CD8+ TEFF, associated with better clinical outcome in HBV+ ESCC patients. Lastly, we found CD8+ TEFF from HBV+ ESCC patients showing higher fraction of Exhaustionhi T than their HBV- counterpart. In summary, anti-PD-1 therapy on HBV+ non-liver cancer patients is safe and achieves better outcomes than that on HBV- non-liver cancer patients, potentially because HBV+ patients had higher fraction of Exhaustionhi T, which made them more efficiently respond to anti-PD-1 therapy.

Comprehensive mapping of alternative polyadenylation site usage and its dynamics at single-cell resolution.

Alternative polyadenylation (APA) plays an important role in posttranscriptional gene regulation such as transcript stability and translation efficiency. However, our knowledge about APA dynamics at the single-cell level is largely unexplored. Here, we developed single-cell polyadenylation sequencing, a strand-specific approach for sequencing the 3' end of transcripts, to investigate the landscape of APA at the single-cell level. By analyzing several cell lines, we found many genes using multiple polyA sites in bulk data are prone to use only one polyA site in each single cell. Interestingly, cell cycle genes were significantly enriched in genes with high variation in polyA site usages. Furthermore, the 414 genes showing a polyA site usage switch after cell synchronization enriched cell cycle genes, while the differentially expressed genes after cell synchronization did not enrich cell cycle genes. We further identified 812 genes showing polyA site usage changes between neighboring cell cycles, which were grouped into six clusters, with cell phase-specific functional categories enriched in each cluster. Deletion of one polyA site in MSL1 and SCCPDH results in slower and faster cell cycle progression, respectively, supporting polyA site usage switch played an important role in cell cycle. These results indicate that APA is an important layer for cell cycle regulation.

Perfluorooctane Sulfonamide (PFOSA) Induces Cardiotoxicity via Aryl Hydrocarbon Receptor Activation in Zebrafish.

Perfluorooctane sulfonamide (PFOSA), a precursor of perfluorooctanesulfonate (PFOS), is widely used during industrial processes, though little is known about its toxicity, particularly to early life stage organisms that are generally sensitive to xenobiotic exposure. Here, following exposure to concentrations of 0.01, 0.1, 1, 10, and 100 µg/L PFOSA, transcriptional, morphological, physiological, and biochemical assays were used to evaluate the potential effects on aquatic organisms. The top Tox functions in exposed zebrafish were related to cardiac diseases predicted by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and Ingenuity Pathway Analysis (IPA) analysis. Consistent with impacts predicted by transcriptional changes, abnormal cardiac morphology, disordered heartbeat signals, as well as reduced heart rate and cardiac output were observed following the exposure of 0.1, 1, 10, or 100 µg/L PFOSA. Furthermore, these PFOSA-induced cardiac effects were either prevented or alleviated by supplementation with an aryl hydrocarbon receptor (AHR) antagonist or ahr2-morpholino knock-down, uncovering a seminal role of AHR in PFOSA-induced cardiotoxicity. Our results provide the first evidence in fish that PFOSA can impair proper heart development and function and raises concern for PFOSA analogues in the natural environment.

Reinvestigation of Classic T Cell Subsets and Identification of Novel Cell Subpopulations by Single-Cell RNA Sequencing.

Classic T cell subsets are defined by a small set of cell surface markers, while single-cell RNA sequencing (scRNA-seq) clusters cells using genome-wide gene expression profiles. The relationship between scRNA-seq clustered populations (scCPops) and cell surface marker-defined classic T cell subsets remains unclear. In this article, we integrated six bead-enriched T cell subsets with 62,235 single-cell transcriptomes from human PBMCs and clustered them into nine scCPops. Bead-enriched CD4+/CD45RA+/CD25- naive T and CD8+/CD45RA+ naive T cells were mainly clustered into their scCPop counterparts, while cells from the other T cell subsets were assigned to multiple scCPops, including mucosal-associated invariant T cells and NKT cells. The multiple T cell subsets forming one scCPop exhibit similar expression patterns, but not vice versa, indicating scCPop is a more homogeneous cell population with similar cell states. Interestingly, we discovered and named IFN signaling-associated gene (ISAG) high T (ISAGhi T) cells, a T cell subpopulation that highly expressed ISAGs. We further enriched ISAGhi T cells from human PBMCs by FACS of BST2 for scRNA-seq analyses. The ISAGhi T cell cluster disappeared on t-distributed stochastic neighbor embedding plot after removing ISAGs, whereas the ISAGhi T cell cluster showed up by analysis of ISAGs alone, indicating ISAGs are the major contributor of the ISAGhi T cell cluster. BST2+ and BST2- T cells showing different efficiencies of T cell activation indicate that a high level of ISAGs may contribute to quick immune responses.

Microscopic Afterglow Bioimaging by Ultralong Organic Phosphorescent Nanoparticles in Living Cells and Zebrafish.

Compared with short-lived emission probes featuring fluorescence imaging , the use of phosphorescent probes imparts the advantage of long-lived signal persistence that distinguishes against background fluorescence interference. However, the realization of ultralong organic phosphorescent (UOP) probes with an ultralong emission lifetime in an aqueous medium is still a challenge. Here, we present a rational strategy for obtaining UOP nanoparticles (NPs) in an air-saturated aqueous medium prepared using an organic phosphor (PDBCz) and a surfactant polymer (PVP), named PDBCz@PVP, showing an ultralong emission lifetime of 284.59 ms and a phosphorescence quantum efficiency of 7.6%. The excellent phosphorescence properties and water solubility of PDBCz@PVP make it a promising candidate for biological imaging. The as-prepared PDBCz@PVP NPs possess excellent luminescence intensity as well as illustrious biocompatibility both in vitro and in vivo. We demonstrate their use as an efficient phosphorescent nanoprobe both in living cells and zebrafish by capturing their afterglow emission signals under microscopy observation for the first time, realizing convenient and fast bioimaging with low cost, which allows for anti-fluorescence interference and shows promise for the future theragnostic applications in nanomedicine.

Targeting RNA helicase DHX33 blocks Ras-driven lung tumorigenesis in vivo.

Ras has been found to be mutated in 30% of non-small cell lung cancers, and its mutation has been regarded as a causal factor underlying tumorigenesis. However, no successful medicine has been developed so far to inhibit Ras for lung cancer treatment. We have previously identified DHX33 as a Ras downstream effector, promoting cell cycle progression and cell growth. In this study, with the K-Ras (G12D);DHX33 (lox/lox) mouse model, we discovered that genetic ablation of DHX33 inhibited tumor development. We further found that ablation of DHX33 altered the expression of nearly 2000 genes which are critical in cancer development such as cell cycle, apoptosis, glycolysis, Wnt signaling, and cell migration. Our study for the first time demonstrates the pivotal role of the DHX33 in Ras-driven lung cancer development in vivo and highlights that pharmacological targeting DHX33 can be a feasible option in treating Ras-mutant lung cancers.

Theacrine, a purine alkaloid from kucha, protects against Parkinson's disease through SIRT3 activation.

BACKGROUND: Oxidative damage of dopaminergic neurons is the fundamental causes of Parkinson's disease (PD) that has no standard cure at present. Theacrine, a purine alkaloid from Chinese tea Kucha, has been speculated to benefit the neurodegeneration in PD, through similar actions to its chemical analogue caffeine, albeit excluding side effects. Theacrine has nowadays gained a lot of interest for its multiple benefits, while the investigations are weak and insufficient. HYPOTHESIS/PURPOSE: It is well-known that tea has a wide range of functions, especially in the prevention and treatment of neurodegenerative diseases. Theacrine is an active monomer compound in Camellia assamica var. kucha Hung T. Chang & H.S.Wang (Kucha), which appears to be effective and safe in PD therapy. The aim of this study is to examine its actions in diverse PD models and explore the mechanisms. STUDY DESIGN: For determination of theacrine's effects, we employed diverse oxidative damage-associated PD models, including 6-OHDA-treated rats, MPTP-treated mice/zebrafish and MPP+-treated SH-SY5Y cells, and using caffeine, selegiline and depranyl as positve control. For investigation and verification of the mechanisms, we utilized approaches testing mitochondrial function-related parameters and enzyme activity as well as applied gene knockdown and overexpression. METHODS: We employed behavioral tests including spontaneous activity, pole, swimming, rotarod and gait, immunohistochemistry, HPLC, flow cytometry, immunohistochemistry, Western blot, gene knockdown by siRNA and overexpression by plasmid in this study. RESULTS: Theacrine is demonstrated to retrieve the loss of dopaminergic neurons and the damages of behavioral performance in multiple animal models of PD (6-OHDA-treated rats and in MPTP-treated mice and zebrafish). The followed data of MPP+-treated SH-SY5Y cells indicate that theacrine relieves apoptosis resulted from oxidative damage and mitochondrial dysfunction. Further investigations illustrate that theacrine activates SIRT3 directly. It is of advantage to prevent apoptosis through SIRT3-mediated SOD2 deacetylation that reduces ROS accumulation and restores mitochondrial function. This concept is elaborated by 3TYP that inhibits SIRT3 enzyme activity and knockdown/overexpression of SIRT3 gene, demonstrating a crucial role of SIRT3 in theacrine-benefited dopaminergic neurons. CONCLUSION: Theacrine prevents apoptosis of dopaminergic neurons through directly activating SIRT3 which deacetylating SOD2 and restoring mitochondrial functions.

Oxyphylla A Promotes Degradation of α-Synuclein for Neuroprotection via Activation of Immunoproteasome.

Parkinson's disease (PD), the second most common neurodegenerative disorder, is neuropathologically characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the presence of Lewy bodies in surviving neurons. α-synuclein (α-syn) is the major component of Lewy bodies and its deposition in neurons is critical pathological event in the pathogenesis of PD. Herein, we reported that Oxyphylla A, a novel lead compound from the fruit of Alpinia oxyphylla, significantly promoted α-syn degradation in a cellular PD model. When exploring the molecular pathways, we found that Oxyphylla A promoted α-syn degradation in a ubiquitin proteasome system (UPS)-dependent and autophagy-independent manner. We further confirmed that Oxyphylla A enhanced UPS activity by upregulating 20S subunit PSMB8 expression. A mechanism study revealed that Oxyphylla A activated the PKA/Akt/mTOR pathway to trigger PSMB8 expression and enhance UPS activity. Finally, we illustrated that Oxyphylla A alleviated the accumulation of both Triton-soluble and Triton-insoluble forms of α-syn and protected against α-syn-induced neurotoxicity in A53T α-syn transgenic mice. These findings suggest that the activation of UPS, via small molecular UPS enhancers including Oxyphylla A, may be a therapeutic strategy for intervention against PD and related diseases.

Transcriptomic Responses of Bisphenol S Predict Involvement of Immune Function in the Cardiotoxicity of Early Life-Stage Zebrafish (Danio rerio).

Bisphenol S (BPS), an alternative for bisphenol A (BPA) that is present in thermal paper and numerous consumer products, has been linked to estrogenic, cytotoxic, genotoxic, neurotoxic, and immunotoxic responses. However, the mechanisms of BPS toxicity remain poorly understood. Here, following exposure to environmentally relevant concentrations ranging from 0.1 to 100 µg/L BPS, transcriptional changes evaluated by enriched gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Ingenuity Pathway Analysis (IPA) predicted cardiac disease and impairment of immune function in zebrafish at the embryo-to-larvae stage. Consistent with impacts predicted by transcriptional changes, significant sublethal impacts were observed ranging from reduced heart rate [8.7 ± 2.4% reductions at 100 µg/L BPS treatment; P < 0.05] to abnormal cardiac morphology [atrial/ventricle area significantly increased; 36.2 ± 9.6% at 100 µg/L BPS treatment; P < 0.05]. RNA-sequencing analysis results also indicated changes in nitric oxide synthetase (NOS2) and interleukin 12 (IL12) after BPS treatment, which was confirmed at the protein level. Increased expression of other cytokine genes was observed in larvae, suggesting inflammatory responses may be contributing to cardiac impairment by BPS. BPS caused cardiotoxicity, which temporally corresponded with inflammatory responses as predicted from RNA sequencing and confirmed at the protein and cellular levels of biological organization. Additional study is needed to find causal linkages between these responses.

The Bruton's Tyrosine Kinase Inhibitor Ibrutinib Impairs the Vascular Development of Zebrafish Larvae.

Ibrutinib is an orally bioavailable, irreversible selective Bruton's tyrosine kinase inhibitor that has demonstrated impressive therapeutic effects in patients with B cell malignancies. However, adverse effects, such as bleeding and hypertension, are also reported, implying that studies on the toxicological effect of ibrutinib on living organisms are needed. Here, we have used zebrafish, a successful model organism for studying toxicology, to investigate the influence of ibrutinib during embryogenesis. We found that ibrutinib had potent toxicity on embryonic development, especially vascular development in zebrafish embryos. We also revealed that ibrutinib perturbed vascular formation by suppressing angiogenesis, rather than vasculogenesis. In addition, ibrutinib exposure led to the collapse of the vascular lumen, as well as reduced proliferation and enhanced apoptosis of vascular endothelial cells. Moreover, the expression of vascular development-related genes was also altered in ibrutinib-treated embryos. To our knowledge, this is the first study to describe the vascular toxicity of ibrutinib in an animal model, providing a theoretical basis for clinical safety guidelines in ibrutinib treatment.

Tetramethylpyrazine Analogue T-006 Exerts Neuroprotective Effects against 6-Hydroxydopamine-Induced Parkinson's Disease In Vitro and In Vivo.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and there is no cure for it at present. We have previously reported that the tetramethylpyrazine (TMP) derivative T-006 exhibited beneficial effects in Alzheimer's disease (AD) models. However, its effect on PD remains unclear. In the present study, we investigated the neuroprotective effects and underlying mechanisms of T-006 against 6-hydroxydopamine- (6-OHDA-) induced lesions in in vivo and in vitro PD models. Our results demonstrated that T-006 alleviated mitochondrial membrane potential loss and restored the energy metabolism and mitochondrial biogenesis that were induced by 6-OHDA in PC12 cells. In addition, animal experiments showed that administration of T-006 significantly attenuated the 6-OHDA-induced loss of tyrosine hydroxylase- (TH-) positive neurons in the SNpc, as well as dopaminergic nerve fibers in the striatum, and also increased the concentration of dopamine and its metabolites (DOPAC, HVA) in the striatum. Functional deficits were restored following T-006 treatment in 6-OHDA-lesioned mice, as demonstrated by improved motor coordination and rotational behavior. In addition, we found that the neuroprotective effects of T-006 were mediated, at least in part, by the activation of both the PKA/Akt/GSK-3ß and CREB/PGC-1α/NRF-1/TFAM pathways. In summary, our findings demonstrate that T-006 could be developed as a novel neuroprotective agent for PD, and the two pathways might be promising therapeutic targets for PD.

Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9.

Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antisense morpholino-mediated knockdown embryos. Altogether, this work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and our bioinformatics analysis also provides some useful guidance to design gene-specific gRNAs for successful gene editing.

Light-Induced Protein Degradation with Photocaged PROTACs.

Induction of protein degradation is emerging as a powerful strategy to modulate protein functions and alter cellular signaling pathways. Proteolysis-targeting chimeras (PROTACs) have been used to degrade a range of diverse proteins in vitro and in vivo. Here we present a type of photo-caged PROTACs (pc-PROTACs) to induce degradation activity with light. Photo-removable blocking groups were added to a degrader of Brd4, and the resulting molecule pc-PROTAC1 showed potent degradation activity in live cells only after light irradiation. Furthermore, this molecule efficiently degraded Brd4 and induced expected phenotypic changes in zebrafish. Additionally, this approach was successfully applied to construct pc-PROTAC3 of BTK. Thus, a general strategy to induce protein degradation with light was established to augment the chemists' toolbox to study disease-relevant protein targets.

Tetramethylpyrazine Analogue T-006 Promotes the Clearance of Alpha-synuclein by Enhancing Proteasome Activity in Parkinson's Disease Models.

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide and is characterized in part by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). The main pathological hallmark of PD is the intraneuronal accumulation of misfolded α-synuclein (α-syn) aggregates. Mutations in the SNCA gene (encoding α-syn) and variations in its copy number are associated with some forms of familial PD. In the present study, T-006, a new tetramethylpyrazine (TMP) derivative with recently reported anti-Alzheimer activity, is shown to significantly promote α-syn degradation in a cellular PD model. Moreover, we illustrate that T-006 inhibits the accumulation of both Triton-soluble and -insoluble forms of α-syn and protects against α-syn-induced neurotoxicity in A53T-α-syn transgenic mice. The mechanism of action of T-006 was verified by evaluation of a potential protein degradation pathway. We found that T-006 promotes α-syn degradation in a proteasome-dependent and autophagy-independent manner. We further confirmed that T-006 enhances proteasome activity by upregulating 20S proteasome subunit ß5i (LMP7) protein expression. A functional study revealed that T-006 activates the PKA/Akt/mTOR/p70S6K pathway to trigger LMP7 expression and enhance chymotrypsin-like proteasomal activity. These findings indicate that T-006 is a potent proteasome activator and a potential therapeutic agent for the prevention and treatment of PD and related diseases.

Rapamycin and FK506 derivative TH2849 could ameliorate neurodegenerative diseases through autophagy with low immunosuppressive effect.

Autophagy is an essential cellular process concern with cellular homeostasis down-regulated by mTOR, whose activity can be modulated by rapamycin, a kind of lipophilic macrolide antibiotic, through forming a complex with immunophilin FKBP12 essential for mTOR regulation to induce autophagy. Therefore, rapamycin is normally used as a neuron protective agent. The immunophilin FKBP12 binding ligand FK506 is well known as an immunosuppressive agent by inhibiting the calcineurin expression. In this study, we synthesized a series of modified compounds based on the FKBP12 binding moiety to as same as the binding structure of rapamycin and FK506 particularly. We removed the other binding regions of the complex that has the property of immunosuppression. We found that a novel small molecule named TH2849 from these derivative compounds has a significant binding connection with mTOR by comparing to calcineurin. The effects of TH2849 on calcineurin/NFAT were not as significant as FK506, and weak effects on IL2/p34cdc2 /cyclin signaling pathway were also found. Moreover, TH2849 also shows mitochondrial protective effect through stabilizing the mitochondrial structure and transmembrane potential (ΔΨm) and could rescue dopaminergic neurons in MPTP-treated zebrafishes as well as mice models with less immunosuppressive effect. Our present study shows that TH2849 works as a neuroprotective agent possibly by inducing autophagy and low immunosuppressive effect.

Central role of autophagic UVRAG in melanogenesis and the suntan response.

UV-induced cell pigmentation represents an important mechanism against skin cancers. Sun-exposed skin secretes α-MSH, which induces the lineage-specific transcriptional factor MITF and activates melanogenesis in melanocytes. Here, we show that the autophagic tumor suppressor UVRAG plays an integral role in melanogenesis by interaction with the biogenesis of lysosome-related organelles complex 1 (BLOC-1). This interaction is required for BLOC-1 stability and for BLOC-1-mediated cargo sorting and delivery to melanosomes. Absence of UVRAG dispersed BLOC-1 distribution and activity, resulting in impaired melanogenesis in vitro and defective melanocyte development in zebrafish in vivo. Furthermore, our results establish UVRAG as an important effector for melanocytes' response to α-MSH signaling as a direct target of MITF and reveal the molecular basis underlying the association between oncogenic BRAF and compromised UV protection in melanoma.

miR-216b enhances the efficacy of vemurafenib by targeting Beclin-1, UVRAG and ATG5 in melanoma.

Autophagy maintains cells survival in many stressful conditions including starvation, growth factor deprivation and misfolded protein accumulation. Additionally, autophagic survival mechanisms are used by transformed tumor cells to inhibit cell death, limit drug effectiveness and possibly generate drug resistance. However, the mechanism of how cells utilize autophagy during drug resistance is not fully understood. Here, we demonstrate that miR-216b plays an important role in alleviating drug resistance by regulating autophagy in melanoma. We show that miR-216b attenuates autophagy by directly targeting three key autophagy genes Beclin-1, UVRAG and ATG5. Overexpression of these genes from miRNA immune cDNA constructs rescue autophagic activity in the presence of miR-216b. Antagomir-mediated inactivation of endogenous miR-216b led to an increase of Beclin-1, UVRAG, ATG5, and subsequent autophagic activity. More importantly, we have discovered that BRAF(V600E) inhibitor vemurafenib suppresses miR-216b activity, which in turn activates autophagy to generate drug resistance in both BRAFi-sensitive and -resistant cells. Strikingly, ectopic expression of miR-216b increases the efficacy of vemurafenib both in vitro and in vivo. Taken together, these data indicate that miR-216b regulates autophagy by suppressing three key autophagy genes, and enhances the antitumor activity of vemurafenib in BRAF(V600E) melanoma cells.

Evaluation of the Hair Cell Regeneration in Zebrafish Larvae by Measuring and Quantifying the Startle Responses.

The zebrafish has become an established model organism for the study of hearing and balance systems in the past two decades. The classical approach to examine hair cells is to use dye to conduct selective staining, which shows the number and morphology of hair cells but does not reveal their function. Startle response is a behavior closely related to the auditory function of hair cells; therefore it can be used to measure the function of hair cells. In this study, we developed a device to measure the startle response of zebrafish larvae. By applying various levels of stimulus, it showed that the system can discern a 10 dB difference. The hair cell in zebrafish can regenerate after damage due to noise exposure or drug treatment. With this device, we measured the startle response of zebrafish larvae during and after drug treatment. The results show a similar trend to the classical hair cell staining method. The startle response was reduced with drug treatment and recovered after removal of the drug. Together it demonstrated the capability of this behavioral assay in evaluating the hair cell functions of fish larvae and its potential as a high-throughput screening tool for auditory-related gene and drug discovery.

DHX33 Transcriptionally Controls Genes Involved in the Cell Cycle.

The RNA helicase DHX33 has been shown to be a critical regulator of cell proliferation and growth. However, the underlying mechanisms behind DHX33 function remain incompletely understood. We present original evidence in multiple cell lines that DHX33 transcriptionally controls the expression of genes involved in the cell cycle, notably cyclin, E2F1, cell division cycle (CDC), and minichromosome maintenance (MCM) genes. DHX33 physically associates with the promoters of these genes and controls the loading of active RNA polymerase II onto these promoters. DHX33 deficiency abrogates cell cycle progression and DNA replication and leads to cell apoptosis. In zebrafish, CRISPR-mediated knockout of DHX33 results in downregulation of cyclin A2, cyclin B2, cyclin D1, cyclin E2, cdc6, cdc20, E2F1, and MCM complexes in DHX33 knockout embryos. Additionally, we found the overexpression of DHX33 in a subset of non-small-cell lung cancers and in Ras-mutated human lung cancer cell lines. Forced reduction of DHX33 in these cancer cells abolished tumor formation in vivo Our study demonstrates for the first time that DHX33 acts as a direct transcriptional regulator to promote cell cycle progression and plays an important role in driving cell proliferation during both embryo development and tumorigenesis.

Kaempferol Identified by Zebrafish Assay and Fine Fractionations Strategy from Dysosma versipellis Inhibits Angiogenesis through VEGF and FGF Pathways.

Natural products are a rich resource for the discovery of therapeutic substances. By directly using 504 fine fractions from isolated traditional Chinese medicine plants, we performed a transgenic zebrafish based screen for anti-angiogenesis substances. One fraction, DYVE-D3, was found to inhibit the growth of intersegmental vessels in the zebrafish vasculature. Bioassay-guided isolation of DYVE-D3 indicates that the flavonoid kaempferol was the active substance. Kaempferol also inhibited the proliferation and migration of HUVECs in vitro. Furthermore, we found that kaempferol suppressed angiogenesis through inhibiting VEGFR2 expression, which can be enhanced by FGF inhibition. In summary, this study shows that the construction of fine fraction libraries allows efficient identification of active substances from natural products.