Discovery of TCP-(MP)-caffeic acid analogs as a new class of agents for treatment of osteoclastic bone loss.

Inhibition of LSD1 was proposed as promising and attractive therapies for treating osteoporosis. Here, we synthesized a series of novel TCP-(MP)-Caffeic acid analogs as potential LSD1 inhibitors to assess their inhibitory effects on osteoclastogenesis by using TRAP-staining assay and try to explore the preliminary SAR. Among them, TCP-MP-CA (11a) demonstrated osteoclastic bone loss both in vitro and in vivo, showing a significant improvement in the in vivo effects compared to the LSD1 inhibitor GSK-LSD1. Additionally, we elucidated a mechanism that 11a and its precursor that 11e directly bind to LSD1/CoREST complex through FAD to inhibit LSD1 demethylation activity and influence its downstream IκB/NF-κB signaling pathway, and thus regulate osteoclastic bone loss. These findings suggested 11a or 11e as potential novel candidates for treating osteoclastic bone loss, and a concept for further development of TCP-(MP)-Caffeic acid analogs for therapeutic use in osteoporosis clinics.

Promising properties of cytochrome P450 BM3 reconstituted from separate domains by split intein.

Recombinant cytochrome P450 monooxygenases possess significant potential as biocatalysts, and efforts to improve heme content, electron coupling efficiency, and catalytic activity and stability are ongoing. Domain swapping between heme and reductase domains, whether natural or engineered, has thus received increasing attention. Here, we successfully achieved split intein-mediated reconstitution (IMR) of the heme and reductase domains of P450 BM3 both in vitro and in vivo. Intriguingly, the reconstituted enzymes displayed promising properties for practical use. IMR BM3 exhibited a higher heme content (>50 %) and a greater tendency for oligomerization compared to the wild-type enzyme. Moreover, these reconstituted enzymes exhibited a distinct increase in activity ranging from 165 % to 430 % even under the same heme concentrations. The reproducibility of our results strongly suggests that the proposed reconstitution approach could pave a new path for enhancing the catalytic efficiency of related enzymes.

Gamma irradiation-engineered macrophage-derived exosomes as potential immunomodulatory therapeutic agents.

The modulation of macrophage polarization is a promising strategy for maintaining homeostasis and improving innate and adaptive immunity. Low-dose ionizing radiation has been implicated in macrophage immunomodulatory responses. However, studies on the relationship between exosomes and regulation of macrophage polarization induced by ionizing radiation are limited. Therefore, this study investigated the alterations in macrophages and exosomes induced by gamma irradiation and elucidated the underlying mechanisms. We used the mouse macrophage cell line RAW 264.7 to generate macrophages and performed western blot, quantitative reverse transcription-PCR, and gene ontology analyses to elucidate the molecular profiles of macrophage-derived exosomes under varying treatment conditions, including 10 Gy gamma irradiation. Exosomes isolated from gamma-irradiated M1 macrophages exhibited an enhanced M1 phenotype. Irradiation induced the activation of NF-κB and NLRP3 signaling in M1 macrophages, thereby promoting the expression of pro-inflammatory cytokines. Cytokine expression was also upregulated in gamma-irradiated M1 macrophage-released exosomes. Therefore, gamma irradiation has a remarkable effect on the immunomodulatory mechanisms and cytokine profiles of gamma-irradiated M1 macrophage-derived exosomes, and represents a potential immunotherapeutic modality.

Tumor Pre-Targeting System Using Streptavidin-Expressing Bacteria.

PURPOSE: A major obstacle to targeted cancer therapy is identifying suitable targets that are specifically and abundantly expressed by solid tumors. Certain bacterial strains selectively colonize solid tumors and can deliver genetically encoded cargo molecules to the tumor cells. Here, we engineered bacteria to express monomeric streptavidin (mSA) in tumors, and developed a novel tumor pre-targeting system by visualizing the presence of tumor-associated mSA using a biotinylated imaging probe. PROCEDURES: We constructed a plasmid expressing mSA fused to maltose-binding protein and optimized the ribosome binding site sequence to increase solubility and expression levels. E. coli MG1655 was transformed with the recombinant plasmid, expression of which is driven by the pBAD promotor. Expression of mSA was induced by L-arabinose 4 days after injection of bacteria into mice bearing CT26 mouse colon carcinoma cells. Selective accumulation of mSA in tumor tissues was visualized by optical imaging after administration of a biotinylated fluorescent dye. Counting of viable bacterial cells was also performed. RESULTS: Compared with a conventional system, the novel expression system resulted in significantly higher expression of mSA and sustained binding to biotin. Imaging signals in tumor tissues were significantly stronger in the mSA-expressing group than in non-expressing group (P = 0.0005). Furthermore, the fluorescent signal in tumor tissues became detectable again after multiple inductions with L-arabinose. The bacterial counts in tumor tissues showed no significant differences between conditions with and without L-arabinose (P = 0.45). Western blot analysis of tumor tissues confirmed expression and binding of mSA to biotin. CONCLUSIONS: We successfully engineered tumor-targeting bacteria carrying a recombinant plasmid expressing mSA, which was targeted to, and expressed in, tumor tissues. These data demonstrate the potential of this novel tumor pre-targeting system when combined with biotinylated imaging probes or therapeutic agents.

Construction and characterization of a functional variant hFGF7 with enhanced properties by circular permutation.

Human fibroblast growth factor 7 (hFGF7) is a member of the paracrine-acting FGF family and mediates various reactions such as wound healing, tissue homeostasis, and liver regeneration. These activities make it a plausible candidate for pharmaceutical applications as a drug. However, the low expression level and stability of the recombinant hFGF7 were known to be major hurdles for further applications. Here, the expression level and stability of hFGF7 were attempted to improve by changing the order of amino acids through circular permutation (CP), thereby expecting an alternative fate according to the N-end rule. CP-hFGF7 variants were constructed systematically by using putative amino acid residues in the loop region that avoided the disruption of the structural integrity especially in the functional motif. Among them, cp-hFGF7115-114 revealed a relatively higher expression level in the soluble fraction than the wild-type hFGF7 and was efficiently purified (7 mg L-1) to apparent homogeneity. The activity and stability of the purified variant cp-hFGF7115-114 were comparable or superior to that of the wild-type hFGF7, thereby strongly suggesting that CP could be an alternative tool for the functional expression of hFGF7 in Escherichia coli.

A short guide on blue fluorescent proteins: limits and perspectives.

The advent of the so-called colorful biology era is in line with the discovery of fluorescent proteins (FPs), which can be widely used to detect the intracellular locations of macromolecules or to determine the abundance of metabolites in organelles. The application of multiple FPs that emit different spectra and colors could be implemented to precisely evaluate cellular events. FPs were initially established with the emergence of the green fluorescent protein (GFP) from jellyfish. Red fluorescent proteins (RFPs) from marine anemones and several corals adopt fluorescent chromophores that are similar to GFP. Chromophores of GFP and GFP-like FPs are formed through the oxidative rearrangement of three chromophore-forming residues, thereby limiting their application to only oxidative environments. Alternatively, some proteins can be fluorescent upon their interaction with cellular prosthetic cofactors and, thus, work in aerobic and anaerobic conditions. The modification of an NADPH-dependent blue fluorescent protein (BFP) also expanded its application to the quantization of NADPH in the cellular environment. However, cofactor-dependent BFPs have an intrinsic weakness of poor photostability with a high fluorescent background. This review explores GFP-derived and NADPH-dependent BFPs with a focus on NADPH-dependent BFPs, which might be technically feasible in the near future upon coupling with two-photon fluorescence microscopy or nucleic acid-mimickers. KEY POINTS: • Oxidation-dependent GFP-like BFPs and redox-free NADPH-dependent BFPs • GFPs of weak photostability and intensity with a high fluorescent background • Real-time imaging using mBFP under two-photon fluorescence microscopy.

Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

Enhanced production of difficult-to-express proteins through knocking down rnpA gene expression.

Escherichia coli has been employed as a workhorse for the efficient production of recombinant proteins. However, some proteins were found to be difficult to produce in E. coli. The stability of mRNA has been considered as one of the important factors affecting recombinant protein production. Here we report a generally applicable and simple strategy for enhancing mRNA stability, and consequently improving recombinant protein production in E. coli. RNase P, a ribozyme comprising an RNA subunit (RnpB) and a protein subunit (RnpA), is involved in tRNA maturation. Based on the finding that purified RnpA can digest rRNA and mRNA in vitro, it was reasoned that knocking down the level of RnpA might enhance recombinant protein production. For this, the synthetic small regulatory RNA-based knockdown system was applied to reduce the expression level of RnpA. The developed RnpA knockdown system allowed successful overexpression of 23 different recombinant proteins of various origins and sizes, including Cas9 protein, antibody fragment, and spider silk protein. Notably, a 284.9-kDa ultra-high molecular weight, highly repetitive glycine-rich spider silk protein, which is one of the most difficult proteins to produce, could be produced to 1.38 g L-1 , about two-fold higher than the highest value previously achieved, by a fed-batch culture of recombinant E. coli strain employing the RnpA knockdown system. The RnpA-knockdown strategy reported here will be generally useful for the production of recombinant proteins including those that have been difficult to produce.

One-step metal affinity purification of recombinant hFGF19 without using tags.

Human fibroblast growth factor 19 (hFGF19) belongs to the endocrine FGF19 superfamily and is considered a potential agent to treat severe or relapsing nonalcoholic fatty liver disease. Numerous studies have confirmed the beneficial effects of this hormone on the related symptoms of the disease and attempts at producing recombinant proteins in various hosts are steadily proliferating. Recently, we reported that authentic hFGF19 can be solubly expressed through combining synonymous codon substitutions and co-expression with disulfide-bond isomerase (DsbC) in Escherichia coli. However, during purification, hFGF19 without the His-tag occasionally co-eluted with His-tagged DsbC when using metal affinity chromatography, thereby requiring auxiliary purification steps to achieve apparent homogeneity. This phenomenon provides evidence that hFGF19 specifically interacts with immobilized Ni2+, which can thus be used as an alternative tool for the purification of hFGF19. Consequently, we could simply and reproducibly purify hFGF19 from cell lysates by using Ni2+-immobilized metal affinity chromatography and stepwise gradient elution with imidazole.

Expression and Characterization of Monomeric Recombinant Isocitrate Dehydrogenases from Corynebacterium glutamicum and Azotobacter vinelandii for NADPH Regeneration.

The enzymatic transformation of various chemicals, especially using NADPH-dependent hydroxylase, into more soluble and/or high value-added products has steadily garnered increasing attention. However, the industrial application of these NADPH-dependent hydroxylases has been limited due to the high cost of the cofactor NADPH. As an alternative, enzymatic NADPH-regeneration systems have been developed and are frequently used in various fields. Here, we expressed and compared two recombinant isocitrate dehydrogenases (IDHs) from Corynebacterium glutamicum and Azotobacter vinelandii in Escherichia coli. Both enzymes were hyper-expressed in the soluble fraction of E. coli and were single-step purified to apparent homogeneity with yields of more than 850 mg/L. These enzymes also functioned well when paired with NADPH consumption systems. Specifically, NADPH was regenerated from NADP+ when an NADPH-consuming cytochrome P450 BM3 from Bacillus megaterium was incorporated. Therefore, both enzymes could be used as alternatives to the commonly used regeneration system for NADPH. These enzymes also have promising potential as genetic fusion partners with NADPH-dependent enzymes due to the monomeric nature of their quaternary structure, thereby resulting in self-sufficient biocatalysts via NADPH regeneration in a single polypeptide with NADPH-dependent activity.

A designed fusion tag for soluble expression and selective separation of extracellular domains of fibroblast growth factor receptors.

Fibroblast growth factor receptors (FGFRs) generate various transduction signals by interaction with fibroblast growth factors (FGFs) and are involved in various biological functions such as cell proliferation, migration, and differentiation. Malfunction of these proteins may lead to the development of various diseases, including cancer. Accordingly, FGFRs are considered an alternative therapeutic target for protein and/or gene therapy. However, the screening of antagonists or agonists of FGFRs is challenging due to their complex structural features associated with protein expression. Herein, we conducted the development of a protease-free cleavable tag (PFCT) for enhancing the solubility of difficult-to express protein by combining maltose-binding protein (MBP) and the C-terminal region of Npu intein. To validate the availability of the resulting tag for the functional production of extracellular domains of FGFRs (Ec_FGFRs), we performed fusion of PFCT with the N-terminus of Ec_FGFRs and analyzed the expression patterns. Almost all PFCT-Ec_FGFR fusion proteins were mainly detected in the soluble fraction except for Ec_FGFR4. Upon addition of the N-terminal region of Npu intein, approximately 85% of the PFCT-Ec_FGFRs was separated into PFCT and Ec_FGFR via intein-mediated cleavage. Additionally, the structural integrity of Ec_FGFR was confirmed by affinity purification using heparin column. Taken together, our study demonstrated that the PFCT could be used for soluble expression and selective separation of Ec_FGFRs.

Growth Suppression of a Gingivitis and Skin Pathogen Cutibacterium (Propionibacterium) acnes by Medicinal Plant Extracts.

Propionibacterium acnes, newly reclassified as Cutibacterium acnes, is an anaerobic Gram-positive bacterium causing acne, found mainly on the skin. In addition, P. acnes is responsible for inflammation of the gums (gingivitis) and blood vessels, consequently leading to various diseases in the human body. In recent years, the evolution of microorganisms, such as P. acnes, that have become resistant to many commercial antibiotics due to the widespread use of antimicrobial drugs in the treatment of infectious diseases has emerged as a major clinical problem. We here analyzed the potential use of 37 medicinal plant extracts as plausible candidates for treating P. acnes, in terms of total phenolic and flavonoid contents, antioxidants scavenging and antimicrobial activity. Consequently, methanol extracts from 14 medicinal plants showed promising antimicrobial activities against P. acnes. In particular, as the extracts from Chrysosplenium flagelliferum F. and Thuja orientalis L. exhibited distinct antimicrobial activities in both the broth dilution and disc diffusion assay, they could be effectively used as active ingredients for preventing or treating inflammatory periodontal diseases, such as periodontitis.

Soluble Expression and Efficient Purification of Recombinant Class I Hydrophobin DewA.

Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic-hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts.

A new, reliable, and high-throughput strategy to screen bacteria for antagonistic activity against Staphylococcus aureus.

BACKGROUND: Antibiotic-resistant Staphylococcus aureus clones have emerged globally over the last few decades. Probiotics have been actively studied as an alternative to antibiotics to prevent and treat S. aureus infections, but identifying new probiotic bacteria, that have antagonistic activity against S. aureus, is difficult since traditional screening strategies are time-consuming and expensive. Here, we describe a new plasmid-based method which uses highly stable plasmids to screen bacteria with antagonistic activity against S. aureus. RESULTS: We have created two recombinant plasmids (pQS1 and pQS3) which carry either gfpbk or mCherry under the control of a S. aureus quorum-sensing (QS) promoter (agrP3). Using this recombinant plasmid pair, we tested 81 bacteria isolated from Holstein dairy milk to identify bacteria that had growth-inhibiting activity against S. aureus and suggest potential explanations for the growth inhibition. The stability test illustrated that pQS1 and pQS3 remained highly stable for at least 24 h in batch culture conditions without selection pressure from antibiotics. This allowed co-culturing of S. aureus with other bacteria. Using the newly developed pQS plasmids, we found commensal bacteria, isolated from raw bovine milk, which had growth-inhibiting activity (n = 13) and quorum-quenching (QQ) activity (n = 13) towards both S. aureus Sa25 (CC97) and Sa27 (CC151). The pQS-based method is efficient and effective for simultaneously screening growth-inhibiting and QQ bacteria against S. aureus on agar media. CONCLUSIONS: It was shown that growth-inhibiting and QQ activity toward pQS plasmid transformants of S. aureus can be simultaneously monitored by observing the zone of growth inhibition and reporter protein inhibition on agar plates. Newly identified antagonistic bacteria and their functional biomolecules are promising candidates for future development of probiotic drugs and prophylactics/therapeutics for bacterial infections including S. aureus. Furthermore, this new approach can be a useful method to find bacteria that can be used to prevent and treat S. aureus infections in both humans and animals.

Verrucomicrobial methanotrophs grow on diverse C3 compounds and use a homolog of particulate methane monooxygenase to oxidize acetone.

Short-chain alkanes (SCA; C2-C4) emitted from geological sources contribute to photochemical pollution and ozone production in the atmosphere. Microorganisms that oxidize SCA and thereby mitigate their release from geothermal environments have rarely been studied. In this study, propane-oxidizing cultures could not be grown from acidic geothermal samples by enrichment on propane alone, but instead required methane addition, indicating that propane was co-oxidized by methanotrophs. "Methylacidiphilum" isolates from these enrichments did not grow on propane as a sole energy source but unexpectedly did grow on C3 compounds such as 2-propanol, acetone, and acetol. A gene cluster encoding the pathway of 2-propanol oxidation to pyruvate via acetol was upregulated during growth on 2-propanol. Surprisingly, this cluster included one of three genomic operons (pmoCAB3) encoding particulate methane monooxygenase (PMO), and several physiological tests indicated that the encoded PMO3 enzyme mediates the oxidation of acetone to acetol. Acetone-grown resting cells oxidized acetone and butanone but not methane or propane, implicating a strict substrate specificity of PMO3 to ketones instead of alkanes. Another PMO-encoding operon, pmoCAB2, was induced only in methane-grown cells, and the encoded PMO2 could be responsible for co-metabolic oxidation of propane to 2-propanol. In nature, propane probably serves primarily as a supplemental growth substrate for these bacteria when growing on methane.

Differences in diagnostic impact of dual-tracer PET/computed tomography according to the extrahepatic metastatic site in patients with hepatocellular carcinoma.

OBJECTIVES: We compared the diagnostic performance of C-11 acetate and F-18 fluorodeoxyglucose (FDG) PET/computed tomography (CT) for the detection of extrahepatic metastasis in patients with hepatocellular carcinoma (HCC) and evaluated whether the improvement in the diagnostic performance of dual tracer PET/CT differs by the metastatic site. METHODS: Fifty-eight patients who had extrahepatic metastasis on either C-11 acetate or F-18 FDG PET/CT were enrolled, and 193 metastatic lesions were analyzed in this retrospective study. The metastatic lesions were categorized based on six sites of involvement. According to each involved site, the tracer avidity of the metastatic lesions was compared using the maximum standardized uptake value (SUVmax). RESULTS: Bone was the most frequent categorized metastatic site (44.8%), followed by lymph node (39.7%), lung (34.5%), soft tissue (27.6%), adrenal gland (6.9%), and vascular category (3.4%). C-11 acetate PET/CT showed a higher SUVmax than F-18 FDG PET/CT in metastatic bone lesions (P = 0.003). F-18 FDG uptake was significantly higher than C-11 acetate uptake in metastatic lymph node lesions (P < 0.001). The detection rate of dual tracer PET/CT was significantly higher in the metastatic lung (93.6%) and soft tissue (100%) lesions. However, the diagnostic performance of dual tracer PET/CT was limited in the metastatic bone and lymph node lesions because each tracer's detection rate was very high (bone: 94.6% in C-11 acetate, lymph node: 94.1% in F-18 FDG). CONCLUSIONS: The tracer avidity of metastatic lesions differed according to the involved site. This difference affected the complementary role of dual tracer PET/CT in the diagnosis of extrahepatic metastases in patients with HCC.

Soluble Expression of hFGF19 without Fusion Protein through Synonymous Codon Substitutions and DsbC Co-Expression in E. coli.

Human fibroblast growth factor 19 (hFGF19) is a difficult-to-express protein that is frequently fused with another protein for soluble expression. However, residual amino acids after cleavage with protease represent one of the major problems in therapeutic protein development. Here, we introduced synonymous codon substitutions in the N-terminal region encoding sequence of hFGF19 and co-expressed disulfide bond isomerase (ΔssDsbC) to functionally express hFGF19 without any fusion protein. Synonymous codon substitution significantly increased hFGF19 expression. Subsequent co-expression of ΔssDsbC with a selected variant of hFGF19 (scvhFGF19) further increased the proportion of soluble hFGF19 expression in Escherichia coli XL1-Blue. Both total and soluble scvhFGF19 expression increased remarkably in the alternative host, E. coli Origami 2 with mutated thioredoxin reductase and glutathione reductase. scvhFGF19 purification by anion exchange and heparin affinity chromatography resulted in a yield of 6.5 mg/L under normal induction conditions in flask culture. As such, a high cell density culture is expected to achieve an even higher yield. The biological activities of purified scvhFGF19 were assessed based on its ability to activate ERK1/2 signaling pathway in HepG2 hepatocarcinoma cells. In conclusion, the strategy described here may represent an efficient alternative process for the production of hFGF19 and/or related proteins.

Biocatalytic Production of a Potent Inhibitor of Adipocyte Differentiation from Phloretin Using Engineered CYP102A1.

In this study, we investigated an efficient enzymatic strategy for producing potentially valuable phloretin metabolites from phlorizin, a glucoside of phloretin that is rich in apple pomace. Almond ß-glucosidase efficiently removed phlorizin's glucose moiety to produce phloretin. CYP102A1 engineered by site-directed mutagenesis, domain swapping, and random mutagenesis catalyzed the highly regioselective C-hydroxylation of phloretin into 3-OH phloretin with high conversion yields. Under the optimal hydroxylation conditions of 15 g cells L-1 and a 20 mM substrate for whole-cell biocatalysis, phloretin was regioselectively hydroxylated into 3.1 mM 3-OH phloretin each hour. Furthermore, differentiation of 3T3-L1 preadipocytes into adipocytes and lipid accumulation were dramatically inhibited by 3-OH phloretin but promoted by phloretin. Consistent with these inhibitory effects, the expression of adipogenic regulator genes was downregulated by 3-OH phloretin. We propose a platform for the sustainable production and value creation of phloretin metabolites from apple pomace capable of inhibiting adipogenesis.

Real-time monitoring of NADPH levels in living mammalian cells using fluorescence-enhancing protein bound to NADPHs.

Nicotinamide adenine nucleotide phosphate (NADPH) has been known to be involved in the multiple pathways of cell metabolism. However, conventional quantification assays for NADPH have required breaking down the cell membranes of around one million cells per assay, and monitoring NADPH flux in living cells has been limited by a few available tools. Here, we visualized NADPH levels in human cervical cancer cells HeLa using metagenome-derived blue fluorescent protein (mBFP), which specifically binds to NADPH and enhances the intrinsic fluorescence of NADPH up to 10-fold when imaged by two-photon microscopy to reduce photodamage. Adding an oxidizing agent such as diamide to HeLa cells that expressed mBFP led to an immediate decrease of intracellular NADPH depending on glucose availability in culture media. Furthermore, inhibiting glucose-6-phosphate dehydrogenase (G6PD) in the pentose phosphate pathway with dehydroandrosterone (DHEA) and knockdown of G6PD transcripts gradually decreased NADPH when diamide was added to living cells. These results demonstrate that introducing a bacterial mBFP gene into mammalian cells is a straightforward approach to monitoring intracellular NADPH flux in real time at the single-cell level. Moreover, this strategy can be expanded to tracking the spatio-temporal changes in NADPH even in single-cell organelles such as mitochondria and chloroplasts, which will allow us to more precisely assess the efficacy of biochemically or biophysically metabolic perturbations in animal and plant cells.

Soluble overexpression of a flagellin derivative from Salmonella enterica using synonymous codon substitutions of 5'-coding region in Escherichia coli.

OBJECTIVE: To obtain a recombinant flagellin derivative CBLB502, expressed in functionally soluble form, the technology of library construction and screening of synonymous codon variants was employed, and its expression, solubility, and activity were assessed. RESULTS: We screened several synonymous codon variants scvCBLB502s with the enhanced solubility from the constructed library, harboring the random substitutions of the first ten amino acid residues of the parental CBLB502 with synonymous codons. Among them, scvCBLB502-5 was purified (> 8.4 mg/l) by single step procedure using an affinity chromatography without any ancillary treatment with protease inhibitor cocktail solution and/or boiling at 90 °C. Subsequent study showed that the recombinant protein scvCBLB502-5 distinctly induced the TLR5 (Toll-Like Receptor 5)-mediated NF-κB activation and also IL-8 production in HEK293-hTLR5 cells. CONCLUSION: Results showed that scvCBLB502-5, engineered through the synonymous codon substitutions, was easily expressed in functionally soluble form and maintained the proper folding to be recognized by TLR5, as an inducer for pathogen-associated molecular pattern (PAMP).