A versatile approach for the site-specific modification of recombinant antibodies using a combination of enzyme-mediated bioconjugation and click chemistry.

A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.

Single-chain antibody conjugated to a cage amine chelator and labeled with positron-emitting copper-64 for diagnostic imaging of activated platelets.

Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain antibody (scFvanti-LIBS) conjugated to a positron emitting copper-64 complex of a cage amine sarcophagine chelator (MeCOSar) is reported. This tracer was compared in vitro to a (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) rapidly under mild conditions and with higher specific activity than scFvanti-LIBS-DOTA. The utility of scFvanti-LIBS-MeCOSar as a diagnostic agent was assessed in vivo in a mouse model of acute thrombosis. The uptake of scFvanti-LIBS-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar is an excellent tool for highly sensitive in vivo detection of activated platelets in PET and has the potential to be used for early diagnosis of acute thrombotic events.

Fully automated synthesis and coupling of [(18) F]FBEM to glutathione using the iPHASE FlexLab module.

Site-specific radiolabelling of peptides or antibodies using [(18) F]FBEM is often preferred over non-site-specific radiolabelling with [(18) F]SFB because it does not affect the affinity of the antibody to its target. Unfortunately, the synthesis of [(18) F]FBEM and its conjugation to thiol containing macromolecules requires some manual intervention, which leads to radiation exposure of the radiochemist. In this publication, we report on the complete automation of [(18) F]FBEM production and its subsequent conjugation to glutathione using a slightly modified iPHASE FlexLab module. [(18) F]FBEM was produced in 1.185 ± 0.168 GBq (15-20%; n = 10; 0.75 ± 0.106 GBq non-decay corrected) with a specific activity of 57 ± 10 GBq/µmol. Radiochemical purity was 97 ± 1% and the synthesis time including HPLC purification and reformulation was 70 min. After evaporation to dryness, [(18) F]FBEM was conjugated to glutathione in PBS buffer pH 7.4 in quantitative yields. This fully automated method does not require any manual intervention and therefore reduces the radiation exposure to the operator.

Liver histology as predictor of outcome in patients with acute liver failure.

Acute liver failure (ALF) is a clinical syndrome associated with significant morbidity and mortality with a highly unpredictable outcome. We retrospectively analyzed 71 ALF patients (53 males; mean age = 27.5 ± 15.6 years) that underwent transjugular liver biopsy (TJLB) at our institution. The aims of this study are (i) to report our experience with TJLB in these patients, and (ii) to examine the role of liver histology in predicting their outcome. We also compared the histopathological findings between TJLB and explanted liver specimens in 31 patients who underwent liver transplantation (LT). Biopsy specimens were satisfactory for histopathological analyses in 69 (97.1%) patients, confirmed the clinical diagnosis in 56 (81.2%) patients, and altered the diagnosis in 13 (18.8%) patients. Minor complications were encountered in four (5.6%) patients. Percentage of hepatocyte necrosis was the only histological parameter that has significant discriminatory prognostic value, with no survivors having >75% necrosis without LT. In conclusions, TJLB is a safe technique for obtaining liver tissue in both adult and pediatric patients with ALF. Histological characteristics, mainly etiological diagnosis and degree of hepatocyte necrosis may assist in clinical decision-making for need of LT in these patients.

High-throughput analysis of ammonia oxidiser community composition via a novel, amoA-based functional gene array.

Advances in microbial ecology research are more often than not limited by the capabilities of available methodologies. Aerobic autotrophic nitrification is one of the most important and well studied microbiological processes in terrestrial and aquatic ecosystems. We have developed and validated a microbial diagnostic microarray based on the ammonia-monooxygenase subunit A (amoA) gene, enabling the in-depth analysis of the community structure of bacterial and archaeal ammonia oxidisers. The amoA microarray has been successfully applied to analyse nitrifier diversity in marine, estuarine, soil and wastewater treatment plant environments. The microarray has moderate costs for labour and consumables and enables the analysis of hundreds of environmental DNA or RNA samples per week per person. The array has been thoroughly validated with a range of individual and complex targets (amoA clones and environmental samples, respectively), combined with parallel analysis using traditional sequencing methods. The moderate cost and high throughput of the microarray makes it possible to adequately address broader questions of the ecology of microbial ammonia oxidation requiring high sample numbers and high resolution of the community composition.

Giant cell tumor of the pancreas: a pathological diagnosis with poor prognosis.

BACKGROUND: Giant cell tumors are rare and highly malignant tumors of the pancreas. Based on two distinct cell populations, they have been divided into two subtypes corresponding to the osteoclast-like giant cell tumor and the pleomorphic giant cell carcinoma of the pancreas. Distinctive imaging features of the tumors remain uncharacterized. Surgical removal is the only appropriate treatment for them, but responses to chemotherapy or radiotherapy remain undocumented. METHODS: Clinical, radiological, histopathologic, and immunohistochemical features of two cases of giant cell tumor of the pancreas are presented along with a brief review of the literature. RESULTS: En-bloc resection was done successfully in both cases. The patient with an osteoclast-like giant cell tumor remained disease-free with no clinical or radiological evidence of recurrence at 6 months after surgery. However, the patient with the pleomorphic type died 4 months later due to diffuse pulmonary metastasis. CONCLUSIONS: En-bloc surgical resection is the only appropriate treatment for giant cell tumors. The overall prognosis of these tumors is poorer than that of pancreatic ductal adenocarcinoma, especially the pleomorphic type. More studies are required to document the management and outcomes of the tumors.

A hepatic metastasis from pleomorphic adenoma of salivary gland: an unusual presentation.

Metastasizing pleomorphic adenoma (PA) is a rare condition displaying benign histological features as of PA in the metastatic lesion. The present study report a case of 46 years old male presented with space occupying lesion in the right lobe of liver. Patient underwent extended right hepatectomy for space occupying lesion in liver and the histological features were suggestive of metastatic benign pleomorphic adenoma. Surgical resection is the treatment of choice for both primary and metastatic lesion.

Isolation and characterisation of a high-efficiency desaturase and elongases from microalgae for transgenic LC-PUFA production.

The production of long-chain polyunsaturated fatty acids from precursor molecules linoleic acid (LA; 18:2omega6) and alpha-linolenic acid (ALA; 18:3omega3) is catalysed by sequential desaturase and elongase reactions. We report the isolation of a front-end Delta6-desaturase gene from the microalgae Ostreococcus lucimarinus and two elongase genes, a Delta6-elongase and a Delta5-elongase, from the microalga Pyramimonas cordata. These enzymes efficiently convert their respective substrates when transformed in yeast (39-75% conversion for omega3 substrate fatty acids), and the Delta5-elongase in particular displays higher elongation efficiency (75% for conversion of eicosapentaenoic acid (20:5omega3) to docosapentaenoic acid (22:5omega3)) than previously reported genes. In addition, the Delta6-desaturase is homologous with acyl-CoA desaturases and shows a strong preference for the omega3 substrate ALA.

The physical movement of urine from the kidneys to the urinary bladder and bladder compliance in two anurans.

Urine movement from the ureters to the bladder was accomplished by a combination of gravity and pressures resulting from lung inflation, buccal pumping, and smooth-muscle contraction. Urine movement from the cloaca to the urinary bladder was the result of pressure generated by cloacal smooth-muscle contraction and opposed by urinary bladder pressure, resulting from compliance, lung inflation, and buccal movements. The frequency of cloacal smooth-muscle contraction measured in Chaunus marinus (4.9 min(-1)) and Lithobates grylio (2.8 min(-1)) generated cloacal pressures that averaged 0.67 and 0.59 kPa, respectively. Chaunus marinus had a mean internal bladder pressure of 0.29 kPa, while Lithobates catesbeiana had a mean internal bladder pressure of 0.16 kPa. Using direct urinary bladder infusion and physiological pressure transducers on live anurans, it was determined that urinary bladder compliance was lower (P < 0.05) in the aquatic frog L. grylio (2,050 mL kPa(-1) kg(-1)) than in the terrestrial toad C. marinus (4,440 mL kPa(-1) kg(-1)). Taken together, these data suggest that bladder filling is a result of pressures generated in the ureters and the cloaca that are greater than internal bladder pressures and that the more aquatic species had a less compliant urinary bladder compared with that of the terrestrial species.

An unusual cause of S1 radicular pain presenting as early phantom pain in a transfemoral amputee: a case report.

Recent epidemiologic studies have shown back pain to be a significant cause of pain in lower-limb amputees, but only a handful of cases have reported sciatica in amputees. The symptoms are usually described as a phantom pain or neuropathic pain in the residual limb that is often refractory to conventional treatments. These symptoms typically occur with back pain and are distinct from the patient's usual symptoms. Interestingly, back pain is not a universal finding. We present a patient with presumed phantom limb pain subsequently discovered to be caused by an S1 radiculopathy. This patient's supposed phantom pain persisted despite multiple medication trials. Initial work-up revealed a sciatic neuroma at the stump. Treatments targeting this neuroma were unsuccessful. Further evaluation found that a sacroiliac joint screw placed to stabilize a pelvic fracture had intruded into the S1 neuroforamen. A diagnostic S1 nerve block temporarily relieved the patient's pain, and the screw was removed. Pain persisted and a spinal cord stimulator was placed resulting in improvement of his pain. Because conventional diagnostic tests are limited, including physical exam and electromyography, a fluoroscopically guided selective spinal nerve block proved to be a useful diagnostic tool in this patient.

Two-photon absorption cross-sections and time-resolved fluorescence imaging using porphyrin photosensitisers.

Three porphyrin systems have been characterised for use in two-photon fluorescence imaging of biological samples. We have determined the two-photon absorption cross sections (sigma(2)) of the di-cation, free-base and metallated forms of hematoporphyrin derivative (HpD), hematoporphyrin IX (Hp9) and a boronated protoporphyrin (BOPP) using the open-aperture Z-scan and the two-photon induced fluorescence (TPIF) techniques at an excitation wavelength of 800 nm. The insertion of either protons or a metal ion into the macrocycle is shown not to significantly influence the sigma(2) of the porphyrins. Two-photon time-resolved fluorescence images of C6 glioma cells transfected with a free-base form of the BOPP have been obtained as a function of the porphyrin concentration. These studies reveal a maximum useful porphyrin concentration for fluorescence imaging purposes of approximately 30 microg mL(-1).

Dietary fat and fat types as early determinants of childhood obesity: a reappraisal.

BACKGROUND: The growing prevalence of childhood overweight and obesity has renewed interest in determining the influence of the maternal and infant diet on the risk of developing excess fat mass later in life. APPROACH: Review of available human and animal data reporting the effects of dietary fat and fat types early in life on adipose development. RESULTS: Rodent studies tend to show that maternal high-fat feeding during pregnancy and lactation results in increased adiposity of the offspring. Nevertheless, today there is a lack of population-based studies investigating this potential detrimental effect of maternal high-fat intake. Most epidemiological studies, performed so far, do not find any association between the level of dietary fat intake of infants and children and body weight and/or fatness. Regarding fat types exposure to high levels of dietary n-6 fatty acids during gestation and post-natal life, has been shown to promote obesity in mice. Nevertheless, other rodent studies do not demonstrate such an effect. CONCLUSION: There is no evidence supporting a restriction of fat intake during the first two post-natal years but the potential detrimental effects of maternal high-fat intake during gestation should be further investigated. The role of dietary fat types as early determinants of childhood obesity has so far been poorly studied. Robust evidence to support the adipogenic effects of n-6 fatty acids enriched-diets is currently lacking but this hypothesis is of importance and should be further evaluated in different animal models as well as in longitudinal human studies.

Alkane C-H bond activation in zeolites: evidence for direct protium exchange.

The mechanism of alkane C-H bond activation in heterogeneous acid catalysis is unknown. (1)H solid-state NMR techniques have been used to simultaneously detect the reactivity of both catalyst and alkane reactant protons in a true in-situ experimental design. Specifically, the activation of isobutane C-H bonds by the solid acid zeolite HZSM-5 is directly observed, and the rate of proton transfer between the solid catalyst surface and gaseous isobutane is quantitatively measured using isotopic (1)H/(2)H exchange methods. An observable adsorption complex forms between the isobutane and the primary Bronsted acid site of ZSM-5, which leads to proton exchange between the zeolite surface and the isobutane methyl groups at temperatures (273 K) much lower than previously reported. The secondary acid site in ZSM-5 is less accessible to or less reactive with the isobutane molecule. Simultaneous detection of protium loss from the Bronsted acid site and protium gain by perdeuterated isobutane reveals a common rate constant equal to 4.1-4.6 x 10(-4) s(-1) at 298 K, but at lower temperatures, the transition between this and a much slower rate process is resolved. The measured activation energy for isobutane H/D exchange is 57 kJ/mol. In all experiments, the isobutane reagent was purified to eliminate any unsaturated impurities that might serve as initiators for carbenium-ion mechanisms, and the active catalyst was free of any organic contaminants that might serve as a source of unsaturated initiators. In total, our results are consistent with direct proton exchange between the zeolite surface and the methyl groups of isobutane.

[The course of Helicobacter pylori-associated duodenal ulcer concurrent with chronic opisthorchiasis].

The purpose of the investigation was to study the specific features and the course of Helicobacter pylori-associated duodenal ulcer (DU) concurrent with chronic opisthorchiasis (CO) A hundred and twenty-seven patients with H. pylori-associated DU were examined. Of them 78 patients were found to have H. pylori-associated DU concurrent with CO. In mixed pathology, asthenovegetative disorders were observed in most patients, one third had eosinophilia. With an over 10-year history of CO, acid production and pepsin secretion substantially decreased in both phases of gastric secretion. Mucosal atrophic changes were found mainly in the antral portion of the stomach, which frequency correlated with the duration of Opisthorchis invasion. The incidence of atrophic duodenitis tripled. All the patients received eradication therapy by the classical first-line treatment regimen including omeprazole and the antibiotics clarithromycin and amoxicillin. In the mixed pathology group, the time of ulcerative defect scarring increased by an average of 5 days. Fifty-eight with pathology continuously receiving the antisecretoty agent underwent dehelminthization with the anthelminthic bilthricide in 33 patients and with the plant anthelminthic dry aspen bark extract in 25. Evaluation of the efficiency of eradication therapy indicated that the best results of H. pylori eradication had been achieved in the patients dehelminthized with bilthricide. Assessment of long-term results showed that after multimodality therapy (eradication followed by bilthricide dehelminthization), there were no relapses for 2 years and there was a drop in the number of patients resorting to on-request therapy in patients with H. pylori-associated DU concurrent with CO.

Metabolic engineering of Arabidopsis to produce nutritionally important DHA in seed oil.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are nutritionally important long-chain (≥ C20) omega-3 polyunsaturated fatty acids (ω3 LC-PUFA) currently obtained mainly from marine sources. A set of genes encoding the fatty acid chain elongation and desaturation enzymes required for the synthesis of LC-PUFA from their C18 PUFA precursors was expressed seed-specifically in Arabidopsis thaliana. This resulted in the synthesis of DHA, the most nutritionally important ω3 LC-PUFA, for the first time in seed oils, along with its precursor EPA and the ω6 LC-PUFA arachidonic acid (ARA). The assembled pathway utilised Δ5 and Δ6 desaturases that operate on acyl-CoA substrates and led to higher levels of synthesis of LC-PUFA than previously reported with acyl-PC desaturases. This demonstrates the potential for development of land plants as alternative sources of DHA and other LC-PUFA to meet the growing demand for these nutrients.

Identification of an adsorption complex between an alkane and zeolite active sites.

Direct observation of the Bronsted acid site signal in an active zeolite catalyst following adsorption of stoichiometric quantities of isobutane reveals the presence of a specific adsorption complex. Independent polarization transfer experiments in which magnetization originates with either the catalyst or the adsorbed isobutane confirm this assignment. The initial steps in alkane reactivity are poorly defined, and this experimentally verified complex is proposed as a route to C-H bond activation in solid acids.

Similarity of insulin detemir pharmacokinetics, safety, and tolerability profiles in healthy caucasian and Japanese american subjects.

The objective of this study was to compare the pharmacokinetics of insulin detemir in three ascending doses in healthy Japanese and Caucasian subjects. This was an open-label, single-center, parallel-group design evaluating 30 subjects (15 Japanese and 15 Caucasians). Subjects received a total of three subcutaneous injections (one injection per visit) of insulin detemir (0.19, 0.38, 0.75 U/kg [1 U = 24 nmol]) in ascending order. Following drug administration, subjects received intravenous glucose in 0.5-mg/kg/min increments every 30 minutes, followed by a constant rate of 2.0 mg/kg/min for up to 12 hours. For pharmacokinetic evaluations, serial blood sampling was performed over a period of 30 hours after dosing. Of the subjects, 36 were enrolled, and 30 completed the study. There was a linear dose-response relationship between the three ascending insulin detemir doses and serum insulin detemir AUC values for both the Japanese and Caucasian subjects. The two dose-response regression lines had equivalent slopes but slightly different intercepts (although not statistically significant). This difference may be due to variation in AUC, body weight differences, or chance. Six subjects discontinued the study, 2 as a result of adverse events (blood draw-related ecchymosis and hypoglycemia). The most frequent treatment-emergent adverse events (TEAE) were headache, dizziness, and reactions related to blood draws/infusion sites. All TEAEs were mild to moderate in severity. The results show that an increase in insulin detemir dose will result in a similar increase in insulin detemir concentration in the two ethnic groups. Therefore, therapeutic dosing of insulin detemir is expected to be similar in both ethnic groups, with no special dose adjustment or algorithm based on race. Insulin detemir at 0.19, 0.38, and 0.75 U/kg was generally well tolerated in both Japanese and Caucasian subjects.

Myocardial effects of ethanol consumption in the rat with streptozotocin-induced diabetes.

BACKGROUND: Rats with streptozotocin (STZ)-induced diabetes exhibit alterations in cardiac function, ventricular remodeling, and changes in cell signaling, which includes protein kinase C (PKC) isoforms. Moderate consumption of ethanol has a beneficial effect on cardiovascular outcomes in the general population, an effect that has recently been found to extend to patients with diabetes mellitus. We studied the effect of low-dose ethanol consumption on cardiac function, geometry, and PKC isoforms in the rat with STZ-induced diabetes. METHODS: Four groups of rats were studied over 8 to 10 weeks: control, STZ-induced diabetes, 12% (v/v) ethanol consumption, and STZ-induced diabetes plus 4% (v/v) ethanol consumption. Invasive hemodynamic measurements were performed; myocardial tissue was obtained for analysis for total PKC and cytosolic and membrane protein content of PKC-alpha, PKC-delta, and PKC-epsilon, and two-dimensional and M-mode echocardiograms were obtained. RESULTS: Compared with rats with diabetes alone, consumption of 4% ethanol prevented the decrease in left ventricular dP/dt seen with diabetes alone, as well as the increase in left ventricular internal dimension. Up-regulation of PKC-alpha, -delta, and -epsilon occurring in the diabetic animals was also prevented by ethanol consumption, whereas ethanol alone had no effect on PKC isoform pattern. CONCLUSIONS: These data suggest that STZ-induced cardiac remodeling and dysfunction are associated with increases in PKC activity, particularly PKC-alpha, -delta, and -epsilon, and that consumption of ethanol can prevent these changes.

Effect of human recombinant leptin on lipid handling by fully differentiated Caco-2 cells.

It has been established that leptin displays a number of effects on peripheral tissues. We have investigated the effect of the hormone on lipid synthesis, apolipoprotein biogenesis and lipoprotein secretion in Caco-2 cells. Immunocytochemistry revealed the presence of leptin receptors (Ob-Rb) on the basolateral membrane. Incubation of cells with 200 nM leptin resulted in a decreased export of triglycerides in the basolateral medium without affecting monoglyceride, diglyceride and cholesterol ester lipid classes. It also significantly reduced the output of de novo-synthesized apolipoprotein (Apo)B-100 and ApoB-48 as well as that of newly formed chylomicrons and of low-density lipoproteins. It also enhanced that of ApoA-I, ApoA-IV and ApoE. Our results support the hypothesis that leptin can affect energy balance at the gut level by reducing lipid release into the circulation.