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NPC1 gene encodes a transmembrane glycoprotein on the late endosome/lysosomal membrane. Its mutation leads to a rare and aggravated autosomal recessive neurovisceral condition, termed Niemann-Pick disease type C1 (NPC1), which is characterized by progressive neurodegeneration, visceral symptoms, and premature death. To investigate the influence of NPC1 gene deletion on cell morphology, adhesion, proliferation, and apoptosis, CRISPR-Cas9 technology was used to knockout the NPC1 gene in HEK 293 T cells. Sanger sequencing, western blotting, and immunofluorescence were used to confirm successful NPC1 ablation. Filipin staining results indicated that deletion of NPC1 gene led to accumulation of unesterified cholesterol in HEK 293 T cells. Phalloidin staining results revealed cell aggregation, synapse shortening, nuclear enlargement, and cytoskeleton filamentous actin thinning in HEK 293 T cells with NPC1 gene mutation. Furthermore, NPC1 gene mutated HEK 293 T cell showed enhanced cell adhesion, inhibited cell proliferation, and increased cell apoptosis. In addition, NPC1 gene mutations significantly increased the protein expression levels of E-cadherin and γ-catenin and significantly decreased the protein expression levels of Wnt 3a, c-Myc, and cyclin D1. These results suggest that NPC1 may regulate cell adhesion by affecting the cadherin-catenin complex through E-cadherin, and that the classical Wnt signaling pathway may be inhibited by restricting β-catenin from entering the nucleus to inhibit cell proliferation.

Hypoxia-inducible factor 1 (HIF-1) functions as a master regulator of the cellular response to hypoxic stress. Two HIF-1α paralogs, HIF-1αA and HIF-1αB, were generated in euteleosts by the specific, third round of genome duplication, but one paralog was later lost in most families with the exception of cyprinid fish. How these duplicates function in mitochondrial regulation and whether their preservation contributes to the hypoxia tolerance demonstrated by cyprinid fish in freshwater environments is not clear. Here we demonstrated the divergent function of these two zebrafish Hif-1a paralogs through cellular approaches. The results showed that Hif-1aa played a role in tricarboxylic acid cycle by increasing the expression of Citrate synthase and the activity of mitochondrial complex II, and it also enhanced mitochondrial membrane potential and ROS production by reducing free Ca2+ in the cytosol. Hif-1ab promoted intracellular ATP content by up-regulating the activity of mitochondrial complexes I, III and IV and the expression of related genes. Furthermore, both the two zebrafish Hif-1a paralogs promoted mitochondrial mass and the expression level of mtDNA, contributing to mitochondrial biogenesis. Our study reveals the divergent functions of Hif-1aa and Hif-1ab in cellular mitochondrial regulation.

N-cadherin, a member of the cadherin family, plays an important role in neural development. In addition, N-cadherin has been reported to be crucial in neuronal migration, axonal outgrowth, and axonal path-finding. However, the mechanism underlying the effects of N-cadherin in neuronal migration is not entirely clear. In this study, we investigated the overexpression or knockdown of N-cadherin in the optic tectum during chicken embryo development, and then analyzed the effect of N-cadherin on neuronal migration. The results showed that compared with the control group, in the N-cadherin knockdown group, the neuronal migration of the optic tectum was significantly affected and could not arrive at destination. The stratum griseum central layer of the optic tectum mainly includes multipolar neurons, which could not be formed after the knockdown of N-cadherin, and more neurons form the bipolar or monopolar neurons compared with the control group. Compared with the control group, more cells stayed in the neuroepithelium layer. The axonal length in the optic tectum was significantly (P

To study the effects of CTNNB1 gene knockout by CRISPR-Cas9 technology on cell adhesion, proliferation, apoptosis, and Wnt/β-catenin signaling pathway. CTNNB1 gene of HEK 293T cells was knocked out by CRISPR-Cas9. This was confirmed by sequencing and western blotting. Methylthiazolyl-tetrazolium bromide assays indicated that deletion of β-catenin significantly weakened adhesion ability and inhibited proliferation rate (P

Usnic acid (UA) is the main secondary metabolite isolated from lichens, with moderate anticancer activity. A small group of (+)-UA derivatives characterized with flavanone moiety was designed and synthesized, and their anticancer activities were evaluated in leukemia cells. It was demonstrated that (+)-UA derivatives 6a–6g inhibited the proliferation of leukemia cells HL-60 and K562 with low micromolar IC50 values. Mechanisms of action were investigated to find that 6g induced apoptosis in HL-60 and K562 cell lines, and affected the expression of MNK/eIF4E axis-related proteins, such as Mcl-1, p-eIF4E, p-4E-BP1. Finally, kinase inhibition assay suggested 6g was a potential inhibitor of pan-Pim kinases. Meanwhile, the blocking of phosphorylation of BAD and 4E-BP1 by 6g, together with the proposed binding mode of 6g with Pim-1 further confirmed its Pim inhibition effects. Our finding provides the sight towards the potential mechanism of (+)-UA derivatives 6g as anti-leukemia agents.

The clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated proteins (Cas) belong to the crucial adaptive immune system, which exist in archaea and bacteria. Currently, CRISPR-Cas9 system has been modified and widely used to edit genome. In this review, we summarized the discovery, classification and mechanism of CRISPR-Cas system and further discussed the application of CRISPR-Cas9 in gene therapy, mainly in disease models.

A novel series of 6-hydroxy-4-methoxy-3-methylbenzofuran-7-carboxamide derivatives featured with various C-2 substituents were designed and synthesized as Mnks inhibitors through fragment-based drug design. Among them, 5b, 5i, 5o and 8k showed the best Mnk2 inhibitory activity with IC50 values of 1.45, 1.16, 3.55 and 0.27 μM, respectively. And these compounds inhibited the activity of Mnk1 at the same time. Furthermore, compounds 5o and 8k exhibited anti-proliferative effects to human leukemia cancer THP-1 and MOLM-13 cell lines and colon cancer HCT-116 cell line. Moreover, Western blot assay suggested that 8k could decrease the levels of p-eIF4E in a dose-dependent manner in HCT-116 cells. Docking studies demonstrated strong interactions between 8k and Mnk2. Therefore, this unique benzofuran scaffold demonstrated great potential to be further explored as potent Mnks inhibitors with improved potency.

One of the most important events in vertebrate evolutionary history is the water-to-land transition, during which some morphological and physiological changes occurred in concert with the loss of specific genes in tetrapods. However, the molecular mechanisms underlying this transition have not been well explored. To explore vertebrate adaptation to different oxygen levels during the water-to-land transition, we performed comprehensive bioinformatics and experimental analysis aiming to investigate the NAMPT family in vertebrates. NAMPT, a rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, is critical for cell survival in a hypoxic environment, and a high level of NAMPT significantly augments oxidative stress in normoxic environments. Phylogenetic analysis showed that NAMPT duplicates arose from a second round whole-genome duplication event. NAMPTA existed in all classes of vertebrates, whereas NAMPTB was only found in fishes and not tetrapods. Asymmetric evolutionary rates and purifying selection were the main evolutionary forces involved. Although functional analysis identified several functionally divergent sites during NAMPT family evolution, in vitro experimental data demonstrated that NAMPTA and NAMPTB were functionally conserved for NAMPT enzymatic function in the NAD+ salvage pathway. In situ hybridization revealed broad NAMPTA and NAMPTB expression patterns, implying regulatory functions over a wide range of developmental processes. The morpholino-mediated knockdown data demonstrated that NAMPTA was more essential than NAMPTB for vertebrate embryo development. We propose that the retention of NAMPTB in water-breathing fishes and its loss in air-breathing tetrapods resulted from vertebrate adaptation to different oxygen levels during the water-to-land transition.

Short interspersed elements (SINEs) are moderately repetitive DNA sequences in eukaryotic genomes. Although eukaryotic genomes contain numerous SINEs copy, it is very difficult and laborious to isolate and identify them by the reported methods. In this study, the inverse PCR was successfully applied to isolate SINEs from Opsariichthys bidens genome in Eastern Asian Cyprinid. A group of SINEs derived from tRNA(Ala) molecular had been identified, which were named Opsar according to Opsariichthys. SINEs characteristics were exhibited in Opsar, which contained a tRNA(Ala)-derived region at the 5' end, a tRNA-unrelated region, and AT-rich region at the 3' end. The tRNA-derived region of Opsar shared 76 % sequence similarity with tRNA(Ala) gene. This result indicated that Opsar could derive from the inactive or pseudogene of tRNA(Ala). The reliability of method was tested by obtaining C-SINE, Ct-SINE, and M-SINEs from Ctenopharyngodon idellus, Megalobrama amblycephala, and Cyprinus carpio genomes. This method is simpler than the previously reported, which successfully omitted many steps, such as preparation of probes, construction of genomic libraries, and hybridization.

The Clarias fuscus is an important economic fish in China and distributed widely in south China, e.g. Yangtze river, Pearl River, and Min River, even Hainan island. It is also distributed in Southeast Asia and Africa, so it is a good model for study population genetics and geological changes of these region. In this study, the complete mitochondrial genome sequence of C. fuscus has been obtained with PCR. The gene composition and arrangement of mitochondrial genome sequence of C. fuscus are similar to most of other vertebrates, which contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a non-coding control region with total length of 16,518 bp. Most of genes are encoded on Heavy-Strand (H-strand), except for eight tRNA and ND6 genes. Like most vertebrates, the bias of G and C has universality in different region (genes). The complete mitochondrial genome sequence of C. fuscus would contribute for better understanding of population genetics, conservation, biogeography, evolution of this species.

The study had mainly investigated the synthesis of sucrose-6-acetate (s-6-a) in fructosyltransferase action. The synthesis reaction of s-6-a was performed between sucrose and glucose-6-acetate (g-6-a), and identified by high performance liquid chromatography (HPLC). According to the reaction of s-6-a catalyzed by fructosyltransferase from Aspergillus oryzae, the effect factors of reaction, such as the ratio of g-6-a to sucrose, temperature, time, pH, substrate and enzyme concentration in the reaction, were investigated. All results indicated that the fructosyltransferase could catalyze the s-6-a synthesis, and the optimal conditions of fructosyltransferase in reaction were 50°C, pH 6.2, 48h reaction time, 60% sucrose, 1:3 ratio of g-6-a to sucrose and 4.0mg/L concentration of enzyme. This study plays the important role in sucralose synthesis, because it is very cumbersome in the reported methods.

This study was designed to express the onion fructosyltransferase by Escherichia coli DH5alpha, and obtain the optimal conditions of FST-1 activity. Thereby, fructosyltransferase gene was obtained by RT-PCR from onion in this experiment, and named FST-1. The expressed proteins were analyzed by SDS-PAGE. FST-1 activity was identified by the high performance liquid chromatography (HPLC). The optimal conditions of FST-1 were analyzed by the dinonylnaphthalene sulfonic acid (DNS) and orthogonal test. Results revealed that FST-1 was identified to 98% similarity with fructosyltransferase mRNA of onion (accession number: AJ006066). FST-1 was successfully expressed in E. coli DH5alpha. HPLC results indicated that the expressed protein from FST-1 had a good transferring activity for fructose. The optimal conditions of FST-1 in catalyzing reaction were the pH 5.0, 45 degrees C and 60% sucrose substrate. The results in this experiment would lay the foundation for the large-scale of kestose by bio-catalysis method.