Downregulation of microRNA-214 and overexpression of FGFR-1 contribute to hepatocellular carcinoma metastasis

miR-214 is one of the most significantly downregulated microRNAs (miRNAs) in hepatocellular carcinoma (HCC). Fibroblast growth factor receptor 1 (FGFR-1) is a miR-214 target gene implicated in the progression of HCC. However, the roles of miR-214 and FGFR-1 in HCC are not fully understood. Here, we analyzed the expression of miR-214 and FGFR-1 in 65 cases of HCC and paired non-neoplastic tissue specimens using real-time PCR and Western blot (WB), respectively. Our data indicated that miR-214 was downregulated and FGFR-1 was overexpressed in HCC compared to the paired non-neoplastic tissues. The low miR-214 expression was correlated with portal vein invasion (p = 0.016) and early recurrence (p = 0.045) in HCC patients. Moreover, the low miR-214 expression was correlated with high positive rate of FGFR-1 in HCC cases (p = 0.020). Our data further demonstrated that miR-214 overexpression in SK-HEP1 and HepG2 cells downregulated FGFR-1 expression and inhibited liver cancer cell invasion. The Luciferase assay results further demonstrated the targeted regulation of FGFR-1 by miR-214. In conclusion, our data indicate that the downregulation of miR-214 in HCC and the upregulation of its target gene FGFR-1 is associated with HCC progression. Therefore, miR-214 and FGFR-1 are potential prognostic markers and therapeutic targets in HCC.     

Functional expression and structural characterization of ORF cDNA encoding chitinase of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)

Chitin is an important component of the exoskeleton and peritrophic matrix in insects. Its bio-degradation is initiated by the endo-splitting chitinase. We cloned an ORF cDNA encoding chitinase from the last instar larva of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), into E. coli to confirm its functionality. Its amino acid sequence was compared with previously described lepidopteran chitinases. S. exigua chitinase expression enhanced cell growth approx. 1.5 fold in transformed E. coli than in the wild strain in a 1% colloidal chitin-containing medium with insufficient regular nutrients. Compared with the wild strain, the two intracellular chitin degradation derivatives, glucosamine and N-acetylglucosamine, increased approx. 5.8 and 1.5 fold, respectively, and extracellular chitinase activity in the transformed strain was about 1.6 fold higher. The ORF of S. exigua chitinase-encoding cDNA including stop codon was composed of 1674 bp nucleotides and the calculated molecular weight of the deduced 557 amino acid residues was about 62.6 kDa. The ORF consisted of an N-terminal leading signal peptide (AA 1-20), a catalytic domain (AA 21-392), a linker region (AA 393-493), and a C-terminal chitin-binding domain (AA 494-557) showing a typical molting fluid chitinase structure. Phylogenetic analysis determined that all 5 noctuid chitinases were grouped together, while two bombycid enzymes and one tortricid enzyme mapped together in one lineage. In the noctuid group, the sub-lineages reflected their taxonomic relationships at the Genus level.     

Light responsiveness of clock genes, Per1 and Per2, in the olfactory bulb of mice

The olfactory bulb (OB) of rodents has been suggested to possess a self-sustaining circadian oscillator which functions independent from the master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. However, neither histology nor physiology of this extra-SCN clock is studied yet. In the present study, we examined circadian variation of major clock gene expressions in the OB and responsiveness to single photic stimuli. Here we show significant circadian variation in the expression of clock genes, Per1, Per2 and Bmal1 in the OB. Per1 and PER2 were mainly expressed in the mitral cell and granular cell layers of the OB. Light responsiveness of Per1 and Per2 expression was different in the OB from that in the parietal cortex. Both Per1 and Per2 are expressed in the OB only by l000 lux light pulse, whereas 100 lux light was enough to induce Per1 mRNA in the parietal cortex. Interestingly, even 1000 lux light failed to induce Per2 mRNA in the parietal cortex. These clock gene-specific and brain region-dependent responses to lights in the OB and parietal cortex suggest that single light stimulus induces various physiological functions in different brain areas via specific clock gene.     

Syntenin-1-mediated small extracellular vesicles promotes cell growth,migration, and angiogenesis by increasing onco-miRNAs secretion in lung cancer cells

Small extracellular vesicles (sEVs) play a pivotal role in tumor progression by mediating intercellular communication in the tumor microenvironment (TME). Syntenin-1 induces malignant tumor progression in various types of human cancers, including human lung cancer and regulates biogenesis of sEVs. However, the function of syntenin-1-regulated sEVs and miRNAs in sEVs remains to be elucidated. In the present study, we aimed to demonstrate the role of oncogenic Ras/syntenin-1 axis in the release of sEVs and elucidate the function of syntenin-1-mediated miRNAs in sEVs in lung cancer progression. The results revealed that oncogenic Ras promoted the release of sEVs by inducing syntenin-1 expression; disruption of syntenin-1 expression impaired the release of sEVs as well as sEV-mediated cancer cell migration and angiogenesis. Moreover, we identified three miRNAs, namely miR-181a, miR-425-5p, and miR-494-3p, as onco-miRNAs loaded into syntenin-1-dependent sEVs. Remarkably, miR-494-3p was highly abundant in sEVs and its release was triggered by syntenin-1 expression and oncogenic Ras. Ectopic expression of the miR-494-3p mimic enhanced the migration and proliferation of lung cancer cells as well as tube formation in endothelial cells; however, the miR-494-3p inhibitor blocked sEV-mediated effects by targeting tyrosine-protein phosphatase nonreceptor type 12 (PTPN12), a tumor suppressor. sEVs promoted tumor growth and angiogenesis by downregulating PTPN12 expression; however, the miR-494-3p inhibitor significantly suppressed these effects in vivo, confirming that miR-494-3p acts as a major onco-miRNA loaded into lung cancer cell-derived sEVs. Eventually, the oncogenic Ras/syntenin-1 axis may induce cancer progression by increasing miR-494-3p loading into sEVs in lung cancer cells in the TME.Subject terms: Cancer microenvironment, Non-small-cell lung cancer, Oncogenesis     

Leishmania (Viannia) peruviana (MHOM/PE/LCA08): Comparison of THP-1 cell and murine macrophage susceptibility to axenic amastigotes for the screening of leishmanicidal compounds

This study, undertaken to compare the susceptibility of THP-1 cells and murine peritoneal macrophages to Leishmania peruviana amastigotes, obtained THP-1 infection with 10 parasites/cell compared to 2 parasites/murine macrophage. The parasite burden was maximal at 72 h post-infection (h.p.i.) for THP-1 cells, while it was still increasing at 120 h.p.i. for murine macrophages. Since in both cases the infection with L. peruviana affected cell viability, we recommend evaluating any leishmanicidal activity at 72 h.p.i. Amphotericin B reduced Leishmania infection by 50% at concentrations of 0.1 μM in THP-1 and murine macrophages at 72 h.p.i.Our results demonstrate that amastigotes of L. peruviana can infect THP-1 cells and murine macrophages and indicate the suitability of this model to screen compounds for leishmanicidal activity.     

Giardia lamblia: biochemical characterization of an ecto-ATPase activity

In this work, we describe the ability of living trophozoites of Giardia lamblia to hydrolyze extracellular ATP. In the absence of any divalent cations, a low level of ATP hydrolysis was observed (0.78 ± 0.08 nmol Pi × h⁻¹ × 10⁻⁶ cells). The ATP hydrolysis was stimulated by MgCl₂ in a dose-dependent manner. Half maximum stimulation of ATP hydrolysis was obtained with 0.53 ± 0.07 mM. ATP was the best substrate for this enzyme. The apparent K_m for ATP was 0.21 ± 0.04 mM. In the pH range from 5.6 to 8.4, in which cells were viable, this activity was not modified. The Mg²⁺-stimulated ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A₁ (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The impermeant agent DIDS and suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases, decreased the enzymatic activity in a dose-dependent manner, confirming the external localization of this enzyme. Besides ATP, trophozoites were also able to hydrolyse ADP and 5´ AMP, but the hydrolysis of these nucleotides was not stimulated by MgCl₂. Our results are indicative of the occurrence of a G. lamblia ecto-ATPase activity that may have a role in parasite physiology.     

Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli

A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3.From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 °C. Specific activity of StmPr2 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 17 ± 2 U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.     

Strategies for improving extracellular lipolytic enzyme production by Thermus thermophilus HB27

In Thermus thermophilus HB27 cultures the localisation of lipolytic activity is extracellular, intracellular and membrane bound, with low percentage for the former. Therefore, the extracellular secretion must be increased in order to simplify the downstream process and to reduce the economic cost. This study focuses on the design of an innovative operational strategy to increase extracellular lipolytic enzyme production by T. thermophilus HB27 at bioreactor scale. In order to favour its secretion, the effect of several operational variables was evaluated. Among them, the presence of oils in the culture medium leads to improvements in growth and lipolytic enzyme activity. Sunflower oil is the most efficient inducer showing better results when added after 10 h of growth. On the other hand, although surfactants lead to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. Thus, by addition of surfactants at the stationary phase, a release of intracellular and membrane enzyme which increases the extracellular enzyme proportion is detected. Based on these results, strategies with successive addition of oil and surfactant in several culture phases in shake flask are developed and verified in a laboratory scale stirred tank bioreactor.     

Trypanosoma rangeli: A possible role for ecto-phosphatase activity on cell proliferation

Here we demonstrate for the first time that growth of Trypanosoma rangeli, a protozoa parasite, is strongly dependent on the presence of inorganic phosphate (Pi) in the culture medium and that the replacement of the inorganic phosphate in the culture medium by β-glycerophosphate, a substrate for phosphatases lead the cells to achieve its maximal growth. The ecto-phosphatase activity present on the external surface of T. rangeli decreased during the growth phase of the parasite, suggesting that this enzyme could be important for the development. Accordingly, the inhibition of this ecto-phosphatase activity by sodium orthovanadate also inhibited the proliferation of T. rangeli. Parasites maintained in a Pi-starved culture medium (2 mM Pi) had 4-fold more ecto-phosphatase activity as compared to parasites maintained in a Pi-supplemented culture medium (50 mM Pi). Altogether, these results presented here suggest that this ecto-phosphatase activity leads to hydrolysis of phosphorylated compounds present in the extracellular medium, which could contribute to the acquisition of inorganic phosphate during the development of T. rangeli epimastigotes.     

Coenzyme A and its thioester pools in fasted and fed rat tissues

The possibility of elevating the omega-3 fatty acid contents in mammalian cells using the sdd17 gene from Saprolegnia diclina was investigated in the current study. The nucleotide sequence of the sdd17 gene was optimized and the pSDD17-IRES-GFP plasmid was introduced into murine 3T3 fibroblast cells by electroporation, following which its heterologous expression was evaluated by fatty acid analysis. Evaluation of GFP co-expression and RT-PCR analysis indicated that sdd17 could be expressed at very high levels in mammalian cells. Total cellular lipid analysis of transformed cells fed with arachidonic acid (20:4 n − 6) as a substrate showed that the sdd17 expression resulted in an 82-155% (p < 0.05) increase in eicosapentaenoic acid (20:5 n − 3) compared with the control. This expression also reduced the arachidonic acid/(eicosapentaenoic + docosapentaenoic + docosahexaenoic acid) ratio from approximately 4:1 in control cells to 1.5:1 in sdd17-transformed cells (p < 0.05). This study demonstrated that the foreign sdd17 gene from EPA-rich fungus was expressed at a high efficiency and caused the omega-3 fatty acid contents in mammalian cells to be elevated. It also provided a basis for potential applications of this gene in animal transgenesis.     

Optimized production of scygonadin in Pichia pastoris and analysis of its antimicrobial and antiviral activities

The crab antimicrobial peptide scygonadin is confirmed to have antimicrobial activity against bacteria and it is probably associated with the reproductive immunity in Scylla paramamosain. To obtain large quantity of scygonadin for further biological assays, a 306 bp cDNA sequence encoding the mature peptide of scygonadin was cloned into a secretion vector of pPIC9K, and a high-level of the recombinant scygonadin was achieved in Pichia pastoris. The optimal expression condition was determined as incubation with 0.5% methanol for 48 h at 28 °C under pH 6.0, and a total of 70 mg scygonadin was expressed in 1 L culture medium. The recombinant product was purified and 97% pure scygonadin was obtained using immobilized metal affinity chromatography with a yield of 46 mg/L. The recombinant scygonadin was confirmed using SDS-PAGE analysis and MS-fingerprinting. P. pastoris-derived scygonadin exhibited relatively higher antimicrobial activities against bacteria than Escherichia coli-derived scygonadin. The antimicrobial activity of the recombinant scygonadin against pathogenic Aeromonas hydrophila showed salt resistant and the killing kinetics of A. hydrophila was time dependent. Besides, the antiviral assay demonstrated that scygonadin could interfere with white spot syndrome virus (WSSV) replication in vitro-cultured crayfish haematopoietic (Hpt) cells. Taken together, this is the first report on the heterologous expression of scygonadin in P. pastoris, and P. pastoris is an effective expression system for producing large quantities of biological active scygonadin for both research and agricultural application.     

Circadian expression of clock genes in human peripheral leukocytes

In mammals, behavioral and physiological processes display 24-h rhythms that are regulated by a circadian system. In the present study, we investigated the possibility that the expression of clock genes in peripheral leukocytes can be used to assess the circadian clock system. We found that Per1 and Per2 exhibit circadian oscillations in mRNA expression in mouse peripheral leukocytes. Furthermore, the rhythms of Per1 and Per2 mRNA expression in peripheral leukocytes are severely blunted in homozygous Cry1/2 double-deficient mice that are known to have an abolished biological clock. We have examined the circadian expression of clock genes in human leukocytes and found that Per1 mRNA exhibits a robust circadian expression while Per2 and Bmal1 mRNA showed weak rhythm. These observations suggest that monitoring Per1 mRNA expression in human leukocytes may be useful for investigating the function of the circadian system in physiological and pathophysiological states.     

Cellular and molecular responses of haemocytes from Ostrea edulis during in vitro infection by the parasite Bonamia ostreae

Bonamia ostreae is a protozoan, affiliated to the order Haplosporidia and to the phylum Cercozoa. This parasite is intracellular and infects haemocytes, cells notably involved in oyster defence mechanisms. Bonamiosis due to the parasite B. ostreae is a disease affecting the flat oyster, Ostrea edulis. The strategies used by protozoan parasites to circumvent host defence mechanisms remain largely unknown in marine bivalve molluscs. In the present work, in vitro experiments were carried out in order to study the interactions between haemocytes from O. edulis and purified parasite, B. ostreae. We monitored cellular and molecular responses of oyster haemocytes by light microscopy, flow cytometry and real-time PCR 1, 2, 4 and 8 h p.i. Light microscopy was used to measure parasite phagocytosis by oyster haemocytes. Parasites were observed inside haemocytes 1 h p.i. and the parasite number increased during the time course of the experiment. Moreover, some bi-nucleated and tri-nucleated parasites were found within haemocytes 2 and 4 h p.i., respectively, suggesting that the parasite can divide inside haemocytes. Host responses to B. ostreae were investigated at the cellular and molecular levels using flow cytometry and real-time PCR. Phagocytosis capacity of haemocytes, esterase activity and production of radical oxygen species appeared modulated during the infection with B. ostreae. Expression levels of expressed sequence tags selected in this study showed variations during the experiment as soon as 1 h p.i. An up-regulation of galectin (OeGal), cytochrome p450 (CYP450), lysozyme, omega GST (OGST), super oxide dismutase Cu/Zn (Oe-SOD Cu/Zn) and a down-regulation of the extracellular super oxide dismutase SOD (Oe-EcSOD) were observed in the presence of the parasite. Finally, the open reading frames of both SODs (Oe-SOD Cu/Zn and Oe-EcSOD) were completely sequenced. These findings provide new insights into the cellular and molecular bases of the host-parasite interactions between the flat oyster, O. edulis, and the parasite, B. ostreae.