Long non-coding RNA LINC00488 facilitates thyroid cancer cell progression through miR-376a-3p/PON2

Objective: Long non-coding RNAs (lncRNAs) recently have been identified as influential indicators in a variety of malignancies. The aim of the present study was to identify a functional lncRNA LINC00488 and its effects on thyroid cancer in the view of cell proliferation and apoptosis.Methods: In order to evaluate the effects of LINC00488 on the cellular process of thyroid cancer, we performed a series of in vitro experiments, including cell counting kit-8 (CCK-8) assay, EdU (5-ethynyl-2′-deoxyuridine) assay, flow cytometry, transwell chamber assay, Western blot and RT-qPCR. The target gene of LINC00488 was then identified by bioinformatics analysis (DIANA and TargetScan). Finally, a series of rescue experiments was conducted to validate the effect of LINC00488 and its target genes on proliferation, migration, invasion and apoptosis of thyroid cancer.Results: Our findings revealed that LINC00488 was highly expressed in thyroid cancer cell lines (BCPAP, BHP5-16, TPC-1 and CGTH-W3) and promoted the proliferation, migration and invasion, while inhibited the apoptosis of thyroid cancer cells (BCPAP and TPC-1). The results of bioinformatics analysis and dual luciferase reporter gene assay showed that LINC00488 could directly bind to miR-376a-3p and down-regulated the expression level of miR-376a-3p. In addition, Paraoxonase-2 (PON2) was a target gene of miR-376a-3p and negatively regulated by miR-376a-3p. Rescue experiment indicated that LINC00488 might enhance PON2 expression by sponging miR-376a-3p in thyroid cancer.Conclusion: Taken together, our study revealed that lncRNA LINC00488 acted as an oncogenic gene in the progression of thyroid cancer via regulating miR-376a-3p/PON2 axis, which indicated that LINC00488-miR-376a-3p-PON2 axis could serve as novel biomarkers or potential targets for the treatment of thyroid cancer.     

Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L⁻¹ l-lysine from mannitol at a yield of 0.26 mol mol⁻¹ and a maximum productivity of 2.1 g L⁻¹ h⁻¹. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol⁻¹ using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol⁻¹. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.     

Correlates of pentraxin 3 serum concentration in men and women with type 2 diabetes

Elevated levels of plasma pentraxin 3 (PTX3), a marker of inflammation, are associated with the risk of developing cardiovascular diseases in the general population, as well as in patients with type 2 diabetes (DM2). In this study, we aimed to determine factors associated with PTX3 serum concentrations in men and women with DM2. The study included 116 consecutive patients (67 men and 49 women) with DM2 from an outpatient diabetic clinic. Men were characterised by lower age and higher uric acid, creatinine and bilirubin concentrations and waist/hip ratio than women. In women, low-density lipoprotein cholesterol (LDL-C) levels were higher than in men. In men, median (interquartile range) values of PTX3 concentration were 4.02 (1.99), and in women they were 4.53 (3.31) ng/ml (NS). In men, PTX3 concentrations correlated with total cholesterol (TC), triglycerides, apolipoprotein (Apo) C3, Apo B48, Glc and creatinine levels. In women, PTX3 correlated significantly with TC and LDL-C and Apo B100. Partial regression analysis revealed that after adjusting for age, PTX3 concentrations in men were significantly associated with TC, LDL-C, triglycerides, creatinine, Apo C3 and Apo B48, while in women they were associated with TC, LDL-C and Apo B100. The results could be of importance in sex-specific prevention of vascular complications in DM2 patients.     

Degradation of WTAP blocks antiviral responses by reducing the m6A levels of IRF3 and IFNAR1 mRNA

N ⁶‐methyladenosine (m⁶A) is a chemical modification present in multiple RNA species and is most abundant in mRNAs. Studies on m⁶A reveal its comprehensive roles in almost every aspect of mRNA metabolism, as well as in a variety of physiological processes. Although some recent discoveries indicate that m⁶A can affect the life cycles of numerous viruses as well as the cellular antiviral immune response, the roles of m⁶A modification in type I interferon (IFN‐I) signaling are still largely unknown. Here, we reveal that WT1‐associated protein (WTAP), one of the m⁶A “writers”, is degraded via the ubiquitination‐proteasome pathway upon activation of IFN‐I signaling. With the degradation of WTAP, the m⁶A levels of IFN‐regulatory factor 3 (IRF3) and interferon alpha/beta receptor subunit 1 (IFNAR1) mRNAs are reduced, leading to translational suppression of IRF3 and instability of IFNAR1 mRNA. Thus, the WTAP‐IRF3/IFNAR1 axis may serve as negative feedback pathway to fine‐tune the activation of IFN‐I signaling, which highlights the roles of m⁶A in the antiviral response by dictating the fate of mRNAs associated with IFN‐I signaling.     

UNC-16 alters DLK-1 localization and negatively regulates actin and microtubule dynamics in Caenorhabditis elegans regenerating neurons

Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. Our study shows that UNC-16, a Caenorhabditis elegans JIP3 homolog, inhibits axonal regeneration by regulating initiation and rate of regrowth. This occurs through the inhibition of the regeneration-promoting activity of the long isoform of DLK-1 and independently of the inhibitory short isoform of DLK-1. We show that UNC-16 promotes DLK-1 punctate localization in a concentration-dependent manner limiting the availability of the long isoform of DLK-1 at the cut site, minutes after injury. UNC-16 negatively regulates actin dynamics through DLK-1 and microtubule dynamics partially via DLK-1. We show that post-injury cytoskeletal dynamics in unc-16 mutants are also partially dependent on CEBP-1. The faster regeneration seen in unc-16 mutants does not lead to functional recovery. Our data suggest that the inhibitory control by UNC-16 and the short isoform of DLK-1 balances the intrinsic growth-promoting function of the long isoform of DLK-1 in vivo. We propose a model where UNC-16’s inhibitory role in regeneration occurs through both a tight temporal and spatial control of DLK-1 and cytoskeletal dynamics.     

Global fold and backbone dynamics of the hepatitis C virus E2 glycoprotein transmembrane domain determined by NMR

E1 and E2 are two hepatitis C viral envelope glycoproteins that assemble into a heterodimer that is essential for membrane fusion and penetration into the target cell. Both extracellular and transmembrane (TM) glycoprotein domains contribute to this interaction, but study of TM–TM interactions has been limited because synthesis and structural characterization of these highly hydrophobic segments present significant challenges. In this NMR study, by successful expression and purification of the E2 transmembrane domain as a fusion construct we have determined the global fold and characterized backbone motions for this peptide incorporated in phospholipid micelles. Backbone resonance frequencies, relaxation rates and solvent exposure measurements concur in showing this domain to adopt a helical conformation, with two helical segments spanning residues 717–726 and 732–746 connected by an unstructured linker containing the charged residues D728 and R730 involved in E1 binding. Although this linker exhibits increased local motions on the ps timescale, the dominating contribution to its relaxation is the global tumbling motion with an estimated correlation time of 12.3 ns. The positioning of the helix–linker–helix architecture within the mixed micelle was established by paramagnetic NMR spectroscopy and phospholipid-peptide cross relaxation measurements. These indicate that while the helices traverse the hydrophobic interior of the micelle, the linker lies closer to the micelle perimeter to accommodate its charged residues. These results lay the groundwork for structure determination of the E1/E2 complex and a molecular understanding of glycoprotein heterodimerization.     

Muscarinic activation of mitogen-activated protein kinase in PC12 cells

Muscarinic acetylcholine receptors (mAChRs) activate many downstream signaling pathways, some of which can lead to mitogen-activated protein kinase (MAPK) phosphorylation and activation. MAPKs play roles in regulating cell growth, differentiation, and synaptic plasticity. Here, the activation of MAPK was examined in PC12 cells endogenously expressing mAChRs. Western blot analysis using a phosphospecific MAPK antibody revealed a dose-dependent and atropine-sensitive increase in MAPK phosphorylation in cells stimulated with carbachol (CCh). The maximal response occurred after 5 min and was rapidly reduced to baseline. To investigate the receptors responsible for CCh activation of MAPK in PC12 cells, the mAChR subtypes present were determined using RT-PCR and immunoprecipitation. RT-PCR was used to amplify fragments of the appropriate sizes for m1, m4, and m5, and the identities of the bands were confirmed with restriction digests. Immunoprecipitation using subtype-specific antibodies showed that approximately 95% of the expressed receptors were m4, whereas the remaining approximately 5% were m1 and m5. A highly specific m1 toxin completely blocked MAPK phosphorylation in response to CCh stimulation. The mAChR-induced MAPK activation was abolished by protein kinase C down-regulation and partially inhibited by pertussis toxin. Although m1 represents a small proportion of the total mAChR population, pharmacological evidence suggests that m1 is responsible for MAPK activation in PC12 cells.     

Plasmin-sensitive dibasic sequences in the third fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate homomultimerization and concomitant integrin recruitment

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including alpha(v)beta(3) and alpha(5)beta(1). A pep- tide derived from the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV(852)) also forms trimeric complexes and supports alpha(v)beta(3) and alpha(5)beta(1) binding. Substitution of the dibasic RK(841) and KR(845) sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RK( downward arrow)HSK( downward arrow)RH(846). The integrin alpha(9)beta(1), which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell-cell contact. We propose that distal receptor ligation events at the cell-cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.     

Expression of a novel receptor tyrosine kinase gene and a paired-like homeobox gene provides evidence of differences in patterning at the oral and aboral ends of hydra

Axial patterning of the aboral end of the hydra body column was examined using expression data from two genes. One, shin guard, is a novel receptor protein-tyrosine kinase gene expressed in the ectoderm of the peduncle, the end of the body column adjacent to the basal disk. The other gene, manacle, is a paired-like homeobox gene expressed in differentiating basal disk ectoderm. During regeneration of the aboral end, expression of manacle precedes that of shin guard. This result is consistent with a requirement for induction of peduncle tissue by basal disk tissue. Our data contrast with data on regeneration of the oral end. During oral end regeneration, markers for tissue of the tentacles, which lie below the extreme oral end (the hypostome), are detected first. Later, markers for the hypostome itself appear at the regenerating tip, with tentacle markers displaced to the region below. Additional evidence that tissue can form basal disk without passing through a stage as peduncle tissue comes from LiCl-induced formation of patches of ectopic basal disk tissue. While manacle is ectopically expressed during formation of basal disk patches, shin guard is not. The genes examined also provide new information on development of the aboral end in buds. Although adult hydra are radially symmetrical, expression of both genes in the bud's aboral end is initially asymmetrical, appearing first on the side of the bud closest to the parent's basal disk. The asymmetry can be explained by differences in positional information in the body column tissue that evaginates to form a bud. As predicted by this hypothesis, grafts reversing the orientation of evaginating body column tissue also reverse the orientation of asymmetrical gene expression.