An archaeal transcription factor EnfR with a novel 'eighth note' fold controls hydrogen production of a hyperthermophilic archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1, a hyperthermophilic carboxydotrophic archaeon, produces H2 through CO oxidation catalyzed by proteins encoded in a carbon monoxide dehydrogenase (CODH) gene cluster. TON_1525 with a DNA-binding helix-turn-helix (HTH) motif is a putative repressor regulating the transcriptional expression of the codh gene cluster. The T55I mutation in TON_1525 led to enhanced H2 production accompanied by the increased expression of genes in the codh cluster. Here, TON_1525 was demonstrated to be a dimer. Monomeric TON_1525 adopts a novel 'eighth note' symbol-like fold (referred to as 'eighth note' fold regulator, EnfR), and the dimerization mode of EnfR is unique in that it has no resemblance to structures in the Protein Data Bank. According to footprinting and gel shift assays, dimeric EnfR binds to a 36-bp pseudo-palindromic inverted repeat in the promoter region of the codh gene cluster, which is supported by an in silico EnfR/DNA complex model and mutational studies revealing the implication of N-terminal loops as well as HTH motifs in DNA recognition. The DNA-binding affinity of the T55I mutant was lowered by ∼15-fold, for which the conformational change of N-terminal loops is responsible. In addition, transcriptome analysis suggested that EnfR could regulate diverse metabolic processes besides H2 production.

Transcription activation of two clusters for exopolysaccharide biosynthesis by phosphorylated DctD in Vibrio vulnificus.

NtrC-mediated production of exopolysaccharides (EPS), essential components for Vibrio vulnificus biofilms, is highly increased in the presence of dicarboxylic or tricarboxylic acids. Gel-shift assays showed that regulation of the EPS-gene cluster I (EPS-I cluster) by NtrC was direct via binding of phosphorylated NtrC (p-NtrC) to the regulatory region of the EPS-I cluster. In contrast, p-NtrC did not bind to the EPS-II and EPS-III clusters, suggesting that NtrC regulation was not direct and another transcription factor belonging to an NtrC-regulon might play a role in activating their transcription. A candidate transcription factor, DctD, of which expression was induced by NtrC, activated the expression of the EPS-II and EPS-III clusters via direct binding to their upstream regions. Under growth conditions with either dicarboxylic or tricarboxylic acids, the expression of NtrC was induced and the transcription of dctD was activated. Furthermore, DctD exhibited higher transcriptional activity under the conditions with dicarboxylic acids than with tricarboxylic acids. Therefore, this study demonstrates that under dicarboxylate-rich conditions, both the abundance and activity of DctD were markedly induced, which activates the expression of two EPS clusters to maximize biosynthesis of EPS facilitating biofilm maturation in V. vulnificus.

Polar landmark protein HubP recruits flagella assembly protein FapA under glucose limitation in Vibrio vulnificus.

How motile bacteria recognize their environment and decide whether to stay or navigate toward more favorable location is a fundamental issue in survival. The flagellum is an elaborate molecular device responsible for bacterial locomotion, and the flagellum-driven motility allows bacteria to move themselves to the appropriate location at the right time. Here, we identify the polar landmark protein HubP as a modulator of polar flagellation that recruits the flagellar assembly protein FapA to the old cell pole, thereby controlling its activity for the early events of flagellar assembly in Vibrio vulnificus. We show that dephosphorylated EIIAGlc of the PEP-dependent sugar transporting phosphotransferase system sequesters FapA from HubP in response to glucose and hence inhibits FapA-mediated flagellation. Thus, flagellar assembly and motility is governed by spatiotemporal control of FapA, which is orchestrated by the competition between dephosphorylated EIIAGlc and HubP, in the human pathogen V. vulnificus.

Increased Innate Lymphoid Cell 3 and IL-17 Production in Mouse Lamina Propria Stimulated with Giardia lamblia.

Innate lymphoid cells (ILCs) are key players during an immune response at the mucosal surfaces, such as lung, skin, and gastrointestinal tract. Giardia lamblia is an extracellular protozoan pathogen that inhabits the human small intestine. In this study, ILCs prepared from the lamina propria of mouse small intestine were incubated with G. lamblia trophozoites. Transcriptional changes in G. lamblia-exposed ILCs resulted in identification of activation of several immune pathways. Secretion of interleukin (IL)-17A, IL-17F, IL-1ß, and interferon-γ was increased, whereas levels of IL-13, IL-5, and IL22, was maintained or reduced upon exposure to G. lamblia. Goup 3 ILC (ILC3) was found to be dominant amongst the ILCs, and increased significantly upon co-cultivation with G. lamblia trophozoites. Oral inoculation of G. lamblia trophozoites into mice resulted in their presence in the small intestine, of which, the highest number of parasites was detected at the 5 days-post infection. Increased ILC3 was observed amongst the ILC population at the 5 days-post infection. These findings indicate that ILC3 from the lamina propria secretes IL-17 in response to G. lamblia, leading to the intestinal pathology observed in giardiasis.

Repression of VvpM Protease Expression by Quorum Sensing and the cAMP-cAMP Receptor Protein Complex in Vibrio vulnificus.

Septicemia-causing Vibrio vulnificus produces at least three exoproteases, VvpE, VvpS, and VvpM, all of which participate in interactions with human cells. Expression of VvpE and VvpS is induced in the stationary phase by multiple transcription factors, including sigma factor S, SmcR, and the cAMP-cAMP receptor protein (cAMP-CRP) complex. Distinct roles of VvpM, such as induction of apoptosis, lead us to hypothesize VvpM expression is different from that of the other exoproteases. Its transcription, which was found to be independent of sigma S, is induced at the early exponential phase and then becomes negligible upon entry into the stationary phase. SmcR and CRP were studied regarding the control of vvpM expression. Transcription of vvpM was repressed by SmcR and cAMP-CRP complex individually, which specifically bound to the regions -2 to +20 and +6 to +27, respectively, relative to the vvpM transcription initiation site. Derepression of vvpM gene expression was 10- to 40-fold greater in an smcR crp double mutant than in single-gene mutants. Therefore, these results show that the expression of V. vulnificus exoproteases is differentially regulated, and in this way, distinct proteases can engage in specific interactions with a host.IMPORTANCE An opportunistic human pathogen, Vibrio vulnificus produces multiple extracellular proteases that are involved in diverse interactions with a host. The total exoproteolytic activity is detected mainly in the supernatants of the high-cell-density cultures. However, some proteolytic activity derived from a metalloprotease, VvpM, was present in the supernatants of the low-cell-density cultures sampled at the early growth period. In this study, we present the regulatory mechanism for VvpM expression via repression by at least two transcription factors. This type of transcriptional regulation is the exact opposite of those for expression of the other V. vulnificus exoproteases. Differential regulation of each exoprotease's production then facilitates the pathogen's participation in the distinct interactions with a host.

Cyclo-(l-Phe-l-Pro), a Quorum-Sensing Signal of Vibrio vulnificus, Induces Expression of Hydroperoxidase through a ToxR-LeuO-HU-RpoS Signaling Pathway To Confer Resistance against Oxidative Stress.

Vibrio vulnificus, an opportunistic human pathogen, produces cyclo-(l-Phe-l-Pro) (cFP), which serves as a signaling molecule controlling the ToxR-dependent expression of innate bacterial genes, and also as a virulence factor eliciting pathogenic effects on human cells by enhancing intracellular reactive oxygen species levels. We found that cFP facilitated the protection of V. vulnificus against hydrogen peroxide. At a concentration of 1 mM, cFP enhanced the level of the transcriptional regulator RpoS, which in turn induced expression of katG, encoding hydroperoxidase I, an enzyme that detoxifies H2O2 to overcome oxidative stress. We found that cFP upregulated the transcription of the histone-like proteins vHUα and vHUß through the cFP-dependent regulator LeuO. LeuO binds directly to upstream regions of vhuA and vhuB to enhance transcription. vHUα and vHUß then enhance the level of RpoS posttranscriptionally by stabilizing the mRNA. This cFP-mediated ToxR-LeuO-vHUαß-RpoS pathway also upregulates genes known to be members of the RpoS regulon, suggesting that cFP acts as a cue for the signaling pathway responsible for both the RpoS and the LeuO regulons. Taken together, this study shows that cFP plays an important role as a virulence factor, as well as a signal for the protection of the cognate pathogen.

Preoperative Pain Sensitization Is Associated With Postoperative Pillar Pain After Open Carpal Tunnel Release.

BACKGROUND: Postoperative pillar pain (deep-seated wrist pain worsened by leaning on the heel of the hand) sometimes occurs after carpal tunnel release (CTR), leading to weakness in the hand and delayed return to work. Increased pain sensitivity has been found to be associated with worse symptoms and poorer treatment response in a number of chronic musculoskeletal conditions, but few studies have investigated the association of pain sensitization with pillar pain after CTR. QUESTIONS/PURPOSES: (1) Is preoperative pain sensitization in patients with carpal tunnel syndrome (CTS) associated with increased severity of pillar pain after open CTR? (2) What other demographic, electrophysiological, or preoperative clinical characteristics are associated with pillar pain after CTR? METHODS: Over a 35-month period, one surgeon performed 162 open carpal tunnel releases. Patients were eligible if they had sufficient cognitive and language function to provide informed consent and completed a self-reported questionnaire; they were not eligible if they had nerve entrapment other than CTR or if the surgery was covered by workers compensation insurance. Based on these criteria, 148 (91%) were approached for this study. Of those, 17 (9%) were lost to followup before 12 months, leaving 131 for analysis. Their mean age was 54 years (range, 32-78 years), and 81% (106 of 131) were women; 34% (45 of 131) had less than a high school education. We preoperatively measured pain sensitization by assessing the patients' pressure pain thresholds by stimulating pressure-induced pain in the pain-free volar forearm and administering a self-reported Pain Sensitivity Questionnaire minor subscale, an instrument that assesses pain intensity in daily life situations. We evaluated postoperative pillar pain using the "table test" (having the patient lean on a table with their weight on their hands placed on the table's edge with elbows straight) with an 11-point ordinal scale at 3, 6, and 12 months after their surgical procedures. We conducted bivariate and multivariable analyses to determine whether the patients' clinical, demographic, and pain sensitization factors were associated with their postoperative pillar pain severity after CTR. RESULTS: After controlling for relevant confounding variables such as age, education level, and functional states, we found that increased pillar pain severity was associated with the pressure pain threshold (ß = -1.02 [-1.43 to -0.61], partial R = 11%, p = 0.021) and Pain Sensitivity Questionnaire minor (ß = 1.22 [0.73-1.71], partial R = 17%, p = 0.013) at 3 months, but by 6 months, only Pain Sensitivity Questionnaire minor (ß = 0.92 [0.63-1.21], partial R = 13%, p = 0.018) remained an associated variable for pillar pain. Additionally, gender (women) was associated with increased pain severity at 3 (ß = 0.78 [0.52-1.04], partial R = 9%, p = 0.023) and 6 months (ß = 0.72 [0.41-1.01], partial R = 8%, p = 0.027). At 3 months, pressure pain threshold, Pain Sensitivity Questionnaire minor, and gender (women) collectively accounted for 37% of the variance in pillar pain severity; at 6 months, Pain Sensitivity Questionnaire minor and gender (women) accounted for 21% of the variance, but no relationship between those factors and pillar pain was observed at 12 months. CONCLUSIONS: Gender (women) and preoperative pain sensitization measured by pressure pain threshold and self-reported Pain Sensitivity Questionnaire were associated with pillar pain severity up to 3 and 6 months after CTR, respectively. However, the influence of pain sensitization on pillar pain was diminished at 6 months and it did not show persistent effects beyond 12 months. Pain sensitization seems to be more important in the context of recovery from surgical intervention (in the presence of a pain condition) than in healthy states, and clinicians should understand the role of pain sensitization in the postoperative management of CTS. Future research may be needed to determine if therapeutic interventions to reduce sensitization will decrease the risk of pillar pain. LEVEL OF EVIDENCE: Level III, prognostic study.

The Effect of Inter-Departmental Stroke Meetings on Rehabilitation in a Comprehensive Cerebrovascular Center.

BACKGROUND: Stroke is the number one cause of adulthood disability in Korea. Rehabilitation after stroke can minimize functional disability, enhance recovery toward independence, and optimize community reintegration. The inter-departmental stroke meeting (IDSM) is a potential method to improve rehabilitation outcomes in patients with stroke. We aimed to analyze the effect of IDSM on rehabilitation after acute ischemic stroke management. METHODS: Medical records of 753 patients with acute ischemic stroke admitted to the neurology department of our medical center between January and December 2014 were reviewed retrospectively. In May 2014, weekly IDSMs were initiated. All physicians responsible for the patient's care reviewed patient treatment, methods of secondary prevention, and future rehabilitation plans. RESULTS: The transfer rate significantly increased after initiation of IDSM (phase 2, 3) and the length of stay (LOS) before transfer to the rehabilitation department decreased significantly from 9.68 ± 8.50 days to 5.75 ± 2.12 days. There was a reduction in the total LOS from 52 ± 28.57 days to 35 ± 27.21 days after IDSMs were introduced. In non-transferred patients also, the total LOS reduced significantly. The transfer rate increased significantly and the LOS before transfer to the rehabilitation department decreased significantly after implementation of IDSM in a subgroup of patients with moderate to severe stroke. CONCLUSION: The introduction of IDSM was significantly correlated with improvements in transfer rates and reduction of LOS in hospital. This finding shows that IDSMs are an important intervention to improve therapeutic progress and outcomes for patients with stroke.

Phosphorylation of Serine 148 in Giardia lamblia End-binding 1 Protein is Important for Cell Division.

Giardia lamblia is a unicellular organism, showing a polarity with two nuclei and cytoskeletal structures. Accurate positioning of these organelles is essential for division of G. lamblia, which is poorly understood. Giardia lamblia end-binding 1 (GlEB1) protein and G. lamblia aurora kinase (GlAK) have been shown to modulate microtubule (MT) distribution during cytokinesis. A direct association between GlEB1 and GlAK was demonstrated. Like GlEB1, GlAK was also found at nuclear envelopes and median bodies of G. lamblia. In vitro kinase assays using Giardia lysates immunoprecipitated with anti-GlAK antibodies or recombinant GlAK suggested that GlEB1 is a substrate of GlAK. Site-directed mutagenesis indicated that threonine-205 in GlAK was auto-phosphorylated and that GlAK phosphorylated serine (Ser)-148 in GlEB1. Ectopic expression of a mutant GlEB1 (with conversion of Ser-148 into alanine of GlEB1) resulted in an increased number of Giardia cells with division defects. Treatment of G. lamblia with an AK inhibitor triggered cytokinesis defects, and ectopic expression of a phospho-mimetic mutant GlEB1 (with conversion of Ser-148 into aspartate) rescued the defects in Giardia cell division caused by the AK inhibitor. These results suggested that phosphorylation of GlEB1 played a role in cytokinesis in G. lamblia.

Stationary-phase induction of vvpS expression by three transcription factors: repression by LeuO and activation by SmcR and CRP.

An exoprotease of Vibrio vulnificus, VvpS, exhibits an autolytic function during the stationary phase. To understand how vvpS expression is controlled, the regulators involved in vvpS transcription and their regulatory mechanisms were investigated. LeuO was isolated in a ligand-fishing experiment, and experiments using a leuO-deletion mutant revealed that LeuO represses vvpS transcription. LeuO bound the extended region including LeuO-binding site (LBS)-I and LBS-II. Further screening of additional regulators revealed that SmcR and cyclic adenosine monophosphate-receptor protein (CRP) play activating roles in vvpS transcription. SmcR and CRP bound the regions overlapping LBS-I and -II, respectively. In addition, the LeuO occupancy of LBS-I and LBS-II was competitively exchanged by SmcR and CRP, respectively. To examine the mechanism of stationary-phase induction of vvpS expression, in vivo levels of three transcription factors were monitored. Cellular level of LeuO was maximal at exponential phase, while those of SmcR and CRP were maximal at stationary phase and relatively constant after the early-exponential phase, respectively. Thus, vvpS transcription was not induced during the exponential phase by high cellular content of LeuO. When entering the stationary phase, however, LeuO content was significantly reduced and repression by LeuO was relieved through simultaneous binding of SmcR and CRP to LBS-I and -II, respectively.

Deacylated lipopolysaccharides inhibit biofilm formation by Gram-negative bacteria.

The extracellular polysaccharides of Vibrio vulnificus play different roles during biofilm development. Among them, the effect of lipopolysaccharide (LPS), which is crucial for bacterial adherence to surfaces during the initial stage of biofilm formation, on the formation process was examined using various types of LPS extracts. Exogenously added LPS strongly inhibited biofilm formation in a dose-dependent manner. In addition, the exogenous addition of a deacylated form of LPS (dLPS) also inhibited biofilm formation. However, an LPS fraction extracted from a mutant not able to produce O-antigen polysaccharides (O-Ag) did not have an inhibitory effect. Furthermore, biofilm formation by several Gram-negative bacteria was inhibited by dLPS addition. In contrast, biofilm formation by Gram-positive bacteria was not influenced by dLPS but was affected by lipoteichoic acid. Therefore, this study demonstrates that O-Ag in LPS is important for inhibiting biofilm formation and may serve an efficient anti-biofilm agent specific for Gram-negative bacteria.

Age-related differences in control of a visuomotor coordination task: a preliminary study.

[Purpose] The purpose of the current study was to examine age-related differences in control of a perception-action coordination skill. We adapted a visuomotor tracking experiment requiring various coordination patterns between a limb's motion and an external signal. [Subjects and Methods] A total of 12 subjects (6 elderly and 6 young) voluntarily participated in the study. The experimental session consisted of 3 trials for 3 different relative phase patterns: 0°, 90°, and 180°, defined by the relationship between the online visual feedback of the joystick motion and the white dot signal. [Results] The 0° and 180° tracking patterns were stable compared with the 90° tracking pattern for both age groups. The present results also showed that the elderly subjects were less stable than were young subjects for all tracking patterns. [Conclusion] The intrinsic coordination dynamics predicted by the Haken-Kelso-Bunz (HKB) mathematical model did not change with age, whereas utilization of visual feedback information declined overall. Further research is needed regarding methods for increasing utilization of visual feedback information from the perspective of rehabilitation.

Role of AcsR in expression of the acetyl-CoA synthetase gene in Vibrio vulnificus.

BACKGROUND: VarS/VarA is one of the global factors regulating diverse aspects of the metabolism and virulence of bacteria including pathogenic Vibrio spp. An experiment to identify the VarS/VarA-regulon in V. vulnificus revealed that a putative LuxR-type transcriptional regulator was down-regulated in ΔvarA mutant. To investigate the roles of this regulatory cascade, the target gene regulated by a LuxR-regulator was identified and its expression was characterized. RESULTS: Transcriptomic analysis of the mutant deficient in this LuxR-type regulator showed that the acsA gene encoding acetyl-CoA synthetase was down-regulated. Thus, this regulator was named AcsR for "regulator of acetyl-CoA synthetase". A putative histidine kinase gene, acsS, was located five ORFs downstream of the acsR gene. Expression of an acsA::luxAB transcriptional fusion was decreased in both ΔacsR and ΔacsS mutants. Similar to a ΔacsA mutant, strains carrying deletions either in acsR or acsS grew slowly than wild type in a minimal medium with acetate as a sole carbon source. Growth defect of the ΔacsR strain in acetate-minimal medium was restored by complementation. To investigate if AcsR directly regulates acsA expression, in vitro-gel shift assays were performed using the recombinant AcsR and the regulatory region of the acsA gene, showing that AcsR specifically bound the upstream region of the acsA ORF. CONCLUSION: This study indicates that the VarS/VarA system plays a role in V. vulnificus metabolism via regulating AcsR, which in turn controls acetate metabolism by activating the transcription of the acetyl-CoA synthetase gene.

Role of VcrD1 protein in expression and secretion of flagellar components in Vibrio parahaemolyticus.

VcrD1 protein is a component of type III secretion system (T3SS) 1 in Vibrio parahaemolyticus. A comparative analysis of secretomes of wild-type and ΔvcrD1 strains revealed that the mutant was defective in secretion of diverse proteins including several flagellar components. Western blot analyses using specific antibodies confirmed that the secretion of at least four flagellar components, such as FlaA, FlgL, FlgE, and FlgM, was affected by the vcrD1 mutation, which was consistent with decreased motility on soft agar plates and the non-flagellated morphology of the mutant. The ΔexsA mutant, another T3SS1 mutant, did not showed reduced motility, but became non-motile phenotype with the additional ΔvcrD1 mutation. Complementation of wild-type vcrD1 gene into ΔvcrD1 mutant resulted in restored motility. Fractionation of bacterial cytoplasm from the periplasm and membrane revealed lower levels of FlaA and FlgM in the cytoplasm of the ΔvcrD1 mutant, indicating that VcrD1 might regulate the expression of flagellar genes in addition to the secretion of flagellar components in V. parahaemolyticus. Quantitative RT-PCR assays of seven representative flagellar genes in the wild-type and ΔvcrD1 mutant strains demonstrated that transcript levels of two early flagellar genes, flaK and flaL, were not reduced by the vcrD1 mutation, whereas the middle and late flagellar genes were expressed at a lower level in the vcrD1 mutant. This study raises a possibility that VcrD1 plays a role in flagellar morphogenesis in V. parahaemolyticus by regulating the expression and secretion of flagellar components.

Posterosuperior lesion has a high risk of lateral and central nodal metastasis in solitary papillary thyroid cancer.

BACKGROUND: Preoperative nodal assessment of papillary thyroid cancer (PTC) is very important because 60 to 70 % of all disease recurrence in the neck can occur in the lymph nodes. This study explored the association between ultrasonographic intrathyroidal location and the nodal metastasis pattern in solitary PTC. METHODS: Data from 218 patients who underwent total thyroidectomy with or without neck dissection for previously untreated PTC between 2006 and 2010 were retrospectively analyzed. Only patient data for which both preoperative ultrasound findings and postoperative pathologic reports were available were included. Multifocal cases, cases with extrathyroidal extension, and distant metastasis were excluded. The association between nodal metastasis pattern and clinical or pathologic features of solitary PTCs was analyzed, as was the association between ultrasonographic intrathyroidal location and central or lateral nodal metastasis in solitary PTC. RESULTS: Mass size larger than 2 cm (p < 0.001, Odds ratio (OR) 4.117) and central nodal metastasis (p < 0.001, OR 3.984) were related with lateral neck metastasis in multivariate analysis. Male sex (p = 0.001, OR 3.012) and capsular invasion (p < 0.001, OR 4.720) were related with central neck metastasis in multivariate analysis. When analyzing ultrasonographic location of intrathyroidal solitary lesion, posterosuperiorly located lesion was strongly associated with both lateral and central neck metastasis. (p < 0.001 and p = 0.002, respectively). CONCLUSIONS: Posterosuperior location of intrathyroidal solitary PTC has a high risk of lateral and central nodal metastasis when compared to other locations. For such patients, careful preoperative evaluation of nodal status should be done.

Evolutionary conservation of Nkx2.5 autoregulation in the second heart field.

The cardiac homeobox gene Nkx2.5 plays a key and dosage-sensitive role in the differentiation of outflow tract and right ventricle from progenitors of the second heart field (SHF) and Nkx2.5 mutation is strongly associated with human outflow tract congenital heart disease (OFT CHD). Therefore defining the regulatory mechanisms controlling Nkx2.5 expression in SHF populations serves an important function in understanding the etiology of complex CHD. Through a comparative analysis of regulatory elements controlling SHF expression of Nkx2.5 in the chicken and mouse, we have found evidence that Nkx2.5 autoregulation is important for maintaining Nkx2.5 expression during SHF differentiation in both species. However the mechanism of Nkx2.5 maintenance differs between placental mammals and non-mammalian vertebrates: in chick Nkx2.5 binds directly to a genomic enhancer element that is required to maintain Nkx2.5 expression in the SHF. In addition, it is likely that this is true in other non-mammalian vertebrates given that they possess a similar genomic organization. By contrast, in placental mammals, Nkx2.5 autoregulation in the SHF functions indirectly through Mef2c. These data underscore a tight relationship in mammals between Nkx2.5 and Mef2c in SHF transcriptional regulation, and highlight the potential for evolutionary cis-regulatory analysis to identify core, conserved components of the gene networks controlling heart development.

Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus: repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases.

VvhA produced by Vibrio vulnificus exhibits cytolytic activity to human cells including erythrocytes. Since haemolysis by VvhA may provide iron for bacterial growth and pathogenicity, we investigated the expression of VvhA to elucidate the regulatory roles of Fur, a major transcription factor controlling iron-homeostasis. Fur repressed the transcription of vvhBA operon via binding to the promoter region. However, haemolysin content and haemolytic activity were lowered in cell-free supernatant of fur mutant. This discrepancy between the levels of vvhA transcript and VvhA protein in fur mutant was caused by exoproteolytic activities of the elastase VvpE and another metalloprotease VvpM, which were also regulated by Fur. vvpE gene expression was repressed by Fur via binding to the Fur-box homologous region. Regulation of VvpM expression by Fur did not occur at the level of vvpM transcription. In vitro proteolysis assays showed that both proteases efficiently degraded VvhA. In addition, the extracellular levels of VvhA were higher in culture supernatants of vvpE or vvpM mutants than in the wild type. Thus this study demonstrates that Fur regulates haemolysin production at the transcription level of the vvhBA operon and at the post-translation level by regulating the expressions of two VvhA-degrading exoproteases, VvpE and VvpM.

Role of capsular polysaccharide (CPS) in biofilm formation and regulation of CPS production by quorum-sensing in Vibrio vulnificus.

Extracellular polysaccharides, such as lipopolysaccharide and loosely associated exopolysaccharides, are essential for Vibrio vulnificus to form biofilms. The role of another major component of the V. vulnificus extracellular matrix, capsular polysaccharide (CPS), which contributes to colony opacity, has been characterized in biofilm formation. A CPS-deficient mutant, whose wbpP gene encoding UDP-GlcNAc C4-epimerase was knocked out, formed significantly more biofilm than wild type, due to increased hydrophobicity of the cell surface, adherence to abiotic surfaces and cell aggregation. To elucidate the direct effect of CPS on biofilm structure, extracted CPS and a CPS-degrading enzyme, α-N-acetylgalactosaminidase, were added in biofilm assays, resulting in reduction and increment of biofilm sizes respectively. Therefore, it is suggested that CPS play a critical role in determining biofilm size by restricting continual growth of mature biofilms. Since CPS is required after maturation, CPS biosynthesis should be controlled in a cell density-dependent manner, e.g. by quorum-sensing (QS) regulation. Analysing transcription of the CPS gene cluster revealed that it was activated by SmcR, a QS master regulator, via binding to the upstream region of the cluster. Therefore, CPS was produced when biofilm cell density reached high enough to turn on QS regulation and limited biofilms to appropriate sizes.

Comparison of 2 cuff inflation methods of laryngeal mask airway Classic for safe use without cuff manometer in adults.

PURPOSE: This single-center, prospective, randomized, double-blind, 2-arm, parallel group comparison trial was performed to establish whether the adult-sized laryngeal mask airway (LMA) Classic (The Laryngeal Mask Company Ltd, Henley-on-Thames, UK) could be used safely without any consideration of cuff hyperinflation when a cuff of the LMA Classic was inflated using half the maximum inflation volume or the resting volume before insertion of device. BASIC PROCEDURES: Eighty patients aged 20 to 70 years scheduled for general anesthesia using the LMA Classic were included. Before insertion, the cuff was partially filled with half the maximum inflation volume in the half volume group or the resting volume created by opening the pilot balloon valve to equalize with atmospheric pressure in the resting volume group. Several parameters regarding insertion, intracuff pressure, airway leak pressure, and leakage volume/fraction were collected after LMA insertion. MAJOR FINDINGS: The LMA Classic with a partially inflated cuff was successfully inserted in all enrolled patients. Both groups had the same success rate of 95% at the first insertion attempt. The half volume group had a lower mean intracuff pressure compared with the resting volume group (54.5 ± 16.1 cm H2O vs 61.8 ± 16.1 cm H2O; P = .047). There was no difference in airway leak pressure or leakage volume/fraction between the 2 groups under mechanical ventilation. CONCLUSIONS: The partially inflated cuff method using half the maximum recommended inflation volume or the resting volume is feasible with the adult-sized LMA Classic, resulting in a high success rate of insertion and adequate range of intracuff pressures.