The Endoscopic Retrograde Cholangiopancreatography and Endoscopic Ultrasound Connection: Unity Is Strength, or the Endoscopic Ultrasonography Retrograde Cholangiopancreatography Concept.

Endoscopic ultrasound (EUS) and endoscopic retrograde cholangiopancreatography (ERCP) are both crucial for the endoscopic management of biliopancreatic diseases: the combination of their diagnostic and therapeutic potential is useful in many clinical scenarios, such as indeterminate biliary stenosis, biliary stones, chronic pancreatitis and biliary and pancreatic malignancies. This natural and evident convergence between EUS and ERCP, which by 2006 we were calling the "Endoscopic ultrasonography retrograde colangiopancreatography (EURCP) concept", has become a hot topic in the last years, together with the implementation of the therapeutic possibilities of EUS (from EUS-guided necrosectomy to gastro-entero anastomoses) and with the return of ERCP to its original diagnostic purpose thanks to ancillary techniques (extraductal ultrasound (EDUS), intraductal ultrasound (IDUS), cholangiopancreatoscopy with biopsies and probe-based confocal laser endomicroscopy (pCLE)). In this literary review, we retraced the recent history of EUS and ERCP, reported examples of the clinical applicability of the EURCP concept and explored the option of performing the two procedures in only one endoscopic session, with its positive implications for the patient, the endoscopist and the health care system. In the last few years, we also evaluated the possibility of combining EUS and ERCP into a single endoscopic instrument in a single step, but certain obstacles surrounding this approach remain.

A Surface-Enhanced Raman Spectroscopy-Based Biosensor for the Detection of Biological Macromolecules: The Case of the Lipopolysaccharide Endotoxin Molecules.

The development of sensitive methods for the detection of endotoxin molecules, such as lipopolysaccharides (LPS), is essential for food safety and health control. Conventional analytical methods used for LPS detection are based on the pyrogen test, plating and culture-based methods, and the limulus amoebocyte lysate method (LAL). Alternatively, the development of reliable biosensors for LPS detection would be highly desirable to solve some critical issues, such as high cost and a long turnaround time. In this work, we present a label-free Surface-Enhanced Raman Spectroscopy (SERS)-based method for LPS detection in its free form. The proposed method combines the benefits of plasmonic enhancement with the selectivity provided by a specific anti-lipid A antibody (Ab). A high-enhancing nanostructured silver substrate was coated with Ab. The presence of LPS was quantitatively monitored by analyzing the changes in the Ab spectra obtained in the absence and presence of LPS. A limit of detection (LOD) and quantification (LOQ) of 12 ng/mL and 41 ng/mL were estimated, respectively. Importantly, the proposed technology could be easily expanded for the determination of other biological macromolecules.

The Porcine Odorant-Binding Protein as a Probe for an Impedenziometric-Based Detection of Benzene in the Environment.

Odorant-binding proteins (OBPs) are a group of small and soluble proteins present in both vertebrates and insects. They have a high level of structural stability and bind to a large spectrum of odorant molecules. In the environmental field, benzene is the most dangerous compound among the class of pollutants named BTEX (benzene, toluene, ethylbenzene, and xylene). It has several effects on human health and, consequently, it appears to be important to monitor its presence in the environment. Commonly, its detection requires the use of very sophisticated and time-consuming analytical techniques (GC-MS, etc.) as well as the presence of specialized personnel. Here, we present the application of an odorant-binding protein (pOBP) isolated from pigs as a molecular recognition element (MRE) for a low-energy impedenziometric biosensor for outdoor and real-time benzene detection. The obtained results show that the biosensor can detect the presence of 64 pM (5 µg/m3) benzene, the limit value of exposure for human health set by the European Directive 2008/50/EC.

The porcine odorant-binding protein as molecular probe for benzene detection.

In recent years, air pollution has been a subject of great scientific and public interests for the strong impact on human health. Air pollution is due to the presence in the atmosphere of polluting substances, such as carbon monoxide, sulfur and nitrogen oxides, particulates and volatile organic compounds (VOCs), derived predominantly from various combustion processes. Benzene is a VOC belonging to group-I carcinogens with a toxicity widely demonstrated. The emission limit values and the daily exposure time to benzene (TLV-TWA) are 5µg/m3 (0.00157 ppm) and 1.6mg/m3 (0.5 ppm), respectively. Currently, expensive and time-consuming analytical methods are used for detection of benzene. These methods require to perform a few preliminary steps such as sampling, and matrices pre-treatments. In addition, it is also needed the support of specialized personnel. Recently, single-walled carbon nanotube (SWNTs) gas sensors with a limit detection (LOD) of 20 ppm were developed for benzene detection. Other innovative bioassay, called bio-report systems, were proposed. They use a whole cell (Pseudomona putida or Escherichia coli) as molecular recognition element and exhibit a LOD of about 10 µM. Here, we report on the design of a highly sensitive fluorescence assay for monitoring atmospheric level of benzene. For this purpose, we used as molecular recognition element the porcine odorant-binding protein (pOBP). 1-Aminoanthracene was selected as extrinsic fluorescence probe for designing a competitive fluorescence resonance energy transfer (FRET) assay for benzene detection. The detection limit of our assay was 3.9µg/m3, a value lower than the actual emission limit value of benzene as regulated by European law.

Cloning and bacterial expression systems for recombinant human heparanase production: Substrate specificity investigation by docking of a putative heparanase substrate.

Human heparanase (HPSE) is an enzyme that degrades the extracellular matrix. It is implicated in a multiplicity of physiological and pathological processes encouraging angiogenesis and tumor metastasis. The protein is a heterodimer composed of a subunit of 8 kDa and another of 50 kDa. The two protein subunits are noncovalently associated. The cloning and expression of the two protein subunits in Escherichia coli and their subsequent purification to homogeneity under native conditions result in the production of an active HPSE enzyme. The substrate specificity of the HPSE was studied by docking of a putative substrate that is a designed oligosaccharide with the minimum recognition backbone, with the additional 2-N-sulfate and 6-O-sulfate groups at the nonreducing GlcN and a fluorogenic tag at the reducing extremity GlcN. To develop a quantitative fluorescence assay with this substrate would be extremely useful in studies on HPSE, as the HPSE cleavage of fluorogenic tag would result in a measurable response.

A Fluorescence Polarization Assay To Detect Steroid Hormone Traces in Milk.

Steroids are a class of hormones improperly used in livestock as growth-promoting agents. Due to their high risk for human health, the European Union (EU) has strictly forbidden the administration of all natural and synthetic steroid hormones to food-producing animals, and the development of new rapid detection methods are greatly encouraged. This work reports a novel fluorescence polarization assay, ready to use, capable of detecting 17ß-estradiol directly in milk samples with a low limit of detection of <10 pmol. It is based on the coupling of monospecific antibodies against 17ß-estradiol and fluorophores, capable of modulating the fluorescence polarization emission on the basis of the specific binding of antibodies to fluorescence-labeled 17ß-estradiol derivative. The successful detection of 17ß-estradiol has disclosed the development of an efficient method, easily extensible to any food matrix and having the potential to become a milestone in food quality and safety.

Role of p16(INK4a) cytology testing as an adjunct to enhance the diagnostic specificity and accuracy in human papillomavirus-positive women within an organized cervical cancer screening program.

OBJECTIVE: We evaluated the performance of cytologic p16(INK4a) (p16) immunostaining within a cervical cancer screening program for the categories of atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LS after triage with high-risk human papillomavirus (HR-HPV) testing and atypical squamous cells, cannot exclude high-grade intraepithelial squamous lesion (ASC-H) and high-grade squamous intraepithelial lesion (HSIL). We also verified whether the routine introduction of p16 staining might enhance the specificity and positive predictive value (PPV) for cervical intraepithelial neoplasia grade 2 or higher (CIN2+) lesions predicted by a cytological screening test. STUDY DESIGN: Performance of the p16 cytology test was estimated in 578 cytological samples, of which 213 were HR-HPV+ ASC-US, 186 were HR-HPV+ LSIL, 74 were ASC-H, 56 were HSIL-CIN2 and 49 were HSIL-CIN3. All samples had histological follow-up. RESULTS: In the ASC-US category, p16 sensitivity was 91% for CIN2+ and 100% for CIN3, while specificity was 64 and 58%, respectively, negative predictive value (NPV) was 96 and 100%, respectively, and PPV was 39%. In the LSIL category, sensitivity was 77 and 75%, respectively, for CIN2+ and CIN3, while specificity was 64 and 57%, NPV was 93 and 98% and PPV was 30%. Sensitivity for ASC-H and HSIL-CIN3 was 100% for CIN2+ and CIN3, while for HSIL-CIN2 it was 91 and 95%, respectively; NPV for ASC-H was 100%, and for HSIL-CIN2 it was 43 and 86%, respectively. Follow-up examinations of 8 cases diagnosed as p16+ ASC-H and HSIL-CIN3, but histologically negative or CIN1 on the first biopsy, showed 4 CIN2 and 4 CIN3 lesions. CONCLUSIONS: Sensitivity, specificity, PPV and NPV confirm the importance of the utilization of p16 in the categories ASC-US and LSIL after triage with an HR-HPV test. In the ASC-H and HSIL-CIN3 lesions, p16 was shown to be an excellent marker for picking up CIN2+ lesions, especially in cases with cytohistological discordance.

Absorption into fluorescence. A method to sense biologically relevant gas molecules.

In this work we present an innovative optical sensing methodology based on the use of biomolecules as molecular gating nano-systems. Here, as an example, we report on the detection of analytes related to climate change. In particular, we focused our attention on the detection of nitric oxide (NO) and oxygen (O2). Our methodology builds on the possibility of modulating the excitation intensity of a fluorescent probe used as a transducer and a sensor molecule whose absorption is strongly affected by the binding of an analyte of interest used as a filter. The two simple conditions that have to be fulfilled for the method to work are: (a) the absorption spectrum of the sensor placed inside the cuvette, and acting as the recognition element for the analyte of interest, should strongly change upon the binding of the analyte and (b) the fluorescence dye transducer should exhibit an excitation band which overlaps with one or more absorption bands of the sensor. The absorption band of the sensor affected by the binding of the specific analyte should overlap with the excitation band of the transducer. The high sensitivity of fluorescence detection combined with the use of proteins as highly selective sensors makes this method a powerful basis for the development of a new generation of analytical assays. Proof-of-principle results showing that cytochrome c peroxidase (CcP) for NO detection and myoglobin (Mb) for O2 detection can be successfully used by exploiting our new methodology are reported. The proposed technology can be easily expanded to the determination of different target analytes.

Air-dried smears for optimal diagnostic immunocytochemistry.

OBJECTIVE: To exploit formalin-postfixed, air-dried smears for diagnostic immunocytochemistry (ICC). STUDY DESIGN: A series of 144 cases of diagnostic fine needle cytology samples in which air-dried, supplementary smears were available was used to exploit postfixation in the process of antigenic stabilization and rescue for immunocytochemical staining. RESULTS: Postfixation with formalin and with a formalin/ethanol solution gave comparable results as far as recovery and immunocytochemical detection of most monoclonal and polyclonal antibodies. The visualization of the antibody reactions was often superior to that obtained with wet-fixed slides, probably due to the interaction of slow cell dehydration with their consequent optimal flattening observed with formalin postfixation after short rehydration in physiologic saline. CONCLUSION: Although wet fixation of cytopathologic slides in 95% ethanol represents a common standard for ICC, the usage of formalin-postfixed air-dried smears proved reliable and efficient for antigenic rescue and may enter routine usage in cytopathology laboratories. Moreover, in some instances, the visual evaluation of results was easier in the larger, well-flattened cells obtained in air-dried cells.

The tryptophan phosphorescence of porcine and mutant bovine odorant-binding proteins: a probe for the local protein structure and dynamics.

Vertebrate odorant-binding proteins (OBPs) are small extracellular proteins belonging to the lipocalin superfamily. They have been supposed to play a role in events of odorant molecules detection by carrying, deactivating, and/or selecting odorant molecules. The OBPs share a conserved folding pattern, an eight-stranded beta-barrel flanked by an alpha-helix at the C-terminal end of the polypeptide chain. The beta-barrel creates a central nonpolar cavity whose role is to bind and transport hydrophobic odorant molecules. These proteins reversibly bind odorant molecules with dissociation constants ranging from nanomolar to micromolar range. In this work, we have studied the structural features of the OBP from pig and from cow by phosphorescence spectroscopy. The obtained results demonstrate that the indolic phosphorescence of the two studied proteins can be readily detected at ambient temperature solutions and that it is owed exclusively to the internal tryptophan residue located next to the ligand binding cavity, which is generally conserved in the mammalian OBPs. In addition, while both the phosphorescence spectrum and the lifetime yield a picture of the fold of the studied protein in good agreement with the protein crystallographic structures, the triplet probe points out that in solution the polypeptide structure of the both investigated OBPs exists as a multiplicity of slowly interconverting protein conformations. Finally, this work also demonstrates that it is possible to directly detect the binding of the ligands to OBPs as variations of the protein luminescence features, thus, representing the very first observation reported in the literature so far that a fast and direct assay can be used for monitoring the binding of ligands to OBPs.

The differences in the microenvironment of the two tryptophan residues of the glutamine-binding protein from Escherichia coli shed light on the binding properties and the structural dynamics of the protein.

Glutamine-binding protein (GlnBP) from Escherichia coli is a monomer (26 kDa) that is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. GlnBP consists of two domains (termed large and small) linked by two antiparallel beta-strands. The large domain is similar to the small domain but it contains two additional alpha-helices and three more short antiparallel beta-strands. The deep cleft formed between the two domains contains the ligand-binding site. The binding of L-glutamine leads to cleft closing and a significant structural change with the formation of the so-called "closed form" structure. The protein contains two tryptophan residues (W32 and W220) and 10 tyrosine residues. We used phosphorescence spectroscopy measurements to characterize the role of the two tryptophan residues in the protein structure in the absence and the presence of glutamine. Our results pointed out that the phosphorescence of GlnBP is easily detected in fluid solutions where the emission of the two tryptophan residues is readily discriminated by the drastic difference in the phosphorescence lifetime allowing the assignments of the short lifetime to W220 and the long lifetime to W32. In addition, our results showed that the triplet lifetime of the superficial W220 is unusually short because of intramolecular quenching by the proximal Y163. On the contrary, the lifetime of W32 is several hundred milliseconds long, implicating a well-ordered, compact fold of the surrounding polypeptide. The spectroscopic data were analyzed and discussed together with a detailed inspection of the 3D structure of GlnBP.

A strategic fluorescence labeling of D-galactose/D-glucose-binding protein from Escherichia coli helps to shed light on the protein structural stability and dynamics.

The D-glucose/D-galactose-binding protein (GGBP) of Escherichia coli serves as an initial component for both chemotaxis toward D-galactose and D-glucose and high-affinity active transport of the two sugars. GGBP is a monomer with a molecular weight of about 32 kDa that binds glucose with micromolar affinity. The sugar-binding site is located in the cleft between the two lobes of the bilobate protein. In this work, the local and global structural features of GGBP were investigated by a strategic fluorescence labeling procedure and spectroscopic methodologies. A mutant form of GGBP containing the amino acid substitution Met to Cys at position 182 was realized and fluorescently labeled to probe the effect of glucose binding on the local and overall structural organization of the protein. The labeling of the N-terminus with a fluorescence probe as well as the protein intrinsic fluorescence were also used to obtain a complete picture of the GGBP structure and dynamics. Our results showed that the binding of glucose to GGBP resulted in no stabilizing effect on the N-terminus portion of GGBP and in a moderate stabilization of the protein matrix in the vicinity of the ligand-binding site. On the contrary, it was observed that the binding of glucose has a strong stabilization effect on the C-terminal domain of the GGBP structure.

High-affinity binding of cadmium ions by mouse metallothionein prompting the design of a reversed-displacement protein-based fluorescence biosensor for cadmium detection.

Reported in this study are the experimental design and results of a protein-based biosensor for the detection of the cadmium in water using a reversed-displacement format. This reversed-displacement biosensor methodology has successfully measured cadmium in water by direct injection, eliminating the need for preconcentration or pretreatment of samples. A column containing Chelex resin saturated with Zn2+ and a rodhamine-labeled metallothionein (MT) comprised the assay reactive chamber. In fact, MT are small cysteine-rich proteins that bind heavy metals such as zinc, cadmium, copper, and mercury. Since the affinity for cadmium is higher than zinc and mercury, we used this intrinsic feature of MT to develop a fluorescence biosensor. The rodhamine-labeled MT was incubated with the Zn2+-saturated Chelex resin until binding equilibrium was reached. Under a constant flow, samples containing cadmium were introduced into the flow stream displacing the rodhamine-labeled MT. Limits of detection were lower than 0.5 microM for cadmium in water. Importantly, the addition of 1.0 microM Cu2+, 1.0 microM Zn2+, 1.0 microM Mg2+, or 1.0 microM Ca2+ did not cause the displacement of the rodhamine-labeled MT, indicating that the presence of these ions do not affect the specificity of the biosensor. Furthermore, we also demonstrated that the reversed-displacement format can be used to screen water samples containing cadmium, remains effective after dozens of cycles, and provides significant fluorescence response before regeneration is required.

Concomitant occurrence of facial cutaneous and parotid gland metastases from rectal cancer after preoperative chemoradiotherapy.

BACKGROUND: Unusual sites of metastasis to the parotid gland and face are reported in patients with colorectal cancer, but the localization to both sites at the same time has never been described so far. CASE REPORT: Here, we describe the case of a 76-year-old woman with a T3N1M0 G2 rectal adenocarcinoma, treated with preoperative chemoradiotherapy performed at suboptimal dosages due to unacceptable toxicity. At the end of the program, the re-staging demonstrated the presence of metastasis in both the left parotid gland and subcutaneously on the frontal region of her face while the primary locoregional tumour manifestation was radiologically down-staged (reduction in N staging from N1 to N0). The patient did not respond to any other treatment and died due to disease progression 15 months after the diagnosis. CONCLUSIONS: Metastatic tumours in the parotid gland are uncommon with a higher incidence of primary sites of the head and neck. Parotid involvement of rectal adenocarcinoma is also extremely rare, and concomitant involvement of both the parotid gland and the face was not previously described. In this case, we cannot rule out the hypothesis that the delay of surgical removal of the primary tumour and/or a specific action of concomitant chemoradiotherapy on the tumour cell phenotype could promote cancer cell spreading to unusual sites.

Spindle cell neuroendocrine carcinoma of the lung: report of a case with fine needle aspiration cytology and differential diagnostic considerations.

BACKGROUND: Spindle cell neuroendocrine carcinomas of the lung may be frequently observed on fine needle cytology (FNC) samples and often pose stimulating differential diagnostic problems. CASE: The cytopathologic findings from FNC performed on a long-standing coin lesion of the lung in a 54-year-old woman were analyzed in view of the data and long clinical history. CONCLUSION: A final diagnosis of low grade spindle cell neuroendocrine carcinoma was reached by combining cytopathologic and immunocytochemical information. The main lesions considered in the differential diagnosis were intrapulmonary inflammatory myofibroblastic tumor (fibrohistiocytic variant) and spindle cell thymoma.

Vinorelbine plus 3-weekly trastuzumab in metastatic breast cancer: a single-centre phase 2 trial.

BACKGROUND: After two studies reporting response rates higher than 70% in HER2-positive metastatic breast cancer with weekly trastuzumab and vinorelbine, we planned a phase 2 study to test activity of the same combination, with trastuzumab given every 3 weeks. METHODS: Patients with HER2-positive metastatic breast cancer (3+ at immunohistochemistry or positive at fluorescence in situ hybridization), PS < or =2, normal left-ventricular ejection fraction (LVEF) and no more than one chemotherapy line for metastatic disease were eligible. Vinorelbine (30 mg/m2) was given on days 1 & 8 every 21 and trastuzumab (8 mg/kg day 1, then 6 mg/kg) every 21 days). A single-stage phase 2 design, with p0 = 0.45, p1 = 0.65, type I and II error = 0.10, was applied; 22 objective responses were required in 39 patients. RESULTS: From Nov 2002 to May 2005, 50 patients were enrolled, with a median age of 54 years (range 31-81). Among 40 patients eligible for response assessment, there were 7 complete and 13 partial responses (overall response rate 50%; 95% exact CI 33.8-66.2); 11 patients had disease stabilization, lasting more than 6 months in 10 cases. Response rate did not vary according to patients and tumor characteristics, type and amount of previous chemotherapy. Within the whole series, median progression-free survival was 9.6 months (95% CI 7.3-12.3), median overall survival 22.7 months (95% CI 19.5-NA). Fifteen patients (30%) developed brain metastases at a median time of 12 months (range 1-25). There was one toxic death due to renal failure in a patient receiving concomitant pamidronate. Twenty-three patients (46%) had grade 3-4 neutropenia, 2 (4%) grade 3 anemia, 4 (8%) febrile neutropenia. Two patients stopped treatment because of grade 2 decline of LVEF and one patient because of grade 2 liver toxicity concomitant with a grade 1 decline of LVEF. One patient stopped trastuzumab after 50 cycles because of grade 1 decline of LVEF. CONCLUSION: Although lower than in initial studies, activity of 3-weekly trastuzumab plus vinorelbine fell within the range of results reported with weekly schedules. Toxicity was prevalently manageable. This combination is safe and active for metastatic breast cancer patients who received adjuvant taxanes with anthracyclines.

Conformational stability and domain coupling in D-glucose/D-galactose-binding protein from Escherichia coli.

The monomeric D-glucose/D-galactose-binding protein (GGBP) from Escherichia coli (M(r) 33000) is a periplasmic protein that serves as a high-affinity receptor for the active transport and chemotaxis towards both sugars. The effect of D-glucose binding on the thermal unfolding of the GGBP protein at pH 7.0 has been measured by differential scanning calorimetry (DSC), far-UV CD and intrinsic tryptophanyl residue fluorescence (Trp fluorescence). All three techniques reveal reversible, thermal transitions and a midpoint temperature (T(m)) increase from 50 to 63 degrees C produced by 10 mM D-glucose. Both in the absence and presence of D-glucose a single asymmetric endotherm for GGBP is observed in DSC, although each endotherm consists of two transitions about 4 degrees C apart in T(m) values. In the absence of D-glucose, the protein unfolding is best described by two non-ideal transitions, suggesting the presence of unfolding intermediates. In the presence of D-glucose protein, unfolding is more co-operative than in the absence of the ligand, and the experimental data are best fitted to a model that assumes two ideal (two-state) sequential transitions. Thus D-glucose binding changes the character of the GGBP protein folding/unfolding by linking the two domains such that protein unfolding becomes a cooperative, two two-state process. A K(A)' value of 5.6x10(6) M(-1) at 63 degrees C for D-glucose binding is estimated from DSC results. The domain with the lower stability in DSC measurements has been identified as the C-terminal domain of GGBP from thermally induced Trp fluorescence changes.

A novel fluorescence competitive assay for glucose determinations by using a thermostable glucokinase from the thermophilic microorganism Bacillus stearothermophilus.

We describe the use of a thermostable glucokinase in a novel competitive fluorescence assay for glucose. Glucokinase from Bacillus stearothermophilus (BSGK) was found to retain enzymatic activity in solution for over 20 days. The single cysteine residue in BSGK, which is near the active site, was labeled with a fluorescent probe, 2-(4-iodoacetamidoanilino)naphthalene-6-sulfonic acid. The ANS-labeled BSGK displayed a modest 25% decrease in the emission intensity upon binding glucose but no change in lifetime. To obtain a larger spectral change we developed a competitive assay for glucose using the intrinsic tryptophan fluorescence from BSGK and a resonance energy transfer (RET) acceptor-labeled sugar. The sugar-labeled acceptor quenched the BSGK tryptophan emission, and the quenching was reversed upon addition of glucose. The use of RET as the sensing mechanism can be easily extended to longer wavelengths for a more practical glucose sensor.