Strategies for delivery of faecal occult blood test kits and participation to colorectal cancer screening in the Emilia-Romagna Region of Italy.

There is a lack of agreement about which routine invitation strategy should be adopted in colorectal cancer screening. We conducted an observational study to assess the impact of three invitation strategies on participation. Invitation records for the years 2005-2009 were evaluated. There were 2,234,276 invitations from 1,230,683 individuals. Among first invitations, participation associated with direct mailing of the faecal occult blood test kits was slightly lower (relative risk, RR 0.985; 95% confidence interval 0.979-0.990) than that of the reference invitation strategy, that is, the distribution of the test kits by pharmacies. In repeated invitations/previous non-responders, the participation associated with the direct mailing of the test kits was even lower (RR 0.914; 95% confidence interval 0.895-0.933) and this was also the case for the distribution of the test kits by primary care centres (RR 0.983; 95% confidence interval 0.971-0.995). In contrast, in repeated invitations/previous responders, the impact of primary care centres and direct mailing of the test kits was greater than the use of pharmacies, showing only modest RRs: 1.021 (95% confidence interval 1.019-1.023) and 1.029 (95% confidence interval 1.025-1.033) respectively. The faecal occult blood test mailing strategy modestly increased participation in previous responders.

Furan-PNA: a mildly inducible irreversible interstrand crosslinking system targeting single and double stranded DNA.

We here report on the design and synthesis of tailor-made furan-modified peptide nucleic acid (PNA) probes for covalent targeting of single stranded DNA through a crosslinking strategy. After introducing furan-containing building blocks into a PNA sequence, hybridization and furan-oxidation based crosslinking to DNA is investigated. The structure of the crosslinked products is characterized and preliminary investigations concerning the application of these systems to double stranded DNA are shown.

Occurrence of deoxynivalenol and its 3-beta-D-glucoside in wheat and maize.

Deoxynivalenol-3-beta-D-glucoside (D3G), a phase II plant metabolite of the mycotoxin deoxynivalenol (DON), occurs in naturally Fusarium-contaminated cereals. In order to investigate the frequency of occurrence as well as the relative and absolute concentrations of D3G in naturally infected cereals, 23 wheat samples originating from fields in Austria, Germany and Slovakia as well as 54 maize samples from Austrian fields were analysed for DON and D3G by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both analytes were detected in all the 77 field samples. DON was found at levels from 42 to 4130 ng g(-1) (977 +/- 1000 ng g(-1) on average). The D3G concentrations in all cereal samples were in the range 10-1070 ng g(-1) (216 +/- 253 ng g(-1) on average), corresponding to about 5-46 mol% of their DON concentrations (15 +/- 8 mol% on average).

Complexation of the mycotoxin zearalenone with ß-cyclodextrin: Study of the interaction and first promising applications.

This work reports the study of the interactions between native and substituted ß-cyclodextrins and zearalenone and its derivatives α- and ß-zearelonol. The data obtained by fluorescence and NMR experiments suggested that zearalenone, α- and ß-zearalenol and cyclodextrins give rise to host-guest complexation, with the inclusion of the phenolic moiety inside the cyclodextrin cavity. The high stability of these complexes induces a high fluorescence enhancement upon complexation. These results have been successfully applied to the spectrofluorimetric determination of zearalenone in maize raw samples, without any chromatographic separation.

Development of a peptide nucleic acid polymerase chain reaction clamping assay for semiquantitative evaluation of genetically modified organism content in food.

In the present study a peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method was developed and applied to the detection of genetically modified organisms (GMO), to test PCR products for band identity and to obtain a semiquantitative evaluation of GMO content. The minimal concentration of PNA necessary to block the PCR was determined by comparing PCRs containing a constant amount of DNA in the presence of increasing concentration of target-specific PNA. The lowest PNA concentration at which specific inhibition took place, by the inhibition of primer extension and/or steric hindrance, was the most efficient condition. Optimization of PCR clamping by PNA was observed by testing five different PNAs with a minimum of 13 bp to a maximum of 15 bp, designed on the target sequence of Roundup Ready soybean. The results obtained on the DNA extracted from Roundup Ready soybean standard flour were verified also on DNA extracted from standard flours of maize GA21, Bt176, Bt11, and MON810. A correlation between the PNA concentration necessary for inducing PCR clamping and the percentage of the GMO target sequence in the sample was found.

Design and synthesis of fluorescent beta-cyclodextrins for the enantioselective sensing of alpha-amino acids.

Fluorescent monofunctionalized beta-cyclodextrins bearing a copper(II) binding side arm and a dansyl group (CD-NH-AA-CH(2)CH(2)NH-DNS) were designed as enantioselective sensors for unmodified alpha-amino acids. The side arm was derived from amino acid synthons (AA = L- and D-phenylalanine (1 and 2), L- and D-phenylglycine (3 and 4), L-proline (5), and L-cyclohexylglycine (6)) and was chosen in order to contain an amide, an amine, and a sulphonamide group. Enantioselectivity was evaluated by addition of copper(II) complexes of D- or L-valine and D- or L-proline. Chiral discrimination in the fluorescence response was observed in all cases, due to a ligand exchange process. The best conditions for these experiments were found to be the use of an excess (10:1) of the copper complex. The cyclodextrin 4 containing a D-phenylglycine unit was found to be poorly enantioselective, as found for 2, suggesting that the best design can be obtained by using L-amino acids. All L-amino acid containing cyclodextrins showed good enantioselectivities, some of which were higher than those already reported for 1. Other analytes related to amino acids were studied using cyclodextrins 1 and 3. Enantiomers of alpha,alpha-disubstituted amino acids, N-methylamino acids, and amino acid amides were found to be discriminated, while beta-phenylalanine and other molecules bearing a poor anchoring group at the alpha-carbon gave poor enantioselectivity. On the basis of the present data a model for the recognition process, based on the formation of ternary diastereomeric complexes, is proposed.

Case Reports. Chronic and acute Aspergillus meningitis.

Cerebral aspergillosis usually occurs in severely immunocompromized hosts, is difficult to diagnose, and has a poor prognosis. After 14 months of chronic meningitis, ventriculitis, choroid plexitis, and lumbar arachnoiditis, which was complicated by acute hydrocephalus, Aspergillus, suspected to be from the candidus group, was isolated from the cerebrospinal fluid (CSF) of a previously healthy man. Thereafter Aspergillus antigen was found in stored plasma and CSF samples. He was treated with voriconazole and itraconazole. In a haemodialysis patient affected by an acute meningococcal meningitis, following a 3-day symptom-free interval, symptoms and signs of acute meningitis had reappeared and were unresponsive to a broad antimicrobial coverage. However, they resolved within 5 days after liposomal amphotericin B treatment had been started. From his CSF Aspergillus-DNA was identified and Aspergillus fumigatus isolated by culture. These two different clinical cases show that Aspergillus-DNA and antigen detection tests represent an advance in the diagnosis and liposomal amphotericin B, voriconazole, and itraconazole are an advance in the treatment of Aspergillus meningitis.

Peptide nucleic acids and biosensor technology for real-time detection of the cystic fibrosis W1282X mutation by surface plasmon resonance.

In this paper we demonstrate that peptide nucleic acids (PNAs) are excellent probes able to detect the W1282X point mutation of the cystic fibrosis (CF) gene when biospecific interaction analysis (BIA) by surface plasmon resonance (SPR) and biosensor technologies is performed. The results reported here suggest that BIA is an easy, fast, and automatable approach for detecting mutations of CF, allowing real-time monitoring of hybridization between 9-mer CF PNA probes and target biotinylated PCR products generated from healthy, heterozygous subjects and homozygous W1282X samples and immobilized on streptavidin-coated sensor chips. This method is, to our knowledge, the first application of PNAs, BIA, and SPR to a human hereditary mutation, and demonstrates the feasibility of these approaches for discriminating between normal and mutated target DNA. We like to point out that the procedure described in this paper is rapid and informative; results are obtained within a few minutes. This could be of great interest for molecular pre-implantation diagnosis to discriminate homozygous CF embryos from heterozygous and healthy embryos. Other advantages of the methodology described in the present paper are (a) that it is a nonradioactive methodology and (b) that gel electrophoresis and/or dot-spot analysis are not required. More importantly, the demonstration that SPR-based BIA could be associated with microarray technology allows us to hypothesize that the method described in the present paper could be used for the development of a protocol employing multispotting on SPR biosensors of many CF-PCR products and a real-time simultaneous analysis of hybridization to PNA probes. These results are in line with the concept that SPR could be an integral part of a fully automated diagnostic system based on the use of laboratory workstations, biosensors, and arrayed biosensors for DNA isolation, preparation of PCR reactions, and identification of point mutations.

Enantiomeric separation of hydroxy acids and carboxylic acids by diamino-beta-cyclodextrins (AB,AC,AD) in capillary electrophoresis.

Selectively modified 6,6'-dideoxy-6,6'-L-diamino-beta-cyclodextrins (AB, AC, AD) were successfully used as chiral selectors for the enantiomeric separation of hydroxy acids and carboxylic acids (in particular, phenoxyalkanoic acid herbicides) in capillary electrophoresis (CE). Chiral separations were obtained at a low selector concentration (1 mM) with good enantioselectivity and resolution factors. Separations were optimized as a function of pH. The different position of the charged groups on the upper rim greatly influenced the separation, accounting for electrostatic interactions between the protonated amino groups of the cyclodextrins (CDs) and the carboxylate of the selectands. The best enantiomeric separation of hydroxy acids was obtained with the AC regioisomer, whereas carboxylic acids were well resolved only by the AB regioisomer. A recognition model is proposed, based on two-dimensional nuclear magnetic resonance (2-D NMR) experiments, in which the orientation of the guest in the inclusion complex is determined by the electrostatic interactions between the selectand and the CD upper rim.

Chiral separation of amino acids by copper(II) complexes of tetradentate diaminodiamido-type ligands added to the eluent in reversed-phase high-performance liquid chromatography: a ligand exchange mechanism.

In this paper we report a study on the mechanism of the enantiomeric separation of unmodified D,L-amino acids in RP-HPLC by copper(II) complexes of two tetradentate diaminodiamido ligands, (S,S)-N,N'-bis(phenylalanyl)ethanediamine (PheNN-2) and (S,S)-N,N'-bis(methylphenylalanyl)ethanediamine (Me2PheNN-2), added to the eluent. The aim is to investigate whether and how a copper(II) complex with no free equatorial positions can perform chiral discrimination of bidentate analytes such as unmodified amino acids. The problem is approached in a systematic way by: (a) varying the different chromatographic parameters (pH, selector concentration, eluent polarity); (b) performing chiral separation with the selector adsorbed on the stationary phase; (c) studying the ternary complex formation of these ligands with D- and L-amino acids in solution by glass electrode potentiometry and electrospray ionization MS. All the experimental data are consistent with a mechanism of chiral recognition, based on ligand exchange, which involves as selectors the species [Cu2L2H(-2)]2+ and [CuLH(-2)] and proceeds by displacement of two binding sites from the equatorial positions, giving rise to the ternary species [CuLA]+ and [CuLH(-1) A]. The most important factor responsible for chiral discrimination seems to be the affinity of the diastereomeric ternary complexes for the stationary phase since no enantioselectivity is observed in solution.

Recognition and strand displacement of DNA oligonucleotides by peptide nucleic acids (PNAs). High-performance ion-exchange chromatographic analysis.

Peptide nucleic acids (PNAs) are oligonucleotide mimics containing a pseudopeptide chain, which are able to bind complementary DNA tracts with high affinity and selectivity. Two mixed-sequence PNA undecamers (1 and 2) were synthesized and their double-stranded adducts with the complementary oligonucleotides (3 and 4) were revealed by the appearance of the corresponding peak in anion-exchange HPLC. A DEAE column was used and elution was performed with aqueous Tris buffer (pH 8) and an ionic strength gradient (0-0.5 M NaCl). The same effect was not observed with non-complementary oligonucleotides. The stability of the PNA-DNA adducts under the conditions used in the chromatographic system was studied as a function of temperature. Furthermore, in competition experiments double-stranded oligonucleotides were challenged by a PNA complementary to one strand: the formation of the PNA-DNA hybrid and the displacement of the non-complementary strand were observed with high specificity. The results suggest a possible use of ion-exchange HPLC for studying PNA-DNA interactions, and indicate the efficiency of PNA probes in the chromatographic analysis of DNA.

Inhibition of RNA polymerase III elongation by a T10 peptide nucleic acid.

The terminator elements of eukaryotic class III genes strongly contribute to overall transcription efficiency by allowing fast RNA polymerase III (pol III) recycling. Being constituted by a run of thymidine residues on the coding strand (a poly(dA) tract on the transcribed strand), pol III terminators are expected to form highly stable triple-helix complexes with oligothymine peptide nucleic acids (PNAs). We analyzed the effect of a T10 PNA on in vitro transcription of three yeast class III genes (coding for two different tRNAs and the U6 small nuclear RNA) having termination signals of at least ten T residues. At nanomolar concentrations, the PNA almost completely inhibited transcription of supercoiled, but not linearized, templates in a sequence-specific manner. The total RNA output of the first transcription cycle was not affected by PNA concentrations strongly inhibiting multiple round transcription. Thus, an impairment of pol III recycling fully accounts for the observed inhibition. As revealed by the size and the state (free or transcription complex-associated) of the RNAs produced in PNA-inhibited reactions, pol III is "roadblocked" by the DNA-PNA adduct before reaching the terminator region. On different templates, the distance between the active site and the leading edge of the arrested polymerase ranged from 10 to 20 base pairs. Given their ability to efficiently block pol III elongation, oligothymine PNAs lend themselves as potential cell growth inhibitors interfering with eukaryotic class III gene transcription.

Histamine-modified cationic beta-cyclodextrins as chiral selectors for the enantiomeric separation of hydroxy acids and carboxylic acids by capillary electrophoresis.

The enantiomeric separation of alpha-hydroxy acids and carboxylic acids was successfully performed by using 6-deoxy-6-N-histamino-beta-cyclodextrin (CD-hm), a monosubstituted positively charged beta-cyclodextrin (beta-CD) bearing a histamine moiety linked to the C6 of a glucose unit in the upper CD rim via the amino group. Good results were obtained at a low selector concentration (1 mM). The number of positive charges on the upper rim may be modulated as a function of pH, because of the different pKa of the amino and the imidazolyl groups, and was found to affect both the enantioselectivity and resolution factors. With the analogous 6-deoxy-[4-(2-aminoethyl)imidazolyl]-beta-cyclodextrin (CD-mh) bearing the histamine moiety linked to the C6 via the imidazolyl group, very poor results were obtained, showing that the proximity of the positive charge to the cavity plays an important role in the enantiomeric recognition. The complexation mode was studied by electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional nuclear magnetic resonance (2-D NMR) ROESY experiments: the recognition model is consistent with an inclusion complexation of the aromatic ring of the analyte within the CD cavity coupled to electrostatic interactions between the carboxylate and the protonated amino group of the cyclodextrin.

Herpes simplex virus infections of the central nervous system in human immunodeficiency virus-infected patients: clinical management by polymerase chain reaction assay of cerebrospinal fluid.

A duplex polymerase chain reaction (PCR) assay for DNA from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) was applied to cerebrospinal fluid (CSF) from 918 human immunodeficiency virus (HIV)-infected patients with neurological symptoms. HSV-1 or HSV-2 (HSV-1/2) DNA was found in 19 patients (2%). For the 258 patients for whom a diagnosis was confirmed at autopsy, the sensitivity and specificity of PCR analysis for the diagnosis of HSV-1/2 encephalitis were 100% and 99.6%, respectively. Three patients with CD4+ cell counts of > or = 170/microL had HSV-1 central nervous system (CNS) infections (two) or HSV-2 meningitis (one). Sixteen patients with CD4+ cell counts of < 40/microL had HSV-1 CNS infections (two) or mixed HSV-1/2 and cytomegalovirus encephalitis (14). The response to antiviral treatment, which was assessed clinically and by CSF PCR analysis, was variable in the patients with the highest CD4+ cell counts and poor in those with more severe immunosuppression. CSF PCR analysis is of value for the diagnosis and follow-up of treatment of HSV-1/2 CNS infections in HIV-infected patients.

Histamine-modified beta-cyclodextrins for the enantiomeric separation of dansyl-amino acids in capillary electrophoresis.

Two novel monosubstituted beta-cyclodextrins (CD) bearing the histamine moiety linked to the upper CD rim, either through the amino group or the imidazole nitrogen 1N, CD-hm and CD-mh, were successfully used as chiral selectors in capillary electrophoresis for the enantiomeric discrimination of dansyl (Dns)-amino acids. Good results were obtained by using low concentrations of the selectors (1-3 mM). The effect of pH on the chiral discrimination was studied in order to modulate the number and the position of the positive charges. By increasing the pH from 5 to 7.5, chiral discrimination decreased along with the deprotonation of the imidazolyl moiety. Inversion of the migration order was observed with the two CDs, depending on the relative position of the charged moieties on the upper rim. Ion pair interaction coupled to inclusion complexation seems to account for the discrimination process. The effects of the temperature, CD concentration and capillary length on chiral resolution were also examined.

Orellanine poisoning: rapid detection of the fungal toxin in renal biopsy material.

BACKGROUND: Mushroom poisoning by some species of the Cortinarius (Agaricales) often lead to irreversible renal failure caused by the nephrotoxin orellanine. In 1994 and 1995, six poisoning outbreaks involving ten individuals in Northern Italy and in Austria were investigated. METHODS: A total of 87 clinical samples (urine and blood samples including renal biopsy material of three patients) were examined for the presence of orellanine by thin layer chromatography. RESULTS: Orellanine can be detected after a relatively long period following poisoning by performing a simple thin layer chromatography technique using small quantities of renal biopsy material. No toxin was found in urine or blood samples. CONCLUSIONS: Orellanine is rapidly concentrated in the kidneys in a relatively soluble form and cannot be detected in urine, blood and dialysis fluids at the time when first symptoms appear.

Chiral recognition by the copper(II) complex of 6-deoxy-6-N-(2-methylaminopyridine)-beta-cyclodextrin.

A modified beta-cyclodextrin bearing a 2-aminomethylpyridine binding site for copper(II) (6-deoxy-6-[N-(2-methylamino)pyridine)]-beta-cyclodextrin, CDampy) was synthesized by C6-monofunctionalization. The acid-base properties of the new ligand in aqueous solution were investigated by potentiometry and calorimetry, and its conformations as a function of pH were studied by NMR and circular dichroism (c.d.). The formation of binary copper(II) complexes was studied by potentiometry, EPR, and c.d.. The copper(II) complex was used as chiral selector for the HPLC enantiomeric separation of underivatized aromatic amino acids. Enantioselectivity in the overall stability constants of the ternary complexes with D- or L-Trp was detected by potentiometry, whereas the complexes of the Ala enantiomers did not show and difference in stability. These results were consistent with a preferred cis coordination of the amino group of the ligand and of the amino acid in the ternary complexes ("cis effect"), which leads to the inclusion of the aromatic side chain of D-Trp, but not of that of L-Trp. In Trp-containing ternary complexes, the two enantiomers showed differences in the fluorescence lifetime distribution, consistent with only one conformer of D-Trp and two conformers of L-Trp, and the latter were found to be more accessible to fluorescence quenching by acrylamide and KI.