A bundle that includes active surveillance, contact precaution for carriers, and cefazolin-based antimicrobial prophylaxis prevents methicillin-resistant Staphylococcus aureus infections in clean orthopedic surgery.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of orthopedic surgical site infections (SSIs). The aim of this study was to evaluate the effect of a bundle approach in the prevention of orthopedic MRSA SSIs. MATERIAL AND METHODS: MRSA active surveillance and decolonization were performed preoperatively at our institution from July 2004 until 2007. In January 2008, a bundle approach comprising contact precautions for MRSA-positive patients and cefazolin-based antimicrobial prophylaxis (AMP) stewardship was implemented. Data on the prevalence of MRSA SSIs, antimicrobial use density, duration of AMP, and the use of an alcohol antiseptic agent (L/1,000 patient-days) were evaluated during 2 periods: July 2004-December 2007 (period A) and January 2008-December 2012 (period B). RESULTS AND DISCUSSION: The MRSA SSI rate during period B (0.97%; 19 out of 1,966) was significantly lower than that during period A (2.17%; 29 out of 1,333; P = .003). The infection rate correlated negatively with both the cefazolin antimicrobial use density (r = -0.76; P = .0002) and the use of an alcohol antiseptic agent (r = -0.68; P = .002). CONCLUSIONS: An infection-prevention bundle consisting of contact precautions for carriers and AMP stewardship in addition to active surveillance was associated with a significant decrease in the incidence of orthopedic MRSA SSIs.

Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.

Lack of the consensus sequence necessary for tryptophan prenylation in the ComX pheromone precursor.

ComX, an oligopeptide pheromone that stimulates the natural genetic competence controlled by quorum sensing in Bacillus subtilis and related bacilli, contains a prenyl-modified tryptophan residue. Since ComX is the only protein known to contain prenylated tryptophan, the universality of this unique posttranslational modification has yet to be determined. Recently, we developed a cell-free assay system in which the tryptophan residue in the ComX(RO-E-2) pheromone precursor derived from B. subtilis strain RO-E-2 can be geranylated by the ComQ(RO-E-2) enzyme. We report here our attempt to identify the consensus sequence surrounding the geranylated tryptophan residue by using the cell-free system with various ComX(RO-E-2) pheromone precursor analogs. We found that [47-58]ComX(RO-E-2), corresponding to the C-terminal 12-residue peptide of the pheromone precursor, contained a short sequence essential for geranylation. We also found that the length of the sequence between the tryptophan residue and the C-terminus was important for geranylation, and that some [47-58]ComX(RO-E-2) pheromone precursor amino acids were involved in the geranylation reaction. However, we could not identify a consensus sequence surrounding the geranylated tryptophan. Our evidence suggests that, like Rab which lacks a consensus sequence yet is geranylgeranyl-modified on a cysteine residue, the ComX pheromone and its precursor also lack a consensus sequence.

Synthesis, reactivity, and spectroscopic properties of meso-triaryl-5-oxaporphyrins.

[meso-Triaryl-21,23-didehydro-23H-5-oxaporphyrinato](trifluoroacetato)zinc(II) was prepared by the reaction of meso-triarylbilindione with acetic anhydride and zinc acetate, and it was isolated as a trifluoroacetate salt. The X-ray crystallographic study demonstrated that the trifluoroacetate anion was coordinated to the zinc ion. [21,23-Didehydro-10,15,20-tris(4-methoxycarbonylphenyl)-23H-5-oxaporphyrinato](trifluoroacetato)zinc(II) 3a was dissolved in various organic solvents such as toluene, chloroform, diethyl ether, ethyl acetate, acetone, acetonitrile, methanol, DMSO, and DMF, although it readily reacted with alcohols and DMF to yield linear tetrapyrroles. The solubility of 3a in toluene was 4.2 ± 0.1 g dm(-3) at room temperature. 3a showed characteristic UV-vis absorption at 649 nm and fluorescence emission at 657 nm in chloroform. The fluorescence quantum yields of 3a, [21,23-didehydro-10,15,20-triphenyl-23H-5-oxaporphyrinato](trifluoroacetato)zinc(II) (3c), and [21,23-didehydro-10,15,20-tris(4-methoxyphenyl)-23H-5-oxaporphyrinato](trifluoroacetato)zinc(II) (3b) were 0.071, 0.071, and 0.050, respectively. Reaction of 3a with EtOH afforded the zinc complex of 19-ethoxybilinone, and it proceeded 2 orders of magnitude faster than that of [ß-octaalkyl-21,23-didehydro-23H-5-oxaporphyrinato]zinc(II). The reaction with alcohols was sensitive to steric bulk of the alcohols; the rate of reaction with i-PrOH was 2700 times faster than that of t-BuOH at 303 K. The reaction of [meso-triaryl-21,23-didehydro-23H-5-oxaporphyrinato]zinc(II) with water proceeded 3 orders of magnitude slower than that with EtOH.

Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system.

ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.

Implication of substance P neuronal system in the amygdala as a possible mechanism for hypergravity-induced motion sickness.

We previously reported that motion sickness was prevented in rats with amygdala lesion and that provocative motion stimuli increased the number of Fos-positive neurons in the amygdala, suggesting that the amygdala is one of the neural substrates involved in the development of motion sickness. NK-1 receptors in the brain stem and amygdala are thought to play an important role in emesis and affective disorders, respectively. In the present study, to elucidate a role of substance P neuronal system and NK-1 receptors in the brain stem and amygdala in the development of motion sickness, we measured changes in gene expression of NK-1 receptors and preprotachykinin, a precursor of substance P, using quantitative real-time PCR methods in solitary tract nucleus and amygdala in rats after provocative motion stimuli induced by 2G hypergravity load. Effects of systemic administration of CP-99,994, an antagonist for NK-1 receptors, on hypergravity-induced motion sickness were also examined using pica behavior, eating non-nutritive substances such as kaolin, as an index of motion sickness in rats. Hypergravity-induced motion sickness was inhibited by CP-99,994 with a dose-dependent and enantioselective manner. Preprotachykinin mRNA expression was increased in basolateral nucleus of amygdala and solitary tract nucleus after hypergravity load for 3h, whereas NK-1 receptor mRNA expression was not changed by hypergravity in amygdala and solitary tract nucleus. Present results suggest that 2G hypergravity load activated the substance P neuronal system in amygdala as well as in the brain stem and this activation would be related to the development of motion sickness.

Laparoscopic resection of epidermoid cyst arising from an intrapancreatic accessory spleen: a case report with a review of the literature.

We describe a rare case of epidermoid cyst arising in an intrapancreatic accessory spleen that presented as a cystic mass in the tail of the pancreas, and for which laparoscopic distal pancreatectomy was performed successfully. A 36-year-old woman with a cystic mass in the tail of the pancreas, which had been discovered incidentally at a medical checkup, was referred to our department for further examination. Endoscopic retrograde cholangiopancreatography, endoscopic ultrasonography and positron emission tomography demonstrated a multilocular cyst in the tail of the pancreas without any evidence of malignancy, although differential diagnosis was extremely difficult because of the neoplasm-like appearance of the lesion. Therefore, we performed laparoscopic distal pancreatectomy under a preoperative diagnosis of mucinous cystic neoplasm. Postoperative pathologic examination demonstrated an epidermoid cyst arising from a heterotopic spleen within the pancreas. This is the first report of successful laparoscopic distal pancreatectomy for an epidermoid cyst arising in an intrapancreatic accessory spleen. One virtually has no chance to diagnose an epidermoid cyst in an accessory spleen on the basis of preoperative diagnostic workup, and consequently the type of surgical resection (open vs. laparoscopic) would be conditioned by factors other than the clinical entity suspected at the preoperative period.

Analogs of the CLV3 peptide: synthesis and structure-activity relationships focused on proline residues.

CLAVATA3 (CLV3) is a plant peptide hormone in which the proline residues are post-translationally hydroxylated and glycosylated. CLV3 plays a key role in controlling the stem cell mass in the shoot meristem of Arabidopsis thaliana. In a previous report, we identified a dodecapeptide (MCLV3) from CLV3-overexpressing Arabidopsis calli; MCLV3 was the smallest functional peptide derived from the CLV3 precursor. Here, we designed a series of MCLV3 analogs in which proline residues were substituted with proline derivatives or N-substituted glycines (peptoids). Peptoid substitution at Pro9 decreased bioactivity without affecting specific binding to the CLV1-related protein in cauliflower membrane. These findings suggest that peptoid-substituted peptides would be lead compounds for developing potential agonists and antagonists of CLV3.

A novel approach for sentinel lymph node identification using fluorescence imaging and image overlay navigation surgery in patients with breast cancer.

BACKGROUND: We reported a novel technique of sentinel lymph node (SLN) identification using fluorescence imaging of indocyanine green injection. Furthermore, to obtain safe and accurate identification of SLN during surgery, we introduce the image overlay navigation surgery and evaluate its efficacy. METHODS: This study enrolled 50 patients with a tumors <2 cm in diameter. Initially, we obtained three-dimensional (3-D) imaging from multidetector-row computed tomography (MD-CT) by volume rendering. It was projected on the patient's operative field with the clear visualization of lymph node (LN) through projector. Then, the dye of indocyanine green (ICG) was injected subdermally in the areola. Subcutaneous lymphatic channels draining from the areola to the axilla were visible by fluorescence imaging immediately. Lymphatic flow was reached after LN revealed on 3-D imaging. After incising the axillary skin on the point of LN mapping, SLN was then dissected under the guidance of fluorescence imaging with adequate adjustment of sensitivity and 3-D imaging. RESULTS: Lymphatic channels and SLN were successfully identified by Photodynamic eye (PDE) in all patients. And the sites of skin incision also were identical with the LN being demonstrated by 3-D imaging in all patients. The mean number of SLN was 3.7. The image overlay navigation surgery was visually easy to identify the location of SLN from the axillary skin. There were no intra- or postoperative complications associated with SLN identification. CONCLUSIONS: This combined navigations of fluorescence and 3-D imaging revealed more easy and effective to detect SLN intraoperatively than fluorescence imaging alone.

Novel solid-phase refolding method for preparation of scFv-immobilized polystyrene plates with high-antigen-binding activity.

In the present study, we demonstrated site-specific immobilization and solid-phase refolding of single-chain Fv antibodies on hydrophilic polystyrene (phi-PS) plates that was mediated by novel polystyrene binding peptides (PS-tags: RIIIRRIRR), which were originally isolated and optimized in previous studies. Three PS-tag-fused scFvs, namely scFv-PS, scFv-(PS), and scFv-PSII, which were over-expressed in the insoluble fraction of Escherichia coli cells were denatured and site-specifically immobilized onto hydrophilic PS plates in the presence of 0.5-4 M urea and 0.1% Tween 20. The antigen-binding activity of the scFvs was efficiently recovered by washing the surface of the plate with PBS that contained 0.1% Tween 20 (PBST). The solid-phase refolding mediated by PS-tag was successfully applied to several scFvs such as mouse anti-CRP antibodies and an anti-RNase antibody, although further investigation of the versatility of scFv-PSII is needed. The maximal density of PS-tag-fused scFvs was increased more than 15-fold compared with a whole monoclonal antibody (mAb) immobilized on Maxisorp and, consequently, the sensitivity of PS-tag-fused scFvs for CRP in a sandwich ELISA was increased 25-fold. Thus, the novel, solid-phase, refolding method mediated by a PS-tag will be very useful for preparation of solid supports coated with recombinant antibody fragments, which can be used in immunoassays and immuno-separation.

Intraoperative exploration of biliary anatomy using fluorescence imaging of indocyanine green in experimental and clinical cholecystectomies.

BACKGROUND/PURPOSE: We evaluated the usefulness of intraoperative exploration of the biliary anatomy using fluorescence imaging with indocyanine green (ICG) in experimental and clinical cholecystectomies. METHODS: The experimental study was done using two 40-kg pigs and the clinical study was done in 12 patients for whom cholecystectomy was planned from January 2009 to June 2009. Initially we used a laparoscopic approach for the evaluation of fluorescence imaging of the biliary system in the two pigs. Then the clinical study was started on the basis of these experimental results. ICG (1.0 ml/body of 2.5 mg/ml ICG) was infused 1-2 h before surgery. With the subjects under general anesthesia we observed in real time the condition of the biliary tract under the guidance of fluorescence imaging employing an infrared camera or a prototype laparoscope. ICG was added intravenously to observe the location or flow condition of the cystic artery. RESULTS: We obtained a clear view of the biliary tract and the location of the cystic duct in the two pigs. Local compression with a transparent hemispherical plastic device was effective for offering a clearer view. The biliary tract, except for the gallbladder, was clearly recognized in all clinical subjects. Local compression with a transparent hemispherical plastic device for open cholecystectomy and a flat plastic device for laparoscopy provided clearer visualization of the confluence between the cystic duct and common bile duct or common hepatic duct. The location of the cystic artery was revealed after division of the connective tissues, and the flow condition of the cystic artery was confirmed 7-10 s after intravenous re-infusion of ICG. There were no adverse events related to the intraoperative procedure or the ICG itself. CONCLUSIONS: This method is safe and easy for the identification of the biliary anatomy, without requiring cannulation into the cystic duct, X-ray equipment, or the use of radioactive materials. Although fluorescence imaging is still at an early stage of application in comparison with ordinary intraoperative cholangiography, we expect that this method will become routine, offering a lower degree of invasiveness that will help avoid bile duct injury.

Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag).

Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one- and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.

Undifferentiated carcinoma of the esophagus with rapid growth of lymph node. A case report and review of the literature.

We present a case of undifferentiated carcinoma of the esophagus (UCE) treated with chemotherapy consisting of 5-fluorouracil plus nedaplatin and radiotherapy. The patient developed rapid growth of lymph nodes and died of massive hematemesis 2 months after the diagnosis. UCE is rare but highly malignant. Since there is no established treatment for UCE, its clinical outcome is invariably poor. We also reviewed the effectiveness of chemotherapy against UCE.

Progesterone receptor antagonists with a 3-phenylquinazoline-2,4-dione/2-phenylisoquinoline-1,3-dione skeleton.

Novel non-steroidal progesterone receptor antagonists with a 3-phenylquinazoline-2,4-dione/2-phenylisoquinoline-1,3-dione skeleton were developed and their structure-activity relationships were investigated. Among the prepared compounds, 4-(4,4-diethyl-3,4-dihydro-1,3-dioxoquinolin-2(1H)-yl)benzonitrile (DEPIQ-4CN) showed the most potent activity, with IC(50) values of 74-78nM in alkaline phosphatase activity and reporter gene assays.

Intraoperative identification of sentinel lymph nodes by near-infrared fluorescence imaging in patients with breast cancer.

We present a novel method for sentinel lymph node (SLN) identification by fluorescence imaging that provides a high detection rate and a low false-negativity rate. Twenty-five breast cancer patients with tumors less than 3 cm in diameter were enrolled. A combination of indocyanine green and indigo carmine was injected subdermally in the areola. Subcutaneous lymphatic channels draining from the areola to the axilla were immediately showed by fluorescence imaging. After incising the axillary skin near the point of disappearance of the fluorescence, the SLN was dissected under fluorescence guidance. In all patients, the lymphatic channels and SLN were successfully visualized. The mean number of fluorescent SLN and blue-dyed SLN were 5.5 and 2.3. Eight patients were found to have lymph node metastases pathologically. All of them were recognized by fluorescence imaging. This method is feasible and safe for intraoperative detection of SLN allowing real-time observation without any need for training.

External jugular Groshong catheter is associated with fewer complications than a subclavian Argyle catheter.

BACKGROUND: To demonstrate the efficacy and safety of insertion of a Groshong catheter via the external jugular vein (EJV) for central vein access. METHODS: Central venous access was done by either insertion of a Groshong catheter via the EJV or an Argyle catheter via the subclavian vein with single puncture. RESULTS: Eighty patients (group 1) were treated with 146 subclavian venous catheters for 2,770 catheter-days, and 98 patients (group 2) were treated with 147 external jugular venous catheters for 2,381 catheter-days. Fever appeared in 36.3% (53/146) and 16.3% (24/147) of the patients in groups 1 and 2, respectively (p < 0.01). The malposition and pneumothorax rates were 17.1% (25/146) and 2.0% (3/147; p < 0.01), and 2.7% (4/146) and 0% (0/147; p < 0.05) in the two groups, respectively. CONCLUSIONS: Insertion of a Groshong catheter via the EJV is more acceptable for central venous access than insertion of a conventional subclavian venous catheter.

Preoperative flurbiprofen for pain prevention after tonsillectomy in adults.

STUDY OBJECTIVE: To investigate the analgesic efficacy of preoperative flurbiprofen on postoperative pain after tonsillectomy. DESIGN: Prospective, randomized, nonblinded, non-placebo-controlled study. SETTING: Municipal hospital. PATIENTS: Twenty-five ASA physical status I patients older than 20 years of age, who were scheduled for tonsillectomy. INTERVENTIONS: Patients were randomly allocated to two groups to receive preoperative intravenous (IV) 50 mg flurbiprofen (group F) or not (group C). Anesthesia was induced with IV propofol two mg/kg and maintained with nitrous oxide and sevoflurane. MEASUREMENTS: Pain scores at rest and at swallowing, intraoperative bleeding, vital signs during the postanesthetic period, interval until diclofenac sodium suppository rescue, and the total dose required for 12 hours postoperatively were all recorded. MAIN RESULTS: Pain scores at rest as well as those recorded after swallowing 30 minutes after tonsillectomy were significantly lower in group F than in group C. During the first postoperative 1.5 hours, significantly fewer patients in group F required rescue diclofenac suppository than did group C patients. However, total dose of required rescue during the postoperative 12 hours in group F did not significantly differ from that of group C. There were no significant differences in intraoperative bleeding or in any vital signs during the postanesthetic period either. CONCLUSION: Preoperative flurbiprofen suppressed immediate postoperative pain after tonsillectomy. The analgesic effect, however, disappeared in a few hours and was insufficient for overnight pain relief.

Improvement of survival of skin flaps by combined gene transfer of hepatocyte growth factor and prostacyclin synthase.

BACKGROUND: Increasing the local blood flow is a critical factor for long-term survival of skin flaps. Thus, a molecular therapy to increase the blood flow by means of an angiogenic factor is considered to be a useful strategy to improve skin flap survival. We focused on a combined strategy to stimulate not only angiogenesis, but also vasodilation of local microvessels, using co-transfection of the hepatocyte growth factor (HGF) and prostacyclin synthase (PGIS) genes to enhance the survival of random-pattern skin flaps. METHODS AND RESULTS: A 2 x 8 cm full thickness cranial pedicled random-pattern flap was made on the back of each 12-week-old male rat. At 3 days before operation, 400 microg of human HGF and PGIS naked plasmid DNA or control plasmid was transfected into the flaps by needle-less injection using a Shima Jet, resulting in successful expression of human HGF and PGIS in the skin flaps. Transfection of both genes into the distal half of skin flaps at 3 days prior to operation significantly increased the survival rate of skin flaps, while transfection all over the flaps did not. In addition, transfection prior to operation was more effective than simultaneous treatment. Moreover, co-transfection of these genes improved the survival area of skin flaps, accompanied by an increase in blood flow of skin flaps, even in a diabetic model. CONCLUSIONS: Overall, these results indicate that combination treatment with HGF and PGIS genes by Shima Jet could be an effective strategy to improve skin flap survival.