Sequence determinants as key regulators in gene expression of T cells.

T cell homeostasis, T cell differentiation, and T cell effector function rely on the constant fine-tuning of gene expression. To alter the T cell state, substantial remodeling of the proteome is required. This remodeling depends on the intricate interplay of regulatory mechanisms, including post-transcriptional gene regulation. In this review, we discuss how the sequence of a transcript influences these post-transcriptional events. In particular, we review how sequence determinants such as sequence conservation, GC content, and chemical modifications define the levels of the mRNA and the protein in a T cell. We describe the effect of different forms of alternative splicing on mRNA expression and protein production, and their effect on subcellular localization. In addition, we discuss the role of sequences and structures as binding hubs for miRNAs and RNA-binding proteins in T cells. The review thus highlights how the intimate interplay of post-transcriptional mechanisms dictate cellular fate decisions in T cells.

Effect of alkaline cooking of maize on the content of fumonisins B1 and B2 and their hydrolysed forms.

The effect of nixtamalization on the content of fumonisins (FBs), hydrolysed (HFBs) and partially hydrolysed (PHFBs) fumonisins in maize was investigated at laboratory-scale. Maize naturally contaminated with FBs and PHFBs was cooked with lime. Starting raw maize, steeping and washing waters and final masa fractions were analysed for toxin content. Control-cooking experiments without lime were also carried out. The nixtamalization reduced the amount of FBs and PHFBs in masa and converted them to HFBs. However, the three forms of fumonisins collected in all fractions amounted to 183%, indicating that nixtamalization made available forms of matrix-associated fumonisins that were then converted to their hydrolysed forms. Control-cooking enhanced FBs and PHFBs reduction, due to the solubility of fumonisins in water during the steeping process, but did not form HFBs. These findings indicate that benefits associated with enhancing the nutritional value of nixtamalized maize are also associated with a safer product in terms of fumonisin contamination.

Quantitative analysis of mycotoxins in cereal foods by collision cell fragmentation-high-resolution mass spectrometry: performance and comparison with triple-stage quadrupole detection.

A liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in wheat flour, barley flour and crisp bread was developed. Mycotoxin fragmentation patterns obtained by high-energy collision dissociation (HCD) were investigated to obtain quantitative and confirmatory information (two characteristic masses per mycotoxin) using Orbitrap™-based high-resolution mass spectrometry. LC-HRMS (full-scan) detection carried out by HCD allows the monitoring of the pseudo-molecular ion and an additional characteristic fragment (for each mycotoxin) with mass accuracy in the range 0.1-3.9 ppm, meeting current European regulatory requirements for LC-MS confirmatory analysis. A sample preparation procedure based on polymeric solid-phase extraction cartridges was applied, allowing recoveries higher than 74% for nine mycotoxins, with a relative standard deviation lower than 13%. Detection limits in the range 0.5-3.4 µg kg(-1) were obtained for three cereal matrices. A critical comparison between the proposed method and a validated method based on triple quadrupole mass spectrometry showed similar performance in terms of detection limits, recoveries and repeatability, and matrix effects. Based on an efficient sample extraction and clean-up, the LC-HCD-HRMS method reported here represents a reliable and robust alternative tool for mycotoxin analysis in food matrices as compared with well-established triple quadrupole-based approaches.

What is the sensitivity of mammography and dynamic MR imaging for DCIS if the whole-breast histopathology is used as a reference standard?

PURPOSE: Our purpose was to compare mammography and dynamic contrast-enhanced magnetic resonance imaging (MRI) in the detection of ductal carcinoma in situ (DCIS). MATERIALS AND METHODS: Ninety patients (aged 58.6+/-16.1 years) who were candidates for unilateral (n=81) or bilateral (n=9) mastectomy underwent mammography and dynamic contrast-enhanced breast MRI using a coronal three-dimensional gradient-echo sequence with slice thickness < or =3 mm before and after intravenous injection of gadoteridol (0.1 mmol/kg). Mammographic and MR images were evaluated by two offsite readers working in consensus. Pathological examination performed on 5-mm sections covering the whole breast was used as a reference standard. RESULTS: Out of 99 breasts, pathology revealed 26 DCIS in 14 breasts of 14 patients, aged 52.0 +/- 9.6 years. Lesion diameter at pathology was <5 mm (n=4); > or =5 and <10 mm (n=7); > or =10 and <20 mm (n=3); > or =20 mm (n=2); not assessed (n=10). Sensitivity was 35% (9/26) for mammography and 38% (10/26) for MRI (not significant difference, McNemar test). Both mammography and MRI provided a true positive result in seven cases (four of them measured at pathology, with a diameter of 20.0+/-12.9 mm; median 20 mm) and a false negative result in 14 cases (10 of them measured at pathology, with a diameter of 4.2+/-1.9 mm; median 4.6 mm) (p=0.024, Mann-Whitney U test). Only 46% (12/26) of DCIS were detected at mammography and/or MRI; the remaining 54% (14/26) were diagnosed only at pathological examination. CONCLUSIONS: When the whole breast is used as the histopathological reference standard, both mammography and MRI show low sensitivity for DCIS.

Determination of trichothecenes in cereals and cereal-based products by liquid chromatography-tandem mass spectrometry.

A sensitive, accurate and precise method for the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in different food matrices, including wheat, maize, barley, cereal-based infant foods, snacks, biscuits and wafers, has been developed. The method, using liquid chromatography coupled with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC-APCI-MS/MS), allowed unambiguous identification of the selected trichothecenes at low microg per kg levels in such complex food matrices. A clean-up procedure, based on reversed phase SPE Oasis HLB columns, was used, allowing good recoveries for all studied trichothecenes. In particular, NIV recoveries significantly improved compared to those obtained by using Mycosep #227 columns for clean-up of the extracts. Limits of detection in the various investigated matrices ranged 2.5-4.0 microg kg(-1) for NIV, 2.8-5.3 microg kg(-1) for DON, 0.4-1.7 microg kg(-1) for HT-2 and 0.4-1.0 microg kg(-1) for T-2. Mean recovery values, obtained from cereals and cereal products spiked with NIV, DON, HT-2 and T-2 toxins at levels from 10 to 1000 microg kg(-1), ranged from 72 to 110% with mean relative standard deviation lower than 10%. A systematic investigation of matrix effects in different cereals and cereal products was also carried out by statistically comparing the slopes of standard calibration curve with matrix-matched calibration curve for each of the four toxins and the eight matrices tested. For seven of the eight matrices tested, statistically significant matrix effects were observed, indicating that, for accurate quantitative analysis, matrix-matched calibration was necessary. The method was applied to the analysis of 57 samples of ground wheat originated from South Italy and nine cereal food samples collected from retail markets.

Electrosynthesized poly(pyrrole)/poly(2-naphthol) bilayer membrane as an effective anti-interference layer for simultaneous determination of acethylcholine and choline by a dual electrode amperometric biosensor.

Several neural diseases appear related to the neurotransmitter acethylcholine (ACh) and its metabolite choline (Ch) brain levels so that their simultaneous determination is essential. A cross-talk and interference free dual electrode amperometric biosensor for the simultaneous determination of both analytes has been developed. Acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized by glutaraldehyde co-crosslinking with bovine serum albumin. A very efficient rejection of electroactive interferents has been achieved by a novel electrosynthesized polymeric bilayer membrane composed by overoxidised poly(pyrrole) and poly(2-naphthol) films. Sensitivities towards several electroactive interferents ranged from ca. 0.04% (e.g. ascorbate) to ca. 0.3% (e.g. dopamine) of those relevant to ACh and Ch (11 and 15 microA/microM, respectively). Detection limits (at S/N=3) in flow injection analysis were ca. 100 nM for both ACh and Ch at the ChO-AChE electrode and ca. 40 nM for Ch at the ChO sensor. Biosensor performances appear more than adequate for brain tissue homogenates and cerebrospinal fluids analysis where average levels in the low micromolar range are typically found.

[Subareolar injection for sentinel lymph node biopsy in multiple breast cancer]. / L'iniezione subareolare per la biopsia del linfonodo sentinella nei tumori multipli della mammella

In this study we performed subdermal injection of 99mTc-labeled albumin combined with subareolar (SA) injection of blue dye to axillary lymphatic mapping and sentinel lymph node biopsy (SNLB) in patients with multifocal and multicentric breast cancer (MC) to evaluate the feasibility and accuracy of this technique. We compared the results with a group of patients with unifocal breast cancer. From January 1999 to March 2006 axillary lymph node mapping and SLNB was performed on 250 patients followed by a complete axillary lymph node dissection. Retrospective analysis showed that 32 (12.8%) of these patients have MC on final histopathologic examination and 218 (87.2%) have unifocal cancer. In statistical analysis tumor size shows a significant difference (p=.01) with largest lesions in MC. In MC often histological type is invasive lobular with or without in situ cancer (p= .001). Metastatic lymph node involvement was significantly higher in the MC group compared to unifocal cancer group (p=.001). False negative (FN) rate was 5.8% in MC and 9.6% in unifocal cancers. The overall accuracy of lymphatic mapping was 96.8% in MC and 97.6% in unifocal cancers. Sensitivity was 94.4% in MC and 91.2% in unifocal cancers. In this study we provide further evidence that lymphatic mapping may be reliable even in patients with MC. SA injection technique demonstrates a high sentinel lymph node identification rate and low FN rate; therefore this technique should been recommended to SLNB in patients with MC of the breast.

Role of endogenous lipids in the chromophore regeneration of bacteriorhodopsin.

The regeneration method of Khorana [J. Biol. Chem. 262 (1987) 9271] has been modified in order to study the effect of endogenous archaeabacterial lipids and, in particular, of glycocardiolipin (GlyC) in the refolding and chromophore regeneration of bacteriorhodopsin (BR). BR refolding and chromophore regeneration could be obtained in the presence of endogenous lipid mixtures containing or not containing glycocardiolipin; however, the kinetics of bacteriorhodopsin regeneration in the presence of glycocardiolipin was faster than in its absence. These results show for the first time that the interaction of glycocardiolipin with bacteriorhodopsin favours its refolding from the denaturated state and the chromophore regeneration.

[The stereotaxic core breast biopsy using the Mammotome: an alternative to intraoperative examination]. / La core-biopsy mammaria stereotassica con Mammotome: un'alternativa all'esame intraoperatorio.

The aim of this work was to evaluate the accuracy of vacuum-assisted biopsy by comparing it with frozen biopsy. 141 stereotaxic biopsies were performed by Mammotome (Ethicon Endo-Surgery, Hamburg) from January 2000 to March 2001. Biopsies were performed for microcalcifications (n = 105, 74.5%), irregular opacities (n = 20, 14.2%), regular opacities (n = 6, 4.2%), stellate lesions (n = 10, 7.1%). Histological analysis showed 85 (60.3%) benign lesions, 46 (32.6%) malignant lesions including (21 cases of carcinoma in situ and 25 invasive carcinomas) and 10 (7.1%) atypical lesions. All malignant lesions were subjected to surgery. In three cases (1 in situ and 2 invasive), core biopsy was excisional and no residual lesion was observed. Two of the carcinomas in situ revealed invasive features on the surgical biopsy. One of the atypical lesions was underestimated and the final diagnosis was "well differentiated carcinoma in situ." Only three of benign lesions underwent surgery after Mammotome biopsy. Among the 55 frozen-section biopsies of mammographically detected breast lesions performed in the same period, were one false-positive and 3 false-negative cases, while in 4 cases the diagnosis was deferred after paraffin embedding. Our results confirmed Mammotome biopsy as an effective alternative and a more reliable method than frozen-section examination.

Low temperature metabolism of apple phenolics and quiescence of Phlyctaena vagabunda.

The content of chlorogenic acid, (+)-catechin, (-)-epicatechin, phloretin glycosides, and quercetin glycosides in fresh and stored Golden Delicious apples (Malus domestica Borkh) was determined. The relative amount of phenolics in the peel, with the exception of chlorogenic acid and (-)-epicatechin, was higher than that in the flesh. In addition, quercetin glycosides were detected only in the skin. These compounds were tested for fungicidal activity against Phlyctaena vagabunda Desm., the causal agent of a postharvest rot. Chlorogenic acid only inhibited P. vagabunda spore germination and mycelial growth in vitro. Changes of apple phenolics and polyphenol oxidase activity during cold storage and the biological activity of these phenolics have also been analyzed with reference to the development of quiescent infections during cold storage plus shelf life at room temperature. The results obtained suggested that phloridzin and chlorogenic acid in combination with polyphenol oxidase activity could function to arrest P. vagabunda in quiescent infections associated with immature and ripening apple fruit.

Role of endogenous flavonoids in resistance mechanism of Vigna to aphids.

Cultivated and wild species of the genus Vigna were screened for their flavonoid content. Flavonoid HPLC analyses clearly showed that cultivated lines of cowpea (Vigna unguiculata L. Walp.) are very similar from a qualitative point of view, always showing three flavonoid aglycons: quercetin, kaempferol, and isorhamnetin. In addition, a positive relationship between resistance/susceptibility characteristics against aphids and flavonoid glycoside content of cowpea lines was found. The resistant lines showed a flavonoid content higher than that of susceptible ones. In vitro bioassays proved that, among endogenous flavonoids, quercetin and isorhamnetin possess a good inhibitory aphid reproduction rate. Flavonoid HPLC analyses of wild Vigna species supported evidence for the existence of different flavonoid chemotypes in some species of section Vigna. There are kaempferol chemotypes, kaempferol being the main aglycon detected, quercetin chemotypes, containing quercetin glycosides only, and two isorhamnetin chemotypes. When the resistance characteristics to aphids in different chemotypes of the same species were tested, it became evident that quercetin or isorhamnetin chemotypes showed a higher level of resistance compared to kaempferol chemotypes in the same species, thus demonstrating a direct involvement of quercetin or isorhamnetin in the resistance mechanism. These results can provide useful information for further studies on gene expression of resistance factors.

[Blood acceleration is the principal factor in coronary flow increase during exercise test]. / L'accelerazione ematica è il principale fattore di aumento del flusso coronarico durante test da sforzo.

Continuous wave Doppler evaluation of the blood flow velocity in the internal mammary artery anastomosed to the left anterior descending coronary artery is a technique for assessing human coronary flow that shows a good reproducibility and lower change in Doppler angle between resting and supine exercise conditions. In this study the reciprocal changes which occur in coronary blood flow, coronary stroke volume, coronary resistance and blood pressure have been studied. During supine exercise coronary stroke volume remains constant and the increase of blood cells acceleration is necessary to have the shortening of cardiac cycle. This parameter is inversely dependent on the coronary resistances and directly dependent on blood pressure. As a consequence it plays the main role in increasing coronary blood flow during exercise.

Observer variation in mammary thermography: results of a teaching file test carried out in four different centers.

To evaluate observer variation in mammary thermography, a teaching file test of 159 thermographies was worked out by 4 senology centers. The evaluation of accuracy and the K index of variability demonstrated a significant variability among centers. The reliability of thermography in senology seems to be too poor for it to be able to direct any therapeutic decision, either diagnostic or prognostic.