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Mitogen-activated protein kinase-interacting kinases (MNKs) and provirus integration in maloney murine leukemia virus kinases (PIMs) are downstream enzymes of cell proliferation signaling pathways associated with the resistance of tyrosine kinase inhibitors. MNKs and PIMs have complementary effects to regulate cap-dependent translation of oncoproteins. Dual inhibitors of MNKs and PIMs have not been developed. We developed a novel 4,6-disubstituted pyrido[3,2-d]pyrimidine compound 21o with selective inhibition of MNKs and PIMs. The IC50's of 21o to inhibit MNK1 and MNK2 are 1 and 7 nM and those to inhibit PIM1, PIM2, and PIM3 are 43, 232, and 774 nM, respectively. 21o inhibits the growth of myeloid leukemia K562 and MOLM-13 cells with GI50's of 2.1 and 1.2 μM, respectively. 21o decreases the levels of p-eIF4E and p-4EBP1, the downstream products of MNKs and PIMs, as well as cap-dependent proteins c-myc, cyclin D1, and Mcl-1. 21o inhibits the growth of MOLM-13 cell xenografts without causing evident toxicity. 21o represents an innovative dual MNK/PIM inhibitor with a good pharmacokinetic profile.

In this study, we established a mouse model of epilepsy and analysed abnormal neuronal damage and inflammation in the hippocampus of mice with kainic acid (KA)-induced epilepsy to provide the basis for the pathogenesis of epilepsy. C57 mice, aged 4 weeks, were injected intraperitoneally in the KA group with 20 mg/kg of KA and in the sham experimental group with normal saline. The whole brain and hippocampus of mice in the sham experimental group and KA epilepsy model group were collected on days 7, 14, 21 and 28 after injection. The difference in the protein expression in the hippocampus was detected using fluorescence immunohistochemistry. The hippocampal tissue was also collected and frozen to detect protein expression by western blot. The results of the haematoxylin and eosin (HE) and Nissl staining showed that the mouse model of temporal lobe epilepsy could be established by intraperitoneal injection of KA, and the success rate of the model was 53.8%. The expression of DCX-, β-catenin-, GFAP- and Iba-1-labelled glial cells in the KA-induced epilepsy model group were higher than those in the sham group. The results of western blotting showed that the expression of DCX and β-catenin in the KA-induced epilepsy model group was higher than that in the sham experimental group, while the expression of N-cadherin and Iba-1 on days 14 and 28 was significantly (P < .05) higher than that in the sham experimental group. In KA-induced epilepsy model group, the expression of Bcl-2 was decreased, while the expression of Bad and PUMA was increased.

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.

Epilepsy is a nervous system disease caused by abnormal discharge of brain neurons, which is characterized by recurrent seizures. The factors that induce epilepsy include genetic and environmental factors. Genetic factors are important pathogenic factors of epilepsy, such as epilepsy caused by protocadherin-19 (PCDH-19) mutation, which is an X-linked genetic disease. It is more common in female heterozygotes, which are caused by mutations in the PCDH-19 gene. Epilepsy caused by environmental factors is mainly caused by brain injury, which is commonly caused by brain tumors, brain surgery, or trauma to the brain. In addition, the pathogenesis of epilepsy is closely related to abnormalities in some signaling pathways. The Wnt/β-catenin signaling pathway is considered a new target for the treatment of epilepsy. This review summarizes these factors inducing epilepsy and the research hypotheses regarding the pathogenesis of epilepsy. The focus of this review centers on cadherins and the pathogenesis of epilepsy. We analyzed the pathogenesis of epilepsy induced by N-cadherin and PCDH-19 in the cadherin family members. Finally, we expect that in the future, new breakthroughs will be made in the study of the pathogenesis and mechanism of epilepsy at the cellular and molecular levels.

Mitogen-activated protein kinase-interacting kinases (MNKs) and provirus integration in maloney murine leukemia virus kinases (PIMs) are downstream enzymes of cell proliferation signaling pathways associated with the resistance of tyrosine kinase inhibitors. MNKs and PIMs have complementary effects to regulate cap-dependent translation of oncoproteins. Dual inhibitors of MNKs and PIMs have not been developed. We developed a novel 4,6-disubstituted pyrido[3,2

Neurological disorders are primarily diseases with sophisticated etiology that are always refractory and recrudescent. The major obstruction to effective therapies for neurological disorders is the poor understanding of their pathogenic mechanisms. CRISPR-Cas9 technology, which allows precise and effective gene editing in almost any cell type and organism, is accelerating the pace of basic biological research. An increasing number of groups are focusing on uncovering the molecular mechanisms of neurological disorders and developing novel therapies using the CRISPR-Cas9 system. This review highlights the application of CRISPR-Cas9 technology in the treatment of neurological disorders, including Alzheimer's disease, amyotrophic lateral sclerosis and/or frontotemporal dementia, Duchenne muscular dystrophy, Dravet syndrome, epilepsy, Huntington's disease, and Parkinson's disease. Hopefully, it will improve our understanding of neurological disorders and give insights into future treatments for neurological disorders.

Rap1 and N-cadherin regulate glia-independent translocation of cortical neurons. It remains unclear how Rap1 regulates N-cadherin-mediated neuronal migration. Here, we overexpressed Rap1gap in mouse brains (embryonic day 16) to inactivate Rap1, and observed that neurons did not migrate to the outer layer. We confirmed that Rap1 was involved in the regulation of late neurons in vivo. Rap1gap overexpression and Rap1 suppression in CHO cells decreased the expression of cytoskeletal proteins such as tubulin. Changes in the expression of cell morphology regulators, such as N-cadherin and β-catenin, were also observed. Inhibition of N-cadherin in mouse brains prevented neuronal migration to the outer layer. The morphology of CHO cells was changed after overexpression of Rap1gap. We propose that Rap1 regulates the expression of N-cadherin during embryonic development, which affects β-catenin expression. Beta-catenin in turn regulates cytoskeletal protein expression, ultimately affecting neuronal morphology and migration.

Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)‐positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 μm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30‐mm culture dish and 1 ml of slice culture media was added. We show that during serum‐free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured‐tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum‐free medium cultured‐tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.

In this paper, the complete moment convergence for the partial sum of moving average processes $\{X_{n}=\sum_{i=-\infty }^{\infty }a_{i}Y_{i+n},n\geq 1\}$ { X n = ∑ i = − ∞ ∞ a i Y i + n , n ≥ 1 } is established under some mild conditions, where $\{Y_{i},-\infty < i { Y i , − ∞ < i < ∞ } is a sequence of m-widely orthant dependent (m-WOD, for short) random variables which is stochastically dominated by a random variable Y, and $\{a_{i},-\infty < i { a i , − ∞ < i < ∞ } is an absolutely summable sequence of real numbers. These conclusions promote and improve the corresponding results from m-extended negatively dependent (m-END, for short) sequences to m-WOD sequences.

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

Recently, menstrual blood-derived endometrial stem cells (MenSCs) have become attractive for stem cell based therapy due to their abundance, easy and non-invasive extraction and isolation process, high proliferative capacity, and multi-lineage differentiation potential. MenSC-based therapies for various diseases are being extensively researched. However, the high death rate and poor engraftment in sites of damaged tissues reduce the therapeutic value of these stem cells for transplantation. In theory, periodic stem cell transplantation is an alternative strategy to overcome the challenge of the loss of beneficial stem cell-derived effects due to the rapid disappearance of the stem cells in vivo. However, periodic stem cell transplantation requires sufficient amounts of the desired stem cells with a low number of subculture passages. Our previous results have demonstrated that primary MenSCs mainly reside in the deciduous endometrium, and considerable amounts of deciduous endometrium intertwined with menstrual blood clots were discarded after conventional density gradient centrifugation (DGC). Therefore, the aim of this study was to determine whether primary MenSCs exist in the sedimentation of the deciduous endometrium after DGC and further to evaluate the isolation of MenSCs by direct red blood cell lysis treatment. As expected, our results confirmed that substantial amounts of primary MenSCs still remain in the sedimentation after DGC and indicated that MenSC isolation by directly lysing the red blood cells not only guaranteed substantial amounts of superior MenSCs with a low number of subculture passages, but also was time efficient and economical, providing a solid support for extensive clinical application., Summary: MenSC isolation by directly lysing the red blood cells not only guarantees substantial amounts of superior MenSCs with low passage number, but also is time efficient and economical.

Peptidyl-prolyl cis/trans isomerase Pin1 plays a key role in amplifying and translating multiple oncogenic signaling pathways during oncogenesis. The blockade of Pin1 provided a unique way of disrupting multiple oncogenic pathways and inducing apoptosis. Aiming to develop potent Pin1 inhibitors, a series of benzimidazole derivatives were designed and synthesized. Among the derivatives, compounds 6h and 13g showed the most potent Pin1 inhibitory activity with IC50 values of 0.64 and 0.37 &mu, M, respectively. In vitro antiproliferative assay demonstrated that compounds 6d, 6g, 6h, 6n, 6o and 7c exhibited moderate antiproliferative activity against human prostate cancer PC-3 cells. Taken together, these unique benzimidazole derivatives exhibited great potential to be further explored as potent Pin1 inhibitors with improved potency.

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.

Background/aims Menstrual blood-derived endometrial stem cells (MenSCs) emerge as an ideal source for cell-based treatment in regenerative medicine and immunotherapy. However, the major obstacle is the low survival rate in tissues and the limited expansion number. Autophagy is an intracellular metabolic self-degradative process which plays important roles in normal cellular division and survival, and the present study aimed to explore the related mechanisms between autophagy and survival of MenSCs in vitro and in vivo. Methods The MenSCs were obtained from menstrual blood procured from healthy female donors. In vitro, MenSCs were exposed to rapamycin and Earle’s balanced salts solution (EBSS). We evaluated the MenSCs immunophenotypic cell cycle distribution by propidium iodide (PI) staining and cell apoptosis by Annexin V/PI staining as well as their proliferative potential by the MTT assay. We also assessed the expression of genes associated with the cell cycle and Gsk3β signaling pathway by western blot analysis. We depressed Atg5 and Gsk3β expression by short hairpin RNA (shRNA) and undertook the experiments. Moreover, the labeled MenSCs were observed and counted with DiI after transplantation into the mice via the tail vein by microscopy in vivo. Results In vitro, rapamycin and starvation induced autophagy of MenSCs. Hyperactive autophagy significantly induced G0/G1 arrest and slightly promoted apoptosis of MenSCs. Meanwhile, autophagy could stimulate p-GSK3β expression in MenSCs. Further, knockdown GSK3β can accelerate the proliferation of MenSCs by shRNA and CHIR99021. Moreover, the shGSK3β MenSCs showed strong proliferative activity in vitro and in vivo. Conclusions Our results indicate that autophagy induced G0/G1 arrest and apoptosis of MenSCs via GSK3β/β-catenin pathway. Inhibiting autophagy or reduced GSK3β levels may improve survival rate in vivo, thus playing roles in MenSCs therapy.

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.

The Yangtze River is the longest river in China and is divided into upstream and mid‐downstream regions by the Three Gorges (the natural barriers of the Yangtze River), resulting in a complex distribution of fish. Dramatic changes to habitat environments may ultimately threaten fish survival; thus, it is necessary to evaluate the genetic diversity and propose protective measures. Species identification is the most significant task in many fields of biological research and in conservation efforts. DNA barcoding, which constitutes the analysis of a short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence, has been widely used for species identification. In this study, we collected 561 COI barcode sequences from 35 fish from the midstream of the Yangtze River. The intraspecific distances of all species were below 2% (with the exception of Acheilognathus macropterus and Hemibarbus maculatus). Nevertheless, all species could be unambiguously identified from the trees, barcoding gaps and taxonomic resolution ratio values. Furthermore, the COI barcode diversity was found to be low (≤0.5%), with the exception of H. maculatus (0.87%), A. macropterus (2.02%) and Saurogobio dabryi (0.82%). No or few shared haplotypes were detected between the upstream and downstream populations for ten species with overall nucleotide diversities greater than 0.00%, which indicated the likelihood of significant population genetic structuring. Our analyses indicated that DNA barcoding is an effective tool for the identification of cyprinidae fish in the midstream of the Yangtze River. It is vital that some protective measures be taken immediately because of the low COI barcode diversity.

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.

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 0.01) of HEK 293T cells. Nevertheless, deletion of β-catenin did not affect apoptosis of HEK 293T cells, which was analyzed by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide double staining. In addition, expression level of GSK-3β, CCND1, and CCNE1 detected by qPCR and expression level of N-Cadherin and cyclin D1 detected by western blotting were significantly decreased (P 0.01) while expression of γ-catenin detected by western blotting was significantly increased (P 0.001).Knockout of CTNNB1 disturbed Wnt/β-catenin signaling pathway and significantly inhibited adhesion and proliferation of HEK 293T cells.

The fructosyltransferase gene was isolated and cloned from Aspergillus oryzae. The gene was 1368 bp, which encoded a protein of 455 amino acids. To analyze the activity of the expressed fructosyltransferase, the pET32a-fructosyltransferase recombined plasmid was transformed into Escherichia coli BL21. The fructosyltransferase gene was successfully expressed by Isopropyl-β-d-thiogalactoside (IPTG) induction. The molecular weight of the expression protein was about 45 kDa. The optimal conditions of protein expression were 25 °C, 0.1 mM IPTG, and 8 h of inducing time. The optimal concentration of urea dealing with inclusion body was 2.5 M. The expressed protein exhibited a strong fructosyl transfer activity. These results showed that the expressed fructosyltransferas owned transferase activity, and could catalyze the synthesis of sucrose-6-acetate.

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.

Tibetan Plateau (TP) had experienced phased uplift, resulting in inhospitable environment of low temperature, hypoxia and high ultraviolet radiation for Tibetan wildlife. Many organisms can well adapt to TP, it is of ecological and evolutionary interest to untangle how organisms adapt to extreme environment on TP through evolution. Previous studies mainly focused on hypoxia and metabolism related genes, but we know little about the evolutionary history of immune genes in Tibetan wildlife. In this study, we first identified 10 interferon regulatory factor (IRF) genes from Tibetan naked carp Gymnocypris przewalskii. Within this gene family, IRF3, IRF5, IRF7 and IRF8 contained positive selection sites. Evidences indicated that positive selection may lead to IRF genes functional alternations, presumably driving genes towards adaptation to the environmental changes. Taken together, our results suggested 4 candidate genes as interesting targets for further experimental confirmation of their functional variations and contributions to high altitude adaptation in Tibet fish. (C) 2016 Elsevier Ltd. All rights reserved.

Background Hypoxia-inducible factor (HIF) is a master regulator that mediates major changes in gene expression under hypoxic conditions. Though HIF family has been identified in many organisms, little is known about this family in schizothoracine fish. Results Duplicated hif-α (hif-1αA, hif-1αB, hif-2αA, and hif-2αB) genes were identified in schizothoracine fish. All the deduced HIF-α proteins contain the main domains (bHLH-PAS, ODDD, and TAD), also found in humans. Evidence suggests a Cyprinidae-specific deletion, specifically, a conserved proline hydroxylation motif LxxLAP, in the NODD domain of schizothoracine fish HIF-1αA. In addition, a schizothoracine-specific mutation was observed in the CODD domain of the specialized and highly specialized schizothoracine fish HIF-1αB, which is the proline hydroxylation motif mutated into PxxLAP. Standard and stochastic branch-site codon model analysis indicated that only HIF-1αB has undergone positive selection, which may have led to changes in function. To confirm this hypothesis, HIF-αs tagged with Myc were transfected into HEK 293 T cells. Each HIF-1αB was found to significantly upregulate luciferase activity under normoxic and hypoxic conditions, which indicated that the HIF-1αB protein was more stable than other HIF-αs. Conclusions All deduced HIF-α proteins of schizothoracine fish contain important domains, like their mammalian counterparts, and each HIF-α is shorter than that of human. Our experiments reveal that teleost-specific duplicated hif-α genes played different roles under hypoxic conditions, and HIF-1αB may be the most important regulator in the adaptation of schizothoracine fish to the environment of the Tibetan Plateau. Electronic supplementary material The online version of this article (doi:10.1186/s12862-014-0192-1) contains supplementary material, which is available to authorized users.

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.