Gene expression regulation by modulating Hfq expression in coordination with tailor-made sRNA-based knockdown in Escherichia coli.

The tailor-made synthetic sRNA-based gene expression knockdown system has demonstrated its efficacy in achieving pathway balancing in microbes, facilitating precise target gene repression and fine-tuned control of gene expression. This system operates under a competitive mode of gene regulation, wherein the tailor-made synthetic sRNA shares the intrinsic intracellular Hfq protein with other RNAs. The limited intracellular Hfq amount has the potential to become a constraining factor in the post-transcription regulation of sRNAs. To enhance the efficiency of the tailor-made sRNA gene expression regulation platform, we introduced an Hfq expression level modulation-coordinated sRNA-based gene knockdown system. This system comprises tailor-made sRNA expression cassettes that produce varying Hfq expression levels using different strength promoters. Modulating the expression levels of Hfq significantly improved the repressing capacity of sRNA, as evidenced by evaluations with four fluorescence proteins. In order to validate the practical application of this system, we applied the Hfq-modulated sRNA-based gene knockdown cassette to Escherichia coli strains producing 5-aminolevulinic acid and L-tyrosine. Diversifying the expression levels of metabolic enzymes through this cassette resulted in substantial increases of 74.6% in 5-aminolevulinic acid and 144% in L-tyrosine production. Tailor-made synthetic sRNA-based gene expression knockdown system, coupled with Hfq copy modulation, exhibits potential for optimizing metabolic fluxes through biosynthetic pathways, thereby enhancing the production yields of bioproducts.

Mimicking descending necrotizing mediastinitis as acute myocardial infarction in a patient with severe coronary artery disease: A case report.

RATIONALE: It is a crucial disease that descending necrotizing mediastinitis need to be treated promptly with proper antibiotics and drainage. The characteristics of its symptoms such as chest pain are difficult to distinguish from acute myocardial infarction. PATIENT CONCERNS: An 80-year-old female presented with severe squeezing chest pain. The cardiac marker was elevated. And coronary angiography showed the significant coronary stenosis. Although the revascularization through percutaneous coronary intervention was completed successfully, the patient still presented chest pain. Computed tomography of neck revealed that hypodense heterogeneous lesions with clear and distinguishable margin extended from the deep neck to mediastinum diffusely. DIAGNOSES: The patient was diagnosed with descending necrotizing mediastinitis. INTERVENTIONS: Percutaneous catheter insertion to patient's abscess lesion at was performed. OUTCOMES: Catheter drainage of descending necrotizing mediastinitis led to an improvement in the patient's condition. LESSON: Descending necrotizing mediastinitis made chest paint with elevated cardiac enzyme mimicked myocardial infarction.

Evaluation of three real-time PCR assays for differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria species in liquid culture media.

We evaluated the analytical performance of M. tuberculosis complex (MTBC)/nontuberculous mycobacteria (NTM) PCR assays for differential identification of MTBC and NTM using culture-positive liquid media. Eighty-five type strains and 100 consecutive mycobacterial liquid media cultures (MGIT 960 system) were analyzed by a conventional PCR assay (MTB-ID(®) V3) and three real-time PCR assays (AdvanSure™ TB/NTM real-time PCR, AdvanSure; GENEDIA(®) MTB/NTM Detection Kit, Genedia; Real-Q MTB & NTM kit, Real-Q). The accuracy rates for reference strains were 89.4%, 100%, 98.8%, and 98.8% for the MTB-ID V3, AdvanSure, Genedia, and Real-Q assays, respectively. Cross-reactivity in the MTB-ID V3 assay was mainly attributable to non-mycobacterium Corynebacterineae species. The diagnostic performance was determined using clinical isolates grown in liquid media, and the overall sensitivities for all PCR assays were higher than 95%. In conclusion, the three real-time PCR assays showed better performance in discriminating mycobacterium species and non-mycobacterium Corynebacterineae species than the conventional PCR assay.

The Significance of Sensitive Interferon Gamma Release Assays for Diagnosis of Latent Tuberculosis Infection in Patients Receiving Tumor Necrosis Factor-α Antagonist Therapy.

OBJECTIVE: We compared two interferon gamma release assays (IGRAs), QuantiFERON-TB Gold In-Tube (QFT-GIT) and T-SPOT.TB, for diagnosis of latent tuberculosis infection (LTBI) in patients before and while receiving tumor necrosis factor (TNF)-α antagonist therapy. This study evaluated the significance of sensitive IGRAs for LTBI screening and monitoring. METHODS: Before starting TNF-α antagonist therapy, 156 consecutive patients with rheumatic diseases were screened for LTBI using QFT-GIT and T-SPOT.TB tests. According to our study protocol, QFT-GIT-positive patients received LTBI treatment. Patients positive by any IGRAs were subjected to follow-up IGRA tests after completing LTBI-treatment and/or during TNF-α antagonist therapy. RESULTS: At the initial LTBI screening, 45 (28.9%) and 70 (44.9%) patients were positive by QFT-GIT and T-SPOT.TB, respectively. The agreement rate between IGRA results was 78.8% (k = 0.56; 95% confidence interval [95% CI] = 0.43 to 0.68). Of 29 patients who were positive only by T-SPOT.TB in the initial screening, 83% (19/23) were persistently positive by T-SPOT.TB, while QFT-GIT testing showed that 36% (9/25) had conversion during TNF-α antagonist therapy. By the end of the follow-up period (218 to 1,264 days), four patients (4/137, 2.9%) developed active tuberculosis (TB) diseases during receiving TNF-α antagonist therapy. Among them, one was Q-T+, one was Q+T-, and the remaining two were Q-T- at the initial screening (Q, QuantiFERON-TB Gold In-Tube; T, T-SPOT.TB; +, positive; -, negative). Two (2/4, 50%) patients with TB reactivation had at least one prior risk factor consistent with previous TB infection. CONCLUSION: This study demonstrated the need to capitalize on sensitive IGRAs to monitor for LTBI in at-risk patients for a more sensitive diagnosis in countries with an intermediate TB burden.

Comparison of the AdvanSure™ real-time RT-PCR and Seeplex(®) RV12 ACE assay for the detection of respiratory viruses.

The AdvanSure™ RV real-time PCR kit (AdvanSure; LG Life Sciences, Korea) is based on multiplex real-time PCR and can simultaneously detect 14 respiratory viruses. We compared the performance of the AdvanSure assay with the Seeplex RV 12 ACE detection kit (Seeplex; Seegene, Seoul, South Korea), a multiplex end-point PCR assay. A total of 454 consecutive respiratory specimens were tested with both AdvanSure and Seeplex assays; AdvanSure detected 153 (33.7%) positive cases and Seeplex detected 145 (31.9%) positive cases. The positive percent agreement, negative percent agreement, and kappa value for the two assays were 87.2% (95% CI, 80.3-92.1), 91.1% (95% CI, 87.2-93.9), and 0.77 (95% CI, 0.70-0.83), respectively. Compared with the Seeplex assay, the AdvanSure assay had a shorter turnaround time (3h vs. 8h) and a shorter hands-on time (<1h vs 2h). In conclusion, the AdvanSure assay demonstrated comparable performance to the Seeplex assay.

Isolation and identification of fungi from a meju contaminated with aflatoxins.

A home-made meju sample contaminated naturally with aflatoxins was used for isolation of fungal strains. Overall, 230 fungal isolates were obtained on dichloran rosebengal chloramphenicol (DRBC) and dichloran 18% glycerol (DG18) agar plates. Morphological characteristics and molecular analysis of a partial beta-tubulin gene and the internal transcribed spacer (ITS) of rDNA were used for the identification of the isolates. The fungal isolates were divided into 7 genera: Aspergillus, Eurotium, Penicillium, Eupenicillium, Mucor, Lichtheimia, and Curvularia. Three strains from 56 isolates of the A. oryzae/flavus group were found to be aflatoxigenic A. flavus, by the presence of the aflatoxin biosynthesis genes and confirmatory aflatoxin production by high-performance liquid chromatography (HPLC). The predominant isolate from DRBC plates was A. oryzae (42 strains, 36.2%), whereas that from DG18 was A. candidus (61 strains, 53.5%). Out of the 230 isolates, the most common species was A. candidus (34.3%) followed by A. oryzae (22.2%), Mucor circinelloides (13.0%), P. polonicum (10.0%), A. tubingensis (4.8%), and L. ramosa (3.5%). A. flavus and E. chevalieri presented occurrence levels of 2.2%, respectively. The remaining isolates of A. unguis, P. oxalicum, Eupenicillium cinnamopurpureum, A. acidus, E. rubrum, P. chrysogenum, M. racemosus, and C. inaequalis had lower occurrence levels of < 2.0%.

Selective cholinesterase inhibitory activities of a new monoterpene diglycoside and other constituents from Nelumbo nucifera stamens.

A new beta-cyclogeraniol diglycoside (5), along with four known components, cycloartenol (1), p-hydroxybenzoic acid (2), vanilloloside (3), and 5'-O-methyladenosine (4), were first isolated from the n-BuOH fraction of Nelumbo nucifera stamens. The chemical structure of 5 was elucidated as 1-hydroxymethyl-2,6,6-trimethyl-1-cyclohexene 9-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (nuciferoside) on the basis of chemical and spectroscopic evidence, including 1D, 2D NMR, and MS. The anti-Alzheimer effects of 1-5 were evaluated via the acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) inhibition assays. Compounds 1-3 and 5 showed good and noncompetitive inhibition against AChE with IC(50) values of 11.89, 20.07, 4.55, and 3.20 microM and K(i)values of 15.71, 25.44, 7.76, and 5.76 microM, respectively. Compounds 1, 2, and 5 also possessed BChE inhibitory activities with IC(50) values of 13.93, 62.29, 205.78, and 83.06 microM, respectively. The selectivity index (SI) values of 1, 2, 3, and 5, calculated from IC(50) values of BChE and AChE, were 1.2, 3.1, 45.7, and 26.0. However, all isolated compounds lacked BACE1 inhibition up to 100 microM. Therefore, N. nucifera stamens-derived compounds could potentially exert their primary anti-Alzheimer effects as AChE inhibitors rather than BACE1 inhibitors.

Inhibitory effects of Nelumbo nucifera leaves on rat lens aldose reductase, advanced glycation endproducts formation, and oxidative stress.

The preventive and therapeutic potency against oxidative stress and diabetic complications of Nelumbo nucifera were evaluated via the 1,1-diphenyl-2-picrylhydrazyl (DPPH), Trolox equivalent antioxidant capacity (TEAC), and total reactive oxygen species (ROS) assays, as well as the rat lens aldose reductase (RLAR) and advanced glycation endproducts (AGE) assays. The leaf extract of N. nucifera exerted potent antioxidant effects as well as marked inhibitory effects for RLAR and AGE formation, corresponding to high values for total phenolic content (TPC) and total flavonoid content (TFC). Among several solvent fractions, the EtOAc and n-BuOH fractions, having prominent TPC and TFC values, showed significant antioxidant effects in the DPPH and TEAC assays. Moreover, the EtOAc fraction exhibited superior inhibitory effects in the total ROS, RLAR, and AGE assays, with IC(50) values of 9.4, 2.4, and 28.2microg/ml, respectively. Also, the HPLC profiles of the active EtOAc fraction indicated that quercetin 3-O-beta-d-glucopyranoside (Qc-3-Glc) and Qc 3-O-beta-d-glucuronopyranoside (Qc-3-Gln) were two of its major components, as well as Qc 3-O-beta-d-galactopyranoside (Qc-3-Gal) as a minor compound. Therefore, the results suggest that two key antioxidant flavonoids, Qc-3-Glc and Qc-3-Gln, may play important roles in the antioxidant and RLAR inhibitory effects of N. nucifera leaves. Also, the leaves, and the flavonoids contained within them, would clearly have potential uses in the development of therapeutic or preventive agents for diabetic complications and oxidative stress-related diseases.

Cloning of miniature pig HIF-1alpha and its responses to immunosuppressive agents.

Miniature pigs are worth notice as candidate donors for xenotransplantation. However, donor organs are inevitably subjected to hypoxia, which causes vascular endothelial dysfunction. In this respect, hypoxia-inducible factor 1alpha (HIF-1alpha), the key factor for hypoxic adaptation, should be expressed in grafts. However, some immunosuppressive drugs have been reported to suppress HIF-1alpha in rat cells. Here, we first identified the cDNA and protein structures of miniature pig HIF-1alpha, and next investigated the effects of cyclosporine and FK506 on HIF-1alpha expression in endothelial cells of miniature pig. Thus, we conclude that FK506, rather than cyclosporine, may be recommended for xenotransplantation using miniature pig organs.

Hypolipidemic effects of Sophora flavescens and its constituents in poloxamer 407-induced hyperlipidemic and cholesterol-fed rats.

In this study, we investigated the hypolipidemic effects of Sophora flavescens in poloxamer 407-induced hyperlipidemic and cholesterol-fed rats. The MeOH extract and 4 fractions of S. flavescens were administered at doses of 250 and 100 mg/kg body weight, respectively, once a day for 3 d to the poloxamer 407-induced hyperlipidemic rats. Serum lipid levels such as total cholesterol (TC), triglycerides (TG), and low-density lipoprotein-cholesterol (LDL-C) were markedly elevated in the poloxamer 407-induced hyperlipidemic control rats, while lipid levels were significantly decreased in the rats administered the MeOH extract or 4 fractions of S. flavescens. In addition, serum high-density lipoprotein-cholesterol (HDL-C) was reduced in the poloxamer 407-induced hyperlipidemic control rats. However, oral administration of both the MeOH extract and 4 fractions significantly increased HDL-C levels. Of the tested fractions, the EtOAc fraction showed the strongest lipid-lowering effect, as well as a high antiatherogenic potential with atherogenic index (A.I.) values of less than 1.92. We also investigated the hypolipidemic effects of the main compounds of the EtOAc fraction, kurarinol and kuraridinol, using the hyperlipidemic and hypercholesterolemic animal models. Here, elevated TC, TG, and LDL-C levels in the poloxamer 407-induced hyperlipidemic and cholesterol-fed rats were significantly reduced after oral administration of the compounds, and HDL-C levels had a significant increase. Furthermore, A.I. values were lowered by administering kurarinol and kuraridinol. In particular, kuraridinol exhibited stronger protective activities against hyperlipidemia than kurarinol. These results suggest that S. flavescens and its constituents may be effective cholesterol-lowering agents and useful for preventing hypercholesterolemic atherosclerosis.

Preclinical evaluation of YC-1, a HIF inhibitor, for the prevention of tumor spreading.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is believed to promote tumor growth, and thus, is viewed as one of the most compelling cancer therapy targets. YC-1 is widely used as a potent inhibitor of HIF-1alpha both in vitro and in vivo, and is also being developed as a novel anticancer drug. However, little is known about the effects of YC-1 on tumor invasion or metastasis. In the present study, we found that the Hep3B cell migration-stimulatory effect of hypoxia was abolished by HIF-1alpha siRNA or YC-1. YC-1 also significantly inhibited the migrations of other cancer cells. Furthermore, YC-1 effectively inhibited cell invasion through Matrigel. In nude mice, GFP-expressing stable cell-lines of Hep3B or H1299 were inoculated into spleens to induce liver metastasis or into the pleural cavity to induce lung invasion. In untreated mice, many tumor lesions emitting strong fluorescence were found in livers or lungs, and fluorescence intensities and tumor lesion numbers were markedly reduced in YC-1-treated mice. These results suggest that YC-1 effectively inhibits tumor invasion and metastasis, and imply that YC-1 is worth while to further develop as a multipurpose anticancer drug.

FK506: an immunosuppressive agent preserving HIF-1 activity.

During transplantation, donor organs or cells are subjected to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is essential for cellular adaptation to hypoxia. Immunosuppressive agents should be used for preventing graft rejection, but of these, rapamycin and cyclosporine A have been reported to inhibit HIF-1. We examined whether or not another important immunosuppressant, FK506, inhibits HIF-1. In contrast to cyclosporine A, FK506 neither inhibits HIF-1alpha expression in 8 different cell lines, nor represses the transcriptional activity of HIF-1. Compared with cyclosporine A, FK506 significantly reduced the apoptotic cell death by hypoxia. FK506 could preserve HIF-1 activity in donor organs subjected to hypoxia.

Rat lens aldose reductase inhibitory constituents of Nelumbo nucifera stamens.

Aldose reductase, the principal enzyme of the polyol pathway, has been shown to play an important role in the complications associated with diabetes. A methanol extract of the stamens of Nelumbo nucifera Gaertn. was shown to exert an inhibitory effect on rat lens aldose reductase (RLAR), and thus was fractionated using several organic solvents, including dichloromethane, ethyl acetate and n-butanol. The ethyl acetate-soluble fraction, which manifested potent RLAR-inhibitory properties, was then purified further via repeated measures of silica gel and Sephadex LH-20 column chromatography. Thirteen flavonoids: kaempferol (1) and seven of its glycosides (2-9), myricetin 3',5'-dimethylether 3-O-beta-d-glucopyranoside (10), quercetin 3-O-beta-d-glucopyranoside (11) and two isorhamnetin glycosides (12, 13) were isolated from N. nucifera, as well as four non-flavonoid compounds: adenine (14), myo-inositol (15), arbutin (16) and beta-sitosterol glucopyranoside (17). These compounds were all assessed with regard to their RLAR-inhibitory properties. Among the isolated flavonoids, those harboring 3-O-alpha-l-rhamnopyranosyl-(1-->6)-beta-d-glucopyranoside groups in their C rings, including kaempferol 3-O-alpha-l-rhamnopyranosyl-(1-->6)-beta-d-glucopyranoside (5) and isorhamnetin 3-O-alpha-l-rhamnopyranosyl-(1-->6)-beta-d-glucopyranoside (13), were determined to exhibit the highest degree of rat lens aldose reductase inhibitory activity in vitro, evidencing IC(50) values (concentration required for a 50% inhibition of enzyme activity) of 5.6 and 9.0 microm, respectively.

Isorhamnetin glycosides with free radical and ONOO-scavenging activities from the stamens of Nelumbo nucifera.

In this study, we isolated two new isorhamnetin glycosides, designated as nelumboroside A (3) and nelumboroside B (4), as well as the previously-characterized isorhamnetin glucoside (1) and isorhamnetin rutinoside (2), from the n-BuOH fraction of Nelumbo nucifera stamens. The structures of the two new compounds were then determined, using chemical and spectroscopic techniques. All isolated isorhamnetin glycosides 1-4 showed marked antioxidant activities in the DPPH, and ONOO- assays.

Phenolic glycosides from the stem bark of Albizzia julibrissin.

From the stem bark of Albizzia julibrissin DURAZZ (Leguminosae), two new phenolic glycosides (albibrissinosides A and B) were isolated. Their structures were determined by spectroscopic analysis. The albibrissinoside B was found to be a radical scavenger on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.