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Human C-reactive protein (CRP) and lactate dehydrogenase are important markers in clinical laboratory testing-the former is used to detect in vivo inflammation, and the latter is used to detect cell necrosis and tissue destruction. We developed aptamers that bind to human CRP and human lactate dehydrogenase-5 (LDH-5) with high affinities (dissociation constants of 6.2 pM and 235 pM, respectively), applying the systematic evolution of ligands by exponential enrichment (SELEX) method, and by using a modified DNA library containing the following base-appended base modifications: analog adenine derivative at the fifth position of uracil (U ad ), analog guanine derivative at the fifth position of uracil (U gu ), and analog adenine derivative at the seventh position of adenine (A ad ). A potential application of these aptamers as sensor elements includes high-sensitivity target detection in point-of-care testing.

We used base-appended base modification to develop a new adenine analog, which incorporates an adenine derivative at position 7 of adenine. Using the systematic evolution of ligands by exponential enrichment method with a modified DNA library including this analog, we obtained A ad1 , an aptamer that binds strongly to human β-defensin 2, a biomarker of physical stress found in saliva. The dissociation constant of A ad1 with respect to human β-defensin 2 was found to be low (6.8 nM), and was found to bind specifically to human β-defensin 2 in saliva spiked with the protein, as confirmed using pull-down with magnetic beads. To our knowledge, there are no prior reports of nucleic-acid aptamers that bind specifically to human β-defensin 2. However, our results indicated that such adenine analog-containing DNA libraries are extremely effective in the acquisition of high-affinity aptamers., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Importance: Oral propranolol is widely considered to be first-line therapy for complicated infantile hemangioma, but its use in patients with PHACE (posterior fossa malformations, hemangioma, arterial anomalies, cardiac defects, eye anomalies) syndrome has been debated owing to concerns that the cardiovascular effects of the drug may increase the risk for arterial ischemic stroke., Objective: To assess the incidence of adverse events among patients with PHACE syndrome receiving oral propranolol for infantile hemangioma., Design, Setting, and Participants: This multicenter retrospective cohort study assessed the incidence of adverse events among 76 patients with PHACE syndrome receiving oral propranolol for infantile hemangioma at 11 tertiary care, academic pediatric dermatology practices. Medical records from January 1, 2010, through April 25, 2017, were reviewed., Exposures: Patients received oral propranolol, 0.3 mg/kg/dose or more., Main Outcomes and Measures: The main outcome was the rate and severity of adverse events occurring throughout the course of treatment with oral propranolol, as documented in the medical records. Adverse events were graded from 1 to 5 using a scale derived from the Common Terminology Criteria for Adverse Events and were considered to be serious if they were grade 3 or higher., Results: A total of 76 patients (59 girls and 17 boys; median age at propranolol initiation, 56 days [range, 0-396 days]) met the inclusion criteria. There were no reports of serious adverse events (ie, stroke, transient ischemic attack, or cardiovascular events) during treatment with oral propranolol. A total of 46 nonserious adverse events were reported among 29 patients (38.2%); the most commonly reported nonserious adverse events were sleep disturbances and minor gastrointestinal tract and respiratory tract symptoms. In a comparison with 726 infants who received oral propranolol for hemangioma but did not meet criteria for PHACE syndrome, there was no significant difference in the rate of serious adverse events experienced during treatment (0 of 76 patients with PHACE syndrome and 3 of 726 patients without PHACE syndrome [0.4%])., Conclusions and Relevance: This study found that oral propranolol was used to treat infantile hemangioma in 76 patients with PHACE syndrome and that no serious adverse events were experienced. These data provide support for the safety of oral propranolol in this patient population.

The field of care testing toward the analysis of blood and saliva lacks nowadays simple test techniques for biomarkers. In this study, we have developed a novel nucleobase analog, U gu , which is a uracil derivative bearing a guanine base at the 5-position. Moreover, we attempted the development of aptamers that can bind to secretory immunoglobulin A (SIgA), which has been examined as a stress marker in human saliva. It was observed that the acquired aptamer binds strongly and selectively to the SIgA dimer ( K d = 13.6 nM) without binding to the IgG and IgA monomers of human serum. Reduction of the aptamer length (41 mer) successfully improved 4-fold the binding affinity ( K d = 3.7 nM), compared to the original, longer aptamer (78 mer). Furthermore, the development of a simple detection system for human saliva samples by fluorescence polarization was investigated, using the reported human salivary α-amylase (sAA) and the SIgA-binding aptamer. Comparison of the present method with conventional enzyme-linked immunosorbent assay techniques highlighted a significant Pearson's correlation of 0.94 and 0.83 when targeting sAA and SIgA, respectively. It is thus strongly suggested that a new simple test of stress markers in human saliva can be quantified quickly without bound/free (B/F) separation.

We previously demonstrated that administration of norepinephrine, dopamine, and serotonin into the lumbosacral defecation center caused propulsive contractions of the colorectum. It is known that the monoamines in the spinal cord are released mainly from descending neurons in the brainstem. In fact, stimulation of the medullary raphe nuclei, the origin of descending serotonergic neurons, enhances colorectal motility via the lumbosacral defecation center. Therefore, the purpose of this study was to examine the roles of the noradrenergic nucleus locus coeruleus (LC) and dopaminergic nucleus A11 region in the defecation reflex. Colorectal motility was measured with a balloon in anesthetized rats. Electrical stimulation of the LC and A11 region increased colorectal pressure only when a GABA A receptor antagonist was injected into the lumbosacral spinal cord. The effects of the LC stimulation and A11 region stimulation on colorectal motility were inhibited by antagonists of α1-adrenoceptors and D2-like dopamine receptors injected into the lumbosacral spinal cord, respectively. Spinal injection of a norepinephrine-dopamine reuptake inhibitor augmented the colokinetic effect of LC stimulation. The effect of stimulation of each nucleus was abolished by surgical severing of the parasympathetic pelvic nerves. Our findings demonstrate that activation of descending noradrenergic neurons from the LC and descending dopaminergic neurons from the A11 region causes enhancement of colorectal motility via the lumbosacral defecation center. The present study provides a novel concept that the brainstem monoaminergic nuclei play a role as supraspinal defecation centers. NEW & NOTEWORTHY The present study demonstrates that electrical and chemical stimulations of the locus coeruleus or A11 region augment contractions of the colorectum. The effects of locus coeruleus and A11 stimulations on colorectal motility are due to activation of α1-adrenoceptors and D2-like dopamine receptors in the lumbosacral defecation center, respectively. The present study provides a novel concept that the brainstem monoaminergic nuclei play a role as supraspinal defecation centers.

The aim of the present study was to clarify roles of ATP-dependent potassium channels (K ATP channels) in motility of the striated muscle portion in the esophagus. An isolated segment of the rat esophagus was placed in an organ bath and mechanical responses were recorded using a force transducer. Electrical stimulation of the vagus nerve evoked contractile response of striated muscle in the esophageal segment. Application of glibenclamide, an antagonist of K ATP channels, increased amplitude of vagally mediated twitch contractions of the rat esophagus. On the other hand, minoxidil, an agonist of K ATP channels, decreased amplitude of twitch contractions. RT-PCR revealed the expression of subunits of K ATP channels in esophageal tissue. In addition, immunopositivity for subunits of K ATP channels was observed in the striated muscle cells of the esophageal muscle layer. These findings indicate that K ATP channels contribute to motor regulation of striated muscle in the rat esophagus.

Cold-shock proteins are thought to participate in the cold-tolerant nature of hibernating animals. We previously demonstrated that an alternative splicing may allow rapid induction of functional cold-inducible RNA-binding protein (CIRBP) in the hamster heart. The purpose of the present study was to determine the major cause of the alternative splicing in Syrian hamsters. RT-PCR analysis revealed that CIRBP mRNA is constitutively expressed in the heart, brain, lung, liver, and kidney of nonhibernating euthermic hamsters with several alternative splicing variants. In contrast, the short variant containing an open-reading frame for functional CIRBP was dominantly found in the hibernating animals. Keeping the animals in a cold and dark environment did not cause a shift in the alternative splicing. Induction of hypothermia by central administration of an adenosine A 1 -receptor agonist reproduced the shift in the splicing pattern. However, the agonist failed to shift the pattern when body temperature was kept at 37°C, suggesting that central adenosine A 1 receptors are not directly linked to the shift of the alternative splicing. Rapid reduction of body temperature to 10°C by isoflurane anesthesia combined with cooling did not alter the splicing pattern, but maintenance of mild hypothermia (~28°C) for 2 h elicited the shift in the pattern. The results suggest that animals need to be maintained at mild hypothermia for an adequate duration to induce the shift in the alternative splicing. This is applicable to natural hibernation because hamsters entering hibernation show a gradual decrease in body temperature, being maintained at mild hypothermia for several hours.

A nonbenzenoid hydrocarbon, difluoreno[1,9,8- alkj:1',9',8'- gfed]heptalene 1, is synthesized. Experimental and theoretical investigations demonstrate that the planar and symmetric heptalene core within 1 effectively induces the antiaromatic and open-shell character. These properties are not shared by bisanthene 2, a benzenoid isomer of 1.

Background: Esophageal peristalsis is controlled by the brainstem via vago-vagal reflex. However, the precise regulatory mechanisms in the striated muscle portion are largely unknown. The aim of this study was to characterize peristaltic motility in the portion of the esophagus using a novel in vivo method in rats., Methods: A balloon-tipped catheter was placed in the esophagus of a rat anesthetized with urethane. To induce esophageal peristalsis, the balloon was inflated by water injection., Key Results: When the balloon was inflated near the bronchial bifurcation, the balloon was transported in the aboral direction. Vagotomy abolished the peristaltic response. The threshold volume for inducing esophageal peristalsis varied according to the velocity of balloon distention; the volume being effective to induce peristalsis at a low inflation speed was smaller than the threshold volume at a rapid inflation speed. Even in the absence of inflation, keeping the balloon inside the esophagus during an interval period prevented subsequent induction of peristaltic motility. In addition, a nitric oxide synthase inhibitor abolished the induction of esophageal peristalsis., Conclusions and Inferences: Our findings suggest that (a) in addition to the intensity, the velocity of distention is important for activating the mechanosensory mechanism to induce esophageal peristalsis, (b) tonic inputs from afferent fibers located at the mucosa may reduce the excitability of mechanosensors which is necessary for inducing peristalsis, and (c) nitric oxide plays essential roles in the induction of esophageal peristalsis. These results provide novel insights into the regulatory mechanisms of esophageal motility., (© 2018 John Wiley & Sons Ltd.)

Cold-inducible RNA-binding protein (CIRBP) plays important roles in protection against harmful effects of cold temperature. We previously found that several splicing variants of CIRBP mRNA are constitutively expressed in the heart of non-hibernating euthermic hamsters and that one of the variants is predominantly expressed with remarkable reduction in the expression of other variants in hibernating hypothermic hamsters. The aim of this study was to determine whether the regulation of alternative splicing is a common function in a non-hibernator, the mouse. The expression of CIRBP mRNA was assessed by RT-PCR. In euthermic control mice, several splicing variants of CIRBP mRNA were detected in various organs. When hypothermia was induced in mice by using isoflurane anesthesia, the short form variant, which encodes full-length functional CIRBP, was predominantly detected. Keeping body temperature of anesthetized mice at 37°C prevented changes in the splicing pattern. Exposure of mice to a low temperature did not change the splicing pattern, suggesting that endogenous neuronal and/or humoral pathways activated in response to cold stimuli applied to the body surface play minor roles. In agreement with this, the shift in alternative splicing was reproduced in isolated leukocytes in vitro when they were incubated at 28°C. Since application of a TRPM8 or TRPA1 agonist at 37°C failed to promote the shift in the splicing pattern, it seems likely that cold-sensitive channels are not involved in the splicing regulation. Therefore, it is probable that a substantial reduction of temperature is a major cause of the regulation of alternative splicing of CIRBP transcripts. The regulatory system of CIRBP expression at the level of alternative splicing, which was originally discovered in the hibernating hamster, commonly exists in non-hibernators such as mice.

The central regulating mechanisms of defecation, especially roles of the spinal defecation center, are still unclear. We have shown that monoamines including norepinephrine, dopamine, and serotonin injected into the spinal defecation center cause propulsive contractions of the colorectum. These monoamines are the main neurotransmitters of descending pain inhibitory pathways. Therefore, we hypothesized that noxious stimuli in the colorectum would activate the descending monoaminergic pathways projecting to the spinal defecation center and that subsequently released endogenous monoamine neurotransmitters would enhance colorectal motility. Colorectal motility was measured in rats anesthetized with α-chloralose and ketamine. As a noxious stimulus, capsaicin was administered into the colorectal lumen. To interrupt neuronal transmission in the spinal defecation center, antagonists of norepinephrine, dopamine, and/or serotonin receptors were injected intrathecally at the L6-S1 spinal level, where the spinal defecation center is located. Intraluminal administration of capsaicin, acting on the transient receptor potential vanilloid 1 channel, caused transient propulsive contractions. The effect of capsaicin was abolished by surgical severing of the pelvic nerves or thoracic spinal transection at the T4 level. Capsaicin-induced contractions were blocked by preinjection of D2-like dopamine receptor and 5-hydroxytryptamine subtype 2 and 3 receptor antagonists into the spinal defecation center. We demonstrated that intraluminally administered capsaicin causes propulsive colorectal motility through reflex pathways involving the spinal and supraspinal defecation centers. Our results provide evidence that descending monoaminergic neurons are activated by noxious stimulation to the colorectum, leading to facilitation of colorectal motility. NEW & NOTEWORTHY The present study demonstrates that noxious stimuli in the colorectum activates the descending monoaminergic pathways projecting to the spinal defecation center and that subsequently released endogenous monoamine neurotransmitters, serotonin and dopamine, enhance colorectal motility. Our findings provide a possible explanation of the concurrent appearance of abdominal pain and bowel disorder in irritable bowel syndrome patients. Thus the present study may provide new insights into understanding of mechanisms of colorectal dysfunction involving the central nervous system.

Melamine, a nitrogen-rich compound, has been used as a food and milk additive to falsely increase the protein content. However, melamine is toxic, and high melamine levels in food or in milk can cause kidney and urinary problems, or even death. Hence, the detection of melamine in food and milk is desirable, for which numerous detection methods have been developed. Several methods have successfully detected melamine in raw milk; however, they require a sample preparation before the analyses. This study aimed to develop an aptamer-DNAzyme conjugated biosensor for label-free detection of melamine, in raw milk, without any sample preparation. An aptamer-DNAzyme conjugated biosensor was developed via screening using microarray analysis to identify the candidate aptamers followed by an optimization, to reduce the background noise and improve the aptamer properties, thereby, enhancing the signal-to-noise (S/N) ratio of the screened biosensor. The developed biosensor was evaluated via colorimetric detection and tested with raw milk without any sample preparation, using N -methylmesoporphyrin IX for fluorescence detection. The biosensor displayed significantly higher signal intensity at 2 mM melamine (S/N ratio, 20.2), which was sufficient to detect melamine at high concentrations, in raw milk.

The presence of a fecal pellet in the colorectum causes ascending contraction and descending relaxation, propelling the pellet aborally. However, random occurrence of the reflexes at multiple sites would disturb sequential excretion of the pellets, resulting in inefficient defecation. Hence, we postulated that a regulatory mechanism to coordinate peristaltic motility initiated at adjacent portions of the colorectum may exist. Colorectal motility was recorded with balloons located at 2 cm, 5 cm and 7 cm from the anus in vivo in anesthetized rats. The presence of a balloon in the colorectum inhibited motility of the oral side and enhanced motility of the anal side. Both the ascending inhibitory and descending facilitatory actions were unaffected by cutting the pelvic nerves, suggesting little contribution of the lumbosacral defecation center. In contrast, disrupting the continuity of the enteric nervous system abolished the local reflex mechanism. The ascending inhibitory pathway operated in a condition in which facilitatory input from the lumbosacral defecation center was fully activated by intrathecal injection of ghrelin. We also found that functional impairment of the local reflex pathways was evident in rats that recovered from 2,4,6-trinitrobenzensulfonic acid-induced colitis. These results demonstrate that an intrinsic regulatory mechanism to coordinate peristaltic motility initiated at adjacent portions exists in the rat colorectum. The regulation may be beneficial to propel multiple pellets efficiently. In addition, impairment of the local regulatory mechanism might be involved in postinflammatory dysmotility in the colorectum., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Atopic dermatitis is the most common chronic skin disease, and it primarily affects children. Although atopic dermatitis (AD) has the highest effect on burden of skin disease, no high-level studies have defined optimal therapy for severe disease. Corticosteroids have been used to treat AD since the 1950s and remain the only systemic medication with Food and Drug Administration approval for this indication in children, despite published guidelines of care that recommend against this option. Several clinical trials with level 1 evidence have supported the use of topical treatments for mild to moderate atopic dermatitis in adults and children, but these trials have had little consistency in protocol design. Consensus recommendations will help standardize clinical development and trial design for children. The Food and Drug Administration issues guidance documents for industry as a source for "the Agency's current thinking on a particular subject." Although they are nonbinding, industry considers these documents to be the standard for clinical development and trial design. Our consensus group is the first to specifically address clinical trial design in this population. We developed a draft guidance document for industry, Developing Drugs for Treatment of Atopic Dermatitis in Children (≥3 months to <18 years of age). This draft guidance has been submitted to the Food and Drug Administration based on a provision in the Federal Register (Good Guidance Practices)., (© 2018 Wiley Periodicals, Inc.)

Colorectal motility is regulated by two defecation centers located in the brain and spinal cord. In previous studies, we have shown that administration of serotonin (5-HT) in the lumbosacral spinal cord causes enhancement of colorectal motility. Because spinal 5-HT is derived from neurons of the medullary raphe nuclei, including the raphe magnus, raphe obscurus, and raphe pallidus, we examined whether stimulation of the medullary raphe nuclei enhances colorectal motility via the lumbosacral defecation center. Colorectal pressure was recorded with a balloon in vivo in anesthetized rats. Electrical stimulation of the medullary raphe nuclei failed to enhance colorectal motility. Because GABAergic neurons can be simultaneously activated by the raphe stimulation and released GABA masks accelerating actions of the raphe nuclei on the lumbosacral defecation center, a GABA A receptor antagonist was preinjected intrathecally to manifest excitatory responses. When spinal GABA A receptors were blocked by the antagonist, electrical stimulation of the medullary raphe nuclei increased colorectal contractions. This effect of the raphe nuclei was inhibited by intrathecal injection of 5-hydroxytryptamine type 2 (5-HT 2 ) and type 3 (5-HT 3 ) receptor antagonists. In addition, injection of a selective 5-HT reuptake inhibitor in the lumbosacral spinal cord augmented the raphe stimulation-induced enhancement of colorectal motility. Transection of the pelvic nerves, but not transection of the colonic nerves, prevented the effect of the raphe nuclei on colorectal motility. These results demonstrate that activation of the medullary raphe nuclei causes augmented contractions of the colorectum via 5-HT 2 and 5-HT 3 receptors in the lumbosacral defecation center. NEW & NOTEWORTHY We have shown that electrical stimulation of the medullary raphe nuclei causes augmented contractions of the colorectum via pelvic nerves in rats. The effect of the medullary raphe nuclei on colorectal motility is exerted through activation of 5-hydroxytryptamine type 2 and type 3 receptors in the lumbosacral defecation center. The descending serotoninergic raphespinal tract represents new potential therapeutic targets against colorectal dysmotility such as irritable bowel syndrome.

An 86-year-old man was brought in ambulance to our hospital because of sudden hematochezia and abdominal pain during defecation. Intestinal prolapse approximately 80mm from the anus and a type 1 tumor 50mm in size on the mucosal surface were detected. The intestinal prolapse was manually repositioned, and the reduction of the intussusception was confirmed by computed tomography (CT). Following colonoscopy and abdominal-enhanced CT, a sigmoid colon cancer without distant metastases was detected. Elective laparoscopic radical surgery was performed. The present study described a rare case of sigmoid colon cancer with an intussusception prolapsing through the anus and highlighted the treatment strategy by reviewing 48 previous cases. The treatment strategy employed was as follows:first, manual repositioning of the intestinal prolapse was attempted;and second, the presence of intussusception was confirmed by CT. In cases when repositioning of the intussusception was not possible, even with the use of an endoscope or contrast enema, emergency surgery was required.

In order to construct the artificial cells and to understand the physicochemical properties of living cells, it is important to clarify the cell-sized confinement effect on the behaviours of bio-inspired polymers. We report the dynamic behaviours of aqueous hydroxypropyl cellulose (HPC) solution coated with phospholipids in oil (water-in-oil droplets, W/O droplets), accompanied by an increase in the temperature. We directly observed the beginning of phase separation of HPC solution using a fluorescence microscope and confirmed the dependence of such phenomena on droplet size. The results indicate that the start time of phase separation is decreased with an increase in droplet size. The experimental results suggest that the confinement situation accelerates the phase separation of aqueous HPC solutions., Competing Interests: The authors declare no conflict of interest.

Fluorescent proteins are now indispensable tools in molecular research. They have also been adapted for a wide variety of uses in cases involving creative applications, including textiles, aquarium fish, and ornamental plants. Our colleagues have previously cloned a yellow GFP-like protein derived from the marine copepod Chiridius poppei (YGFP), and moreover, succeeded in generating transgenic flowers with clearly visible fluorescence, without the need for high-sensitivity imaging equipment. However, due to the low Stokes shift of YGFP (10 nm), it is difficult to separate emitted light of a labeled object from the light used for excitation; hence, limitations for various applications remain. In this study, which was aimed at developing YGFP mutants with increased Stokes shifts, we conducted stepwise molecular evolution experiments on YGFP by screening random mutations at three key amino acids, based on their fluorescent characteristics and structural stabilities, followed by optimization of their fluorescence output by DNA shuffling of the entire coding sequence. We successfully identified an eYGFPuv that had an excitation maximum in UV wavelengths and a 24-fold increase in fluorescence intensity compared to the previously reported YGFP mutant (H52D). In addition, eYGFPuv exhibited almost 9-fold higher fluorescence intensity compared to the commercially available GFPuv when expressed in human colon carcinoma HCT116 cells and without any differences in cytotoxicity. Thus, this novel mutant with the desirable characteristics of bright fluorescence, long Stokes shift, and low cytotoxity, may be particularly well suited to a variety of molecular and biological applications.

We have attained a chemically modified DNA aptamer against salivary α-amylase (sAA), which attracts researchers' attention as a useful biomarker for assessing human psychobiological and social behavioural processes, although high affinity aptamers have not been isolated from a random natural DNA library to date. For the selection, we used the base-appended base (BAB) modification, that is, a modified-base DNA library containing (E)-5-(2-(N-(2-(N6-adeninyl)ethyl))carbamylvinyl)-uracil in place of thymine. After eight rounds of selection, a 75 mer aptamer, AMYm1, which binds to sAA with extremely high affinity (K d  < 1 nM), was isolated. Furthermore, we have successfully determined the 36-mer minimum fragment, AMYm1-3, which retains target binding activity comparable to the full-length AMYm1, by surface plasmon resonance assays. Nuclear magnetic resonance spectral analysis indicated that the minimum fragment forms a specific stable conformation, whereas the predicted secondary structures were suggested to be disordered forms. Thus, DNA libraries with BAB-modifications can achieve more diverse conformations for fitness to various targets compared with natural DNA libraries, which is an important advantage for aptamer development. Furthermore, using AMYm1, a capillary gel electrophoresis assay and lateral flow assay with human saliva were conducted, and its feasibility was demonstrated.

Background: Serotonin (5-hydroxytryptamine, 5-HT) is a regulatory factor in motility of the gastrointestinal tract including the esophagus. Although we proposed that vagal cholinergic and mast cell-derived non-cholinergic components including serotonin coordinately shorten the esophagus, the precise mechanism of serotonin-induced contractions in the suncus esophagus is still unclear. Therefore, the aims of this study were to determine characteristics of contractile responses induced by serotonin and to identify 5-HT receptor subtypes responsible for regulating motility in the suncus esophagus., Methods: An isolated segment of the suncus esophagus was placed in an organ bath, and longitudinal or circular mechanical responses were recorded using a force transducer., Key Results: Serotonin evoked contractile responses of the suncus esophagus in the longitudinal direction but not in the circular direction. Tetrodotoxin did not affect the serotonin-induced contractions. Pretreatment with a non-selective 5-HT receptor antagonist or double application of 5-HT 1 and 5-HT 2 receptor antagonists blocked the serotonin-induced contractions. 5-HT 1 and 5-HT 2 receptor agonists, but not a 5-HT 3 receptor agonist, evoked contractile responses in the suncus esophagus., Conclusion & Inferences: The findings suggest that serotonin induces contractile responses of the longitudinal smooth muscle in the muscularis mucosae of the suncus esophagus that are mediated via 5-HT 1 and 5-HT 2 receptors on muscle cells. The serotonin-induced contractions might contribute to esophageal peristalsis and emetic response., (© 2016 John Wiley & Sons Ltd.)

Background: There has been a dramatic increase in the off-label use of ophthalmic timolol maleate, a β-blocker used for infantile hemangioma (IH) treatment as a topical counterpart to oral propranolol. Its safety and efficacy in a pediatric population with IH have not been evaluated in a large cohort. Our goal was to retrospectively assess timolol's effectiveness, discern characteristics associated with response, and document reported adverse events., Methods: A multicenter retrospective cohort study of 731 patients treated with topical timolol was completed at 9 centers. Inclusion required an IH suitable for timolol in the treating physician's judgment and access to clinical details including photographs. Logistic regression analysis and descriptive statistics were performed. Primary outcome measures were efficacy assessed by using visual analog scales for color and for size, extent, and volume from review of digital photographs taken as standard of care., Results: Most IHs were localized (80.1%) and superficial (55.3%). Risk of disfigurement was the most common indication for therapy (74.3%). Duration of therapy (P < .0001), initial thinness (P = .008), and subtype (P = .031) were significant predictors of response. Best response occurred in superficial IHs <1 mm thick. Fifty-three (7.3%) required subsequent therapy with systemic β-blocker. Adverse events were mild, occurring in 25 (3.4%) patients. No cardiovascular side effects were documented., Conclusions: Timolol seems to be a well-tolerated, safe treatment option with moderate to good effectiveness, demonstrating best response in thin, superficial IHs regardless of pretreatment size. Timolol can be recommended as an alternative to systemic β-blockers and watchful waiting for many patients., (Copyright © 2016 by the American Academy of Pediatrics.)

Purpose: Platelet-rich fibrin (PRF) has been recently used as the sole grafting material in sinus floor elevation procedures. The aim of this prospective study was to measure the bone gain around the dental implant after using the crestal approach to sinus floor elevation using platelet-rich fibrin as the only grafting material in atrophic posterior maxillae with residual bone height <5 mm., Materials and Methods: Two different types of implants were used: hydroxyapatite (HA) and sandblasted acid-etched (SA) implants. Panoramic radiography and computed tomography were used to measure the endosinus bone gain., Results: Twenty-seven patients with 39 implants (19 HA and 20 SA) were included in this study. The mean residual bone measurements before surgery in the SA and HA groups were 2.85 and 2.68 mm, respectively. The mean average bone gains for 1 year in the SA and HA groups were 4.38 and 4.00 mm, respectively., Conclusion: This prospective study showed that platelet-rich fibrin promoted endosinus bone gain when used as the grafting material in the crestal approach to sinus floor elevation.

Hydrogen sulfide (H2S) is recognized as a gaseous transmitter and has many functions including regulation of gastrointestinal motility. The aim of the present study was to clarify the effects of H2S on the motility of esophageal striated muscle in rats. An isolated segment of the rat esophagus was placed in an organ bath and mechanical responses were recorded using a force transducer. Electrical stimulation of the vagus nerve evoked contractile response in the esophageal segment. The vagally mediated contraction was inhibited by application of an H2S donor. The H2S donor did not affect the contraction induced by electrical field stimulation, which can excite the striated muscle directly, not via vagus nerves. These results show that H2S has an inhibitory effect on esophageal motility not by directly attenuating striated muscle contractility but by blocking vagal motor nerve activity and/or neuromuscular transmissions. The inhibitory actions of H2S were not affected by pretreatment with the transient receptor potential vanniloid-1 blocker, transient receptor potential ankyrin-1 blocker, nitric oxide synthase inhibitor, blockers of potassium channels, and ganglionic blocker. RT-PCR and Western blot analysis revealed the expression of H2S-producing enzymes in esophageal tissue, whereas application of inhibitors of H2S-producing enzymes did not change vagally evoked contractions in the esophageal striated muscle. These findings suggest that H2S, which might be produced in the esophageal tissue endogenously, can regulate the motor activity of esophageal striated muscle via a novel inhibitory neural pathway., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In sinus floor augmentation of an atrophic posterior maxilla, platelet-rich fibrin (PRF) has been used as a graft material. We herein report two cases with histological evaluations of PRF after the surgery. The first case was a 28-year-old female with an atrophic right posterior maxilla who was treated with sinus floor augmentation and simultaneous implant placement using PRF as the sole graft material in our hospital. Twenty-four months after surgery, the implant was unfortunately removed because of occlusal overloading by parafunctional habits. During implant replacement, a tissue sample was obtained from the site of augmentation with PRF and was evaluated histologically. The second case was a 58-year-old man with severe alveolar atrophy of the right maxilla who underwent lateral sinus augmentation using only PRF in a two-stage procedure in our hospital. Samples were obtained at the second-stage surgery and histological examinations were performed. As a result, new bone formation was confirmed histologically in both cases. Our findings show that the use of PRF as a graft material during sinus floor augmentation induces natural bone regeneration.

Objective: Oral mucositis is a common adverse reaction to radiotherapy for head and neck cancer, and there are concerns regarding a decreased quality of life in patients receiving radiotherapy. The purpose of this study was to investigate the relationship between the severity of radiation-induced oral mucositis and the myeloperoxidase (MPO) levels in irradiated tissues., Study Design: Ninety-six F344 rats were divided into the following 4 groups: 10-Gy, 18-Gy, and 30-Gy irradiation groups, and a nonirradiation group. Oral mucositis was induced by the administration of single doses of radiation via exposure. After irradiation, the rats were evaluated on the basis of weight measurements, macroscopic findings according to a grading scale (Oral Mucositis Index [OMI]), and the results of tissue MPO assays., Results: Weights decreased whereas the OMI scores and MPO levels increased, depending on the dose of exposure. The Spearman rank correlation test showed a significant correlation between the OMI scores and the MPO levels in the tissues with a correlation coefficient of 0.824 (P < .01)., Conclusions: In this study, the MPO levels in the irradiated tissue were increased in the cases involving severe radiation-induced oral mucositis evaluated in rats using a grading scale., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The restructuring of a nonfractal particle aggregate in simple shear flow was simulated by a Stokesian dynamics approach. We studied the deformation and the resultant strength change of aggregates by the surrounding flow under the condition that the cohesive strength of an aggregate is comparable to the fluid stress. In particular, we focused on how the aggregate deteriorates because of the fluid stress exerted on it periodically. The image analysis was applied to visualized simulation results for the quantitative estimation of irreversible change in an aggregate configuration. We examined the structural change in the aggregate from various perspectives, i.e., the outer shape, the internal strength, and the fluid stress on the surface of the aggregate. The simulation results show that the aggregate gets squashed after an intricate restructuring process and it elongates along with the streamline as experimentally observed in the previous study. Regarding the internal strength, the weakest point locally develops in the aggregate by periodically varying the fluid stress. A combination of rotation and elongation effects of shear flow is complexly involved in the deterioration of the internal strength of the aggregate.

Purpose: To verify the measurement of cortical bone thickness at the distal radius in vivo using an ultrasonic method., Methods: The method for estimating cortical bone thickness was derived from experiments with in vitro bovine specimens. Propagation time of echo waves and propagation time of slow waves were used for the estimation. The outside diameter of cortical bone and the cortical bone thickness at the distal 5.5 % site of radius were measured with the new ultrasonic bone measurement system, and the results were compared with X-ray pQCT clinical measurements., Results: There was a high positive correlation (r: 0.76) between the cortical bone thickness measured by the new ultrasonic system and the X-ray pQCT results., Conclusion: We will be able to measure not only cancellous bone density but also cortical bone thickness in vivo using ultrasonic waves (without X-ray) safely and repeatedly.

We investigated the actions of probiotics, Streptococcus faecalis 129 BIO 3B (SF3B), in a trinitrobenzenesulfonic acid- (TNBS-) induced colitis model in rats. After TNBS was administered into the colons of rats for induction of colitis, the rats were divided into two groups: one group was given a control diet and the other group was given a diet containing SF3B for 14 days. There were no apparent differences in body weight, diarrhea period, macroscopic colitis score, and colonic weight/length ratio between the control group and SF3B group, suggesting that induction of colitis was not prevented by SF3B. Next, we investigated whether SF3B-containing diet intake affects the restoration of enteric neurotransmissions being damaged during induction of colitis by TNBS using isolated colonic preparations. Recovery of the nitrergic component was greater in the SF3B group than in the control group. A compensatory appearance of nontachykininergic and noncholinergic excitatory components was less in the SF3B group than in the control group. In conclusion, the present study suggests that SF3B-containing diet intake can partially prevent disruptions of enteric neurotransmissions induced after onset of TNBS-induced colitis, suggesting that SF3B has therapeutic potential.

Pharmacists' home visit duties include accepting prescriptions, making inquiries with the attending physician, preparing medication, giving guidance on medication, going on home visits(round trips), and handling insurance claims. In the present study, we measured the time required for each duty and determined work duration per visit. The study took place during September 2013; the sample included 116 subjects and 211 home visits were conducted. Pharmacists' mean work duration per visit was 73 min and 15 s; they spent an average of 36 min and 13 s of this period on round trips involving home visits and providing guidance on medication. Pharmacists are responsible for preparing medication for outpatients; therefore, it can be assumed that they can visit patients at their homes only during the estimated 2-h period after lunch and before the commencement of outpatient service in the evening. Therefore, it is difficult for one pharmacist to visit more than five patients a day. The number of patients that each pharmacist can visit in one week(6 working days)is estimated at 25-30.

We show the bursting process of dioleoylphosphatidylcholine (DOPC) liposomes in response to the addition of acetonitrile, a small toxic molecule widely used in the fields of chemistry and industry. The percentage of destroyed liposomes is reduced upon decreasing the acetonitrile fraction in the aqueous solution and vesicle bursting is not observed at volume ratios of 4:6 and below. This indicates that a high fraction of acetonitrile causes the bursting of liposomes, and it is proposed that this occurs through insertion of the molecules into outer leaflet of the lipid bilayer. The elapsed time between initial addition of acetonitrile and liposome bursting at each vesicle is also measured and demonstrated to be dependent on the volume fraction of acetonitrile and the vesicle size., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: The aim of this study was to evaluate the healing effect of topically applied platelet-rich fibrin (PRF) on experimental oral mucositis induced by chemotherapy in hamsters., Study Design: Oral mucositis was induced in 93 Syrian golden hamsters by an intraperitoneal injection of 5-fluorouracil, which was followed by light scratching of the cheek pouch. The hamsters were randomly divided into a PRF group, a fibrin group, and an untreated control group. The recovery stage of oral mucositis was evaluated through daily weighing, measurements of the ulcer area, histopathologic analysis, and a myeloperoxidase activity assay., Results: The PRF group exhibited significant improvements in the size and histologic features of the ulcer and in the myeloperoxidase activity compared with the control group (P < .05)., Conclusions: The current findings suggest the consideration for future clinical trials in humans., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Plasma BNP levels are predictive of prognosis in hemodialysis patients. However, recent studies showed that the current BNP immunoassay cross-reacts with glycosylated proBNP, and the NT-proBNP assay underestimates glycosylated NT-proBNP. In addition, the recently developed high performance dialyzer removes medium-sized molecular solutes such as β2-microgloburin. We therefore investigated the effects of high performance dialysis on measured levels of glycosylated proBNP, glycosylated NT-proBNP and other BNP-related peptides in end-stage renal disease (ESRD) patients on hemodialysis., Method: The relationships between clinical parameters and BNP-related molecule were also investigated. We used our newly developed immunoassay to measure plasma total BNP and proBNP in 105 normal subjects and 36 ESRD patients before and after hemodialysis. Plasma NT-proBNP was measured using Elecsys II after treatment with or without deglycosylating enzymes. We also measured plasma ANP and cGMP using radioimmunoassays., Results: All the measured BNP-related peptides were significantly higher in ESRD patients than healthy subjects. Total BNP (-38.9%), proBNP (-29.7%), glycoNT-proBNP (-45.5%), nonglycoNT-proBNP (-53.4%), ANP (-50.4%) and cGMP (-72.1%) were all significantly reduced after hemodialysis, and the magnitude of the reduction appeared molecular weight- dependent. Both the proBNP/total BNP and glycoNT-proBNP/nonglycoNT-proBNP ratios were increased after hemodialysis. The former correlated positively with hemodialysis vintage and negatively with systolic blood pressure, while the latter correlated positively with parathyroid hormone levels., Conclusion: These results suggest that hemodialysis using super-flux dialyzer removes BNP-related peptides in a nearly molecular weight-dependent manner. The ProBNP/total BNP and glycoNT-proBNP/nonglycoNT-proBNP ratios appear to be influenced by hemodialysis-related parameters in ESRD patients on hemodialysis.

Many cases of influenza are reported worldwide every year. The influenza virus often acquires new antigenicity, which is known as antigenic shift; this results in the emergence of new virus strains, for which preexisting immunity is not found in the population resulting in influenza pandemics. In the event a new strain emerges, diagnostic tools must be developed rapidly to detect the novel influenza strain. The generation of high affinity antibodies is costly and takes time; therefore, an alternative detection system, aptamer detection, provides a viable alternative to antibodies as a diagnostic tool. In this study, we developed DNA aptamers that bind to HA1 proteins of multiple influenza A virus subtypes by the SELEX procedure. To evaluate the binding properties of these aptamers using colorimetric methods, we developed a novel aptamer-based sandwich detection method employing our newly identified aptamers. This novel sandwich enzyme-linked aptamer assay successfully detected the H5N1, H1N1, and H3N2 subtypes of influenza A virus with almost equal sensitivities. These findings suggest that our aptamers are attractive candidates for use as simple and sensitive diagnostic tools that need sandwich system for detecting the influenza A virus with broad subtype specificities., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Some guanine-rich DNA sequences, which are called DNAzymes, can adopt G-quadruplex structures and exhibit peroxidase activity by binding with hemin. Although known DNAzymes show less activity than horseradish peroxidase, they have the potential to be widely used for the detection of target molecules in enzyme-linked immunosorbent assays if sequences that exhibit higher activity can be identified. However, techniques for achieving this have not yet been described. Therefore, we compared the DNAzyme activities of more than 1000 novelistically designed sequences with that of the original DNAzyme by using an electrochemical detection system on a 12K DNA microarray platform. To the best of our knowledge, this is the first description of an array-based assessment of peroxidase activity of G-quadruplex-hemin complexes. By using this novel assay system, more than 200 different mutants were found that had significantly higher activities than the original DNAzyme sequence. This microarray-based DNAzyme evaluation system is useful for identifying highly active new DNAzymes that might have potential as tools for developing DNA-based biosensors with aptamers.

Background: Recent studies have shown that in addition to brain (or B-type) natriuretic peptide (BNP) and the N-terminal proBNP fragment, levels of intact proBNP are also increased in heart failure. Moreover, present BNP immunoassays also measure proBNP, as the anti-BNP antibody cross-reacts with proBNP. It is important to know the exact levels of proBNP in heart failure, because elevation of the low-activity proBNP may be associated with the development of heart failure., Methodology/principal Findings: We therefore established a two-step immunochemiluminescent assay for total BNP (BNP+proBNP) and proBNP using monoclonal antibodies and glycosylated proBNP as a standard. The assay enables measurement of plasma total BNP and proBNP within only 7 h, without prior extraction of the plasma. The detection limit was 0.4 pmol/L for a 50-µl plasma sample. Within-run CVs ranged from 5.2%-8.0% in proBNP assay and from 7.0%-8.4% in total BNP assay, and between-run CVs ranged from 5.3-7.4% in proBNP assay and from 2.9%-9.5% in total BNP assay, respectively. The dilution curves for plasma samples showed good linearity (correlation coefficients   = 0.998-1.00), and analytical recovery was 90-101%. The mean total BNP and proBNP in plasma from 116 healthy subjects were 1.4 ± 1.2 pM and 1.0 ± 0.7 pM, respectively, and were 80 ± 129 pM and 42 ± 70 pM in 32 heart failure patients. Plasma proBNP levels significantly correlate with age in normal subjects., Conclusions/significance: Our immunochemiluminescent assay is sufficiently rapid and precise for routine determination of total BNP and proBNP in human plasma.

The text mining and full-text searching function were used to analyze the digitalized daily reports of the practical training in pharmacology submitted to the web system in Fukuyama University. Collocations connected to the word "inspection" were searched in the daily reports using the text mining and the full-text search functions of the system. Many collocation groups connected to "inspection" were found in the daily reports and the greatest number of collocations was associated with "preparation of drugs". Practical training in narcotic dispensing has two different aspects: inspection and experience training. The number of people who reported a relation between "inspection" and "narcotics dispensing" in the daily reports was very similar to those who reported a connection between "experience" and "narcotics". Practical training to handle narcotic dispensing is the most fundamental training that the pharmacist must undertake. The progression of team-based medical care has caused medical personnel to recognize the practical training in pharmacology, and the number of the people who reported a relation between "inspection" and "team medical care" in 2011 increased in comparison to 2010. Moreover, the progression of cooperation among hospitals, pharmacies, and local blanches of the Japan Pharmaceutical Association is beneficial to practical training. Practical training in pharmacology is based on the core curriculum, but undergoes periodic modification due to societal circumstances. Therefore, these results suggest that the construction of web system for submitting daily reports is useful for analyzing the daily reports.

Aptamers are functional oligonucleotides of single-stranded RNA or DNA that can selectively recognize their targets with high affinity. Hence, they have been widely developed for analytical, diagnostic, and therapeutic applications. In this review, we have summarized recent advances in the development of aptamer-based detection systems. Aptamers can be amplified exponentially by PCR, which is one of the advantages of aptamers over antibodies. Recently, we have developed immuno-aptamers that bind to mouse or rabbit IgG and constructed a novel sensitive detection system based on a conventional ELISA, called the immuno-aptamer PCR assay. In this article, the aptamer-based ready-to-use sensors and another PCR-based aptamer assays are also described; moreover, we have discussed highly sensitive aptamer-based detection systems., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The prognosis for patients with unresectable intrahepatic cholangiocarcinoma(ICC) is extremely poor. Case 1 was a 65- year-old woman who had an ICC of 9 cm in diameter (mass-forming type) in the right lobe with portal trunk invasion. She was treated with hepatic arterial infusion chemotherapy[cisplatin(CDDP)/5-fluorouracil(5-FU)/l-leucovorin(l-LV)] and radiation therapy (total dose, 50 Gy). After 6 months, abdominal computed tomography (CT) revealed that the tumor had regressed. She survived for 7 years without recurrence of the ICC; subsequently, she died of peritoneal cancer. Case 2 was a 59-year-old woman who had an ICC of 8 cm in diameter (mass-forming type) in the left lobe with lymph node metastasis in the hepatoduodenal ligament; the right hepatic artery was involved by the metastatic lymph nodes. She was treated with hepatic arterial infusion chemotherapy(CDDP/5-FU/l-LV) and radiation therapy(total dose, 30 Gy). After 10 months, abdominal CT revealed that the tumor had disappeared, but paraaortic and mediastinal lymph node metastases were detected. She was therefore treated with systemic chemotherapy. Treatment with systematic chemotherapy enabled her to survive for over 5 years with a good performance status.

Dipeptidyl peptidase III (DPP III), the zinc peptidase, has a unique helix portion in the metal-binding motif (HELLGH). The enzyme activity of the cupric derivative of rat DPP III (Cu(II)-rat DPP III) for Lys-Ala-β-NA is about 30% of that of the wild-type enzyme. On the other hand, the enzyme activity of Cu(II)-rat del-DPP III, in which Leu453 is deleted from the metal-binding motif, possesses only 1-2% of the enzyme activity of rat del-DPP III. The EPR spectra of Cu(II)-rat DPP III in the presence of various concentrations of the substrate, Lys-Ala-β-NA, changed dramatically, showing formation of the enzyme-metal-substrate complex. The EPR spectra of Cu(II)-rat del-DPP III did not change in the presence of excess Lys-Ala-β-NA. The deletion of Leu453 from the HELLGH motif of rat DPP III leads to a complete loss of flexibility in the ligand geometry around the cupric ions. Under the formation of the enzyme-metal-substrate complex, Glu451 of Cu(II)-rat DPP III is sufficiently able to approach the water molecule via a very different orientation from that of the resting state; however, Glu451 of Cu(II)-rat del-DPP III is not able to access the water molecule., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pt nanoparticle is a strong reductant and has been used as an antioxidant in cosmetics and medicine. It was reported to have catalase-like activity, which converts hydrogen peroxide to water and oxygen. However, in this study, freshly prepared Pt nanoparticle was almost inert towards decomposing hydrogen peroxide. The catalase-like activity of Pt nanoparticle increased with increasing weeks of storage at room temperature and became more significant when the Pt nanoparticle was exposed to air. No hydroxyl radical formation was confirmed by several methods such as ESR spin-trapping, dimethyl sulphoxide oxidation, salicylic acid hydroxylation and hydroxytoluene oxidation, indicating that the decomposition of hydrogen peroxide proceeds by the two-electron oxidation/reduction reaction. The oxidatively deteriorated Pt nanoparticle catalytically decomposed ascorbic acid, which is one of the most important biological antioxidants. We found that such oxidation was effectively prevented by the addition of Pd nanoparticle. We also discussed the reaction mechanisms and application of Pt nanoparticle., (© 2012 John Wiley & Sons A/S.)

Unlabelled: DNA or RNA aptamers have gained attention as the next generation antibody-like molecules for medical or diagnostic use. Conventional secondary structure prediction tools for nucleic acids play an important role to truncate or minimize sequence, or introduce limited chemical modifications without compromising or changing its binding affinity to targets in the design of improved aptamers selected by Systematic Evolution of Ligands by EXponential enrichment (SELEX). We describe a novel software package, ValFold, capable of predicting secondary structures with improved accuracy based on unique aptamer characteristics. ValFold predicts not only the canonical Watson-Crick pairs but also G-G pairs derived from G-quadruplex (known structure for many aptamers) using the stem candidate selection algorithm., Availability: The database is available for free at http://code.google.com/p/valfold/

Unlabelled: A reference database for trabecular bone density, cortical thickness, and elastic modulus of trabecular bone for a novel ultrasonic bone densitometry system (LD-100) based on two longitudinal waves (fast and slow) was determined over a wide age range in a normal Japanese population., Introduction: A novel ultrasonic bone densitometry system (LD-100 system) was applied to create a reference database for trabecular bone density (TBD), cortical thickness (CoTh), and elastic modulus of trabecular bone (EMTb) for this device over a wide age range in a normal Japanese population., Methods: In a comparative study between LD-100 and peripheral quantitative computed tomography (pQCT) systems, 52 individuals were examined by both systems at the same radius simultaneously. To create a reference database, a total of 2,380 healthy subjects (1,179 men, 1,201 women), ages 18-99 years, were examined using the LD-100 system., Results: Highly significant correlations between the LD-100 and pQCT systems were found in TBD (r = 0.877, p < 0.001) and CoTh (r = 0.723, p < 0.001). For the reference database, peak values of TBD, CoTh, and EMTb were observed at 30-34 years (255.09 mg/cm(3)), 20-24 years (5.23 mm), and 20-24 years (4.09 GPa) in men, and at 25-29 years (209.24 mg/cm(3)), 25-29 years (3.98 mm), and 20-24 years (3.33 GPa) in women, respectively. The TBD fell significantly (p < 0.05) beginning at 55-59 years in both sexes, with a relatively rapid decrease in women. The CoTh showed a significant decrease beginning at 40-44 years in men and 50-54 years in women. The EMTb showed a significant decrease beginning at 40-44 years in men and 55-59 years in women., Conclusions: The LD-100 system is a useful bone densitometry device and the database of age-related changes in TBD, CoTh, and EMTb established in this study will provide fundamental data for future studies related to bone status.

Sphingosylphosphorylcholine (SPC) is a lysosphingolipid that exerts multiple functions, including acting as a spasmogen, as a mitogenic factor for various types of cells, and sometimes as an inflammatory mediator. Currently, liquid chromatography/tandem mass spectrometry (LC/MS/MS) is used for the quantitation of SPC. However, because of the complicated procedures required it may not be cost effective, hampering its regular usage in a routine practical SPC monitoring. In this report, we have generated RNA aptamers that bind to SPC with high affinity using an in vitro selection procedure and developed an enzyme-linked aptamer assay system using the minimized SPC aptamer that can successfully distinguish SPC from the structurally related sphingosine 1-phosphate (S1P). This is the first case of the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process being performed with a lysosphingolipid. The SPC aptamers would be valuable tools for the development of aptamer-based medical diagnosis and for elucidating the biological role of SPC.

Background/purpose: Liver resection is a widely preferred treatment modality for hepatocellular carcinomas (HCCs). This study aimed to compare the survival impact of anatomical resection with that of limited resection, in patients with single HCCs no larger than 5 cm in diameter., Methods: A cohort study was carried out on 209 consecutive patients who underwent hepatic resection for a single HCC no larger than 5 cm in diameter between January 1994 and March 2007 at Osaka City General Hospital., Results: The cumulative 5-year overall survival and disease-free survival rates in the anatomical resection group (n = 111) were 71 and 40%, respectively, both of which were significantly better than the 48 and 25% seen in the limited resection group (n = 98) (P = 0.0043 and P = 0.0232, respectively). Better effects of the anatomical resection on both overall and disease-free survival were seen in patients having HCC larger than 2 cm in diameter and in patients with moderately or poorly differentiated HCC. But no significant difference in either overall or disease-free survival was seen between the groups in patients with a HCC 2 cm or less in diameter or in the patients with well-differentiated HCC. Using Cox's regression model, anatomical resection was confirmed to be an independent favorable factor for both overall and disease-free survival., Conclusions: Anatomical resection is therefore recommended for histologically advanced single HCCs ranging from 2 to 5 cm in diameter.

Nucleic acid amplification techniques were applied to the enzyme linked immunosorbent assay (ELISA) with an antibody-specific aptamer, R18. This novel detection system is a modification of the original immuno-polymerase chain reaction (immuno-PCR), but oligonucleotide-labeled antibodies are not required in the assay. This method is performed with the usual ELISA protocol, using an RNA aptamer for rabbit IgG instead of the conventional secondary antibody. After the assay plate was washed, quantitative reverse transcription (RT)-PCR was performed. Ribonuclease (RNase) inhibitors are not needed for this method. The detection limit of the quantitative RT-PCR is over 100 times more sensitive than the original ELISA method, even with the same sandwich-antibody combination. Only 1 mg of aptamer is sufficient for more than 10 million assays. This aptamer-based quantitative PCR successfully detected 16 attomoles (16 x 10(-18)) of vascular endothelial growth factor (VEGF). This is a cost-effective and easy method to increase the sensitivity of the rabbit antibody-based ELISA systems. The new method is referred to as immuno-aptamer PCR (iaPCR), to distinguish it from the original immuno-PCR.

A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF), which included pepsin at pH 2. The displayed beta-glucosidase resisted pepsin digestion compared with secreted, free beta-glucosidase. In SDS-PAGE and Western blotting analysis, the secreted beta-glucosidase was immediately digested within 1 min following SGF treatment, although the displayed beta-glucosidase was stable for more than 60 min following SGF treatment. In addition, the residual activity of secreted beta-glucosidase was completely destroyed after 10 min SGF treatment. However, displayed beta-glucosidase retained 14% of its residual activity following the same treatment. These results clearly show that cell surface display technology using enzymes can reveal the protease resistance of a protein of interest under various conditions.

Convulxin (CVX) is a C-type lectin-like protein from the venom of the South American rattlesnake that functions as a potent agonist of the platelet collagen receptor glycoprotein VI (GPVI). Although CVX is widely used as a platelet agonist, the molecular basis for its extremely high potency is not clear. In order to delineate possible mechanisms for CVX-induced GPVI activation, we used analytical ultracentrifugation to determine the assembly state of CVX in solution and surface plasmon resonance in order to understand the affinity, kinetics, and stoichiometry of GPVI binding to CVX. We show here that CVX exists in solution as a dimer of alpha4beta4 rings, yielding eight potential binding sites for GPVI. Binding studies confirm that all eight sites are able to bind GPVI tightly, each with high picomolar or low nanomolar affinity. Reanalysis of previously determined crystal structures of CVX revealed the dimer in both structures. The dimeric nature of CVX and its ability to bind eight GPVI molecules suggest that it might be capable of binding to GPVI expressed on two opposing surfaces. Agglutination assays using GPVI-coated beads confirm that CVX is able to bridge distinct GPVI-coated surfaces and indicate that CVX agglutination of platelets is dependent on GPVI binding. Thus, in addition to clustering up to eight GPVI receptors, CVX may facilitate platelet activation by bridging platelets directly.

The objective of this study is to measure muscle activity during walking in order to estimate the distance that must be traveled for safe evacuation when the effect of the flow velocity changes. In the present study, electromyogram data were measured for six muscles on the right lower extremity, namely, the rectus femoris, vastus lateralis, long head of the biceps femoris, medial gastrocnemius, tibialis anterior, and gluteus medius. The experimental conditions included walking through a flooded hallway with 0.5-m-high water flowing at four different flow velocities (0, 0.47, 0.76, and 1.12 m/s in the direction opposite to the walking direction) and walking on dry ground. From the results, the tibialis anterior exhibited the highest muscle activity of up to 85% MVC with the toe off the ground when the flow velocity was the highest, suggesting that the subject tended to avoid stumbling against water flow during ankle dorsiflexion. The activity of the gluteus medius increased with the flow velocity, suggesting that the subject tried to stabilize the pelvic area to avoid falling. Empirical data for muscle activity during walking under flooded conditions in association with water height and flow velocity will contribute to better understanding safe evacuation from flooded urban underground areas.

Hospital-acquired bacterial infections are an increasingly important cause of morbidity and mortality worldwide. Staphylococcal species are responsible for the majority of hospital-acquired infections, which are often complicated by the ability of staphylococci to grow as biofilms. Biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus requires cell-surface proteins (Aap and SasG) containing sequence repeats known as G5 domains; however, the precise role of these proteins in biofilm formation is unclear. We show here, using analytical ultracentrifugation (AUC) and circular dichroism (CD), that G5 domains from Aap are zinc (Zn(2+))-dependent adhesion modules analogous to mammalian cadherin domains. The G5 domain dimerizes in the presence of Zn(2+), incorporating 2-3 Zn(2+) ions in the dimer interface. Tandem G5 domains associate in a modular fashion, suggesting a "zinc zipper" mechanism for G5 domain-based intercellular adhesion in staphylococcal biofilms. We demonstrate, using a biofilm plate assay, that Zn(2+) chelation specifically prevents biofilm formation by S. epidermidis and methicillin-resistant S. aureus (MRSA). Furthermore, individual soluble G5 domains inhibit biofilm formation in a dose-dependent manner. Thus, the complex three-dimensional architecture of staphylococcal biofilms results from the self-association of a single type of protein domain. Surface proteins with tandem G5 domains are also found in other bacterial species, suggesting that this mechanism for intercellular adhesion in biofilms may be conserved among staphylococci and other Gram-positive bacteria. Zn(2+) chelation represents a potential therapeutic approach for combating biofilm growth in a wide range of bacterial biofilm-related infections.