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FOXC1 is a member of the forkhead family of transcription factors, and whose function is poorly understood. A variety of FOXC1 mutants have been identified in patients diagnosed with the autosomal dominant disease Axenfeld-Rieger syndrome, which is mainly characterized by abnormal development of the eyes, particularly those who also have accompanying congenital heart defects (CHD). However, the role of FOXC1 in CHD, and how these mutations might impact FOXC1 function, remains elusive. Our previous work provided one clue to possible function, demonstrating that zebrafish foxc1a, an orthologue of human FOXC1 essential for heart development, directly regulates the expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells. Abnormal expression of Nkx2-5 leads to CHD in mice and is also associated with CHD patients. Whether this link extends to the human system, however, requires investigation. In this study, we demonstrate that FOXC1 does regulate human NKX2-5 expression in a dose-dependent manner via direct binding to its proximal promoter. A comparison of FOXC1 mutant function in the rat cardiac cell line H9c2 and zebrafish embryos suggested that the zebrafish embryos might serve as a more representative model system than the H9c2 cells. Finally, we noted that three of the Axenfeld-Rieger syndrome FOXC1 mutations tested increased, whereas a fourth repressed the expression of NKX2-5. These results imply that mutant FOXC1s might play etiological roles in CHD by abnormally regulating NKX2-5 in the patients. And zebrafish embryos can serve as a useful in vivo platform for rapidly evaluating disease-causing roles of mutated genes.

2019-Novel Coronavirus (2019-nCoV) is the pathogen of Corona Virus Disease 2019. Nucleic acid detection of 2019-nCoV is one of the key indicators for clinical diagnosis. However, the positive rate is only 30-50%. Currently, fluorescent quantitative RT-PCR technology is mainly used to detect 2019-nCoV. According to “The Laboratory Technical Guidelines for Detection 2019-nCoV (Fourth Edition)” issued by National Health and Commission of China and “The Experts’ Consensus on Nucleic Acid Detection of 2019-nCoV” released by Chinese Society of Laboratory Medicine, the human samples must be placed under 56°C or higher to inactivate the viruses in order to keep the inspectors from virus infection before the nucleic acids were isolated as the template of qRT-PCR. In this study, we demonstrated that the virus inactivation treatment disrupts its genome integrity seriously when using porcine epidemic diarrhea virus (vaccine), a kind of coronavirus, as a model. Our results showed that only 50.11% of the detectable viral templates left after the inactivation of 56 °C for 30 minutes and only 3.36% left after the inactivation of 92 °C for 5 minutes when the samples were preserved by Hank’s solution, one of an isotonic salt solutions currently suggested. However, the detectable templates of viral nucleic acids can be unchanged after the samples were incubated at 56 °C or higher if the samples were preserved with an optimized solution to protect the RNA from being disrupted. We therefore highly recommend to carry out systematic investigation on the impact of high temperature inactivation on the integrity of 2019-nCoV genome and develop a sample preservation solution to protect the detectable templates of 2019-nCoV nucleic acids from high temperature inactivation damage.

Cardiogenesis is a tightly controlled biological process required for formation of a functional heart. The transcription factor Foxc1 not only plays a crucial role in outflow tract development in mice, but is also involved in cardiac structure formation and normal function in humans. However, the molecular mechanisms by which Foxc1 controls cardiac development remain poorly understood. Previously, we reported that zebrafish embryos deficient in foxc1a, an ortholog of mammalian Foxc1, display pericardial edemas and die 9–10 days postfertilization. To further investigate Foxc1a's role in zebrafish cardiogenesis and identify its downstream target genes during early heart development, we comprehensively analyzed the cardiovascular phenotype of foxc1a-null zebrafish embryos. Our results confirmed that foxc1a-null mutants exhibit disrupted cardiac morphology, structure, and function. Performing transcriptome analysis on the foxc1a mutants, we found that the expression of the cardiac progenitor marker gene nkx2.5 was significantly decreased, but the expression of germ layer–patterning genes was unaffected. Dual-fluorescence in situ hybridization assays revealed that foxc1a and nkx2.5 are co-expressed in the anterior lateral plate mesoderm at the somite stage. Chromatin immunoprecipitation and promoter truncation assays disclosed that Foxc1a regulates nkx2.5 expression via direct binding to two noncanonical binding sites in the proximal nkx2.5 promoter. Moreover, functional rescue experiments revealed that developmental stage–specific nkx2.5 overexpression partially rescues the cardiac defects of the foxc1a-null embryos. Taken together, our results indicate that during zebrafish cardiogenesis, Foxc1a is active directly upstream of nkx2.5.

Glioma is the most common type of primary brain tumor and has an undesirable prognosis. Autophagy plays an important role in cancer therapy, but it is effect is still not definite. P53 is an important tumor suppressor gene and protein that is closely to autophagy. Our aim was to study the effect of β-asarone on inhibiting cell proliferation in human glioma U251 cells and to detect the effect of the inhibition on autophagy through the P53 signal pathway. For cell growth, the cells were divided into four groups: the model, β-asarone, temozolomide (TMZ), and co-administration groups. For cell autoghapy and the P53 pathway, the cells were divided into six groups: the model, β-asarone, 3MA, Rapa, Pifithrin-µ, and NSC groups. The counting Kit-8 assay and flow cytometry (FCM) were then used to measure the cell proliferation and cycle. Electron microscopy was used to observe autophagosome formation. Cell immunohistochemistry/-immunofluorescence, FCM and Western blot (WB) were used to examine the expression of Beclin-1 and P53. The levels of P53 and GAPDH mRNA were detected by RT-PCR. Using WB, we determined autophagy-related proteins Beclin-1, LC3-II/I, and P62 and those of the P53 pathway-related proteins P53, Bcl-2, mTOR, P-mTOR, AMPK, P-AMPK, and GAPDH. We got the results that β-asarone changed the cellular morphology, inhibited cell proliferation, and enhanced the expression of P53, LC3-II/I, Beclin-1, AMPK, and pAMPK while inhibiting the expression of P62, Bcl-2, mTOR, and pmTOR. All the data suggested that β-asarone could reduce the cell proliferation and promote autophagy possible via the P53 pathway in U251 cells.

Transcription factors play crucial roles in patterning posterior neuroectoderm. Previously, zinc finger transcription factor znfl1 was reported to be expressed in the posterior neuroectoderm of zebrafish embryos. However, its roles remain unknown. Here, we report that there are 13 copies of znfl1 in the zebrafish genome, and all the paralogues share highly identical protein sequences and cDNA sequences. When znfl1s are knocked down using a morpholino to inhibit their translation or dCas9-Eve to inhibit their transcription, the zebrafish gastrula displays reduced expression of hoxb1b, the marker gene for the posterior neuroectoderm. Further analyses reveal that diminishing znfl1s produces the decreased expressions of pou5f3, whereas overexpression of pou5f3 effectively rescues the reduced expression of hoxb1b in the posterior neuroectoderm. Additionally, knocking down znfl1s causes the reduced expression of sall4, a direct regulator of pou5f3, in the posterior neuroectoderm, and overexpression of sall4 rescues the expression of pou5f3 in the knockdown embryos. In contrast, knocking down either pou5f3 or sall4 does not affect the expressions of znfl1s. Taken together, our results demonstrate that zebrafish znfl1s control the expression of hoxb1b in the posterior neuroectoderm by acting upstream of pou5f3 and sall4.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly, and studies have suggested that β-asarone has pharmacological effects on beta-amyloid (Aβ) injected in the rat hippocampus. However, the effect of β-asarone on autophagy in the APP/PS1 transgenic mouse is unreported. APP/PS1 transgenic mice were randomly divided into six groups (n=10/group): an untreated group, an Aricept-treated group, a 3-MA-treated group, a rapamycin-treated group, an LY294002-treated group, a β-asarone-treated group. The control group consisted of wild-type C57BL/6 mice. All treatments were administered to the mice for 30 days. Spatial learning and memory were assessed by water maze, passive avoidance, and step-down tests. AChE and Aβ42 levels in the hippocampus were determined by ELISA. p-Akt, p-mTOR, and LC3B expression were detected by flow cytometry. The expression of p-Akt, p-mTOR, Beclin-1, and p62 proteins was assessed by western blot. Changes in autophagy were viewed using a transmission electron microscope. APP and Beclin-1 mRNA levels were measured by Real-Time PCR. The learning and memory of APP/PS1 transgenic mice were improved significantly after β-asarone treatment compared with the untreated group. In addition, β-asarone treatment reduced AChE and Aβ42 levels, increased p-mTOR and p62 expression, decreased p-Akt, Beclin-1, and LC3B expression, decreased the number of autophagosomes and reduced APP mRNA and Beclin-1 mRNA levels compared with the untreated group. That is, β-asarone treatment can improve the learning and memory abilities of APP/PS1 transgenic mouse by inhibiting Beclin-1-dependent autophagy via the PI3K/Akt/mTOR pathway.

Parkinson's disease (PD) is a neurodegenerative disease, with genetics and environment contributing to the disease onset. The limited pathological cognize of the disease restrained the approaches to improve the clinical treatment. Recently, studies showed that endoplasmic reticulum (ER) stress played an important role in the pathogenesis of PD. There was a neuroprotective effect partly mediated by modulating ER stress. β-Asarone is the essential constituent of Acorus tatarinowii Schott volatile oil. Our team observed that β-asarone could improve the behavior of parkinsonian rats; increase the HVA, Dopacl, and 5-HIAA levels; and reduce α-synuclein levels. Here we assumed that the protective role of β-asarone on parkinsonian rats was mediated via ER stress pathway. To prove the hypothesis we investigated the mRNA levels of glucose regulated protein 78 (GRP78) and C/EBP homologous binding protein (CHOP) in 6-hydroxy dopamine (6-OHDA) induced parkinsonian rats after β-asarone treatment. Furthermore, the inositol-requiring enzyme 1/X-Box Binding Protein 1 (IRE1/XBP1) ER stress pathway was also studied. The results showed that β-asarone inhibited the mRNA levels of GRP78 and CHOP, accompanied with the delined expressions of phosphorylated IER1 (p-IRE1) and XBP1. We deduced that β-asarone might have a protective effect on the 6-OHDA induced parkinsonian rats via IRE1/XBP1 Pathway. Collectively, all data indicated that β-asarone might be a potential candidate of medicine for clinical therapy of PD.

β-Asarone (1,2,4-trimethoxy-5-[(Z)-prop-1-enyl]benzene) is an essential component of Acorus tatarinowii Schott volatile oil. Previous research has observed that β-asarone effectively attenuated symptoms in parkinsonian rats and improved their performance, but the mechanism of this effect remains unclear. Other research has shown that endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of Parkinson's disease (PD). The protein kinase RNA-like endoplasmic reticulum kinase (PERK) was observed in the nigrostriatal dopaminergic neurons of patients with PD. However, our group observed that ER stress and autophagy occurred in 6-hydroxy dopamine (6-OHDA)-induced parkinsonian rats, and ER stress might induce autophagy. We assume that the protective role of β-asarone in parkinsonian rats is mediated via the ER stress-autophagy pathway. To support this hypothesis, we investigated the expressions of glucose regulated protein 78 (GRP78), PERK phosphorylation (p-PERK), C/EBP homologous binding protein (CHOP), Bcl-2 and Beclin-1 in 6-OHDA-induced parkinsonian rats after β-asarone treatment. The results showed that the β-asarone group and PERK inhibitor group had lower levels of GRP78, p-PERK, CHOP and Beclin-1 while having higher levels of Bcl-2. We deduced that β-asarone might regulate the ER stress-autophagy via inhibition of the PERK/CHOP/Bcl-2/Beclin-1 pathway in 6-OHDA-induced parkinsonian rats.

Both endoplasmic reticulum (ER) stress and autophagy involve in the pathological process of Parkinson's disease (PD). But the relationship between them is not clear in PD. A 6-OHDA-induced parkinsonian rat is recognized as a standard model for many years, and it can be used in experimental study. The glucose regulated protein 78 (GRP78) is a master regulator of ER stress, and the C/EBP homologous binding protein (CHOP) is an indicator of the UPR signaling. Besides, the Beclin-1 is also well known as a regulator of autophagy, and P62 is a specific marker to monitor autophagy. Therefore, we investigated the expressions of GRP78, CHOP, Beclin-1 and P62 in 6-OHDA-induced parkinsonian rat. Unilateral 6-OHDA injection into medial forebrain bundle was used except sham-operated rats. The rats were randomly divided into 6 groups: a sham-operated group; a model group; a 3-methyladenine (3-MA) group, administered 3-MA---autophagy inhibitor; a rapamycin group, administered rapamycin---autophagy inducer; a 4-phenylbutyric acids (4-PBA) group, administered 4-PBA---ER stress inhibitor; a tunicamycin (TM) group, administered TM---ER stress inducer. The results showed that the expressions of GRP78, CHOP and Beclin-1 increased, P62 decreased in model group; the expressions of GRP78 and CHOP were unchanged in 3-MA group and rapamycin group; but the expression of Beclin-1 decreased and P62 increased in 4-PBA group, while the expression of Beclin-1 increased and P62 decreased in TM group. These data suggest that ER stress and autophagy occurred in 6-OHDA-induced parkinsonian rat, and ER stress might induce autophagy. The result is important for the pathological mechanism of PD.

Foxc1a is a member of the forkhead transcription factors. It plays an essential role in zebrafish somitogenesis. However, little is known about the molecular mechanisms underlying its controlling somitogenesis. To uncover how foxc1a regulates zebrafish somitogenesis, we generated foxc1a knock-out zebrafish using TALEN (transcription activator-like effector nuclease) technology. The foxc1a null embryos exhibited defective somites at early development. Analyses on the expressions of the key genes that control processes of somitogenesis revealed that foxc1a controlled early somitogenesis by regulating the expression of myod1. In the somites of foxc1a knock-out embryos, expressions of fgf8a and deltaC were abolished, whereas the expression of aldh1a2 (responsible for providing retinoic acid signaling) was significantly increased. Once the increased retinoic acid level in the foxc1a null embryos was reduced by knocking down aldh1a2, the reduced expression of myod1 was partially rescued by resuming expressions of fgf8a and deltaC in the somites of the mutant embryos. Moreover, a chromatin immunoprecipitation assay on zebrafish embryos revealed that Foxc1a bound aldh1a2 promoter directly. On the other hand, neither knocking down fgf8a nor inhibiting Notch signaling affected the expression of aldh1a2, although knocking down fgf8a reduced expression of deltaC in the somites of zebrafish embryos at early somitogenesis and vice versa. Taken together, our results demonstrate that foxc1a plays an essential role in early somitogenesis by controlling Fgf and Notch signaling through restricting the expression of aldh1a2 in paraxial mesoderm directly.

Meiotic recombination is initiated from the formation of DNA double-strand breaks (DSBs). In Arabidopsis, several proteins, such as AtPRD1, AtPRD2, AtPRD3, AtDFO and topoisomerase (Topo) VI-like complex, have been identified as playing important roles in DSB formation. Topo VI-like complex in Arabidopsis may consist of subunit A (Topo VIA: AtSPO11-1 and AtSPO11-2) and subunit B (Topo VIB: MTOPVIB). Little is known about their roles in Arabidopsis DSB formation. Here, we report on the characterization of the MTOPVIB gene using the Arabidopsis mutant alleles mtopVIB-2 and mtopVIB-3, which were defective in DSB formation. mtopVIB-3 exhibited abortion in embryo sac and pollen development, leading to a significant reduction in fertility. The mtopVIB mutations affected the homologous chromosome synapsis and recombination. MTOPVIB could interact with Topo VIA proteins AtSPO11-1 and AtSPO11-2. AtPRD1 interacted directly with Topo VI–like proteins. AtPRD1 also could interact with AtPRD3 and AtDFO. The results indicated that AtPRD1 may act as a bridge protein to interact with AtPRD3 and AtDFO, and interact directly with the Topo VI-like proteins MTOPVIB, AtSPO11-1 and AtSPO11-2 to take part in DSB formation in Arabidopsis.

Insulinoma-associated1a (insm1a) is a zinc-finger transcription factor playing a series of functions in cell formation and differentiation of vertebrate central and peripheral nervous systems and neuroendocrine system. However, its roles on the development of motor neuron have still remained uncovered. Here, we provided evidences that insm1a was a vital regulator of motor neuron development, and provided a mechanistic understanding of how it contributes to this process. Firstly, we showed the localization of insm1a in spinal cord, and primary motor neurons (PMNs) of zebrafish embryos by in situ hybridization, and imaging analysis of transgenic reporter line Tg(insm1a: mCherry)ntu805. Then we demonstrated that the deficiency of insm1a in zebrafish larvae lead to the defects of PMNs development, including the reduction of caudal primary motor neurons (CaP), and middle primary motor neurons (MiP), the excessive branching of motor axons, and the disorganized distance between adjacent CaPs. Additionally, knockout of insm1 impaired motor neuron differentiation in the spinal cord. Locomotion analysis showed that swimming activity was significantly reduced in the insm1a-null zebrafish. Furthermore, we showed that the insm1a loss of function significantly decreased the transcript levels of both olig2 and nkx6.1. Microinjection of olig2 and nkx6.1 mRNA rescued the motor neuron defects in insm1a deficient embryos. Taken together, these data indicated that insm1a regulated the motor neuron development, at least in part, through modulation of the expressions of olig2 and nkx6.1.

Insulinoma-associated1a (Insm1a) is a zinc-finger transcription factor playing a series of functions in cell formation and differentiation of vertebrate central and peripheral nervous systems and neuroendocrine system. However, its roles on the development of motor neuron have still remained uncovered. Here, we provided evidences thatinsmlawas a vital regulator of motor neuron development and provide a mechanistic understanding of how it contributes to this process. Firstly, we showed the localization ofinsmlain spinal cord and primary motor neurons (PMNs) of zebrafish embryos byin situhybridization and imaging analysis of transgenic reporter lineTg(insmla: mCherry)ntu805. Then we demonstrated that the deficiency ofinsmlain zebrafish larvae lead to the defects of PMNs development, including the reduction of caudal primary motor neurons (CaP) and middle primary motor neurons (MiP), the excessive branching of motor axons, and the disorganized distance between adjacent CaPs. Additionally, knockout ofinsm1impaired motor neuron differentiation in the spinal cord. Locomotion analysis showed thatinsmla-null zebrafish significantly reduced the swimming activity. Furthermore, we proved that theinsmlaloss of function significantly decreased the transcripts levels of botholig2andnkx6.1. Microinjection ofolig2andnkx6.1mRNA rescued the motor neuron defects ininsmladeficient embryos. Taken together, these data indicate thatinsmlaregulates the motor neuron development, at least in part, through modulation of the expressions ofolig2andnkx6.1.

Glioma is the most common primary brain tumor and has an undesirable prognosis due to the blood-brain barrier (BBB) and drug resistance. A thorough investigation of the changes in intracellular drug concentrations is important to observe therapeutic effects and cell resistance. P-glycoprotein (P-gp) is an essential protein of Multi-drug resistance 1 (MDR1). The over-expression of P-gp and MDR1 is associated with poor prognosis and drug-resistance in glioma. However, β-asarone can pass through the BBB easily and increase the drug concentration in the rat brain. Our aim is to study the effect of β-asarone on promoting the entry of temozolomide (TMZ) into human glioma U251 cells. The cells were divided into three groups: model group, TMZ group (300μM) and co-administration group (360μM β-asarone; 300μM TMZ). We further detected P-gp and MDR1 expression in U251 and rat glioma C6 cells in four groups: model group (U251/C6), TMZ group (U251 300μM, C6 420μM), β-asarone group (U251 360μM, C6 450μM) and co-administration group (β-asarone 360μM, TMZ 300μM for U251; β-asarone 450μM, TMZ 420μM for C6). Then, high performance liquid chromatography was used to determine the intracellular and extracellular levels of TMZ. Morphological changes in both cells were observed by the microscope. The Counting Kit-8 assay was used to measure the cell proliferation and toxicity. Cell immunohistochemistry/immunofluorescence, flowcytometry and western blot were synchronously used to examine the expression of P-gp. We also determined the levels of MDR1 mRNA by RT-PCR. The results showed that β-asarone could promote the entry of TMZ into U251 cells through the membrane. The co-administration of β-asarone and TMZ also decreased cell proliferation and the expression of P-gp and MDR1 better than single medication in U251 and C6 cells. All of the data suggest that β-asarone might contribute to treatment by promoting TMZ's entry into glioma cells, thereby contributing to anti-cancer growth and inhibiting P-gp and MDR1 expression.