Damped Oscillating Phosphoryl Transfer Reaction in the Cyanobacterial Circadian Clock.

Most organisms have circadian clocks to ensure the metabolic cycle to resonate with the rhythmic environmental changes without "damping," or losing robustness. Cyanobacteria is the oldest and simplest form of life that is known to harbor this biological intricacy. Its KaiABC-based central oscillator proteins can be reconstituted inside a test tube, and the post-translational modification cycle occurs with 24 h periodicity. KaiC's two major phosphorylation sites, Ser-431 and Thr-432, become phosphorylated and dephosphorylated by interacting with KaiA and KaiB, respectively. Here, we mutate Thr-432 into Ser to find the oscillatory phosphoryl transfer reaction damps. Previously, this mutant KaiC was reported to be arrhythmic in vivo. However, we found that the mutant KaiC gradually loses the ability to run in an autonomous manner and stays constitutively phosphorylated after 3 cycles in vitro.

Shift in Conformational Equilibrium Underlies the Oscillatory Phosphoryl Transfer Reaction in the Circadian Clock.

Oscillatory phosphorylation/dephosphorylation can be commonly found in a biological system as a means of signal transduction though its pivotal presence in the workings of circadian clocks has drawn significant interest: for example in a significant portion of the physiology of Synechococcus elongatus PCC 7942. The biological oscillatory reaction in the cyanobacterial circadian clock can be visualized through its reconstitution in a test tube by mixing three proteins-KaiA, KaiB and KaiC-with adenosine triphosphate and magnesium ions. Surprisingly, the oscillatory phosphorylation/dephosphorylation of the hexameric KaiC takes place spontaneously and almost indefinitely in a test tube as long as ATP is present. This autonomous post-translational modification is tightly regulated by the conformational change of the C-terminal peptide of KaiC called the "A-loop" between the exposed and the buried states, a process induced by the time-course binding events of KaiA and KaiB to KaiC. There are three putative hydrogen-bond forming residues of the A-loop that are important for stabilizing its buried conformation. Substituting the residues with alanine enabled us to observe KaiB's role in dephosphorylating hyperphosphorylated KaiC, independent of KaiA's effect. We found a novel role of KaiB that its binding to KaiC induces the A-loop toward its buried conformation, which in turn activates the autodephosphorylation of KaiC. In addition to its traditional role of sequestering KaiA, KaiB's binding contributes to the robustness of cyclic KaiC phosphorylation by inhibiting it during the dephosphorylation phase, effectively shifting the equilibrium toward the correct phase of the clock.

Anti-biofilm and anti-virulence effects of zerumbone against Acinetobacter baumannii.

Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen that affects patients with a compromised immune system and is becoming increasingly important as a hospital-derived infection. This pathogen is difficult to treat owing to its intrinsic multidrug resistance and ability to form antimicrobial-tolerant biofilms. In the present study, we aimed to assess the potential use of zerumbone as a novel anti-biofilm and/or anti-virulence agent against A. baumannii. The results showed that zerumbone at sub-inhibitory doses decreased biofilm formation and disrupted established A. baumannii biofilms. The zerumbone-induced decrease in biofilm formation was dose-dependent based on the results of microtitre plate biofilm assays and confocal laser scanning microscopy. In addition, our data validated the anti-virulence efficacy of zerumbone, wherein it significantly interfered with the motility of A. baumannii. To support these phenotypic results, transcriptional analysis revealed that zerumbone downregulated the expression of biofilm- and virulence-associated genes (adeA, adeB, adeC and bap) in A. baumannii. Overall, our findings suggested that zerumbone might be a promising bioactive agent for the treatment of biofilm- and virulence-related infections caused by multidrug-resistant A. baumannii.

Antibacterial and antibiofilm effects of α-humulene against Bacteroides fragilis.

The rapid increase in antibiotic resistance has prompted the discovery of drugs that reduce antibiotic resistance or new drugs that are an alternative to antibiotics. Plant extracts have health benefits and may also exhibit antibacterial and antibiofilm activities against pathogens. This study determined the antibacterial and antibiofilm effects of α-humulene extracted from plants against enterotoxigenic Bacteroides fragilis, which causes inflammatory bowel disease. The minimum inhibitory concentration and biofilm inhibitory concentration of α-humulene for B. fragilis were 2 µg/mL, and the biofilm eradication concentration was in the range of 8-32 µg/mL. The XTT reduction assay confirmed that the cellular metabolic activity in biofilm rarely occurred at the concentration of 8-16 µg/mL. In addition, biofilm inhibition by α-humulene was also detected via confocal laser scanning microcopy. Quantitative real-time polymerase chain reaction (qPCR) was also used to investigate the effect of α-humulene on the expression of resistance-nodulation-cell division type multidrug efflux pump genes (bmeB1 and bmeB3). According to the results of qPCR, α-humulene significantly reduced the expression of bmeB1 and bmeB3 genes. This study demonstrates the potential therapeutic application of α-humulene for inhibiting the growth of B. fragilis cells and biofilms, and it expands the knowledge about biofilm medicine.

The Circadian Clock-A Molecular Tool for Survival in Cyanobacteria.

Cyanobacteria are photosynthetic organisms that are known to be responsible for oxygenating Earth's early atmosphere. Having evolved to ensure optimal survival in the periodic light/dark cycle on this planet, their genetic codes are packed with various tools, including a sophisticated biological timekeeping system. Among the cyanobacteria is Synechococcus elongatus PCC 7942, the simplest clock-harboring organism with a powerful genetic tool that enabled the identification of its intricate timekeeping mechanism. The three central oscillator proteins-KaiA, KaiB, and KaiC-drive the 24 h cyclic gene expression rhythm of cyanobacteria, and the "ticking" of the oscillator can be reconstituted inside a test tube just by mixing the three recombinant proteins with ATP and Mg2+. Along with its biochemical resilience, the post-translational rhythm of the oscillation can be reset through sensing oxidized quinone, a metabolite that becomes abundant at the onset of darkness. In addition, the output components pick up the information from the central oscillator, tuning the physiological and behavioral patterns and enabling the organism to better cope with the cyclic environmental conditions. In this review, we highlight our understanding of the cyanobacterial circadian clock and discuss how it functions as a molecular chronometer that readies the host for predictable changes in its surroundings.

Antibiofilm and antibacterial activities of repurposing auranofin against Bacteroides fragilis.

The aim of this study is to evaluate the antibiofilm and antibacterial effects of auranofin against WT-ETBF, rETBF, WT-NTBF and clinically isolated Bacteroides fragilis strains. The minimum inhibitory concentration and biofilm inhibitory concentration of 0.25 µg/ml for auranofin against B. fragilis were determined, and the biofilm eradication concentration was 1 µg/ml. At an auranofin concentration of 0.5 µg/ml, little cellular metabolic activity was found. Confocal laser scanning microcopy results confirmed the inhibition of biofilm by auranofin. The effects of auranofin on the outer membrane protein (ompA) gene and the RND-type efflux pump (bmeB3) gene were investigated using quantitative real-time polymerase chain reaction (qRT-PCR). The qRT-PCR results showed that treatment with auranofin significantly reduced the gene expression compared to controls without auranofin. These data indicate the applicability of auranofin as a repurposed drug due to its inhibitory effect against biofilm formation of B. fragilis. Therefore, our study demonstrates that auranofin, already approved for human use, is a promising drug that has strong antibiofilm and antibacterial activity against B. fragilis.

Magnesium Regulates the Circadian Oscillator in Cyanobacteria.

The circadian clock controls 24-h biological rhythms in our body, influencing many time-related activities such as sleep and wake. The simplest circadian clock is found in cyanobacteria, with the proteins KaiA, KaiB, and KaiC generating a self-sustained circadian oscillation of KaiC phosphorylation and dephosphorylation. KaiA activates KaiC phosphorylation by binding the A-loop of KaiC, while KaiB attenuates the phosphorylation by sequestering KaiA from the A-loop. Structural analysis revealed that magnesium regulates the phosphorylation and dephosphorylation of KaiC by dissociating from and associating with catalytic Glu residues that activate phosphorylation and dephosphorylation, respectively. High magnesium causes KaiC to dephosphorylate, whereas low magnesium causes KaiC to phosphorylate. KaiC alone behaves as an hourglass timekeeper when the magnesium concentration is alternated between low and high levels in vitro. We suggest that a magnesium-based hourglass timekeeping system may have been used by ancient cyanobacteria before magnesium homeostasis was established.

Perioperative administration of propranolol to women undergoing ovarian cancer surgery: A pilot study.

OBJECTIVE: This study was done to evaluate whether perioperative propranolol (ß-blocker) in ovarian cancer patients undergoing debulking surgery reduced perioperative tumor growth induced by surgical stress. METHODS: This was a prospective randomized single institution analysis. The primary objective was to compare the changes in CA 125 level (changes between preoperation day 2 and postoperative day 7). As a study arm, patients received a low dose of propranolol 40 mg/day (4×10 mg) starting two days before surgery and 40 mg twice daily for three days following surgery. RESULTS: Twenty-two patients were enrolled and 16 were evaluable for efficacy. The drug was well tolerated. The mean decrease of CA 125 during the seven perioperative days was 83.1±8.9% in the propranolol group and 72.4±14.7% in the placebo group. The difference was statistically significant (P=0.044). The change of C-reactive protein, cortisol, and anxiety score (State-Trait Anxiety Inventory-X1) were not different between the two groups. CONCLUSION: This preliminary result is the first to directly test the role of perioperative propranolol on tumor growth. Even with the small sample size and short term use of the drug, perioperative propranolol was effective in reducing tumor burden (as measured by CA 125) suggesting its potential benefits in decreasing perioperative tumor growth.

Catamenial hemoptysis accompanied by subcutaneous endometriosis treated with combination therapy.

Extra pelvic endometriosis is considered to be rare. This paper reports a case of catamenial hemoptysis accompanied by subcutaneous endometriosis in 26-year-old woman. A computed tomography scan of the chest revealed a focal ground-glass opacity lesion in the posterior segment of the right upper lobe. Histopathology confirmed the diagnosis of endometriosis of right lung and concurrent subcutaneous endometriosis. She was treated with surgical resection of the endometriosis lesions on two different sites and perioperative gonadotropin-releasing hormone agonist therapy. The 6-month follow-up after combination treatment showed no recurrence. Though long-term follow-up result is needed, aggressive treatment using combination treatment (surgery and perioperative medication) should be considered for symptomatic extra pelvic endometriosis.

Clinical outcomes and neurodevelopmental outcome of prenatally diagnosed agenesis of corpus callosum in single center of Korea.

OBJECTIVE: With recent advances and frequent use of prenatal ultrasound, the antenatal diagnosis of agenesis of the corpus callosum (ACC) is not rare in obstetrics practices. However, information regarding the long-term neurological outcome remains uncertain. The aim of this study was to investigate clinical outcomes of prenatally diagnosed ACC and to analyze postnatal neurodevelopmental outcomes of ACC neonates born in our single center. METHODS: We retrospectively reviewed 56 cases of prenatally suspected ACC referred to our center. RESULTS: Fifty-six fetuses were diagnosed with ACC, and 12 of those were followed-up in our center until delivery. Of the remaining 44, 7 were delivered after being referred back to the original hospital, 23 were lost to follow-up, and 14 had unknown outcomes. Among all 56, 29 were considered to have isolated ACC and 27 were considered to have non-isolated ACC. Of the 10 live fetuses delivered in our center, four had isolated ACC, three had non-isolated ACC, and the rest had outcomes unrelated to ACC. Neurodevelopmental outcome was followed-up until approximately age 3 years. Of the four with isolated ACC, three (75%) had normal neurodevelopmental outcomes. CONCLUSION: Similar to other studies, the results of our single-center study included positive neurodevelopmental outcomes for those with isolated ACC. However, despite our endeavor to counsel patients with prenatally diagnosed ACC, the delivery rate in our center was quite low. Therefore, larger, multicenter, retrospective studies including long-term neurological development outcomes are crucial and urgently needed to provide better counseling.

Korean translation and validation of the WHOQOL-DIS for people with spinal cord injury and stroke.

BACKGROUND: The QOL assessment to measure disabilities has needed to survey studies for a variety of fields. Until now, no Korean translation of the World Health Organization Quality of Life-disability (WHOQOL-DIS) module existed. OBJECTIVE/HYPOTHESIS: We translated and cross-culturally adapted the WHOQOL-DIS module into Korean, testing its reliability and validity with Korean spinal cord injury and stroke patients. METHODS: The translation occurred using the methodology recommended by WHO. Ten participants were cognitively debriefed and the psychometric properties of the Korean WHOQOL-DIS module were evaluated for reliability and validity with 85 patients (SCI = 58, stroke = 27). Concurrent validity was assessed for the WHOQOL-BREF scores. Internal consistency coefficients were analyzed using Cronbach's alpha. Test-retest reliability (n = 30) was measured using a 7-10-day interval and analyzed using Pearson's correlation coefficients. Validity was evaluated using EFA and CFA. RESULTS: There was high internal consistency with the WHOQOL-BREF, (α = 0.603-0.875). Pearson's correlation was significant in the DIS module's three sub-domains (r = 0.759). EFA revealed slightly different models from the original version and there was an internal consistency difference between test and retest. The three-domain structure model with a higher-order factor fit well with the WHOQOL-DIS module's development. CONCLUSIONS: This instrument can help evaluate the quality of life of Koreans with disabilities. Further studies with other disabilities are required for additional evidence of validity and reliability.

Primary ovarian carcinoid tumor showing unusual histology and nuclear accumulation of ß-catenin.

Carcinoid tumor of the ovary is uncommon. We herein report a very rare case of primary ovarian carcinoid tumor with aggressive histology and an unusual immunophenotype. A 21-year-old woman presented with a palpable abdominal mass. Computed tomographic scan revealed a large, extensively necrotic solid mass in the left ovary. The patient underwent a left salpingo-oophorectomy. Grossly, the left adnexa showed a large, vaguely lobulated ovarian tumor measuring 22×15×13 cm. Histologically, the tumor had a readily identifiable neuroendocrine growth pattern, but some areas showed solid growth pattern associated with mild nuclear pleomorphism and multiple foci of punctate necrosis. Furthermore, mitotic figures were recognized in 8 per 10 high-power fields, and a few foci of large coagulative tumor necrosis were also noted. In addition, the tumor tissue exhibited uniform, strong nuclear ß-catenin immunoreactivity, indicating the nuclear accumulation of ß-catenin in the individual tumor cells. In summary, we described the first case of primary ovarian carcinoid tumor with loss of neuroendocrine growth pattern, increased mitotic activity and large areas of coagulative tumor necrosis. According to the WHO classification of pulmonary carcinoid tumor, this case may be classified as "atypical" carcinoid. However, currently, no primary ovarian atypical carcinoid exists in the classification system. Due to its rarity, there are no established diagnostic criteria and clinical data on patient outcomes for ovarian carcinoid tumors with aggressive histology. Additional reports are clearly necessary. We also showed for the first time the nuclear accumulation of ß-catenin in carcinoid tumor cells, suggestive of a role for ß-catenin in the tumorigenesis of ovarian atypical carcinoid tumor or its aggressive histology.

Anti-wrinkle activity of Platycarya strobilacea extract and its application as a cosmeceutical ingredient.

In order to investigate the potential of Platycarya strobilacea fruit extract as an active ingredient for cosmetics, we measured their free-radical scavenging activity, elastase inhibitory activity, the expression of MMP-1 (matrix metalloproteinase-1), and type I collagen synthesis in normal human fibroblast cells. To isolate the main component compounds from P. strobilacea fruit extract, we purified the extract through solvent fractionation, column chromatography, and recrystallization. The component compounds were identified as ellagic acid and 4-O-xyloside of ellagic acid (ellagic acid 4-O-xylopyranoside). P. strobilacea fruit extract and ellagic acid increased the expression of type I collagen mRNA in a dose-dependent manner (up to 37% and 41% at 20 microg/ml and 1.0 microg/ml, respectively), comparable to that of ascorbic acid (up to 39% at 500 muM). A clinical study of measurements using visual evaluation and image analysis showed a statistically significant difference (p < 0.05) between the effects of the test and placebo products. This result suggests that P. strobilacea fruit extract could be used as an active ingredient for antiaging cosmetics.

Prevalence and classification of pathogenic Escherichia coli isolated from fresh beef, poultry, and pork in Korea.

Foodborne diseases occur worldwide, including through the consumption of contaminated meat. This study was conducted to investigate the prevalence of Escherichia coli contamination in fresh beef, poultry, and pork, and to determine whether any isolated E. coli possessed genes associated with pathogenicity. Three thousand meat samples were collected from 2004 to 2006 and were tested for the presence of E. coli. Two hundred and seventy-three E. coli isolates were obtained from beef, poultry, and pork, resulting in an overall isolation rate of 9.1%. Of these isolates, 201 were obtained from 1350 pork samples (14.9%), followed by 41 of 900 poultry samples (4.6%) and 31 of 750 beef samples (4.1%). A total of 39 pathogenic E. coli isolates from the three meat types were categorized into three virulence groups, namely enterotoxigenic E. coli (43.6%), enterohemorrhagic E. coli (EHEC) (35.9%; 22.6% of beef, 7.3% of poultry, and 2.0% of pork), and enteropathogenic E. coli (20.5%). Fourteen strains were identified as belonging to the EHEC, which included O18, O136, O119, O86, O8, O111, O15, O128, and O6. This study demonstrated that pathogenic E. coli are found in meat in Korea, and could act as a transmission vehicle for human infection as suggested by the occurrence and classification of pathogenic E. coli in retail meats. Furthermore, the data from this study could be used in the risk assessment of foodborne illnesses linked to meat consumption.

Inhibitory effect of caprylic acid and mild heat on Cronobacter spp. (Enterobacter sakazakii) in reconstituted infant formula and determination of injury by flow cytometry.

The combined effects of caprylic acid and mild heat were investigated to ascertain their impact on Cronobacter spp. (E. sakazakii) in reconstituted infant formula. Samples containing a mixture of 3 strains of Cronobacter spp. (10(7) to 10(8) CFU/ml) were prepared with various concentrations of caprylic acid (5, 10, 20, and 30 mM) and were then heated to 45, 50, and 55 degrees C. The inhibitory effect of the combined treatment resulted in a synergistic effect, in which Cronobacter spp. numbers were reduced much more rapidly with increased temperatures and concentrations of caprylic acid. When samples were treated with 30 mM caprylic acid, the time required to reduce Cronobacter spp. cell numbers to an approximate reduction of 7.8 log CFU/ml was 60 min at 45 degrees C, 20 min at 50 degrees C, and 10 min at 55 degrees C. In the validation assay using a low population of Cronobacter spp. (approximately 10(3) log CFU/ml), no recovery of injured cells was observed after samples were treated with 10 mM caprylic acid for 20 min at 55 degrees C, 20 mM caprylic acid for 10 min at 50 degrees C and 55 degrees C, and 30 mM caprylic acid for 10 min at 45 degrees C to 55 degrees C. To determine the bactericidal mechanism of caprylic acid, membrane integrity was examined by fluorescent staining followed by flow cytometry and confocal microscopy. Increased cellular inactivation was associated with increased propidium iodide staining, indicating damage to the cell membrane of Cronobacter spp.. Overall, these data indicate that the addition of this natural antimicrobial agent to infant formula may have potential use for controlling microbes prior to consumption at lower heating temperatures. The study also provides a complementary understanding of the mode of action of caprylic acid on Cronobacter spp.

Effect of nonylphenol ethoxylates (NPEs) on barrier functions of epithelial cell membranes: opening of tight junctions and competitive inhibition of P-gp-mediated efflux.

The effect of nonylphenol ethoxylates (NPEs) on selected barrier functions of biological membranes, such as tight junction and P-gp efflux pump of epithelial membranes, against the transport of xenobiotics was examined. The Caco-2 cell line was used to evaluate the transport of mannitol and daunomycin across the cell monolayer as well as the cellular uptake of daunomycin. In the presence of NPEs, the transport of mannitol was increased, with NP-9 showing a maximal effect, and the transepithelial electrical resistance (TEER) was reduced. The onset of this effect of NP-9 was fairly rapid and reversible for a short term (e.g., 2 h) treatment, while irreversible for a long term (e.g., 72 h) treatment. In the presence of NP-9, the apical uptake of daunomycin was increased, suggesting competitive inhibition between NP-9 and daunomycin in the efflux via the P-gp system. However, a 72 h pretreatment of the cells with NP-9 (up to 1000 nM) did not affect the apparent cellular uptake of daunomycin, suggesting no significant effect of NPEs on the expression of P-gp. In conclusion, NPEs appear to rapidly open the tight junction of epithelial cell membranes and to competitively inhibit the efflux of P-gp substrates, thereby reducing the self-protection ability of the organism against xenobiotics or hazardous environmental compounds that are transported via the paracellular pathway (i.e., uptake) or the P-gp system (i.e., efflux).