The Analyses of Cetacean Virus-Responsive Genes Reveal Evolutionary Marks in Mucosal Immunity-Associated Genes.

Viruses are the most common and abundant organisms in the marine environment. To better understand how cetaceans have adapted to this virus-rich environment, we compared cetacean virus-responsive genes to those from terrestrial mammals. We identified virus-responsive gene sequences in seven species of cetaceans, which we compared with orthologous sequences in seven terrestrial mammals. As a result of evolution analysis using the branch model and the branch-site model, 21 genes were selected using at least one model. IFN-ε, an antiviral cytokine expressed at mucous membranes, and its receptor IFNAR1 contain cetacean-specific amino acid substitutions that might change the interaction between the two proteins and lead to regulation of the immune system against viruses. Cetacean-specific amino acid substitutions in IL-6, IL-27, and the signal transducer and activator of transcription (STAT)1 are also predicted to alter the mucosal immune response of cetaceans. Since mucosal membranes are the first line of defense against the external environment and are involved in immune tolerance, our analysis of cetacean virus-responsive genes suggests that genes with cetacean-specific mutations in mucosal immunity-related genes play an important role in the protection and/or regulation of immune responses against viruses.

α-Poly-L-lysine functions as an adipogenic inducer in 3T3-L1 preadipocytes.

α-Poly-L-lysine (PLL) has been used for various purposes such as cell attachment, immunization, and molecular delivery, and is known to be cytotoxic to several cell lines. Here, we studied the effect of PLL on the adipogenesis of 3T3-L1 cells and investigated the underlying mechanism. Differentiation media containing PLL with a molecular weight (MW) greater than 4 kDa enhanced lipid droplet formation and increased adipogenic marker levels, indicating an increase in adipocyte differentiation. PLL with a molecular weight between 30 and 70 kDa was more effective than PLL of other sizes in 3T3-L1 cell differentiation. Moreover, PLL induced 3T3-L1 adipogenesis in insulin-free adipocyte differentiation medium. Incubation with insulin and PLL exhibited greater adipogenesis than insulin treatment only even at a high concentration. PLL stimulated insulin signaling and augmented the signaling pathway when it was added with insulin. While PLL did not activate the glucocorticoid receptor, which is phosphorylated by dexamethasone (DEX), it showed a positive effect on the cAMP signal pathway when preadipocytes were treated with PLL and 3-isobutyl-1-methylxanthine (IBMX). Consistent with these results, incubation with PLL and DEX without IBMX induced adipocyte differentiation. We also observed that the mitotic clonal expansion phase was the critical stage in adipogenesis for inducing the effects of PLL. These results suggest that PLL functions as an adipogenic inducer in 3T3-L1 preadipocytes and PLL has a direct effect on insulin signaling, one of the main regulatory pathways.

The genome of the giant Nomura's jellyfish sheds light on the early evolution of active predation.

BACKGROUND: Unique among cnidarians, jellyfish have remarkable morphological and biochemical innovations that allow them to actively hunt in the water column and were some of the first animals to become free-swimming. The class Scyphozoa, or true jellyfish, are characterized by a predominant medusa life-stage consisting of a bell and venomous tentacles used for hunting and defense, as well as using pulsed jet propulsion for mobility. Here, we present the genome of the giant Nomura's jellyfish (Nemopilema nomurai) to understand the genetic basis of these key innovations. RESULTS: We sequenced the genome and transcriptomes of the bell and tentacles of the giant Nomura's jellyfish as well as transcriptomes across tissues and developmental stages of the Sanderia malayensis jellyfish. Analyses of the Nemopilema and other cnidarian genomes revealed adaptations associated with swimming, marked by codon bias in muscle contraction and expansion of neurotransmitter genes, along with expanded Myosin type II family and venom domains, possibly contributing to jellyfish mobility and active predation. We also identified gene family expansions of Wnt and posterior Hox genes and discovered the important role of retinoic acid signaling in this ancient lineage of metazoans, which together may be related to the unique jellyfish body plan (medusa formation). CONCLUSIONS: Taken together, the Nemopilema jellyfish genome and transcriptomes genetically confirm their unique morphological and physiological traits, which may have contributed to the success of jellyfish as early multi-cellular predators.

Analysis of the Draft Genome of the Red Seaweed Gracilariopsis chorda Provides Insights into Genome Size Evolution in Rhodophyta.

Red algae (Rhodophyta) underwent two phases of large-scale genome reduction during their early evolution. The red seaweeds did not attain genome sizes or gene inventories typical of other multicellular eukaryotes. We generated a high-quality 92.1 Mb draft genome assembly from the red seaweed Gracilariopsis chorda, including methylation and small (s)RNA data. We analyzed these and other Archaeplastida genomes to address three questions: 1) What is the role of repeats and transposable elements (TEs) in explaining Rhodophyta genome size variation, 2) what is the history of genome duplication and gene family expansion/reduction in these taxa, and 3) is there evidence for TE suppression in red algae? We find that the number of predicted genes in red algae is relatively small (4,803-13,125 genes), particularly when compared with land plants, with no evidence of polyploidization. Genome size variation is primarily explained by TE expansion with the red seaweeds having the largest genomes. Long terminal repeat elements and DNA repeats are the major contributors to genome size growth. About 8.3% of the G. chorda genome undergoes cytosine methylation among gene bodies, promoters, and TEs, and 71.5% of TEs contain methylated-DNA with 57% of these regions associated with sRNAs. These latter results suggest a role for TE-associated sRNAs in RNA-dependent DNA methylation to facilitate silencing. We postulate that the evolution of genome size in red algae is the result of the combined action of TE spread and the concomitant emergence of its epigenetic suppression, together with other important factors such as changes in population size.

iMGEins: detecting novel mobile genetic elements inserted in individual genomes.

BACKGROUND: Recent advances in sequencing technology have allowed us to investigate personal genomes to find structural variations, which have been studied extensively to identify their association with the physiology of diseases such as cancer. In particular, mobile genetic elements (MGEs) are one of the major constituents of the human genomes, and cause genome instability by insertion, mutation, and rearrangement. RESULT: We have developed a new program, iMGEins, to identify such novel MGEs by using sequencing reads of individual genomes, and to explore the breakpoints with the supporting reads and MGEs detected. iMGEins is the first MGE detection program that integrates three algorithmic components: discordant read-pair mapping, split-read mapping, and insertion sequence assembly. Our evaluation results showed its outstanding performance in detecting novel MGEs from simulated genomes, as well as real personal genomes. In detail, the average recall and precision rates of iMGEins are 96.67 and 100%, respectively, which are the highest among the programs compared. In the testing with real human genomes of the NA12878 sample, iMGEins shows the highest accuracy in detecting MGEs within 20 bp proximity of the breakpoints annotated. CONCLUSION: In order to study the dynamics of MGEs in individual genomes, iMGEins was developed to accurately detect breakpoints and report inserted MGEs. Compared with other programs, iMGEins has valuable features of identifying novel MGEs and assembling the MGEs inserted.

Molecular characterization of positively selected genes contributing aquatic adaptation in marine mammals.

BACKGROUND: Marine mammals, which have evolved independently into three distinct lineages, share common physiological features that contribute to their adaptation to the marine environment. OBJECTIVE: To identify positively selected genes (PSGs) for adaptation to the marine environment using available genomic data from three taxonomic orders: cetaceans, pinnipeds, and sirenians. METHODS: Based on the genomes within each group of Artiodactyla, Carnivora and Afrotheria, we performed selection analysis using the branch-site model in CODEML. RESULTS: Based on the branch-site model, 460, 614, and 359 PSGs were predicted for the cetaceans, pinnipeds, and sirenians, respectively. Functional enrichment analysis indicated that genes associated with hemostasis were positively selected across all lineages of marine mammals. We observed positive selection signals for the hemostasis and coagulation-related genes plasminogen activator, urokinase (PLAU), multimerin 1 (MMRN1), gamma-glutamyl carboxylase (GGCX), and platelet endothelial aggregation receptor 1 (PEAR1). Additionally, we found out that the sodium voltage-gated channel alpha subunit 9 (SCN9A), serine/arginine repetitive matrix 4 (SRRM4), and Ki-ras-induced actin-interacting protein (KRAP) are under positive selection pressure and are associated with cognition, neurite outgrowth, and IP3-mediated Ca2 + release, respectively. CONCLUSION: This study will contribute to our understanding of the adaptive evolution of marine mammals by providing information on a group of candidate genes that are predicted to influence adaptation to aquatic environments, as well as their functional characteristics.

Structural and biochemical investigation into stable FGF2 mutants with novel mutation sites and hydrophobic replacements for surface-exposed cysteines.

Fibroblast growth factor 2 (FGF2) is an attractive biomaterial for pharmaceuticals and functional cosmetics. To improve the thermo-stability of FGF2, we designed two mutants harboring four-point mutations: FGF2-M1 (D28E/C78L/C96I/S137P) and FGF2-M2 (D28E/C78I/C96I/S137P) through bioinformatics, molecular thermodynamics, and molecular modeling. The D28E mutation reduced fragmentation of the FGF2 wild type during preparation, and the substitution of a whale-specific amino acid, S137P, enhanced the thermal stability of FGF2. Surface-exposed cysteines that participate in oligomerization through intermolecular disulfide bond formation were substituted with hydrophobic residues (C78L/C78I and C96I) using the in silico method. High-resolution crystal structures revealed at the atomic level that the introduction of mutations stabilizes each local region by forming more favorable interactions with neighboring residues. In particular, P137 forms CH-π interactions with the side chain indole ring of W123, which seems to stabilize a ß-hairpin structure, containing a heparin-binding site of FGF2. Compared to the wild type, both FGF2-M1 and FGF2-M2 maintained greater solubility after a week at 45 °C, with their Tm values rising by ~ 5 °C. Furthermore, the duration for FGF2-M1 and FGF2-M2 to reach 50% residual activity at 45 °C extended to 8.8- and 8.2-fold longer, respectively, than that of the wild type. Interestingly, the hydrophobic substitution of surface-exposed cysteine in both FGF2 mutants makes them more resistant to proteolytic cleavage by trypsin, subtilisin, proteinase K, and actinase than the wild type and the Cys → Ser substitution. The hydrophobic replacements can influence protease resistance as well as oligomerization and thermal stability. It is notable that hydrophobic substitutions of surface-exposed cysteines, as well as D28E and S137P of the FGF2 mutants, were designed through various approaches with structural implications. Therefore, the engineering strategies and structural insights adopted in this study could be applied to improve the stability of other proteins.

Heating-mediated purification of active FGF21 and structure-based design of its variant with enhanced potency.

Fibroblast growth factor 21 (FGF21) has pharmaceutical potential against obesity-related metabolic disorders, including non-alcoholic fatty liver disease. Since thermal stability is a desirable factor for therapeutic proteins, we investigated the thermal behavior of human FGF21. FGF21 remained soluble after heating; thus, we examined its temperature-induced structural changes using circular dichroism (CD). FGF21 showed inter-convertible temperature-specific CD spectra. The CD spectrum at 100 °C returned to that at 20 °C when the heated FGF21 solution was cooled. Through loop swapping, the connecting loop between ß10 and ß12 in FGF21 was revealed to be associated with the unique thermal behavior of FGF21. According to surface plasmon resonance (SPR) experiments, in vitro cell-based assays, and model high-fat diet (HFD)-induced obesity studies, heated FGF21 maintained biological activities that were comparable to those of non-heated and commercial FGF21s. Based on sequence comparison and structural analysis, five point-mutations were introduced into FGF21. Compared with the wild type, the heated FGF21 variant displayed improved therapeutic potential in terms of body weight loss, the levels of hepatic triglycerides and lipids, and the degree of vacuolization of liver in HFD-fed mice.

N-benzyl-N-methyldecan-1-amine and its derivative mitigate 2,4- dinitrobenzenesulfonic acid-induced colitis and collagen-induced rheumatoid arthritis.

As our previous study revealed that N-benzyl-N-methyldecan-1-amine (BMDA), a new molecule originated from Allium sativum, exhibits anti-neoplastic activities, we herein explored other functions of the compound and its derivative [decyl-(4-methoxy-benzyl)-methyl-amine; DMMA] including anti-inflammatory and anti-oxidative activities. Pretreatment of THP-1 cells with BMDA or DMMA inhibited tumor necrosis factor (TNF)-α and interleukin (IL)-1ß production, and blocked c-jun terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), MAPKAP kinase (MK)2 and NF-κΒ inflammatory signaling during LPS stimulation. Rectal treatment with BMDA or DMMA reduced the severity of colitis in 2,4-dinitrobenzenesulfonic acid (DNBS)-treated rat. Consistently, administration of the compounds decreased myeloperoxidase (MPO) activity (representing neutrophil infiltration in colonic mucosa), production of inflammatory mediators such as cytokine-induced neutrophil chemoattractant (CINC)-3 and TNF-α, and activation of JNK and p38 MAPK in the colon tissues. In addition, oral administration of these compounds ameliorated collagen-induced rheumatoid arthritis (RA) in mice. The treatment diminished the levels of inflammatory cytokine transcripts, and protected connective tissues through the expression of anti-oxidation proteins such as nuclear factor erythroid-related factor (Nrf)2 and heme oxygenase (HO)1. Additionally, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels did not differ between the BMDA- or DMMA-treated and control animals, indicating that the compounds do not possess liver toxicity. Taken together, these findings propose that BMDA and DMMA could be used as new drugs for curing inflammatory bowel disease (IBD) and RA.

One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies.

We 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.

Fibroblast growth factor 11 inhibits foot-and-mouth disease virus gene expression and replication in vitro.

Foot-and-mouth disease virus (FMDV) causes highly contagious disease of cloven-hoofed animals such as cattle, swine, and sheep. Although FMD vaccine is the traditional way to protect against the disease, the use of FMD vaccines to protect early infection is limited. The alternative strategy of applying antiviral agents is required to control the spread of FMDV in outbreak situations. Fibroblast growth factor 11 (FGF11) is a member of the intracellular FGF. Here, we identified the inhibitory effect of FGF11 on FMDV gene expression through the transcriptional and translational regulation. For the quantitative analysis of FMDV transcription/translation level, we firstly constructed a plasmid reporter system (FMDV five prime untranslated region (5' UTR) -luci) conjugating luciferase encoding gene with FMDV 5' UTR region, which is a non-coding region to control FMDV transcription/translation and includes cis-acting replication element (CRE) and internal ribosome entry site (IRES). FGF11 decreased the gene expression of FMDV 5' UTR-luci reporter in a dose-dependent manner. We further confirmed the inhibitory function of FGF11 on FMDV gene expression a replication in the FMDV-infected pig cells. FGF11 expression inhibited RNA production of FMDV RNA polymerase 3D gene in the FMDV-infected cells. In addition, while FMDV cell infection induced cytopathic effect (CPE) within 24 hr, FGF11 expression dramatically repressed CPE at the basal level. These results indicate that FGF11 inhibits FMDV gene expression and replication in vitro, implicating to provide intervention strategy for FMDV pathogenesis and transmission.

Expression and purification of intracrine human FGF 11 and study of its FGFR-dependent biological activity.

Fibroblast growth factor 11 (FGF11) is one of intracrine FGFs (iFGFs), which function within cells. Unlike canonical FGFs, FGF11 remains intracellularly and plays biological roles in FGF receptor (FGFR)-independent manner. Here, we established an expression system of recombinant FGF11 proteins in E. coli and investigated whether the extracellular administration of FGF11 can activate cellular signaling. Human FGF11 has two isoforms, FGF11a and FGF11b, depending on the presence of nuclear localization sequences (NLSs) in the N-terminus. Because these two isoforms are unstable, we prepared an FGF11a-Mut by substituting three cysteine residues in the NLS with serine and FGF11b-ΔC with C-terminal truncation. The introduction of mutation in the NLS improved the solubility of FGF11 prepared from E. coli. Exogenous addition of FGF11b and FGF11b-ΔC to BALB3T3 increased cell proliferation, while FGF11a-Mut exerted no effect. FGF11b-ΔC showed higher cell proliferation activity and FGFR signaling than FGF11b. The cell-proliferating activities of FGF11b and FGF11b-ΔC were blocked by an FGFR1 inhibitor or a recombinant FGFR1, confirming the FGFR1-dependent extracellular activity of FGF11b. The analysis of circular dichroism suggested that the C-terminus of FGF11 has an α-helical structure, which may affect its interaction with FGFR1. These results suggest that the N-and C-terminus of recombinant FGF11 are involved in the activation of FGFR1. The above results provide novel insights into the function and mechanism of FGF11 that may aid the development of useful ligands for FGFR regulation.

Crystallization and preliminary X-ray crystallographic analysis of the NmrA-like DDB_G0286605 protein from the social amoeba Dictyostelium discoideum.

The DDB_G0286605 gene product from Dictyostelium discoideum, an NmrA-like protein that belongs to the short-chain dehydrogenase/reductase family, has been crystallized by the hanging-drop vapour-diffusion method at 295 K. A 1.64 Šresolution data set was collected using synchrotron radiation. The DDB_G0286605 protein crystals belonged to space group P2(1), with unit-cell parameters a=67.598, b=54.935, c=84.219 Å, ß = 109.620°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 43.25% with 99% probability. Molecular-replacement trials were attempted with three NmrA-like proteins, NmrA, HSCARG and QOR2, as search models, but failed. This may be a consequence of the low sequence identity between the DDB_G0286605 protein and the search models (DDB_G0286605 has a primary-sequence identity of 28, 32 and 19% to NmrA, HCARG and QOR2, respectively).

Crystallization and preliminary X-ray crystallographic analysis of the short-chain dehydrogenase/reductase-type DDB_G0291732 protein from Dictyostelium discoideum.

The DDB_G0291732 gene product from Dictyostelium discoideum, which is a NmrA-like protein that belongs to the short-chain dehydrogenase/reductase superfamily but shows deviations in conserved sequence regions, has been crystallized by the hanging-drop vapour-diffusion method at 295 K. A 1.65 Šresolution data set was collected using synchrotron radiation. The crystals of DDB_G0291732 protein belonged to space group P2(1), with unit-cell parameters a=38.5, b=63.7, c=56.0 Å, ß=91.7°. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 38.1%.

Methylglyoxal accumulation by glutathione depletion leads to cell cycle arrest in Dictyostelium.

Reduced glutathione (GSH) serves as a primary redox buffer and its depletion causes growth inhibition or apoptosis in many organisms. In Dictyostelium discoideum, the null mutant (gcsA(-)) of gcsA encoding gamma-glutamylcysteine synthetase shows growth arrest and developmental defect when GSH is depleted. To investigate the mechanism by which GSH depletion induces growth arrest, a proteomic analysis was performed and aldose reductase (AlrA) was identified as the most prominently induced protein in gcsA(-) cells. Induction of AlrA was dependent on GSH concentration and was repressed by GSH but not effectively by either the reducing agent such as dithiothreitol or overexpression of superoxide dismutase. Methylglyoxal (MG), a toxic alpha-ketoaldehyde, strongly induced alrA expression and AlrA catalysed MG reduction efficiently. The alrA knockdown gcsA(-) cells (gcsA(-)/alrA(as)) exhibited more decreased growth rate than gcsA(-) cells, whereas the gcsA(-) cells overexpressing alrA (gcsA(-)/alrA(oe)) showed the recovery of growth rate. Interestingly, intracellular MG levels were significantly augmented in gcsA(-)/alrA(as) cells compared with gcsA(-) cells following GSH depletion. By contrast, gcsA(-)/alrA(oe) cells showed repression of MG induction. Furthermore, MG treatment inhibited growth of wild-type KAx3 cells, inducing G1 phase arrest. Thus, our findings suggest that MG accumulated by GSH depletion inhibits cell growth in Dictyostelium.

FGF11 influences 3T3-L1 preadipocyte differentiation by modulating the expression of PPARγ regulators.

Fibroblast growth factor 11 (FGF11) is a member of the intracellular fibroblast growth factor superfamily. Here, we identified FGF11 as a novel mediator of adipogenesis. During 3T3-L1 adipocyte differentiation, the expression of FGF11 decreased at the mitotic clonal expansion stage and increased at the terminal differentiation stage. FGF11 knockdown reduced the expression of peroxisome proliferator-activated receptor gamma (PPARγ), a master regulator of adipogenesis, resulting in the inhibition of adipocyte differentiation. Treatment with the PPARγ agonist rosiglitazone restored the inhibition of adipogenesis caused by FGF11 knockdown. We also report that the expression of the PPARγ regulators CCAAT/enhancer-binding protein α, sterol regulatory element-binding protein 1, KLF9, KLF2, GATA binding factor 2, and GATA binding factor 3 was influenced by FGF11. These results suggest that FGF11 indirectly controls the expression of PPARγ through modifying the expression of multiple PPARγ regulators, thereby mediating adipogenesis.

The Draft Genome of an Octocoral, Dendronephthya gigantea.

Coral reefs composed of stony corals are threatened by global marine environmental changes. However, soft coral communities of octocorallian species, appear more resilient. The genomes of several cnidarians species have been published, including from stony corals, sea anemones, and hydra. To fill the phylogenetic gap for octocoral species of cnidarians, we sequenced the octocoral, Dendronephthya gigantea, a nonsymbiotic soft coral, commonly known as the carnation coral. The D. gigantea genome size is ∼276 Mb. A high-quality genome assembly was constructed from PacBio long reads (29.85 Gb with 108× coverage) and Illumina short paired-end reads (35.54 Gb with 128× coverage) resulting in the highest N50 value (1.4 Mb) reported thus far among cnidarian genomes. About 12% of the genome is repetitive elements and contained 28,879 predicted protein-coding genes. This gene set is composed of 94% complete BUSCO ortholog benchmark genes, which is the second highest value among the cnidarians, indicating high quality. Based on molecular phylogenetic analysis, octocoral and hexacoral divergence times were estimated at 544 MYA. There is a clear difference in Hox gene composition between these species: unlike hexacorals, the Antp superclass Evx gene was absent in D. gigantea. Here, we present the first genome assembly of a nonsymbiotic octocoral, D. gigantea to aid in the comparative genomic analysis of cnidarians, including stony and soft corals, both symbiotic and nonsymbiotic. The D. gigantea genome may also provide clues to mechanisms of differential coping between the soft and stony corals in response to scenarios of global warming.

High resolution crystal structure of PedB: a structural basis for the classification of pediocin-like immunity proteins.

BACKGROUND: Pediocin-like bacteriocins, ribosomally-synthesized antimicrobial peptides, are generally coexpressed with cognate immunity proteins in order to protect the bacteriocin-producer from its own bacteriocin. As a step for understanding the mode of action of immunity proteins, we determined the crystal structure of PedB, a pediocin-like immunity protein conferring immunity to pediocin PP-1. RESULTS: The 1.6 A crystal structure of PedB reveals that PedB consists of an antiparallel four-helix bundle with a flexible C-terminal end. PedB shows structural similarity to an immunity protein against enterocin A (EntA-im) but some disparity to an immunity protein against carnobacteriocin B2 (ImB2) in both the C-terminal conformation and the local structure constructed by alpha3, alpha4, and their connecting loop. Structure-inspired mutational studies reveal that deletion of the last seven residues of the C-terminus of PedB almost abolished its immunity activity. CONCLUSION: The fact that PedB, EntA-im, and ImB2 share a four-helix bundle structure strongly suggests the structural conservation of this motif in the pediocin-like immunity proteins. The significant difference in the core structure and the C-terminal conformation provides a structural basis for the classification of pediocin-like immunity proteins. Our mutational study using C-terminal-shortened PedBs and the investigation of primary sequence of the C-terminal region, propose that several polar or charged residues in the extreme C-terminus of PedB which is crucial for the immunity are involved in the specific recognition of pediocin PP-1.

FGF11 induced by hypoxia interacts with HIF-1α and enhances its stability.

Fibroblast growth factor 11 (FGF11) is an intracellular FGF. Although induction of FGF11 by hypoxia has been observed in several cell types, the molecular function of FGF11 is not clearly understood yet. Here, we investigated the role of FGF11 under hypoxia. We identified hypoxia-inducible factor-1α (HIF-1α) as an interacting protein of FGF11 using immunoprecipitation and mass spectrometry. FGF11 knockdown decreased HIF-1α protein, while FGF11 overexpression increased it, without affecting HIF-1α mRNA. Protein stability test and ubiquitination assay showed that FGF11 increased HIF-1α stability by acting upstream of proteasomal degradation. Altogether, these results suggest a cross-regulation between HIF-1α and FGF11, through which hypoxia-induced FGF11 reinforces hypoxia responses by enhancing the stability of HIF-1α.

Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning.

Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature. To overcome this problem, we developed a modified thermo-regulated one-step SLIC approach by testing shorter T4 DNA polymerase treatment durations (5 s-2.5 min) over a wide range of temperatures (25-75°C). The highest cloning efficiency resulted when inserts with homology lengths <20 bases were treated with T4 DNA polymerase for 30 s at 50°C. This briefer T4 polymerase treatment at a higher temperature helps increase cloning efficiency for inserts with strong secondary structures at their ends, increasing the utility of one-step SLIC for the cloning of short fragments.