bWe developed one-step sequence-and ligation-independent cloning (SLIC) as a simple, cost-effective, time-saving, and versatile cloning method. Highly efficient and directional cloning can be achieved by direct bacterial transformation 2.5 min after mixing any linearized vector, an insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room temperature.T he need for high-throughput recombinant DNA technology is rising because of the rapid increase in interest in functional genomic studies following the surge of data generated by nextgeneration sequencing. Construction of a library or conversion of an existing library into a different context requires a highthroughput gene cloning method, but conventional methods suffer from high cost, prolonged manipulation, or sequence restriction.Ligation-independent cloning (LIC) is based on the 3=-to-5= exonuclease activity of T4 DNA polymerase and has been used for 2 decades as a high-throughput method due to its uniformity and cost-effectiveness but requires a specifically designed vector containing a long stretch of sequence that lacks a particular deoxynucleoside triphosphate (1,(3)(4)(5)(6)9). Sequence-and ligation-independent cloning (SLIC) overcomes the sequence restraint of LIC and allows the assembly of multiple overlapping fragments simultaneously, but the cloning efficiency of SLIC in the absence of RecA is rather low (10). Various recombinase-based cloning methods, including Gateway cloning (Invitrogen), Cre-lox recombination, Red/ET recombination (Gene Bridges), In-Fusion (Clontech), Cold Fusion (System Biosciences), and CloneEZ (GenScript), have been developed, but the general use of these methods has been hampered by high cost and restrictions in the sequence or hosts (2, 12-15).We have optimized SLIC to make it comparable to commercial methods in terms of simplicity, time saving, and cloning efficiency. One-step SLIC utilizes only T4 DNA polymerase but shows cloning efficiencies similar to those of the original SLIC method in the presence of RecA and of commercial methods. An overview of one-step SLIC is illustrated in Fig. 1A. First, the vector needs to be linearized by either restriction enzyme digestion or inverse PCR. An insert(s) is prepared by PCR with primers with a 15-bp or longer extension homologous to each end of the linearized vector. Second, the vector and insert(s) are mixed and incubated at room temperature for 2.5 min with T4 DNA polymerase to generate 5= overhangs. For optimal results, a 1:2 to 1:4 molar vector-to-insert ratio is desirable. Third, the reaction mixture is placed on ice for 10 min for single-strand annealing and then competent Escherichia coli cells are transformed with the annealed DNA complex directly. The annealed complex turns into seamless recombinant DNA through homologous recombination in vivo with high efficiency. For a detailed description of the method, see the supplemental material.pUC118-HMG (11) was cleaved with BamHI, and a 1-kb insert containing Ton_0709 from the genomic DNA of Thermococcus onnurineus NA1 ...

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The shift from terrestrial to aquatic life by whales was a substantial evolutionary event. Here we report the whole-genome sequencing and de novo assembly of the minke whale genome, as well as the whole-genome sequences of three minke whales, a fin whale, a bottlenose dolphin and a finless porpoise. Our comparative genomic analysis identified an expansion in the whale lineage of gene families associated with stress-responsive proteins and anaerobic metabolism, whereas gene families related to body hair and sensory receptors were contracted. Our analysis also identified whale-specific mutations in genes encoding antioxidants and enzymes controlling blood pressure and salt concentration. Overall the whale-genome sequences exhibited distinct features that are associated with the physiological and morphological changes needed for life in an aquatic environment, marked by resistance to physiological stresses caused by a lack of oxygen, increased amounts of reactive oxygen species and high salt levels.

Gold compounds are used in the treatment of rheumatoid arthritis. NF-κB is a transcription factor implicated in the expression of many inflammatory genes. NF-κB is activated by signal-induced phosphorylation and subsequent degradation of inhibitory IκB (inhibitory protein that dissociates from NF-κB) proteins, and a multisubunit IκB kinase (IKK) has been identified previously. We tested the effect of various gold compounds on the activation of NF-κB and IKK in LPS-stimulated RAW 264.7 mouse macrophages. A lipophilic gold compound, auranofin, suppressed the LPS-induced increase of nuclear κB-binding activity, degradation of IκB proteins, and IKK activation. Auranofin also blocked IKK activation induced by TNF and PMA/ionomycin, suggesting that the target of auranofin action is common among these diverse signal pathways. In vitro IKK activity was suppressed by addition of hydrophilic gold compounds, such as aurothiomalate, aurothioglucose, and AuCl3. Other thiol-reactive metal ions such as zinc and copper also inhibited IKK activity in vitro, and induction of IKK in LPS-stimulated macrophages. In vitro IKK activity required the presence of reducing agent and was blocked by addition of thiol group-reactive agents. Two catalytic subunits of IKK complex, IKKα and IKKβ, were both inhibited by these thiol-modifying agents, suggesting the presence of a cysteine sulfhydryl group in these subunits, which is critical for enzyme activity. The antiinflammatory activity of gold compounds in the treatment of rheumatoid arthritis may depend on modification of this thiol group by gold.

SUMMARYProin¯ammatory cytokine tumour necrosis factor (TNF) mediates its diverse effects through cell surface receptors. A variety of in¯ammatory signals are known to modulate TNF activities by changing expression and shedding of cell-surface TNF receptors. We have examined the effects of anti-rheumatic drug chloroquine on the expression of cell surface and soluble TNF receptors in human histiocytic U-937 cells. Chloroquine partially reduced production of soluble p55 and p75 TNF receptors in cells stimulated with phorbol 12-myristate 13-acetate (PMA). In these cells, induction of both TNF receptor mRNA was not changed and the levels of cell-associated TNF receptors were rather increased by chloroquine. Flow cytometric analysis revealed that chloroquine does not inhibit the PMAtriggered shedding of TNF receptors from cell surface, while it was suppressed by a metalloproteinase inhibitor BB-3103. Treatment of U-937 cells with chloroquine signi®cantly reduced the level of cell surface TNF receptors and a similar effect was observed with human peripheral blood monocytes. Other weak-base amines, including hydroxychloroquine, ammonium chloride and methylamine, also induced reduction of cell surface TNF receptors, whereas lysosomal proteinase inhibitor, leupeptin, and BB-3013 were without effect. Our results suggest that chloroquine down-regulates cell surface TNF receptors by retarding their transport to the cell surface, while cleavage of cell surface receptors is not inhibited by chloroquine.

Low oxygen or hypoxia can be observed in the central region of solid tumors. Hypoxia is a strong stimulus for new blood vessel formation or angiogenesis, which is essential for tumor growth and progression. Fibroblast growth factor 11 (FGF11) is an intracellular non-secretory FGF whose function has not yet been fully characterized. In the present study, we demonstrated that FGF11 expression is upregulated under hypoxic conditions in human umbilical vein endothelial cells (HUVECs). FGF11 overexpression stimulated capillary-like tube formation, yet did not affect cell migration. Notably, FGF11 markedly increased the levels of tight junction proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5 in HUVECs. The FGF11 promoter contains hypoxia response elements (HREs), and hypoxia-inducible factor-1 (HIF-1) binds to HREs to activate hypoxia-related genes. We demonstrated that hypoxia or HIF-1 expression under normoxic conditions increased the luciferase activity driven by the FGF11 promoter. However, deletion of the HREs from the FGF11 promoter rendered reporter gene activity unresponsive to hypoxia or HIF-1. Taken together, we propose that FGF11 may be involved in the stabilization of capillary-like tube structures associated with angiogenesis and may act as a modulator of hypoxia-induced pathological processes such as tumorigenesis.

Abbreviations: AMPK, AMP-activated protein kinase; HFD, high-fat diet; ND, normal diet; PPARγ, peroxisome proliferator-activated receptor γ; FAS, fatty acid synthase; ACC1, acetyl-CoA carboxylase 1; SCD1, stearyl-CoA desaturase 1; RXRα , retinoid X receptor α ; BADGE, bisphenol A diglycidyl ether; ADD1, adipocyte differentiation and determination factor 1; SREBP1c, sterol regulatory element binding protein 1c; ACO, acyl-CoA oxidase; CPT1, carnitine palmitoyltransferase 1; C/EBP, CCAAT/enhancer-binding protein; AICAR, aminoimidazole carboxamide ribonucleotide AbstractLysimachia foenum-graecum has been used as an oriental medicine with anti-inflammatory effect. The anti-obesity effect of L. foenum-graecum extract (LFE) was first discovered in our screening of natural product extract library against adipogenesis. To characterize its anti-obesity effects and to evaluate its potential as an anti-obesity drug, we performed various obesity-related experiments in vitro and in vivo. In adipogenesis assay, LFE blocked the differentiation of 3T3-L1 preadipocyte in a dose-dependent manner with an IC50 of 2.5 μg/ml. In addition, LFE suppressed the expression of lipogenic genes, while increasing the expression of lipolytic genes in vitro at 10 μ g/ml and in vivo at 100 mg/kg/day. The anti-adipogenic and anti-lipogenic effect of LFE seems to be mediated by the inhibition of PPARγ and C/EBPα expression as shown in in vitro and in vivo, and the suppression of PPARγ activity in vitro. Moreover, LFE stimulated fatty acid oxidation in an AMPK-dependent manner. In high-fat diet (HFD)-induced obese mice (n = 8/group), oral administration of LFE at 30, 100, and 300 mg/kg/day decreased total body weight gain significantly in all doses tested. No difference in food intake was observed between vehicle-and LFE-treated HFD mice. The weight of white adipose tissues including abdominal subcutaneous, epididymal, and perirenal adipose tissue was reduced markedly in LFE-treated HFD mice in a dose-dependent manner. Treatment of LFE also greatly improved serum levels of obesity-related biomarkers such as glucose, triglycerides, and adipocytokines leptin, adiponectin, and resistin. All together, these results showed anti-obesity effects of LFE on adipogenesis and lipid metabolism in vitro and in vivo and raised a possibility of developing LFE as anti-obesity therapeutics.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent joint swelling and progressive destruction of cartilage and bone. Current RA treatments are largely empirical in origin and their precise mechanism of action is uncertain. Increasing evidence shows that chronic inflammatory diseases such as RA are caused by prolonged production of proinflammatory cytokines including tumor necrosis factor (TNF) and interleukin 1 (IL-1). The nuclear factor kappaB (NF-kappaB) plays an essential role in transcriptional activation of TNF and IL-1. NF-kappaB is induced by many stimuli including TNF and IL-1, forming a positive regulatory cycle that may amplify and maintain RA disease process. NF-kappaB and enzymes involved in its activation can be a target for anti-inflammatory treatment. Aspirin and sodium salicylate inhibit activation of NF-KB by blocking IkappaB kinase, a key enzyme in NF-kappaB activation. Glucocorticoids suppress expression of inflammatory genes by binding glucocorticoid receptor with NF-kappaB, and increasing expression of inhibitory protein of NF-kappaB, IkappaBalpha. Sulfasalazine and gold compounds also inhibit NF-kappaB activation. Continuing advances in our understanding of action mechanism of antirheumatic agents will benefit the future development of RA regimens with greater efficacy and less toxicity.