Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity.

Observation of highly dynamic processes inside living cells at the single molecule level is key for a better understanding of biological systems. However, imaging of single molecules in living cells is usually limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination, for example, in a reflected geometry with a high numerical aperture imaging objective, have been developed. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-colour acquisition of z -stacks. We demonstrate fast volume scanning by imaging a two-colour giant unilamellar vesicle (GUV) hemisphere. In addition, the high contrast enabled the imaging and tracking of single lipids in the GUV cap. The enhanced reflected scanning light-sheet microscope enables fast 3D scanning of artificial membrane systems and potentially live cells with single-molecule sensitivity and thereby could provide quantitative and molecular insight into the operation of cells.

High performance passive vibration isolation system for optical tables using six-degree-of-freedom viscous damping combined with steel springs.

Mechanical vibrations in buildings are ubiquitous. Such vibrations limit the performance of sensitive instruments used, for example, for high-precision manufacturing, nanofabrication, metrology, medical systems, or microscopy. For improved precision, instruments and optical tables need to be isolated from mechanical vibrations. However, common active or passive vibration isolation systems often perform poorly when low-frequency vibration isolation is required or are expensive. Furthermore, a simple solution such as suspension from common bungee cords may require high ceilings. Here we developed a vibration isolation system that uses steel springs to suspend an optical table from a common-height ceiling. The system was designed for a fundamental resonance frequency of 0.5 Hz. Resonances and vibrations were efficiently damped in all translational and rotational degrees of freedom of the optical table by spheres, which were mounted underneath the table and immersed in a highly viscous silicone oil. Our low-cost, passive system outperformed several state-of-the-art passive and active systems in particular in the frequency range between 1 and 10 Hz. We attribute this performance to a minimal coupling between the degrees of freedom and the truly three dimensional viscous damping combined with a nonlinear hydrodynamic finite-size effect. Furthermore, the system can be adapted to different loads, resonance frequencies, and dimensions. In the long term, the excellent performance of the system will allow high-precision measurements for many different instruments.

Novel Furin Inhibitors with Potent Anti-infectious Activity.

New peptidomimetic furin inhibitors with unnatural amino acid residues in the P3 position were synthesized. The most potent compound 4-guanidinomethyl-phenylacteyl-Arg-Tle-Arg-4-amidinobenzylamide (MI-1148) inhibits furin with a Ki value of 5.5 pM. The derivatives also strongly inhibit PC1/3, whereas PC2 is less affected. Selected inhibitors were tested in cell culture for antibacterial and antiviral activity against infectious agents known to be dependent on furin activity. A significant protective effect against anthrax and diphtheria toxin was observed in the presence of the furin inhibitors. Furthermore, the spread of the highly pathogenic H5N1 and H7N1 avian influenza viruses and propagation of canine distemper virus was strongly inhibited. Inhibitor MI-1148 was crystallized in complex with human furin. Its N-terminal guanidinomethyl group in the para position of the P5 phenyl ring occupies the same position as that found previously for a structurally related inhibitor containing this substitution in the meta position, thereby maintaining all of the important P5 interactions. Our results confirm that the inhibition of furin is a promising strategy for a short-term treatment of acute infectious diseases.

X-ray structures of human furin in complex with competitive inhibitors.

Furin inhibitors are promising therapeutics for the treatment of cancer and numerous infections caused by bacteria and viruses, including the highly lethal Bacillus anthracis or the pandemic influenza virus. Development and improvement of inhibitors for pharmacological use require a detailed knowledge of the protease's substrate and inhibitor binding properties. Here we present a novel preparation of human furin and the first crystal structures of this enzyme in complex with noncovalent inhibitors. We show the inhibitor exchange by soaking, allowing the investigation of additional inhibitors and substrate analogues. Thus, our work provides a basis for the rational design of furin inhibitors.

Examination of carbohydrate metabolism parameters after simultaneous pancreas-kidney transplantation.

End-stage renal failure, a frequent complication of type 1 diabetes mellitus, requires renal replacement therapy. Our team examined the laboratory parameters of carbohydrate metabolism in 18 patients with type 1 diabetes at 10 to 89 months after simultaneous pancreas-kidney transplantation. We compared these results with those of 17 patients with type 1 diabetes who had formerly received kidney-alone transplantations, and were undergoing insulin treatment, as well as with those of 16 metabolically healthy controls. The hemoglobin A1c (HbA1c) and blood glucose levels of the pancreas-kidney transplant recipients were within the normal ranges, not differing significantly from those of the healthy controls. In contrast, the HbA1c and glucose levels were significantly elevated among kidney transplanted diabetic subjects. However, fasting and 2-hour insulin levels of pancreas-kidney transplant patients were significantly higher than those of the controls, indicating insulin resistance. According to these results, the insulin secretion by the pancreas graft sufficiently compensated for insulin resistance. Thus 10 to 89 months after successful pancreas-kidney transplantation, carbohydrate metabolism by type 1 diabetic patients was well controlled without antidiabetic therapy.

Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases.

Optimization of our previously described peptidomimetic furin inhibitors was performed and yielded several analogs with a significantly improved activity. The most potent compounds containing an N-terminal 4- or 3-(guanidinomethyl)phenylacetyl residue inhibit furin with K(i) values of 16 and 8 pM, respectively. These analogs inhibit other proprotein convertases, such as PC1/3, PC4, PACE4, and PC5/6, with similar potency, whereas PC2, PC7, and trypsin-like serine proteases are poorly affected. Incubation of selected compounds with Madin-Darby canine kidney cells over a period of 96 h revealed that they exhibit great stability, making them suitable candidates for further studies in cell culture. Two of the most potent derivatives were used to inhibit the hemagglutinin cleavage and viral propagation of a highly pathogenic avian H7N1 influenza virus strain. The treatment with inhibitor 24 (4-(guanidinomethyl)phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide) resulted in significantly delayed virus propagation compared with an inhibitor-free control. The same analog was also effective in inhibiting Shiga toxin activation in HEp-2 cells. This antiviral effect, as well as the protective effect against a bacterial toxin, suggests that inhibitors of furin or furin-like proprotein convertases could represent promising lead structures for future drug development, in particular for the treatment of infectious diseases.

Purification of the proprotein convertase furin by affinity chromatography based on PC-specific inhibitors.

In eucaryotes, many secreted proteins and peptides are proteolytically excised from larger precursor proteins by a specific class of serine proteases, the proprotein/prohormone convertases (PCs). This cleavage is essential for substrate activation, making the PCs very interesting pharmacological targets in cancer and infectious disease research. Correspondingly, their structure, function and inhibition are intensely studied - studies that require the respective target proteins in large amounts and at high purity. Here we describe the development of a novel purification protocol of furin, the best-studied member of the PC family. We combined the heterologous expression of furin from CHO cells with a novel purification scheme employing an affinity step that efficiently extracts only active furin from the conditioned medium by using furin-specific inhibitor moieties as bait. Several potential affinity tags were synthesized and their binding to furin characterized. The best compound, Biotin-(Adoa)(2)-Arg-Pro-Arg-4-Amba coupled to streptavidin-Sepharose beads, was used in a three-step chromatographic protocol and routinely resulted in a high yield of a homogeneous furin preparation with a specific activity of ~60 units/mg protein. This purification and the general strategy can easily be adapted to the efficient purification of other PC family members.

New substrate analogue furin inhibitors derived from 4-amidinobenzylamide.

A series of new peptidomimetic furin inhibitors was synthesized, which was derived from our previously described lead structure phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (1). Substitution of Val by other amino acid residues revealed several highly potent furin inhibitors with K(i) values of less than 2nM, containing guanidinoalanine, Ile, Phe or Tyr in the P3 position. The replacement of the P2 Arg by Lys was also well accepted, whereas the incorporation of D-amino acids at various positions resulted in poor inhibitors. The use of the 4-amidinobenzylamide group provides convenient synthetic access to stable proprotein convertase inhibitors and derivatives as biochemical tools and for further studies in cell culture.

Potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated P1 arginine mimetics.

Furin belongs to the family of proprotein convertases (PCs) and is involved in numerous normal physiological and pathogenic processes, such as viral propagation, bacterial toxin activation, cancer, and metastasis. Furin and related furin-like PCs cleave their substrates at characteristic multibasic consensus sequences, preferentially after an arginine residue. By incorporating decarboxylated arginine mimetics in the P1 position of substrate analogue peptidic inhibitors, we could identify highly potent furin inhibitors. The most potent compound, phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (15), inhibits furin with a K(i) value of 0.81 nM and has also comparable affinity to other PCs like PC1/3, PACE4, and PC5/6, whereas PC2 and PC7 or trypsin-like serine proteases were poorly affected. In fowl plague virus (influenza A, H7N1)-infected MDCK cells, inhibitor 15 inhibited proteolytic hemagglutinin cleavage and was able to reduce virus propagation in a long-term infection test. Molecular modeling revealed several key interactions of the 4-amidinobenzylamide residue in the S1 pocket of furin contributing to the excellent affinity of these inhibitors.

Practical guidance on intensification of insulin therapy with BIAsp 30: a consensus statement.

BACKGROUND: Basal insulin and premix insulin are commonly prescribed first-line insulin therapies for patients failing to maintain glycaemic control on oral therapy. When control on these insulins starts to drift, premix analogues, such as biphasic insulin aspart 30/70 (BIAsp 30), are a simple and effective tool for intensification as they can be injected up to three-times daily (TID). However, at present, international recommendations for intensification of insulin therapy using premix analogues are limited and specific guidance on dosing is not available for many scenarios. METHODS: In October 2008, an international expert panel met to review the current guidelines for insulin intensification with BIAsp 30 in patients with type 2 diabetes, with the aim of developing practical guidance for general and specialist practitioners. RESULTS: Simple treatment algorithms have been developed for (i) patients on basal insulin (human or analogue) once daily or twice daily (BID) who need intensification to BIAsp 30 BID, and (ii) patients on BIAsp 30 once daily or BID who can be intensified to BIAsp 30 BID or TID. As well as these algorithms, specific guidance has been provided on dose transfer (from basal insulin to BIAsp 30), dose split (when intensifying from once daily to BID), and combination oral therapies. In addition, a guide to dose titration is included. CONCLUSIONS: The guidelines presented here should enable general or specialist practitioners to use BIAsp 30 to intensify the insulin therapy of patients failing on basal insulin or BIAsp 30 once or twice daily.

Infecciones de orina por gérmenes no E. coli y alteraciones nefrourológicas / Non-E. coli urinary infections and urinary tract anomalies

Introducción: Se ha señalado que las infecciones del tracto urinario (ITU) por gérmenes diferentes a E. Coli se asocian con mas frecuencia a malformaciones vesicoureterales y cicatrices renales. El objetivo de este estudio es analizar las características analíticas y las pruebas de imagen (ECO y CUMS) en lactantes ingresados con el diagnóstico de primera ITU y evaluar si hay diferencias entre aquellas producidas por E. coli y las producidas por otros patógenos. Sujetos y métodos: Estudio retrospectivo en 203 pacientes diagnosticados de primera ITU ingresados en el Servicio de Lactantes de nuestro Hospital entre los años 2003 y 2007. Se recogieron datos de laboratorio al ingreso y se les realizó pruebas de imagen (ECO y CUIMS), junto con un seguimiento posterior en Consultas Externas. Resultados: De los 203 niños incluidos, 174 (85,7%) resultaron positivos para E. coli y 29 (14,3%) para diferentes patógenos no E. coli. Estos últimos presentaron más alteraciones en la ECO que los no E. coli: OR =2,61 (1,12-6,05). Asimismo, presentaron mayor frecuencia de anomalías nefrourológicas, considerando como tal la presencia de alteraciones en la ECO y/o CUMS: OR=4,57 (2,01-10,4). También comprobamos que pacientes cuyos urocultivos fueron positivos para patógenos diferentes a E. coli mostraron más frecuentemente reflujo vesicoureteral grado ≥3 que los E. coli: OR = 7,9 (2,68-27,2), p<0,05. En nuestro estudio no hemos encontrado diferencias en el resto de parámetros analizados (datos epidemiológicos y de laboratorio). Conclusión: De nuestros resultados parece deducirse que aquellos niños que presentan una ITU por gérmenes no E. coli presentan más frecuentemente malformaciones nefrourológicas (AU)

Introduction: It is pointed out that non E. coli urinary tract infections (UTI) are more frequently related to vesicoureteral anomalies and renal scarring. This research aims to analyse the laboratory features and imaging studies (urinary tract ultrasound and cycystourethrography) in hospitalized infants diagnosed as first UTI, as well as evaluating possible differences between those caused by E. coli or other pathogens. Patients and methods: Between 2003 and 2007, medical databases of 203 infants hospitalized in our department for first UTI were reviewed. Initial laboratory data and imaging studies (urinary tract ultrasound and cystourethrography) were collected, together with the follow-up of every infant in outpatient services. Results: Out of the 203 infants included in the research study, 174 (85,7%) were diagnosed as E. coli UTI and 29 (14,3%) as UTI caused by other pathogens. More urinary tract ultrasound anomalies were noted in the latter compared to non E. coli: O.R. = 2.31 (1,12-6,05). In this way they showed higher rates of urinary tract anomalies, if we consider these ones as anomalies in ultrasound and/or cystourethrography, with OR=4.57 (2,01-10,4). We also detected that infants with non E. coli UTI yielded a higher association with ≥3 vesicoureteral reflux compared to E coli UTI: OR = 7,9 (2,68-27,2) P<0,05. We have not detected any other differences between E. coli and non E. Coli UTIs (epidemiologic and laboratory data). Conclusion: According to these results, we assume that infants who suffer form a non- E. coli first UTI are more commonly linked to urinary tract anomalies (AU)

[Detection of antibodies against pancreatic islet cells in clinical practice]. / Hasnyálmirigy-szigetsejt elleni antitestek vizsgálata a klinikai gyakorlatban.

Development of diabetes mellitus caused by pancreatic beta-cell destruction of autoimmune origin is the result of a long lasting process. The most easily examinable feature of this stage is the occurrence of the islet cell antibodies. The sera which are positive for islet cell cytoplasmic antibodies (ICA), examined by indirect immunofluorescence, contain a mixture of antibodies. The glutamic acid decarbocylase (GAD), the tyrosin phosphatase (IA2), the insulin, and the GM2-1 glycolipid can be the targets of these antibodies. One can routinely examine the ICA, the GADA, the IA2 antibodies. The detection of antibodies against insulin (IAA) and GM-2-1 glycolipid is not invented in the routine laboratory work. The aim of the authors was the evaluation of clinical significance of occurrence of islet cell antibodies: one hundred and eighteen nondiabetic children an adult human being without known diabetic first degree relatives and 366 type 1 diabetic children and adult patients served as controls. The authors evaluated the predictive value of the different islet cell antibodies to the development of type 1 diabetes mellitus in 596 nondiabetic children with type 1 diabetic first degree relatives. The authors looked for markers of beta-cell destruction among sera of 320 diabetics manifested after 30 years of age with at least half a year of non-insulin-dependency and in the sera of 68 females suffered from gestational diabetes after 0-14 years of the index pregnancy. Finally the authors report 7 cases in which the examination of islet cell antibodies helped the diagnosis and classification of diabetes mellitus. Indirect immunofluorescence method was used for the detection of ICA, radioimmunoassay for that of GADA and IA2 antibodies. There was no positive reaction for ICA and GADA in the nondiabetic population without diabetic first degree relatives. Among the freshly diagnosed type 1 diabetic children 39% were positive for only ICA, 44% for only GADA and 80% for any antibodies. Among the freshly manifested type 1 diabetic adults ICA positivity only was observed in 21%, GADA positivity only in 7.1% and 93% for any antibodies. From the 595 nondiabetic children with type 1 diabetic first degree relatives 23 were positive for ICA, from whom 5 became diabetic during a two years observation period. These diabetic children had multiplex autoantibodies besides ICA. One child from this group, who was negative for ICA became diabetic, too. Among type 2 diabetic patients 13% were positive for ICA alone, 17% were positive for GADA alone and 27% were positive for any antibodies. The insulin dependency manifested in a short time was associated with antibody positivity. Among the gestational diabetics 10 were found positive for ICA. From them, 7 were type 1 diabetics, and 3 were type 2 diabetics at the time of the detection of antibodies. The authors suggest the need of determination of islet cell antibodies in the group of nondiabetic first degree relatives of type 1 diabetic patients (ICA, GADA, IA2 and IAA), in the group of non-insulin-dependent diabetics (ICA and GADA) as a screening for later insulin dependency, and in gestational diabetes after delivery (ICA) as screening for type 1 diabetes mellitus.

Effect of diet and fluvastatin treatment on the serum lipid profile of kidney transplant, diabetic recipients: a 1-year follow up.

The effect of a cholesterol-lowering diet and subsequent fluvastatin treatment (Lescol, Novartis; 20 mg/day) on serum lipids and lipoproteins was investigated in 21 diabetic patients (eight women, 13 men, age range 31-63 years, BMI 25.9 +/- 4.5 kg/m2) who had undergone successful kidney transplantation. A cholesterol-lowering diet followed for 8 weeks had apparently no effect on serum lipid concentrations. Fluvastatin applied afterwards for 12 months significantly decreased the total cholesterol, triglyceride and LDL cholesterol levels from 7.7 +/- 0.94, 2.84 +/- 0.85 and 4.87 +/- 1.05 mmol/l to 6.40 +/- 0.74, 2.64 +/- 0.86 and 3.52 +/- 0.69 mmol/l, P < 0.001, < 0.05 and < 0.001, respectively, while the level of HDL cholesterol increased from 1.12 +/- 0.28 to 1.52 +/- 0.39 mmol/l, P < 0.001. Serum concentration of lipoprotein(a) remained unchanged. The serum level of apolipoprotein-A1 increased from 1.52 +/- 0.28 to 1.83 +/- 0.29 mmol/l (P < 0.01) and that of lipoprotein-B decreased from 1.37 +/- 0.20 to 1.20 +/- 0.36 mmol/l (P < 0.05). These maximum changes were achieved by the 12th week of fluvastatin treatment, and no further significant change was observed in the remaining part of the year. The other parameters that could have influenced lipid metabolism (doses of diuretics and steroid, daily dose and serum level of cyclosporin, kidney function, degree of proteinuria, HbA1c, etc.) remained unchanged throughout the study. Thus, the improvement in lipid concentrations can be ascribed exclusively to fluvastatin. No side effects were observed during the 1-year follow up. Liver enzymes and CPK remained within the normal reference limits. Fluvastatin proved to be an effective and safe drug for treating the dyslipidaemia of transplanted patients receiving steroid cyclosporin immunosuppression.

Effect of microencapsulation on oxygen distribution in islets organs.

In islet transplantation, limitation of oxygen supply may restrict graft function, particularly when encapsulated tissue is used. Therefore, oxygen tensions (PO2) in isolated islet organs (Brockmann bodies) of Osphronemus gorami were measured. In a thermostatically (37 degrees C) controlled measuring chamber, PO2 values were recorded at subsequent microelectrode positions on a radial track toward the center of the organ. In 2 independent groups, we studied the effect of fluid convection (n = 12) and microencapsulation (n = 12). In both groups, sigmoidal PO2 profiles were found, which permit differentiation in an oxygen-depleted zone surrounding the surface, a steep decline inside the tissue corresponding to the oxygen-consuming rim, and a plateau in the center without oxygen consumption which reflects necrosis. The PO2 values decreased (P < 0.001) when convection was stopped. Compared with starting values, PO2 levels at the surface were 61 +/- 3% with and 41 +/- 4% without convection. Surface values for encapsulated tissue were 44 +/- 5% compared with 64 +/- 4% in nonencapsulated tissue. In the tissue, center oxygen dropped to 27 +/- 5% with convection and to 6 +/- 3% without, and to 11 +/- 3% for encapsulated tissue compared with 22 +/- 4% for nonencapsulated tissue. The thickness of the outer oxygen-depleted zone was 81 +/- 16 microns with and 196 +/- 57 microns without convection (P < 0.001), and 188 +/- 16 microns for encapsulated and 94 +/- 14 microns for nonencapsulated tissue (P < 0.001). The oxygen-consuming rim was 295 +/- 22 microns with and 235 +/- 36 microns without convection (NS), and 216 +/- 15 microns for encapsulated and 315 +/- 24 microns for nonencapsulated tissue (P < 0.01). These results illustrate the special distribution of oxygen in isolated islet tissue and indicate that barium alginate encapsulation may worsen oxygenation mainly by expanding the "unstirred water layer" surrounding the tissue.

Long-term function of porcine islets and single cells embedded in barium-alginate matrix.

The influence of alginate-embedding on the maintenance of functioning and morphological integrity in long-term culture of isolated porcine islets and islet cells was studied. Free-floating islets and islet cells served for control. Function was tested after the 1st, 2nd and 4th week. Basal and glucose-stimulated insulin secretion of embedded islets decreased slightly, but significantly after the first week (from 4.39 +/- 0.64 to 2.87 +/- 0.47 at normal and from 11.96 +/- 1.44 to 4.76 +/- 0.78 microU pro 24 h pro islet at elevated glucose concentration, p < 0.05 and < 0.01, resp.) and remained unchanged thereafter. Glucose-stimulation resulted in significant increases in insulin secretion at all three testings (p < 0.001, < 0.01 and < 0.01). Single cells in alginate matrix had even more stable insulin secretion throughout the whole cultivation with significant increases to glucose challenge (p < 0.01, < 0.01 and < 0.05). In contrast, insulin secretion of free-floating islet cells decreased from 5.70 +/- 1.19 to 2.04 +/- 0.64 and to 1.05 +/- 0.33 at basal conditions (p < 0.01 and < 0.05) and from 11.39 +/- 1.87 to 2.76 +/- 0.76 and to 2.15 +/- 0.71 microU pro 24 h pro islet under stimulation (p < 0.01 and not sign). In addition, the secretory response to glucose challenge was significant only at the first testing (p < 0.05). Non-embedded islets could be tested only at the first week since after this time they dissociated to single cells. Embedded islets and single cells showed intact morphology after four weeks with trypan blue (TPB) positivity of less than 5%.(ABSTRACT TRUNCATED AT 250 WORDS)

[Is it permissible to treat young diabetics with oral antidiabetics?]. / Szabad-e fiatal cukorbetegeket orális antidiabetikumokkal kezelni?

Authors analyzed the case history of 25 young diabetic patients, whose disease has been diagnosed before the age of thirty. The question that has been raised: is it allowed to treat young diabetics with oral drugs? By classifying the patients, they stated followings: In the 1. group they classified 16 verified MODY/NIDDY patients. In the second group they classified 3 young diabetics, whose disease had been evaluated as slowly progressing IDDM (autoimmune form). 3 patients belonged to the 3 group. They had been classified as MODY/NIDDY patients, however an extremely long lasting remission period--due to the short observation time--can not be excluded. The remaining 3 diabetic patients belonged to the IDDM group, with a long remission period. They were treated incorrectly with oral hypoglycemic drugs. Young diabetics can be treated with oral drugs only in case, when they are proven MODY/NIDDY patients. The precise differential diagnosis between this form and autoimmune IDDM, as well as long lasting remission periode, is extremely important.