Identification of multiple plasma lipids as diagnostic biomarkers of hypercholesterolemia and the underlying mechanisms based on pseudo-targeted lipidomics.

RATIONALE: Hypercholesterolemia is an important risk factor for cardiovascular diseases and death. This study performed pseudo-targeted lipidomics to identify differentially expressed plasma lipids in hypercholesterolemia, to provide a scientific basis for the diagnosis and pathogenesis of hypercholesterolemia. METHODS: Pseudo-targeted lipidomic analyses of plasma lipids from 20 patients with hypercholesterolemia and 20 normal control subjects were performed using liquid chromatography-mass spectrometry. Differentially expressed lipids were identified by principal component analysis and orthogonal partial least squares discriminant analysis. Receiver operating characteristic curves were used to identify differentially expressed lipids with high diagnostic value. The Kyoto Encyclopedia of Genes and Genomes pathway database was used to identify enriched metabolic pathways. RESULTS: We identified 13 differentially expressed lipids in hypercholesterolemia using variable importance of projection > 1 and p < 0.05 as threshold parameters. The levels of eight sphingomyelins and cholesterol sulfate were higher and those of three triacylglycerols and lysophosphatidylcholine were reduced in hypercholesterolemia. Seven differentially expressed plasma lipids showed high diagnostic value for hypercholesterolemia. Functional enrichment analyses showed that pathways related to necroptosis, sphingolipid signaling, sphingolipid metabolism, and steroid hormone biosynthesis were enriched. CONCLUSIONS: This pseudo-targeted lipidomics study demonstrated that multiple sphingomyelins and cholesterol sulfate were differentially expressed in the plasma of patients with hypercholesterolemia. We also identified seven plasma lipids, including six sphingomyelins and cholesterol sulfate, with high diagnostic value.

Hypoxic Preconditioning Modulates BDNF and Its Signaling through DNA Methylation to Promote Learning and Memory in Mice.

Hypoxic preconditioning (HPC) as an endogenous mechanism can resist hypoxia/ischemia injury and exhibit protective effects on neurological function including learning and memory. Although underlying molecular mechanisms remain unclear, HPC probably regulates the expression of protective molecules by modulating DNA methylation. Brain-derived neurotrophic factor (BDNF) activates its signaling upon binding to the tropomyosin-related kinase B (TrkB) receptor, which is involved in neuronal growth, differentiation, and synaptic plasticity. Therefore, this study focused on the mechanism by which HPC regulates BDNF and BDNF/TrkB signaling through DNA methylation to influence learning and memory. Initially, the HPC model was established by hypoxia stimulations on ICR mice. We found that HPC downregulated the expression of DNA methyltransferase (DNMT) 3A and DNMT3B. Then, the upregulation of BDNF expression in HPC mice was generated from a decrease in DNA methylation of the BDNF gene promoter detected by pyrophosphate sequencing. Subsequently, upregulation of BDNF activated BDNF/TrkB signaling and ultimately improved learning and spatial memory in HPC mice. Moreover, after mice were intracerebroventricularly injected with the DNMT inhibitor, the restraint of DNA methylation accompanied by an increase of BDNF and BDNF/TrkB signaling was also discovered. Finally, we observed that the inhibitor of BDNF/TrkB signaling prevented HPC from ameliorating learning and memory in mice. However, the DNMT inhibitor promoted spatial cognition in mice. Thus, we suggest that HPC may upregulate BDNF by inhibiting DNMTs and decreasing DNA methylation of the BDNF gene and then activate BDNF/TrkB signaling to improve learning and memory in mice. This may provide theoretical guidance for the clinical treatment of cognitive dysfunction caused by ischemia/hypoxia disease.

Possible Involvement of DNA Methylation in TSC1 Gene Expression in Neuroprotection Induced by Hypoxic Preconditioning.

Background: It has been reported that ischemia and ischemic preconditioning (IPC) have different effects on the expression of tuberous sclerosis complex 1 (TSC1), which may contribute to the tolerance to ischemia/hypoxia with the increase of autophagy. The mechanisms of TSC1 differential expression are still unclear under ischemia/IPC conditions in hippocampal Cornu Ammon 1 (CA1) and Cornu Ammon 3 (CA3) area neuronal cells. While we have shown that 5-Aza-CdR, a DNA methyltransferase inhibitor, can upregulate TSC1 and increase hypoxic tolerance by autophagy in vivo and in vitro, in this study, we examined whether DNA methylation was involved in the differential expression of TSC1 in the CA1 and CA3 regions induced by hypoxic preconditioning (HPC). Methods: Level of rapamycin (mTOR) autophagy, a downstream molecular pathway of TSC1/TSC2 complex, was detected in HPC mouse hippocampal CA1 and CA3 areas as well as in the HPC model of mouse hippocampal HT22 cells. DNA methylation level of TSC1 promoter (-720 bp~ -360 bp) was determined in CA1 and CA3 areas by bisulfite-modified DNA sequencing (BMDS). At the same time, autophagy was detected in HT22 cells transfected with GFP-LC3 plasmid. The role of TSC1 in neuroprotection was measured by cell viability and apoptosis, and the role of TSC1 in metabolism was checked by ATP assay and ROS assay in HT22 cells that overexpressed/knocked down TSC1. Results: HPC upregulated the expression of TSC1, downregulated the level of P-mTOR (Ser2448) and P-p70S6K (Thr389), and enhanced the activity of autophagy in both in vivo and in vitro. The increased expression of TSC1 in HPC may depend on its DNA hypomethylation in the promoter region in vivo. HPC also could reduce energy consumption in HT22 cells. Overexpression and knockdown of TSC1 can affect cell viability, cell apoptosis, and metabolism in HT22 cells exposed to hypoxia. Conclusion: TSC1 expression induced by HPC may relate to the downregulation of its DNA methylation level with the increase of autophagy and the decrease of energy demand.

Overview of Traditional Mongolian Medical Warm Acupuncture.

Mongolian medical warm acupuncture is a traditional therapy of Mongolian medicine and was developed by people living on the Mongolian Plateau. This kind of traditional oriental medicine has a long history. The main characteristics of Mongolian medical warm acupuncture are the acupoints and the needles used. Its theory is based on the human anatomical structure and the distinct local culture. Mongolian medical warm acupuncture has been practiced for centuries and proved to be very effective in the treatment of age-related diseases, including the musculoskeletal and nervous diseases. This paper aims to briefly introduce the history and scope of Mongolian medical warm acupuncture, with a particular focus on age-related diseases, where Mongolian medical warm acupuncture has shown significant beneficial effects.

Tpr Deficiency Disrupts Erythroid Maturation With Impaired Chromatin Condensation in Zebrafish Embryogenesis.

Vertebrate erythropoiesis involves nuclear and chromatin condensation at the early stages of terminal differentiation, which is a unique process to distinguish mature erythrocytes from erythroblasts. However, the underlying mechanisms of chromatin condensation during erythrocyte maturation remain elusive. Here, we reported a novel zebrafish mutant cas7 with erythroid maturation deficiency. Positional cloning showed that a single base mutation in tprb gene, which encodes nucleoporin translocated promoter region (Tpr), is responsible for the disrupted erythroid maturation and upregulation of erythroid genes, including ae1-globin and be1-globin. Further investigation revealed that deficient erythropoiesis in tprb cas7 mutant was independent on HIF signaling pathway. The proportion of euchromatin was significantly increased, whereas the percentage of heterochromatin was markedly decreased in tprb cas7 mutant. In addition, TPR knockdown in human K562 cells also disrupted erythroid differentiation and dramatically elevated the expression of globin genes, which suggests that the functions of TPR in erythropoiesis are highly conserved in vertebrates. Taken together, this study revealed that Tpr played vital roles in chromatin condensation and gene regulation during erythroid maturation in vertebrates.

Surpassing 13% Efficiency for Polythiophene Organic Solar Cells Processed from Nonhalogenated Solvent.

Benefiting from low cost and simple synthesis, polythiophene (PT) derivatives are one of the most popular donor materials for organic solar cells (OSCs). However, polythiophene-based OSCs still suffer from inferior power conversion efficiency (PCE) than those based on donor-acceptor (D-A)-type conjugated polymers. Herein, a fluorinated polythiophene derivative, namely P4T2F-HD, is introduced to modulate the miscibility and morphology of the bulk heterojunction (BHJ)-active layer, leading to a significant improvement of the OSC performance. The Flory-Huggins interaction parameters calculated from the surface energy and differential scanning calorimetry results suggest that P4T2F-HD shows moderate miscibility with the popular nonfullerene acceptor Y6-BO (2,2'-((2Z,2'Z)-((12,13-bis(2-butyloctyl)-3,9-diundecyl-12,13-dihydro-[1,2,5]thiadiazolo[3,4-e]thieno[2',3':4',5']thieno[2',3':4,5]pyrrolo[3,2-g]thieno[2',3':4,5]thieno[3,2-b]indole-2,10-diyl)bis(methanylylidene))bis(5,6-difluoro-3-oxo-2,3-dihydro-1H-indene-2,1-diylidene))dimalononitrile), while poly(3-hexylthiophene) (P3HT) is very miscible with Y6-BO. As a result, the P4T2F-HD case forms desired nanoscale phase separation in the BHJ film while the P3HT case forms a completely mixed BHJ film, as revealed by transmission electron microscopy (TEM) and grazing-incidence wide-angle X-ray scattering (GIWAXS). By optimizing the cathode interface and the morphology of the P4T2F-HD:Y6-BO films processed from nonhalogenated solvents, a new record PCE of 13.65% for polythiophene-based OSCs is demonstrated. This work highlights the importance of controlling D/A interactions for achieving desired morphology and also demonstrates a promising OSC system for potential cost-effective organic photovoltaics.

Exosomal MicroRNA-126 from RIPC Serum Is Involved in Hypoxia Tolerance in SH-SY5Y Cells by Downregulating DNMT3B.

Ischemic tolerance in the brain can be induced by transient limb ischemia, and this phenomenon is termed remote ischemic preconditioning (RIPC). It still remains elusive how this transfer of tolerance occurs. Exosomes can cross the blood-brain barrier, and some molecules may transfer neuroprotective signals from the periphery to the brain. Serum miRNA-126 is associated with ischemic stroke, and exosomal miRNA-126 has shown protective effects against acute myocardial infarction. Therefore, this study aims to explore whether exosomal miRNA-126 from RIPC serum can play a similar neuroprotective role. Exosomes were isolated from the venous serum of four healthy young male subjects, both before and after RIPC. Exosomal miRNA-126 was measured by real-time PCR. The miRNA-126 target sequence was predicted by bioinformatics software. SH-SY5Y neuronal cells were incubated with exosomes, and the cell cycle was analyzed by flow cytometry. The expression and activity of DNA methyltransferase (DNMT) 3B, a potential target gene of miRNA-126, were examined in SH-SY5Y cells. The cell viability of SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD) was also investigated. To confirm the association between miRNA-126 and DNMT3B, we overexpressed miRNA-126 in SH-SY5Y cells using lentiviral transfection. miRNA-126 expression was upregulated in RIPC exosomes, and bioinformatics prediction showed that miRNA-126 could bind with DNMT3B. DNMT levels and DNMT3B activity were downregulated in SH-SY5Y cells incubated with RIPC exosomes. After overexpression of miRNA-126 in SH-SY5Y cells, global methylation levels and DNMT3B gene expression were downregulated in these cells, consistent with the bioinformatics predictions. RIPC exosomes can affect the cell cycle and increase OGD tolerance in SH-SY5Y cells. RIPC seems to have neuroprotective effects by downregulating the expression of DNMTs in neural cells through the upregulation of serum exosomal miRNA-126.

Nd2O3 Nanoparticles Induce Toxicity and Cardiac/Cerebrovascular Abnormality in Zebrafish Embryos via the Apoptosis Pathway.

INTRODUCTION: Rare-earth nanoparticles in the environment and human body pose a potential threat to human health. Although toxic effects of rare-earth nanoparticles have been extensively studied, the effects on the early development are not well understood. In this study, we attempted to explain the toxic effects of neodymium oxide (Nd2O3) nanoparticles on early development. METHODS: We added the Nd2O3 nanoparticles at different concentrations and recorded the mortality and malformation rate per 24 hrs under a microscope. The live embryos treated with Nd2O3 nanoparticles were imaged as movies and Z step lapses with a confocal microscope, and heart rates were counted for 30 s to measure the cardiac function. The live Tg (Flk1:EGFP) transgenic embryos exposed to Nd2O3 nanoparticles were observed under confocal microscope to measure the cerebrovascular development. Subsequently, we extracted the total protein for Western blot at 5 days post-fertilisation (dpf). Embryos were collected to undergo TUNEL staining for apoptosis detection. RESULTS: Nd2O3 nanoparticles disturbed embryo development at high concentrations (>200 µg/mL). The mortality and malformation rate gradually increased in a dose-dependent manner by morphological observation, while the Nd2O3 median lethal concentration (LD50) was 203.4 µg/mL at 120 hrs post-fertilisation (hpf). Furthermore, the Nd2O3-treated embryos showed severe arrhythmia and reduced heart rate. We also observed the markedly cerebrovascular disappearance at middle concentration (100 and 200 µg/mL). The downregulated autophagy flux in brain blood vessels and increased apoptosis level in neurons might affect vessels sprouting and contribute to the vanished cerebrovascular. CONCLUSION: The results suggested that the embryos exposed to Nd2O3 activated the apoptosis pathway and induced toxicity and abnormal cardiac/cerebrovascular development.

5-Aza-2'-deoxycytidine increases hypoxia tolerance-dependent autophagy in mouse neuronal cells by initiating the TSC1/mTOR pathway.

BACKGROUND: Our previous study found that 5-Aza-2'-deoxycytidine (5-Aza-CdR) can repress the expression and activity of protein serine/threonine phosphatase-1γ (PP1γ) in mouse hippocampus. It is well known that PP1γ regulates cell metabolism, which is related to hypoxia/ischaemia tolerance. It has been reported that it can also induce autophagy in cancer cells. Autophagy is important for maintaining cellular homeostasis associated with metabolism. In this study, we examined whether 5-Aza-CdR increases hypoxia tolerance-dependent autophagy by initiating the TSC1/mTOR/autophagy signalling pathway in neuronal cells. METHODS: 5-Aza-CdR was either administered to mice via intracerebroventricular injection (i.c.v) or added to cultured hippocampal-derived neuronal cell line (HT22 cell) in the medium for cell culture. The hypoxia tolerance of mice was measured by hypoxia tolerance time and Perl's iron stain. The mRNA and protein expression levels of tuberous sclerosis complex 1 (TSC1), mammalian target of rapamycin (mTOR) and autophagy marker light chain 3 (LC3) were measured by real-time PCR and western blot. The p-mTOR and p-p70S6k proteins were used as markers for mTOR activity. In addition, the role of autophagy was determined by correlating its intensity with hypoxia tolerance in a time-dependent manner. At the same time, the involvement of the TSC1/mTOR pathway in autophagy was also examined through transfection with TSC1 (hamartin) plasmid. RESULTS: 5-Aza-CdR was revealed to increase hypoxia tolerance and induce autophagy, accompanied by an increase in mRNA and protein expression levels of TSC1, reduction in p-mTOR (Ser2448) and p-p70S6k (Thr389) protein levels, and an increase in the ratio of LC3-II/LC3-I in both mouse hippocampus and hippocampal-derived neuronal cell line (HT22). The fluorescence intensity of hamartin was enhanced in the hippocampus of mice exposed to 5-Aza-CdR. Moreover, HT22 cells that over-expressed TSC1 showed more autophagy. CONCLUSIONS: 5-Aza-CdR can increase hypoxia tolerance by inducing autophagy by initiating the TSC1/mTOR pathway.

Effects of 5-Aza on p-Y1472 NR2B related to learning and memory in the mouse hippocampus.

BACKGROUND: We have previously reported that 5-Aza-2-deoxycytidine (5-Aza-cdR) can repress protein serine/threonine phosphatase-1γ (PP1γ) expression and activity in the mouse hippocampus and affect the behaviour of mice in a water maze. It is well known that the phosphorylation of N-methyl-d-aspartate receptor 2B subunit (NR2B) plays a role in behaviour. In this study, we examined whether 5-Aza-cdR affects NR2B phosphorylation at tyrosine 1472 (p-Y1472 NR2B) and whether it affected the responses of the mice in a passive avoidance test. METHODS: 5-Aza-cdR (10 µM) was administered to mice via intracerebroventricular injection (i.c.v). The learning and memory behaviour of the mice were evaluated by measuring their response in a step-down type passive avoidance test 24 h after the injection. The mRNA level of NR2B was measured by real-time PCR. NR2B and p-Y1472 NR2B protein expression in the mouse hippocampus was detected by western blot and immunofluorescence. CDK5 activity was detected by the ADP-Glo™ + CDK5/p35 Kinase Enzyme System. To further clarify whether the 5-Aza-cdR effects on behaviour were dependent on cellular proliferation or not, the effect of 5-Aza-cdR on the expression level of NR2B, the phosphorylation level of p-Y1472 NR2B, cell viability and the cell cycle were analysed using the immortalized mouse hippocampal neuronal cells neural cell line HT22 treated with 10 µM 5-Aza-cdR compared with an untreated control group. RESULTS: After injection with 5-Aza-cdR, the behaviour of the mice in the step-down test was improved, while their phosphorylation level of p-Y1472 NR2B was increased and their CDK5 activity was decreased in the hippocampus. In vitro experiments showed 10 µM 5-Aza-cdR increased the p-Y1472 NR2B phosphorylation level with inhibition of cell viability and cell cycle arrest. CONCLUSIONS: Our results suggested that the effect of 5-Aza-cdR on behaviour may be related to the increase in phosphorylation of p-Y1472 NR2B in the hippocampus.

Electron Acceptors With a Truxene Core and Perylene Diimide Branches for Organic Solar Cells: The Effect of Ring-Fusion.

In this work, a star-shaped planar acceptor named FTr-3PDI was synthesized via ring-fusion between truxene core and three bay-linked perylene diimide (PDI) branches. Compared to the unfused non-planar acceptor Tr-3PDI, FTr-3PDI exhibits better structural rigidity and planarity, as well as more effective conjugation between truxene core and PDI branches. As a result, FTr-3PDI shows up-shifted energy levels, enhanced light absorption coefficient, increased electron mobility, and more favorable phase separation morphology in bulk-heterojunction (BHJ) blend films as compared to Tr-3PDI. Consequently, FTr-3PDI afforded higher power conversion efficiency (PCE) in BHJ solar cells when blended with a polymer donor PTB7-Th. This work demonstrates that ring-fusion is a promising molecular design strategy to combine the merits of truxene and PDI for non-fullerene acceptors used in organic solar cells.

The histone demethylase Jmjd3 regulates zebrafish myeloid development by promoting spi1 expression.

The histone demethylase Jmjd3 plays a critical role in cell lineage specification and differentiation at various stages of development. However, its function during normal myeloid development remains poorly understood. Here, we carried out a systematic in vivo screen of epigenetic factors for their function in hematopoiesis and identified Jmjd3 as a new epigenetic factor that regulates myelopoiesis in zebrafish. We demonstrated that jmjd3 was essential for zebrafish primitive and definitive myelopoiesis, knockdown of jmjd3 suppressed the myeloid commitment and enhanced the erythroid commitment. Only overexpression of spi1 but not the other myeloid regulators rescued the myeloid development in jmjd3 morphants. Furthermore, preliminary mechanistic studies demonstrated that Jmjd3 could directly bind to the spi1 regulatory region to alleviate the repressive H3K27me3 modification and activate spi1 expression. Thus, our studies highlight that Jmjd3 is indispensable for early zebrafish myeloid development by promoting spi1 expression.

5-Aza-2'-deoxycytidine, a DNA methylation inhibitor, induces cytotoxicity, cell cycle dynamics and alters expression of DNA methyltransferase 1 and 3A in mouse hippocampus-derived neuronal HT22 cells.

Epigenetic processes such as DNA methylation are essential for processes of gene expression in normal mammalian development. DNA methyltransferases (DNMT) are responsible for initiating and maintaining DNA methylation. It is known that 5-Aza-CdR, an inhibitor of DNMT induces cytotoxicity by reducing DNMT activity in various tumor cell lines. However, disturbances in neuronal DNA methylation may also play a role in altered brain functions. Thus, it was of interest to determine whether alterations in DNA methylation might be associated with neuronal functions by using 5-Aza-CdR, on mouse hippocampus-derived neuronal HT22 cell line. In particular, the aim of this study was to investigate the effects of 5-Aza-CdR on cell growth inhibition, cell cycle arrest, apoptosis as well as the expression levels of DNMT in HT22 cells. HT22 cells were incubated with 5 or 20 µmol/L 5-Aza-CdR for 24 h. Data showed that 5-Aza-CdR at both concentrations significantly inhibited proliferation of HT22 cells and exacerbated cytoplasmic vacuolization. Flow cytometry analysis demonstrated that 5-Aza-CdR treatment at both concentrations decreased early apoptosis but enhanced late apoptosis. Cell cycle analysis illustrated that 5-Aza-CdR treatment induced S phase arrest. Further, incubation with 5-Aza-CdR produced a down-regulation in expression of mRNA and protein DNMT1 and 3A but no marked changes were noted in DNMT 3B and p21 expression. In addition, DNMT1 activity was significantly decreased at both 5-Aza-CdR concentrations. Evidence indicates that 5-Aza-CdR induced cytotoxicity was associated with altered mRNA and protein expression of DNMT 1 and 3A associated with reduced DNMT1 activity in HT22 cells which might affect brain functions.

An Open-Circuit Voltage and Power Conversion Efficiency Study of Fullerene Ternary Organic Solar Cells Based on Oligomer/Oligomer and Oligomer/Polymer.

Variations in the open-circuit voltage (Voc ) of ternary organic solar cells are systematically investigated. The initial study of these devices consists of two electron-donating oligomers, S2 (two units) and S7 (seven units), and the electron-accepting [6,6]-phenyl C71 butyric acid methyl ester (PC71 BM) and reveals that the Voc is continuously tunable due to the changing energy of the charge transfer state (Ect ) of the active layers. Further investigation suggests that Voc is also continuously tunable upon change in Ect in a ternary blend system that consists of S2 and its corresponding polymer (P11):PC71 BM. It is interesting to note that higher power conversion efficiencies can be obtained for both S2:S7:PC71 BM and S2:P11:PC71 BM ternary systems compared with their binary systems, which can be ascribed to an improved Voc due to the higher Ect and an improved fill factor due to the improved film morphology upon the incorporation of S2. These findings provide a new guideline for the future design of conjugated polymers for achieving higher performance of ternary organic solar cells.

Effects of 5-Aza-2'-deoxycytidine on expression of PP1γ in learning and memory.

BACKGROUND: Learning and memory is a complex process. Some reports have shown that protein kinases (PKs) and phosphatases (PPs) are important mediators in this process. And it is also well known that protein serine/threonine phosphatase 1 (PP1) and DNA methylation are critically involved in learning and memory. METHODS: In the current study, the mice and cultured cells (NG108-15) were treated with vehicle or 5-Aza-2'-deoxycytidine (5-aza-cdR), a DNA methyltransferase (DNMT) inhibitor. The ability of learning and memory of mice was detected by Morris Water Maze, while the mRNA and protein expression levels of DNMTs and PP1γ in mice hippocampus were measured by real-time PCR and western-blot. To further clarify whether the 5-aza-cdR effects on learning and memory depend on cell proliferation/apoptosis or not, the effects of 5-aza-cdR on the cell proliferation, apoptosis, and PP1γ transcriptional activity were analyzed by using the xCelligence system, flow cytometer and Luciferase reporter assay, respectively. RESULTS: The ability of learning and memory was increased while the expressions of DNMTs and PP1γ were decreased in the hippocampus of mice which were injected with 5-aza-cdR. In vitro experiments showed 10µM 5-aza-cdR inhibited cell proliferation, decreased PP1γ transcription without inducing apoptosis. CONCLUSION: Our data demonstrate that the 5-aza-cdR restrains the expression of PP1γ which is related to learning and memory in the mice.

TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis.

In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II ß binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.

Mutation of kri1l causes definitive hematopoiesis failure via PERK-dependent excessive autophagy induction.

Dysregulation of ribosome biogenesis causes human diseases, such as Diamond-Blackfan anemia, del (5q-) syndrome and bone marrow failure. However, the mechanisms of blood disorders in these diseases remain elusive. Through genetic mapping, molecular cloning and mechanism characterization of the zebrafish mutant cas002, we reveal a novel connection between ribosomal dysfunction and excessive autophagy in the regulation of hematopoietic stem/progenitor cells (HSPCs). cas002 carries a recessive lethal mutation in kri1l gene that encodes an essential component of rRNA small subunit processome. We show that Kri1l is required for normal ribosome biogenesis, expansion of definitive HSPCs and subsequent lineage differentiation. Through live imaging and biochemical studies, we find that loss of Kri1l causes the accumulation of misfolded proteins and excessive PERK activation-dependent autophagy in HSPCs. Blocking autophagy but not inhibiting apoptosis by Bcl2 overexpression can fully rescue hematopoietic defects, but not the lethality of kri1l(cas002) embryos. Treatment with autophagy inhibitors (3-MA and Baf A1) or PERK inhibitor (GSK2656157), or knockdown of beclin1 or perk can markedly restore HSPC proliferation and definitive hematopoietic cell differentiation. These results may provide leads for effective therapeutics that benefit patients with anemia or bone marrow failure caused by ribosome disorders.

DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish.

BACKGROUND: DNA methyltransferase 1 (Dnmt1) regulates expression of many critical genes through maintaining parental DNA methylation patterns on daughter DNA strands during mitosis. It is essential for embryonic development and diverse biological processes, including maintenance of hematopoietic stem and progenitor cells (HSPCs). However, the precise molecular mechanism of how Dnmt1 is involved in HSPC maintenance remains unexplored. METHODS: An N-ethyl-N-nitrosourea (ENU)-based genetic screening was performed to identify putative mutants with defects in definitive HSPCs during hematopoiesis in zebrafish. The expression of hematopoietic markers was analyzed via whole mount in situ hybridization assay (WISH). Positional cloning approach was carried out to identify the gene responsible for the defective definitive hematopoiesis in the mutants. Analyses of the mechanism were conducted by morpholino-mediated gene knockdown, mRNA injection rescue assays, anti-phosphorylated histone H3 (pH3) immunostaining and TUNEL assay, quantitative real-time PCR, and bisulfite sequencing analysis. RESULTS: A heritable mutant line with impaired HSPCs of definitive hematopoiesis was identified. Positional cloning demonstrated that a stop codon mutation was introduced in dnmt1 which resulted in a predicted truncated Dnmt1 lacking the DNA methylation catalytic domain. Molecular analysis revealed that expression of CCAAT/enhancer-binding protein alpha (C/ebpa) was upregulated, which correlated with hypomethylation of CpG islands in the regulation regions of cebpa gene in Dnmt1 deficient HSPCs. Overexpression of a transcriptional repressive SUMO-C/ebpa fusion protein could rescue hematological defects in the dnmt1 mutants. Finally, dnmt1 and cebpa double null embryos exhibited no obvious abnormal hematopoiesis indicated that the HSPC defects triggered by dnmt1 mutation were C/ebpa dependent. CONCLUSIONS: Dnmt1 is required for HSPC maintenance via cebpa regulation during definitive hematopoiesis in zebrafish.

GSTT1 deletion is related to polycyclic aromatic hydrocarbons-induced DNA damage and lymphoma progression.

The interrelationship between genetic susceptibility and carcinogenic exposure is important in cancer development. Polymorphisms in detoxification enzymes of the glutathione-S-transferases (GST) family are associated with an increased incidence of lymphoma. Here we investigated the molecular connection of the genetic polymorphism of GSTT1 to the response of lymphocytes to polycyclic aromatic hydrocarbons (PAH). In neoplastic situation, GSTT1 deletions were more frequently observed in lymphoma patients (54.9%) than in normal controls (42.0%, P = 0.009), resulting in an increased risk for lymphoma in individuals with GSTT1-null genotype (Odds ratio = 1.698, 95% confidence interval = 1.145-2.518). GSTT1 gene and protein expression were accordingly decreased in GSTT1-deleting patients, consistent with activated profile of cell cycle regulation genes. Mimicking environmental exposure using long-term repeat culture with low-dose PAH metabolite Hydroquinone, malignant B- and T-lymphocytes presented increased DNA damage, pCHK1/MYC expression and cell proliferation, which were counteracted by ectopic expression of GSTT1. Moreover, GSTT1 expression retarded xenograft tumor formation of Hydroquinone-treated lymphoma cells in nude mice. In non-neoplastic situation, when zebrafish was exposed to PAH Benzo(a)pyrene, molecular silencing of gstt1 enhanced the proliferation of normal lymphocytes and upregulated myca expression. Collectively, these findings suggested that GSTT1 deletion is related to genetic predisposition to lymphoma, particularly interacting with environmental pollutants containing PAH.

Activated N-Ras signaling regulates arterial-venous specification in zebrafish.

BACKGROUND: The aberrant activation of Ras signaling is associated with human diseases including hematological malignancies and vascular disorders. So far the pathological roles of activated Ras signaling in hematopoiesis and vasculogenesis are largely unknown. METHODS: A conditional Cre/loxP transgenic strategy was used to mediate the specific expression of a constitutively active form of human N-Ras in zebrafish endothelial and hematopoietic cells driven by the zebrafish lmo2 promoter. The expression of hematopoietic and endothelial marker genes was analyzed both via whole mount in situ hybridization (WISH) assay and real-time quantitative PCR (qPCR). The embryonic vascular morphogenesis was characterized both by living imaging and immunofluorescence on the sections with a confocal microscopy, and the number of endothelial cells in the embryos was quantified by flow cytometry. The functional analyses of the blood circulation were carried out by fluorescence microangiography assay and morpholino injection. RESULTS: In the activated N-Ras transgenic embryos, the primitive hematopoiesis appeared normal, however, the definitive hematopoiesis of these embryos was completely absent. Further analysis of endothelial cell markers confirmed that transcription of arterial marker ephrinB2 was significantly decreased and expression of venous marker flt4 excessively increased, indicating the activated N-Ras signaling promotes the venous development at the expense of arteriogenesis during zebrafish embryogenesis. The activated N-Ras-expressing embryos showed atrophic axial arteries and expansive axial veins, leading to no definitive hematopoietic stem cell formation, the blood circulation failure and subsequently embryonic lethality. CONCLUSIONS: Our studies revealed for the first time that activated N-Ras signaling during the endothelial differentiation in vertebrates can disrupt the balance of arterial-venous specification, thus providing new insights into the pathogenesis of the congenital human vascular disease and tumorigenic angiogenesis.