Competitive inhibition assay for the detection of progesterone in dairy milk using a fiber optic SPR biosensor.

Analytical methods that are often used for the quantification of progesterone in bovine milk include immunoassays and chromatographic techniques. Depending on the selected method, the main disadvantages are the cost, time-to-result, labor intensity and usability as an automated at-line device. This paper reports for the first time on a robust and practical method to quantify small molecules, such as progesterone, in complex biological samples using an automated fiber optic surface plasmon resonance (FO-SPR) biosensor. A FO-SPR competitive inhibition assay was developed to determine biologically relevant concentrations of progesterone in bovine milk (1-10 ng/mL), after optimizing the immobilization of progesterone-bovine serum albumin (P4-BSA) conjugate, the specific detection with anti-progesterone antibody and the signal amplification with goat anti-mouse gold nanoparticles (GAM-Au NPs). The progesterone was detected in a bovine milk sample with minimal sample preparation, namely ½ dilution of the sample. Furthermore, the developed bioassay was benchmarked against a commercially available ELISA, showing excellent agreement (R2 = 0.95). Therefore, it is concluded that the automated FO-SPR platform can combine the advantages of the different existing methods for quantification of progesterone: sensitivity, accuracy, cost, time-to-result and ease-of-use.

Real-time PCR melting analysis with fiber optic SPR enables multiplex DNA identification of bacteria.

A fiber optic surface plasmon resonance (FO-SPR) technology was developed that enables simultaneous quantification and identification of multiple DNA targets on the same platform. The bioassay was based on the hybridization/melting of DNA-coated Au nanoparticles on the FO-SPR sensor when targets are present. The multiplex concept was successfully demonstrated on two related bacteria and for detection of multiple mutations in sequences. In conclusion, FO-SPR technology shows a great potential as a next generation in vitro diagnostics tool.

Ara h 1 protein-antibody dissociation study: evidence for binding inhomogeneities on a molecular scale.

The characterization of biomolecular interactions is essential when designing novel biosensors, since the interaction between the bioreceptor and the ligand determines important biosensing parameters such as sensitivity and selectivity. In this paper we study the interaction of the trimeric Ara h 1 protein with a monoclonal anti-Ara h 1 antibody by means of magnetic force-induced dissociation. The proteins were bound to magnetic particles and polystyrene surfaces by EDC/NHS reaction chemistry and by physisorption, respectively. Two different molecular configurations have been investigated, with either the Ara h 1 protein on the particles or the Ara h 1 protein on the polystyrene surface. A model with a Gaussian distribution of energy barriers for dissociation gives an adequate description for the measured multi-exponential decays. We hypothesize that distributions of molecular orientations as well as experimentally induced variations may underlay the observed distributions. The two molecular configurations show a different peak value of the energy distribution. Similarly, SPR experiments for two distinct configurations (either Ara h 1 protein on the surface, or anti-Ara h 1 antibody on the surface) also show clear differences in dissociation behavior. We hypothesize that the multivalency of the involved molecules leads to different modes of binding. The results of this work highlight the importance of molecular inhomogeneities when studying the interaction processes of biomolecular complexes.

Enabling fiber optic serotyping of pathogenic bacteria through improved anti-fouling functional surfaces.

Significant research efforts are continually being directed towards the development of sensitive and accurate surface plasmon resonance biosensors for sequence specific DNA detection. These sensors hold great potential for applications in healthcare and diagnostics. However, the performance of these sensors in practical usage scenarios is often limited due to interference from the sample matrix. This work shows how the co-immobilization of glycol(PEG) diluents or 'back filling' of the DNA sensing layer can successfully address these problems. A novel SPR based melting assay is used for the analysis of a synthetic oligomer target as well as PCR amplified genomic DNA extracted from Legionella pneumophila. The benefits of sensing layer back filling on the assay performance are first demonstrated through melting analysis of the oligomer target and it is shown how back filling enables accurate discrimination of Legionella pneumophila serogroups directly from the PCR reaction product with complete suppression of sensor fouling.

Immunohistochemical detection of cyclin E in transitional cell carcinoma.

PURPOSE: It is known that expression disorders of cell cycle regulators play an important role in the development and prognosis of various malignant tumors. Cyclin expression changes during the cell cycle. This work aimed to analyse the expression of cyclin E in transitional cell carcinoma (TCC) and also to compare the expression of cyclin E with tumor stage and histological grade as well as to determine possible existence of differences in the expression of cyclin E in TCCs of the upper and lower urothelium. METHODS: Twenty-four cases of TCC of the urinary tract were retrospectively analysed (6 cancers of the renal pelvis, 2 of the ureter and 15 of the bladder; 4 were infiltrative). Immunohistochemical staining for cyclin E of the analysed transitional cancer cells was assessed semiquantitatively: diffuse cyclin E expression + + + (> 50% of all cells), expression in larger groups of cells: + + (up to 50% of all cells), expression in individual cells or small cell clusters: + (<10% of all cells), and absence of expression. Tumor stage was based on clinical and morphological criteria. WHO classification (Lyon 2004) was used for determination of the histological grade. RESULTS: Non-parametric Spearman's correlation showed that there was no statistically significant correlation between tumor stage and expression of cyclin E (ρ = -0331, p> 0.05). Also, no statistically significant correlation between grade and the expression of cyclin E (ρ = -0077, p> 0.05) was found. x2 test results showed no statistically significant difference (x2 = 2.136, p = 0.775) in the expression of cyclin E between upper and lower urothelium. CONCLUSION: This study showed non significant decreased expression of cyclin E with poor differentiation, muscle invasion and upper/lower urothelium. Expression of cyclin E decreased with increasing histological grade and stage of the tumor.

[Aorto-enteric fistulas]. / Aorto-entericne fistule.

INTRODUCTION: The aorto-enteric fistula (AEF) is a direct communication between aorta and intestinal lumen. There are primary and secondary forms. Primary AEFs are usually due to erosion of an aortic aneurysm (AAA) into the intestine, while secondary forms are caused by reconstructive procedures on the abdominal aorta. The incidence of primary AEF ranges from 0.1 to 0.8%, and secondary from 0.4% to 2.4% [2-4]. The mortality rate after surgical treatment of secondary AEFs is from 14% to 70% [5]. Therefore, they are of great medical importance. The aim of this paper is the presentation of 9 new cases. METHODS: Over a 33-year period (1966-1999) a retrospective analysis of patients' records identified 9 patients with AEFs. All were males with average age of 66.62 (51-70) years. In Tables 1 and 2 are presented data on our cases. Of the total number of 9 patients, there were 4 primary and 5 secondary AEFs. All primary fistulas were caused by AAA rupture. Secondary AEFs developed after aortic abdominal surgery in the period between one and seven years after the operation. In 7 cases fistula involved the duodenum, in one the sigmoid and in one the transversal colon. The dominant manifestation of fistulas was gastrointestinal bleeding: melaena--8 (89%); haematemesis and melaena--2 (22%); proctorrhagia--1 (11%). In cases of primary AEFs gastrointestinal bleeding was followed by low back pain and haemorrhagic shok, while in cases of secondary AEFs by sepsis (fever, increased leucocytes count, sedimentation). In two cases the final diagnosis was established by gastrography and colonoscopy, while in two patients Duplex ultrasonographic examination suspected AEF. In all other cases the diagnosis was established intraoperatively (Figure 1). After aneurysmal resection in cases of primary AEFs, revascularization of the lower limbs was performed with extra-anatomic axillo-bifemoral bypass graft (one case) and with "in situ" graft placement (three cases) (Figure 2). The duodenal defect was closed transversally with standard two layers suture techniques in two patients without fistula excision, and in two cases after fistulas excision. In one case associated gastero-entero and entero-entero anastomosis was performed. In all cases with secondary AEFs, after removing of the previously implanted aortic graft, the aorta was closed just below the renal arteries root, and wrapped with a vascularized pedicle of omentum, to separate it from the bowel and the contained area. The duodenal defect was closed after fistulas excision using two layers transversal suture technique in two cases, and in one patient with large fistula a partial duodenectomy and Roux's procedure were necessary. In two patients in whom AEFs involved the transversal and sigmoid colon colostoma was performed. In three cases an extra-anatomic axillo-bifemoral bypass graft was performed for lower limbs revascularization, and in one patient bypass from the ascendent aorta to the femoral artery, using retroperitoneal route was carried out. In one patient the revascularization of the lower limbs was not done because of intraoperative death of the patient. RESULTS: Seven of our patients died during the first 15 postoperative days. One died during the operation after massive acute myocardial infarction. In other six cases the mortality causes were: MOFS-3 cases, and secondary enteric fistula-3 cases. Two of our patients survived. One has been followed-up for 15 years, and his axillo-bifemoral bypass is patent. The other with bypass from the ascendent aorta to the femoral artery died 7 years after the operation, also with patent graft. More details are given in Table 3. DISCUSSION: Sir Astley Cooper was the first who described primary AEFs caused by AAA rupture in 1817 [6], and Brock in 1953, first described secondary AEF developed 6 months after aortic homograft implantation [8]. In 1957, Haberer successfully treated primary AEF by suture of the duodenal defect and aneurysmorrhaphy [9]. In our country Stojanovitsh and Vujadinovitsh in 1966, first treated primary AEF [16]. Their patient died due to MOFS. However, in 1984 and 1985, Lotina successfully treated two patients with secondary AEFs [11] (Figure 3, Sheme 1). The authors also analyzed literature data on the aetiology, pathogenesis, clinical manifestations, diagnosis and treatment of AEFs. In conclusion, the authors suggest: 1. "Omega" extra-anatomic bypass from supraceliac artery trough retroperitonely to femoral arteries; 2. "In situ" replacement of the abdominal aorta using cadaveric homografts; 3. Intraoperative control of bleeding with endoluminal balloon occlusive aortic catheter.

Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.

We investigated the effects of various primer-template mismatches on DNA amplification of an HIV-1 gag region by the polymerase chain reaction (PCR). Single internal mismatches had no significant effect on PCR product yield while those at the 3'-terminal base had varied effects. A:G, G:A, and C:C mismatches reduced overall PCR product yield about 100-fold, A:A mismatches about 20-fold. All other 3'-terminal mismatches were efficiently amplified, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches. It should be noted that mismatches of T with either G, C, or T had a minimal effect on PCR product yield. Double mismatches within the last four bases of a primer-template duplex where one of the mismatches is at the 3' terminal nucleotide, in general, reduced PCR product yield dramatically. The presence of a mismatched T at the 3'-terminus, however, allowed significant amplification even when coupled with an adjacent mismatch. Furthermore, even two mismatched Ts at the 3'-terminus allowed efficient amplification.