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In this double-blind two period crossover study, we investigated the effect of indomethacin and sulindac on blood pressure in 25 hypertensive patients being treated with hydrochlorothiazide. The study consisted of seven 4 week periods. In the first and the last period the patients took placebos, in period two, four and six they were treated with hydrochlorothiazide 50 mg once daily alone, and in the third and fifth period hydrochlorothiazide 50 mg once daily was given in combination with either indomethacin 50 mg twice daily or sulindac 200 mg twice daily in double-blind random order. Blood pressure, measured by sphygmomanometer and arteriosonde, and body weight were determined every 2 weeks. Compared with placebo hydrochlorothiazide decreased the mean arterial pressure by 8%. Addition of both indomethacin and sulindac resulted in only slight and generally similar changes of this blood pressure lowering effect. This was found both for the whole group and when both treatment sequence groups were analysed separately. In contrast to sulindac, indomethacin attenuated the hydrochlorothiazide- induced decreases of body weight, plasma potassium and the increase of plasma renin activity. Both non-steroidal anti-inflammatory drugs (NSAID) reduced the 24 h urinary excretion of prostaglandins (PGs), i.e. PGF2 alpha, 6 ketoPGF1 alpha and thromboxane B2 except PGE2. From this study it can be concluded that, in contrast to sulindac, indomethacin attenuated the diuretic action of hydrochlorothiazide, however apparently without consequences for its long-term blood pressure lowering effect. This study does not support the hypothesis that the difference between the two NSAIDs can be explained by different effects on renal PG synthesis. [ABSTRACT FROM AUTHOR]

Fontan circulation is a highly abnormal circulatory state that may affect various organ systems. The effect on body composition is an important factor to assess the condition of the patient. This systematic review assesses body composition and possibly related adverse outcomes in patients with a Fontan circulation, to provide an overview of current insights. Studies evaluating body composition by compartment (either fat mass or lean/muscle mass) in Fontan patients published up to April 2023 were included in this systematic review. Of 1392 potential studies, 18 studies met the inclusion criteria. In total, body composition measurements of 774 Fontan patients were included. Body composition was measured using dual-energy X-ray absorptiometry (DXA) (n = 12), bioelectrical impedance analysis (BIA) (n = 5), computer tomography (CT) (n = 1), or magnetic resonance imaging (MRI) (n = 1). All studies reported a normal body mass index (BMI) in Fontan patients, compared to controls. Five out of nine studies reported significantly higher body fat values, and twelve out of fifteen studies reported significantly lower muscle or lean mass values in the Fontan population compared to the healthy population. Unfavorable body composition in Fontan patients was associated with decreased exercise capacity, worse cardiac function, and adverse outcomes including hospital admissions and death.   Conclusions: Despite having a normal BMI, Fontan patients have an increased fat mass and decreased muscle mass or lean mass compared to the healthy population. This unfavorable body composition was associated with various adverse outcomes, including a decreased exercise capacity and worse cardiac function. What is Known: • Patients with a Fontan circulation have a decreased exercise capacity compared to healthy peers, an unfavorable body composition might be a contributor to their impaired exercise capacity. What is New: • Fontan patients are predisposed to an unfavorable body composition, characterized by increased fat mass and decreased muscle mass accompanied by a normal BMI compared to the healthy population. • Among others, unfavorable body composition was associated with decreased exercise capacity, cardiac function, and increased morbidity in patients with a Fontan circulation., (© 2023. The Author(s).)

Endobronchial metastasis occurs in only 2%-5% of non-pulmonary cancers. Here we report on an 84-year-old woman who presented with breathlessness and light-headedness while on holiday in Australia, 2 years post-treatment for endometrial cancer. Initial CT pulmonary angiogram identified a soft tissue mass in the left hemithorax. A chest radiograph performed after repatriation was consistent with a large left pleural effusion, but bedside ultrasound showed a lobulated mass involving the left hemidiaphragm. A pleural procedure in the traditional 'triangle of safety' would have resulted in inadvertent puncture of the underlying mass. Serial imaging confirmed the mass was rapidly progressing, and metastatic malignant mixed Mullerian endometrial carcinoma was diagnosed by endobronchial biopsy. A tunnelled intrapleural catheter was inserted for symptom relief, and the patient deteriorated and died at home 2 weeks later. To our knowledge, this is the first case of endobronchial metastasis from malignant mixed Mullerian tumour of the uterus., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Triazole ureas constitute a versatile class of irreversible inhibitors that target serine hydrolases in both cells and animal models. We have previously reported that triazole ureas can act as selective and CNS-active inhibitors for diacylglycerol lipases (DAGLs), enzymes responsible for the biosynthesis of 2-arachidonoylglycerol (2-AG) that activates cannabinoid CB 1 receptor. Here, we report the enantio- and diastereoselective synthesis and structure-activity relationship studies. We found that 2,4-substituted triazole ureas with a biphenylmethanol group provided the most optimal scaffold. Introduction of a chiral ether substituent on the 5-position of the piperidine ring provided ultrapotent inhibitor 38 (DH376) with picomolar activity. Compound 38 temporarily reduces fasting-induced refeeding of mice, thereby emulating the effect of cannabinoid CB 1 -receptor inverse agonists. This was mirrored by 39 (DO34) but also by the negative control compound 40 (DO53) (which does not inhibit DAGL), which indicates the triazole ureas may affect the energy balance in mice through multiple molecular targets.

Background: Aging has been associated with increasing common bile duct (CBD) diameter and reported as independently predictive of the likelihood of choledocolithiasis. These associations are controversial with uncertain diagnostic utility in patients presenting with symptomatic disease. The current study examined the relationship between age, CBD size, and the diagnostic probability of choledocolithiasis., Methods: Symptomatic patients undergoing evaluation for suspected choledocolithiasis from January 2008 to February 2011 were reviewed. In the cohort without choledocolithiasis, the relationship between aging and CBD size was examined as a continuous variable and by comparing mean CBD size across stratified age groups. Multivariate analysis examined the relationship between increasing age and diagnostic probability of choledocolithiasis in all patients., Results: Choledocolithasis was diagnosed by MR cholangiopancreatography (MRCP) or endoscopic retrograde (ERCP) in 496 of 1,000 patients reviewed. Mean CBD was 6.0 mm (±2.8 mm) in the 504 of 1,000 patients without choledocolithiasis on ERCP/MRCP. Increasing age had no correlation with CBD size as a continuous variable (r2 = 0.011, p = 0.811). No difference occurred across age groups (Kruskal-Wallis, p = 0.157). Age had no association with diagnostic likelihood of choledocolithiasis (AOR [95% CI] 0.99 [0.98-1.01], adjusted-p = 0.335)., Conclusion: In a large population undergoing investigation for biliary disease, increasing age was neither associated with increasing CBD diameter nor predictive of the likelihood of choledocolithiasis., (© 2017 S. Karger AG, Basel.)

Glucosylceramide metabolism and the enzymes involved have attracted significant interest in medicinal chemistry, because aberrations in the levels of glycolipids that are derived from glucosylceramide are causative in a range of human diseases including lysosomal storage disorders, type 2 diabetes, and neurodegenerative diseases. Selective modulation of one of the glycoprocessing enzymes involved in glucosylceramide metabolism-glucosylceramide synthase (GCS), acid glucosylceramidase (GBA1), or neutral glucosylceramidase (GBA2)-is therefore an attractive research objective. In this study we took two established GCS inhibitors, one based on deoxynojirimycin and the other a ceramide analogue, and merged characteristic features to obtain hybrid compounds. The resulting 39-compound library does not contain new GCS inhibitors; however, a potent (200 nm) GBA1 inhibitor was identified that has little activity toward GBA2 and might therefore serve as a lead for further biomedical development as a selective GBA1 modulator., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this paper, a new synthetic route toward 6-hydroxysphingosine and α-hydroxy ceramide is described. The synthesis employs a cross-metathesis to unite a sphingosine head allylic alcohol with a long-chain fatty acid alkene that also bears an allylic alcohol group. To allow for a productive CM coupling, the sphingosine head allylic alcohol was protected with a cyclic carbonate moiety and a reactive CM catalyst system, consisting of Grubbs II catalyst and CuI, was employed.

Background: Increasing ambient temperature to prevent intraoperative patient hypothermia remains widely advocated despite unconvincing evidence of efficacy. Heat stress is associated with decreased cognitive and psychomotor performance across multiple tasks but remains unexamined in an operative context. We assessed the impact of increased ambient temperature on laparoscopic operative performance and surgeon cognitive stress., Study Design: Forty-two performance measures were obtained from 21 surgery trainees participating in the counter-balanced, within-subjects study protocol. Operative performance was evaluated with adaptations of the validated, peg-transfer, and intracorporeal knot-tying tasks from the Fundamentals of Laparoscopic Surgery program. Participants trained to proficiency before enrollment. Task performance was measured at two ambient temperatures, 19 and 26°C (66 and 79°F). Participants were randomly counterbalanced to initial hot or cold exposure before crossing over to the alternate environment. Cognitive stress was measured using the validated Surgical Task Load Index (SURG-TLX)., Results: No differences in performance of the peg-transfer and intracorporeal knot-tying tasks were seen across ambient conditions. Assessed via use of the six bipolar scales of the SURG-TLX, we found differences in task workload between the hot and cold conditions in the areas of physical demands (hot 10 [3-12], cold 5 [2.5-9], P = .013) and distractions (hot 8 [3.5-15.5], cold 3 [1.5-5.5], P = .001). Participant perception of distraction remained greater in the hot condition on full scoring of the SURG-TLX., Conclusion: Increasing ambient temperature to levels advocated for prevention of intraoperative hypothermia does not greatly decrease technical performance in short operative tasks. Surgeons, however, do report increased perceptions of distraction and physical demand. The impact of these findings on performance and outcomes during longer operative procedures remains unclear., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (D-gluco and L-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted L-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their D-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics.

Background: Thoracoabdominal firearm injuries present major diagnostic and therapeutic challenges because of the risk for potential injury in multiple anatomic cavities and the attendant dilemma of determining the need for and correct sequencing of cavitary intervention. Injury patterns, management strategies, and outcomes of thoracoabdominal firearm trauma remain undescribed across a large population., Methods: All patients with thoracoabdominal firearm injury admitted to a major Level I trauma center during a 16-year period were reviewed., Results: The 984 study patients experienced severe injury burden; 25% (243 of 984) presented in cardiac arrest, and 75% (741 of 984) had an Abbreviated Injury Scale (AIS) score of 3 or greater in both the chest and the abdomen. Operative management occurred in 86% (638 of 741). Of the patients arriving alive, 68% (507 of 741) underwent laparotomy alone, 4% (27 of 741) underwent thoracotomy alone, and 14% (104 of 741) underwent dual-cavitary intervention. Negative laparotomy occurred in 3%. Diaphragmatic injury (DI) occurred in 63%. Seventy-five percent had either DI or hollow viscus injury. Cardiac injury was present in 33 patients arriving alive. Despite the use of trauma bay ultrasound, 44% of the patients with cardiac injury underwent initial laparotomy. In half of this group, ultrasound did not detect pericardial blood. The need for thoracotomy, either alone or as part of dual-cavitary intervention, was the strongest independent risk factor for mortality in those arriving alive., Conclusion: Greater kinetic destructive potential drives the peril of thoracoabdominal firearm trauma, producing clinical challenges qualitatively and quantitatively different from nonfirearm injuries. Severe injury, on both sides of the diaphragm, generates high operative need with low rates of negative exploration. The need for emergent intervention and a high incidence of DI or hollow viscus injury limit opportunity for nonoperative management. Even with ultrasound, emergent preoperative diagnosis remains challenging, as the complex combination of intra-abdominal, thoracic, and diaphragmatic injuries can provoke misinterpretation of both radiologic and clinical data. Successful emergent management requires thorough assessment of all anatomic spaces, integrating ultrasonographic, radiologic, and clinical findings., Level of Evidence: Epidemiologic study, level III.

Introduction: Selective non-operative management (NOM) is standard of care for clinically stable patients with blunt splenic trauma and expectant management approaches are increasingly utilised in penetrating abdominal trauma, including in the setting of solid organ injury. Despite this evolution of clinical practice, little is known about the safety and efficacy of NOM in penetrating splenic injury., Methods: Trauma registry and medical record review identified all consecutive patients presenting to LAC+USC Medical Center with penetrating splenic injury between January 2001 and December 2011. Associated injuries, incidence and nature of operative intervention, local and systemic complications and mortality were determined., Results: During the study period, 225 patients experienced penetrating splenic trauma. The majority (187/225, 83%) underwent emergent laparotomy. Thirty-eight clinically stable patients underwent a deliberate trial of NOM and 24/38 (63%) were ultimately managed without laparotomy. Amongst patients failing NOM, 3/14 (21%) underwent splenectomy while an additional 6/14 (42%) had splenorrhaphy. Hollow viscus injury (HVI) occurred in 21% of all patients failing NOM. Forty percent of all NOM patients had diaphragmatic injury (DI). All patients undergoing delayed laparotomy for HVI or a splenic procedure presented symptomatically within 24h of the initial injury. No deaths occurred in patients undergoing NOM., Conclusions: Although the vast majority of penetrating splenic trauma requires urgent operative management, a group of patients does present without haemodynamic instability, peritonitis or radiologic evidence of hollow viscus injury. Management of these patients is complicated as over half may remain clinically stable and can avoid laparotomy, making them potential candidates for a trial of NOM. HVI is responsible for NOM failure in up to a fifth of these cases and typically presents within 24h of injury. Delayed laparotomy, within this limited time period, did not appear to increase mortality nor preclude successful splenic salvage. In clinically stable patients, diagnostic laparoscopy remains essential to evaluate and repair occult DI. As NOM for penetrating abdominal trauma becomes more common, multi-centre data is needed to more accurately define the principles of patient selection and the limitations and consequences of this approach in the setting of splenic injury., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Penetrating thoracoabdominal trauma, with potential injury to two anatomic cavities, significantly challenges surgical management, yet this injury pattern has not been reviewed across a large patient series., Methods: The trauma registry of a major level 1 center was queried for all adult patients admitted with thoracoabdominal stab wounds between January 1996 and December 2011., Results: The study identified 617 patients; 11% arrived hypotensive (systolic blood pressure < 90 mm Hg), 6.5% had Glasgow Coma Scale (GCS) score less than 8, and 3.6% were in cardiac arrest. Of those arriving alive, 350 (59%) of 595 underwent surgery (88% laparotomy, 3% thoracotomy, and 9% both procedures). Nontherapeutic laparotomy was performed on 12.3% of these patients. Cardiac injury occurred in 71% (29 of 41) of the patients arriving alive undergoing thoracotomy. Among this group, only 1 (2.4%) of 41 had a major thoracic vessel or aortic injury without cardiac trauma. Diaphragmatic injury (DI) occurred in 224 (38%) of 595, with 72 (32.1%) of these 224 demonstrating no computed tomographic evidence of DI. Either hollow viscus injury or DI occurred in 50%. Only 36.8% of liver, 58% of spleen, and 29.8% of kidney injuries required surgical repair. The need for dual-cavitary intervention was associated with a precipitous increase in patient mortality., Conclusion: Patients with thoracoabdominal stab wounds present considerable clinical challenges due to high surgical need, high occult DI incidence, persistently high rates of negative laparotomy, and significant mortality with dual-cavitary intervention. Many patients with solid-organ injuries do not require intervention. High incidence of hollow viscus injury and DI ultimately limits nonoperative management. Laparoscopy is necessary to exclude occult DI. In unstable patients, determination of which anatomic cavity to explore primarily requires exclusion of cardiac injury. In those with equivocal clinical or ultrasonographic evidence of cardiac trauma, laparotomy, with transdiaphragmatic pericardial window, if a causative abdominal injury is not immediately apparent, seems the most effective strategy., Level of Evidence: Epidemiologic study, level III.

Background: We evaluated the potential for QT/corrected QT (QTc) interval prolongation after sugammadex given with propofol or sevoflurane anaesthesia., Methods: This was a two-factorial, randomized, parallel-group study in 132 healthy subjects. Anaesthesia was maintained with sevoflurane or propofol. At ~20 min following sevoflurane/propofol initiation, sugammadex 4 mg/kg or placebo was administered. Neuromuscular blocking agents were not administered. Electrocardiograms were recorded regularly. The primary variable was the time-matched mean difference in the Fridericia-corrected QT interval (QTcF) change from baseline for sugammadex versus placebo when combined with propofol or sevoflurane. No relevant QTcF prolongation was concluded if the upper one-sided 95 % confidence interval (CI) was below the 10 ms margin of regulatory non-inferiority, up to 30 min post-study drug. Blood samples were taken for pharmacokinetic analysis. An exploratory analysis evaluated potential QT/QTc effects of neostigmine 50 μg/kg/glycopyrrolate 10 μg/kg in combination with propofol., Results: The estimated mean QTcF differences between sugammadex and placebo ranged from -2.4 to 0.6 ms when combined with either anaesthetic. The largest upper one-sided 95 % CI for the mean QTcF difference between sugammadex and placebo was 2 ms, occurring 2 min post-dosing. Propofol and sevoflurane resulted in mean QTcF increases exceeding 10 and 30 ms, respectively. On top of these prolongations, the effect of sugammadex was negligible at all timepoints. The mean peak sugammadex concentration was 66.5 μg/mL, with exposure similar in the sevoflurane/propofol groups. The mean QTcF changes from baseline following neostigmine/glycopyrrolate in 10 healthy subjects ranged between -1.4 and 3.6 ms., Conclusion: Sugammadex 4 mg/kg does not cause clinically relevant QTc interval prolongation versus placebo when combined with propofol or sevoflurane.

Background: In asymptomatic patients with penetrating thoracic trauma and a normal initial chest x-ray, successive prospective trials have decreased the minimum observation period required for exclusion of significant injury from 6 to 3 h. Despite the quality of these studies, this interval remains arbitrary and the true requisite observation time for safe discharge remains unknown. The current study evaluates the ability of "early" repeat chest x-ray, at intervals approaching 1 h, to exclude clinically significant injury., Methods: Eighty-eight, asymptomatic patients with penetrating chest trauma and normal initial chest radiographs were prospectively enrolled in this study. All patients received an "early" follow-up chest x-ray, at a median interval of 1 h and 34 min (interquartile range: 1 h 35 min to 2 h 22 min), and a second repeat x-ray at a "delayed" interval no earlier than 3 h postadmission. Radiographic abnormalities in clinically stable patients were followed with serial examination and repeat imaging for a minimum of 6 h. All patients received both "early" and "delayed" repeat CXRs with no patient discharged before full assessment., Results: One of the 88 patients with initially normal chest x-ray underwent tube thoracostomy at the discretion of the attending surgeon before any repeat imaging. Of the remaining patients, 4 of 87 (4.6 %) demonstrated radiographic abnormalities on "early" repeat imaging. Two patients had pneumothoraces, successfully managed without intervention; the remaining two demonstrated evidence of hemothorax, subsequently undergoing tube thoracostomy. Two more patients (2.3 %) developed pneumothoraces on "delayed" imaging, both successfully observed without intervention., Conclusions: In asymptomatic patients with penetrating thoracic trauma and normal initial chest radiographs, "early" repeat chest x-ray, at intervals approaching 1 h, appears sufficient to exclude clinically significant pathology and to allow safe patient discharge.

Aim: Conjugation to antithrombin III ATIII-binding pentasaccharides has been proposed as a novel method to extend the half-life of therapeutic proteins. We aim to validate this technological concept in man by performing a first-in-human study using CarboCarrier® insulin (SCH 900948) as an example. A rising single dose phase 1 study was performed assessing safety, tolerability, pharmacokinetics and relative bioactivity of CarboCarrier® insulin. Safety, tolerability and pharmacokinetics (PK) of single doses of CarboCarrier® insulin in healthy volunteers were explored, and the dose-response relationship and relative bioactivity of CarboCarrier® insulin in subjects with type 2 diabetes were investigated., Methods: After an overnight fast, subjects were randomized to a treatment sequence. PK and pharmacodynamic (glucose, insulin and C-peptide) samples were obtained for up to 72 h post-dose. Effects of CarboCarrier® insulin were compared with those of NPH-insulin., Results: CarboCarrier® insulin was safe and well-tolerated and no consistent pattern of adverse events occurred. CarboCarrier® insulin exposure (Cmax and AUC) increased proportionally with dose. The mean terminal elimination half-life ranged between 3.11 and 5.28 h. All CarboCarrier® insulin dose groups showed decreases in the mean change from baseline of plasma glucose concentrations compared with the placebo group., Conclusions: CarboCarrier® insulin is pharmacologically active showing features of insulin action in man. The elimination half-life of the molecule was clearly extended compared with endogenous insulin, indicating that conjugation to ATIII-binding pentasaccharides is a viable approach to extend the half-life of therapeutic proteins in humans. This is an important step towards validation of the CarboCarrier® technology by making use of CarboCarrier® insulin as an example., (© 2012 MSD Oss B.V., a subsidiary of Merck & Co., Inc., Whitehouse Station, N.J., U.S.A. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.)

Magnetotactic bacteria are a morphologically, metabolically and phylogenetically disparate array of bacteria united by the ability to biomineralize membrane-encased, single-magnetic-domain mineral crystals (magnetosomes) that cause the cell to orientate along the Earth's geomagnetic field. The most commonly observed type of magnetotactic bacteria is the ubiquitous magnetotactic cocci, which comprise their own phylogenetic group. Strain MC-1(T), a member of this group, was isolated from water collected from the oxic-anoxic interface of the Pettaquamscutt Estuary in Rhode Island, USA, and cultivated in axenic culture. Cells of strain MC-1(T) are roughly spherical, with two sheathed bundles of flagella at a single pole (bilophotrichous). Strain MC-1(T) uses polar magnetotaxis, and has a single chain of magnetite crystals per cell. Cells grow chemolithoautotrophically with thiosulfate or sulfide as the electron donors, and chemo-organoheterotrophically on acetate. During autotrophic growth, strain MC-1(T) relies on the reductive tricarboxylic acid cycle for CO2 fixation. The DNA G+C content is 54.2 mol%. The new genus and species Magnetococcus marinus gen. nov., sp. nov. are proposed to accommodate strain MC-1(T) ( = ATCC BAA-1437(T)  = JCM 17883(T)), which is nominated as the type strain of Magnetococcus marinus. A new order (Magnetococcales ord. nov.) and family (Magnetococcaceae fam. nov.) are proposed for the reception of Magnetococcus and related magnetotactic cocci, which are provisionally included in the Alphaproteobacteria as the most basal known lineage of this class.

Clostridium difficile is known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Toxinogenic strains of the bacterium produce toxins A (TcdA) and B (TcdB), which are associated with the pathogenicity. The standard methods for diagnosis of C. difficile infection include the cell cytotoxicity assay and the culture of a toxinogenic strain. Due to the long turnaround time of these methods, more rapid methods are preferred. Enzyme immunoassays are fast, but lack sensitivity. Therefore, real-time PCR methods have been developed. The real-time PCR described in this chapter detects tcdB, the gene coding for toxin B. Since toxin A-negative, toxin B-positive strains have been reported to cause disease as well, these strains can also be detected by this method which uses an automated STAR-MagnaPure method for the optimum isolation of DNA from feces. An internal control is included as well to control for inhibition of the PCR method.

As a group, the extremities are the most commonly injured anatomic region in nonfatal firearm trauma and are associated with high rates of vascular and bony injury. This study examines the epidemiology, incidence and distribution of firearm-related extremity trauma and the relationship between injury pattern and local or systemic complications. Review of the National Trauma Databank identified 6987 patients with isolated extremity firearm injury. Epidemiologic data, injury pattern incidence, and local and systemic complications were reviewed. Multivariate analysis identified the impact of extremity injury pattern on complications. Overall fracture incidence was 22 per cent. Fracture was associated with both vascular (odds ratio [OR], 1.9; 95% confidence interval [CI], 1.5 to 2.4; P < 0.001) and nerve injury (OR, 2.6; 95% CI, 1.9 to 3.5; P < 0.001). Isolated fracture increased risk of compartment syndrome (OR, 2.4; 95% CI, 1.1 to 5.3; P = 0.035). Vascular injury alone increased the risk of compartment syndrome (OR, 11.5; 95% CI, 5.0 to 26.2; P < 0.001) and deep venous thrombosis (OR, 7.9; 95% CI, 2.5 to 25.2; P < 0.001). Fracture and vascular injury together also increased risk of wound infection (OR, 9.7; 95% CI, 3.9 to 23.4; P < 0.001). In patients with extremity trauma, the injury pattern significantly impacts local but not systemic complication rates. Gunshot-related fracture, occurring in one-fifth of patients, increases the risk of vascular and nerve injury. Vascular injury, with or without fracture, is the biggest predictor of local complications.

The feasibility of isotachophoresis in channels of sub micrometer and nanometer dimension is investigated. A sample injection volume of 0.4 pL is focused and separated in a 330 nm deep channel. The sample consists of a biomatrix containing the fluorescently-labeled amino acids glutamate and phenylalanine, 20 attomoles of each. Isotachophoretic focusing is successfully demonstrated in a 50 nm deep channel. Separation of the two amino acids in the 50 nm deep channel however, could not be performed as the maximum applicable voltage was insufficient. This limit is imposed by bubble formation that we contribute to cavitation as a result of the mismatch in electro-osmotic flow, so called electrocavitation. This represents an unexpected limit on the miniaturization of ITP. Nonetheless, we report the smallest isotachophoretic separation and focusing experiment to date, both in terms of controlled sample injection volume and channel height.

Evaluation of patients after penetrating neck injury has evolved over time. Previously, location of injury and symptoms were used to determine management. The contemporary management of penetrating neck injuries relies on physical examination. Patients with hard signs of vascular or aerodigestive tract injury require immediate operation, regardless of location of injury. Those with no signs can be observed. For the remainder with soft signs, multidetector computed tomographic angiography (MDCTA) is a highly sensitive and specific screening modality for evaluating the vasculature and aerodigestive structures in the neck. Utilizing MDCTA, the patient can be safely directed towards operative intervention, observation, or further investigation.

Objectives: To examine the specific injuries, need for operative intervention, and clinical outcomes of patients with blunt thoracoabdominal trauma., Design: Trauma registry and medical record review., Setting: Level I trauma center in Los Angeles, California., Patients: All patients with thoracoabdominal injuries from January 1996 to December 2010., Main Outcome Measures: Injuries, incidence and type of operative intervention, clinical outcomes, and risk factors for mortality., Results: Blunt thoracoabdominal injury occurred in 1661 patients. Overall, 474 (28.5%) required laparotomy, 31 (1.9%) required thoracotomy (excluding resuscitative thoracotomy), and 1146 (69.0%) required no thoracic or abdominal operation. Overall incidence of intraabdominal solid organ injury was 59.7% and hollow viscus injury, 6.0%. Blunt cardiac trauma occurred in 6.3%; major thoracic vessel injury, in 4.6%; and diaphragmatic trauma, in 6.0%. The majority of solid organ injuries were managed nonoperatively (liver, 83.9%; spleen, 68.3%; and kidney, 91.2%). Excluding patients with severe head trauma, mortality ranged from 4.5% with nonoperative management to 18.1% and 66.7% in those requiring laparotomy and dual cavitary exploration, respectively. Age 55 years or older, Injury Severity Score of 25 or more, Glasgow Coma Scale score of 8 or less, initial hypotension, massive transfusion, and liver, cardiac, or abdominal vascular trauma were all independent risk factors for mortality., Conclusions: Most patients with blunt thoracoabdominal trauma are managed nonoperatively. The need for non-resuscitative thoracotomy or combined thoracoabdominal operation is rare. The abdomen contains the overwhelming majority of injuries requiring operative intervention and should be the initial cavity of exploration in the patient requiring emergent surgery without directive radiologic data.

Background and Purpose: Graves' disease (GD) is an autoimmune disease in which the thyroid is overactive, producing excessive amounts of thyroid hormones, caused by thyroid-stimulating hormone (TSH) receptor-stimulating immunoglobulins (TSIs). Many GD patients also suffer from thyroid eye disease (Graves' ophthalmopathy or GO), as TSIs also activate TSH receptors in orbital tissue. We recently developed low molecular weight (LMW) TSH receptor antagonists as a novel therapeutic strategy for the treatment of GD and GO. Here, we determined the molecular pharmacology of a prototypic, nanomolar potent LMW TSH receptor antagonist, Org 274179-0., Experimental Approach: Using CHO cells heterogeneously expressing human TSH receptors and rat FRTL-5 cells endogenously expressing rat TSH receptors, we determined the potency and efficacy of Org 274179-0 at antagonizing TSH- and TSI-induced TSH receptor signalling and its cross-reactivity at related follicle-stimulating hormone and luteinizing hormone receptors. We analysed the allosteric mode of interaction of Org 274179-0 and determined whether it is an inverse agonist at five naturally occurring, constitutively active TSH receptor mutants., Key Results: Nanomolar concentrations of Org 274179-0 completely inhibited TSH (and TSI)-mediated TSH receptor activation with little effect on the potency of TSH, in accordance with an allosteric mechanism of action. Conversely, increasing levels of TSH receptor stimulation only marginally reduced the antagonist potency of Org 274179-0. Org 274179-0 fully blocked the increased basal activity of all the constitutively active TSH receptor mutants tested with nanomolar potencies., Conclusions and Implications: Nanomolar potent TSH receptor antagonists like Org 274179-0 have therapeutic potential for the treatment of GD and GO., (© 2011 Merck Sharp & Dohme Corp. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Non-destructive techniques characterising the mechanical properties of cells, tissues, and biomaterials provide baseline metrics for tissue engineering design. Ultrasonic wave propagation and attenuation has previously demonstrated the dynamics of extracellular matrix synthesis in chondrocyte-seeded hydrogel constructs. In this paper, we describe an ultrasonic method to analyse two of the construct elements used to engineer articular cartilage in real-time, native cartilage explants and an agarose biomaterial. Results indicated a similarity in wave propagation velocity ranges for both longitudinal (1500-1745 m/s) and transverse (350-950 m/s) waveforms. Future work will apply an acoustoelastic analysis to distinguish between the fluid and solid properties including the cell and matrix biokinetics as a validation of previous mathematical models.

Glucosylceramide synthase (GCS) is an approved drug target for the treatment of Gaucher disease and is considered as a valid target for combating other human pathologies, including type 2 diabetes. The clinical drug N-butyldeoxynojirimycin (Zavesca) is thought to inhibit through mimicry of its substrate, ceramide. In this work we demonstrate that, in contrast to what is proposed in this model, the C2-hydroxyl of the deoxynojirimycin core is important for GCS inhibition. Here we show that C6-OH appears of less important, which may set guidelines for the development of GCS inhibitors that have less affinity (in comparison with Zavesca) for other glycoprocessing enzymes, in particular those hydrolases that act on glucosylceramide.

Neurotransmission requires Ca(2+)-dependent release of secretory products through fusion pores that open and reclose (partial membrane distention) or open irreversibly (complete membrane distention). It has been challenging to distinguish between these release modes; however, in the work presented here, we were able to deduce different modes of depolarization-evoked exocytosis in neuroendocrine chromaffin and PC12 cells solely by analyzing amperometric recordings. After we determined the quantal size (Q), event half-width (t(50)), event amplitude (I(peak)), and event decay time constant (τ(decay)), we fitted scatter plots of log-transformed data with a mixture of one- and two-dimensional Gaussian distributions. Our analysis revealed three distinct and differently shaped clusters of secretory events, likely corresponding to different modes of exocytosis. Complete membrane distention, through fusion pores of widely varying conductances, accounted for 70% of the total amount of released catecholamine. Two different kinds of partial membrane distention (kiss-and-run and kiss-and-stay exocytosis), characterized by mode-specific fusion pores with unitary conductances, accounted for 20% and 10%, respectively. These results show that our novel one- and two-dimensional analysis of amperometric data reveals new release properties and enables one to distinguish at least three different modes of exocytosis solely by analyzing amperometric recordings., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Glucosylceramide synthase (GCS) is an important target for clinical drug development for the treatment of lysosomal storage disorders and a promising target for combating type 2 diabetes. Iminosugars are useful leads for the development of GCS inhibitors; however, the effective iminosugar type GCS inhibitors reported have some unwanted cross-reactivity toward other glyco-processing enzymes. In particular, iminosugar type GCS inhibitors often also inhibit to some extent human acid glucosylceramidase (GBA1) and the nonlysosomal glucosylceramidase (GBA2), the two enzymes known to process glucosylceramide. Of these, GBA1 itself is a potential drug target for the treatment of the lysosomal storage disorder, Gaucher disease, and selective GBA1 inhibitors are sought after as potential chemical chaperones. The physiological importance of GBA2 in glucosylceramide processing in relation to disease states is less clear, and here, selective inhibitors can be of use as chemical knockout entities. In this communication, we report our identification of a highly potent and selective N-alkylated l-ido-configured iminosugar. In particular, the selectivity of 27 for GCS over GBA1 is striking.

The chemoenzymatic synthesis of three 1-deoxynojirimycin-type iminosugars is reported. Key steps in the synthetic scheme include a Dibal reduction-transimination-sodium borohydride reduction cascade of reactions on an enantiomerically pure cyanohydrin, itself prepared employing almond hydroxynitrile lyase (paHNL) as the common precursor. Ensuing ring-closing metathesis and Upjohn dihydroxylation afford the target compounds.

The lipophilic iminosugar N-[5-(adamantan-1-ylmethoxy)pentyl]-1-deoxynojirimycin (2, AMP-DNM) potently controls hyperglycemia in obese rodent models of insulin resistance. The reduction of visceral glycosphingolipids by 2 is thought to underlie its beneficial action. It cannot, however, be excluded that concomitant inhibition of intestinal glycosidases and associated buffering of carbohydrate assimilation add to this. To firmly establish the mode of action of 2, we developed a panel of lipophilic iminosugars varying in configuration at C-4/C-5 and N-substitution of the iminosugar. From these we identified the l-ido derivative of 2, l-ido-AMP-DNM (4), as a selective inhibitor of glycosphingolipid synthesis. Compound 4 lowered visceral glycosphingolipids in ob/ob mice and ZDF rats on a par with 2. In contrast to 2, 4 did not inhibit sucrase activity or sucrose assimilation. Treatment with 4 was significantly less effective in reducing blood glucose and HbA1c. We conclude that the combination of reduction of glycosphingolipids in tissue and buffering of carbohydrate assimilation by 2 produces a superior glucose homeostasis.

Three different photoprobes were synthesized to label beta-glucosidases; one probe was based on glucose, two probes on the iminosugar deoxynojirimycin. The affinity of the probes for three different beta-glucosidases was determined. Furthermore, their labeling efficiencies, binding specificities through competition with deoxynojirimycin, and binding specificities in the presence of cell lysate, were evaluated. Especially one showed very high affinity towards non-lysosomal glucoceramidase (IC(50)=20 nM)., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Isolates from patients with Clostridium difficile infection (CDI) usually produce both toxin A (TcdA) and toxin B (TcdB), but an increasing number of reports from Europe and Asia mention infections with TcdA-negative, TcdB-positive (A-/B+) strains, usually characterized as PCR ribotype 017 (type 017). Incidence rates of CDI per 10 000 admissions in a 200-bed Argentinean general hospital were 37, 84, 67, 43, 48 and 42 for the years 2000 to 2005, respectively. The annual percentages of type 017 CDI were 7.7%, 64.6%, 91.4%, 92.0%, 75.0% and 86.4%, respectively. Comparison of 112 017-CDI patients with 41 non-017-CDI patients revealed that 017-CDI patients were more often male (68.8% vs. 46.3%; odds ratio 2.55, 95% confidence interval 1.23-5.50). All type 017 strains tested belonged to toxinotype VIII and had a 1.8-kb deletion in tcdA. In addition, 90% of tested type 017 isolates had high-level resistance to clindamycin and erythromycin, determined by the presence of the ermB gene. Multiple-locus variable-number tandem-repeat analysis (MLVA) was applied to 56 Argentinean isolates and 15 isolates from seven other countries. Country-specific clonal complexes were found in each country. Among 56 Argentinean isolates, four clonal complexes were recognized, accounting for 61% of all isolates. These clonal complexes did not show correlation over time, but seemed to be restricted to specific wards, mainly internal medicine and pulmonology wards. A total of 56% of recurrent infections were caused by a different isolate, despite identification of an identical PCR-ribotype. We conclude that C. difficile type 017 gradually replaced other circulating PCR ribotypes and that MLVA provides detailed insight into nosocomial spread.

In the recent past sugar-derived cyclopropylamines were proposed as structurally new glycosidase inhibitors. In this Letter we report our efforts in the synthesis of a set of alpha-glucose configured oxabicyclo[4.1.0] heptanes, based on this hypothesis, bearing an amine substituent on the propyl ring and reveal that their inhibitory potential towards a range of mammalian glucosidases is modest.

Two complementary strategies for the synthesis of febrifugine are detailed based on previously developed chemoenzymatic approaches to the 3-hydroxypiperidine skeleton. The introduction of the quinazolone-containing side chain in both strategies was based on an N-acyliminium ion-mediated coupling reaction.

Two series of dimeric ligands for a G-protein-coupled receptor were prepared that differ by the interconnecting spacer system. Biological evaluation revealed that both dimeric series exhibit unique biological properties relative to their monomeric counterparts.The luteinizing hormone receptor (LHR), the follicle-stimulating hormone receptor (FSHR), and the thyroid-stimulating hormone receptor (TSHR) belong to the glycoprotein hormone receptor (GpHR) family. A prominent feature of all endogenous glycoprotein ligands is that they share an identical alpha subunit and acquire their selectivity from the unique beta subunit. Recent developments in pro-fertility research have led to the discovery of several low-molecular-weight agonists for the luteinizing hormone/choriogonadotropin receptor that bind to the transmembrane (TM) region of the LHR. Interestingly, some of these agonists are also able to activate the FSHR. Several research groups have shown that ligand dimerization presents a powerful tool to increase the subtype selectivity for structurally related G-protein-coupled receptors. In this work, we applied the dimerization strategy to GpHRs and explored the effect on receptors with closely related TM regions. Two series of dimeric ligands were prepared that differ in the interconnecting spacer system. Biological evaluation revealed that both series exhibit unique selectivity properties for the LHR, originating from either decreased potency or a decreased efficacy toward the FSHR.

In the period April-September 2005, an outbreak of Clostridium difficile infection (CDI) due to PCR ribotype 027 occurred among 50 patients in a 341-bed community hospital in Harderwijk, The Netherlands. A retrospective case-control study was performed to identify risk factors specific for CDI, using a group of patients with CDI (n = 45), a group of randomly selected control patients without diarrhoea (n = 90), and a group of patients with non-infectious diarrhoea (n = 109). Risk factors for CDI and for non-CDI diarrhoea were identified using multiple logistic regression analysis. Independent risk factors for CDI were: age above 65 years (OR 2.6; 95% CI 1.0-5.7), duration of hospitalization (OR 1.04 per additional day; 95% CI 1.0-1.1), and antibiotic use (OR 12.5; 95% CI 3.2-48.1). Of the antibiotics used, cephalosporins and fluoroquinolones were identified as the major risk factors for development of CDI. The risk of developing CDI was particularly high in people receiving a combination of a cephalosporin and a fluoroquinolone (OR 57.5; 95% CI 6.8-483.6). The main factors affecting the risk of non-CDI diarrhoea were proton-pump inhibitors, immunosuppressive drugs, underlying digestive system disease, previous surgery, and gastric tube feeding. The outbreak ended only after implementation of restricted use of cephalosporins and a complete ban on fluoroquinolones, in addition to general hygienic measures, cohorting of patients in a separate ward, education of staff, and intensified environmental cleaning. The results of this study support the importance of appropriate antimicrobial stewardship in the control of hospital outbreaks with C. difficile PCR ribotype 027.

The discovery of the glycosphingolipids is generally attributed to Johan L. W. Thudichum, who in 1884 published on the chemical composition of the brain. In his studies he isolated several compounds from ethanolic brain extracts which he coined cerebrosides. He subjected one of these, phrenosin (now known as galactosylceramide), to acid hydrolysis, and this produced three distinct components. One he identified as a fatty acid and another proved to be an isomer of D-glucose, which is now known as D-galactose. The third component, with an "alkaloidal nature", presented "many enigmas" to Thudichum, and therefore he named it sphingosine, after the mythological riddle of the Sphinx. Today, sphingolipids and their glycosidated derivatives are the subjects of intense study aimed at elucidating their role in the structural integrity of the cell membrane, their participation in recognition and signaling events, and in particular their involvement in pathological processes that are at the basis of human disease (for example, sphingolipidoses and diabetes type 2). This Review details some of the recent findings on the biosynthesis, function, and degradation of glycosphingolipids in man, with a focus on the glycosphingolipid glucosylceramide. Special attention is paid to the clinical relevance of compounds directed at interfering with the factors responsible for glycosphingolipid metabolism.

Since the 1980s the epidemiology of Clostridium difficile infection (CDI) has been investigated by the application of many different typing or fingerprinting methods. To study the epidemiology of CDI, a typing method with a high discriminatory power, typeability, and reproducibility is required. Molecular typing methods are generally regarded as having advantages over phenotypic methods in terms of the stability of genomic markers and providing greater levels of typeability. A growing number of molecular methods have been applied to C. difficile. For the early and rapid detection of outbreak situations, methods such as restriction enzyme analysis, arbitrary primed polymerase chain reaction (PCR), and PCR ribotyping are commonly used. For long-term epidemiology, multilocus sequence typing, multilocus variable number of tandem repeats analysis, and amplified fragment length polymorphism are of interest. Currently, the PCR-ribotyping method and the library of PCR ribotypes in Cardiff are the benchmarks to which most typing studies around the world are compared. Multilocus variable number of tandem repeats analysis is the most discriminative typing method and will contribute significantly to our understanding of the epidemiology of this important nosocomial pathogen.

A stereoselective synthesis of (+)-epiquinamide is presented in combination with determination of the absolute configuration of the natural product. Key steps in the sequence involved chemoenzymatic formation of an enantiomerically pure cyanohydrin, reductive cyclization to the corresponding cyclic N,N-acetal, and subsequent conversion into a suitable N-acyliminium ion precursor to enable construction of the second ring.

Background: Butamben or n-butyl-p-aminobenzoate is a long-acting experimental local anesthetic for the treatment of chronic pain when given as an epidural suspension. We have investigated whether Cav1.2/L-type calcium channels may be a target of this butamben action., Methods: The effect of butamben on these channels was studied in undifferentiated rat PC12-cells with the whole-cell patch-clamp technique in voltage-clamp. Ba(2+) ions were used as the charge carriers in the calcium channel currents, whereas K(+) currents were removed using K(+) free solutions., Results: Butamben 500 microM reversibly suppressed the total whole-cell barium current by 90% +/- 3% (n = 15), whereas 10 microM nifedipine suppressed this barium current by 75% +/- 7% (n = 6). Preexposure to butamben followed by washout decreased the inhibition by nifidepine to 47% +/- 5% (n = 10). These suppressive effects were not due to the measurement procedure and the drug vehicles in the solutions (<0.1% ethanol; n = 6)., Conclusions: Butamben inhibits the total barium current through expressed calcium channel types in PC12 cells, including Cav1.2/L-type channels. Because Cav1.2 channels may also occur in human nociceptive C fibers, this result allows the possibility that these L-type channels are involved in the analgesic action of butamben.

The effect on diagnostic yield of testing sequential stools was assessed during two hospital epidemics of Clostridium difficile. Using a rapid immunoassay, C. difficile-associated disease was diagnosed in 237 diarrhoeal patients, of whom 204 (86%) were diagnosed from the first faeces sample and 12 (5%) were diagnosed from follow-up samples obtained within 1 week. The remaining 21 (9%) patients yielded a positive test from stools obtained >1 week after the initial negative sample. It was concluded that repeated testing of stools for C. difficile toxin is of value in controlling outbreaks of C. difficile infection.

The fact that GPCRs might function in a dimeric fashion is currently well accepted. For GnRHR, a GPCR that regulates gonadotropin release, there is evidence that the receptor also functions as a dimer. We here describe the design and synthesis of a set of dimeric GnRHR antagonists in order to understand the interaction of dimeric ligands to the receptor and to address the question whether GnRHR dimerization is a prerequisite for signalling. Biological evaluation of the compounds shows no discrimination between monomeric and dimeric-ligands in respect to binding affinities, however, the dimeric ligands appear to have different functional properties.

We compared multilocus variable-number tandem-repeat analysis (MLVA) and macrorestriction endonuclease analysis using pulsed-field gel electrophoresis (PFGE) to determine their utility to identify clusters of Clostridium difficile infection (CDI) among 91 isolates of PCR ribotype 027 (NAP1, for North American pulsed-field type 1) from nine hospitals (and 10 general practitioners associated with one institution) in England. We also examined whether mortality in CDI cases was associated with specific MLVA subtypes. PFGE discriminated between ribotype 027 strains at >98% similarity, identifying five pulsovars (I to V) of 1 to 53 isolates. MLVA was markedly more discriminatory, identifying 23 types of 1 to 15 isolates (>71% similarity). PFGE pulsovars I and IV contained 14 and 8 MLVA types, respectively. Twenty-one of twenty-three (91%) of MLVA types were specific to individual PFGE pulsovars. Four CDI clusters were identified in institution A by conventional epidemiological analysis. MLVA typing identified two enlarged and two additional clusters. Thirty of forty-four (68%) patients in institution A with CDI caused by ribotype 027 strains were assigned to seven distinct clusters by a combination of MLVA typing and epidemiological records. Of 33 patients, comprising 14 different MLVA types, nine (27%) died by day 30 (early deaths). Eight of nine (89%) were associated with PFGE type IV C. difficile ribotype 027. Five of nine early deaths were associated with MLVA type 16, which was the dominant type in this cohort (10/33 cases); 4 other distinct MLVA types accounted for the other early deaths. MLVA was far superior to PFGE for analyzing clusters of CDI both within and between institutions. Further study is needed to examine whether subtypes of C. difficile ribotype 027 affect outcome.

We have demonstrated that multiple crude enzyme lysates containing a hydroxynitrile lyase can be used for the enantioselective synthesis of cyanohydrins from aldehydes in microchannels. Using a microreactor setup, two important parameters were efficiently screened consuming only minute amounts of reagents. More importantly, results from the continuous flow reaction were fully consistent with results obtained from larger batchwise processes in which a stable emulsion was formed., (Copyright 2007 Wiley Periodicals, Inc.)

Using 42 isolates contributed by laboratories in Canada, The Netherlands, the United Kingdom, and the United States, we compared the results of analyses done with seven Clostridium difficile typing techniques: multilocus variable-number tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), PCR-ribotyping, restriction endonuclease analysis (REA), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). We assessed the discriminating ability and typeability of each technique as well as the agreement among techniques in grouping isolates by allele profile A (AP-A) through AP-F, which are defined by toxinotype, the presence of the binary toxin gene, and deletion in the tcdC gene. We found that all isolates were typeable by all techniques and that discrimination index scores for the techniques tested ranged from 0.964 to 0.631 in the following order: MLVA, REA, PFGE, slpAST, PCR-ribotyping, MLST, and AFLP. All the techniques were able to distinguish the current epidemic strain of C. difficile (BI/027/NAP1) from other strains. All of the techniques showed multiple types for AP-A (toxinotype 0, binary toxin negative, and no tcdC gene deletion). REA, slpAST, MLST, and PCR-ribotyping all included AP-B (toxinotype III, binary toxin positive, and an 18-bp deletion in tcdC) in a single group that excluded other APs. PFGE, AFLP, and MLVA grouped two, one, and two different non-AP-B isolates, respectively, with their AP-B isolates. All techniques appear to be capable of detecting outbreak strains, but only REA and MLVA showed sufficient discrimination to distinguish strains from different outbreaks.

During a 2-month period in 2005, 13 laboratories participated in a surveillance study of Clostridium difficile-associated disease (CDAD) in 17 hospitals in The Netherlands. The median incidence rate of CDAD was 16/10 000 patient admissions (2.2/10 000 patient-days) and varied from 1 to 46/10 000 patient admissions according to hospital. In total, 81 patients with CDAD were reported; 49 (61%) patients had nosocomial CDAD, and 29 (36%) patients were admitted to hospital when already suffering from diarrhoea. Two (2%) deaths were attributable to CDAD; both of these patients were admitted with severe community-onset CDAD and were aged >80 years. Among 64 toxinogenic isolates, ten (16%) belonged to PCR ribotype 027 and ten (16%) to PCR ribotype 014. Type 027 was identified in ten patients from one hospital during an unrecognised outbreak. Toxinotyping of the 64 isolates revealed the presence of six different toxinogenic types, with 41 (64%) isolates of toxinotype 0, ten (16%) isolates of toxinotype III, and nine (14%) isolates of toxinotype V. Of the 64 toxinogenic isolates, seven (11%) had a 39-bp deletion in the tcdC gene, 11 (17%) had an 18-bp deletion, and one (1%) had a deletion of c. 44 bp. Genes for binary toxin were present in 21 (33%) of the 64 toxinogenic isolates, mainly associated with toxinotypes III and V. It was concluded that the median CDAD incidence rate of 16/10 000 patient admissions in The Netherlands is considerably lower than that in Canada and the USA, and that the emerging type 027 can spread unnoticed. The high proportion (36%) of CDAD cases with a community onset has important implications for future studies of the epidemiology of CDAD.

G protein coupled receptors (GPCRs) are important drug targets in pharmaceutical research. Traditionally, most research efforts have been devoted towards the design of small molecule agonists and antagonists. An interesting, yet poorly investigated class of GPCR modulators comprise the bivalent ligands, in which two receptor pharmacophores are incorporated. Here, we set out to develop a general strategy for the synthesis of bivalent compounds that are projected to bind to the human gonadotropin-releasing hormone receptor (GnRHR). Our results on the dimerisation of a known GnRHR antagonist, with as key step the Huisgen 1,3-cycloaddition, and their ability to bind to and antagonize GnRH-induced GnRHR stimulation, are presented here.