Evaluation of an automatic gas chromatographic system for the identification of bacterial infective agents

The potential clinical application of gas chromatography to microbial identifcation was evaluated. A completely automated system, the MIS (Microbial Identification System; Hewlett- Packard) can analyse and identify pure strains by comparison of their cellular fatty acids patterns (C9-C20) with the reference parameters stored in a library. Three hundred and sixty-seven strains were tested, comparing the gas chromatographic results with those obtained by the traditional microbiological methods in the bacteriology laboratory of our Institute. A standardized extractive procedure was followed to obtain the fatty acid methyl esters (FAMEs), but some modifications to the recommended procedure were introduced in the bacterial growth procedures: colonies harvested not only from the recommended growth media but also from selective media routinely used in the bacteriology laboratory were successfully examined. These modifications did not influence the results but improved the ease for the user; good agreement with the comparison method was observed as far as identifications of genus and species are concerned for 238 cases. The major advantages of this computerized system are a reduction in the time required to obtain the final results, the elimination of human errors by using the autosampler and a better inter-laboratory comparability of results owing to a higher degree of objectivity. On the other hand, the limited throughput of MIS (only 40 samples in 24 h) prevents its use in a large routine laboratory; this technology is appropriate in emergency cases, in taxonomic studies and as a confirmatory method

Myriocin Effect on Tvrm4 Retina, an Autosomal Dominant Pattern of Retinitis Pigmentosa

Tvrm4 mice, a model of autosomal dominant retinitis pigmentosa (RP), carry a mutation of Rhodopsin gene that can be activated by brief exposure to very intense light. Here, we test the possibility of an anatomical, metabolic, and functional recovery by delivering to degenerating Tvrm4 animals, Myriocin, an inhibitor of ceramide de novo synthesis previously shown to effectively slow down retinal degeneration in rd10 mutants (Strettoi et al., 2010; Piano et al., 2013). Different routes and durations of Myriocin administration were attempted by using either single intravitreal (i.v.) or long-term, repeated intraperitoneal (i.p.) injections. The retinal function of treated and control animals was tested by ERG recordings. Retinas from ERG-recorded animals were studied histologically to reveal the extent of photoreceptor death. A correlation was observed between Myriocin administration, lowering of retinal ceramides, and preservation of ERG responses in i.v. injected cases. Noticeably, the i.p. treatment with Myriocin decreased the extension of the retinal-degenerating area, preserved the ERG response, and correlated with decreased levels of biochemical indicators of retinal oxidative damage. The results obtained in this study confirm the efficacy of Myriocin in slowing down retinal degeneration in genetic models of RP independently of the underlying mutation responsible for the disease, likely targeting ceramide-dependent, downstream pathways. Alleviation of retinal oxidative stress upon Myriocin treatment suggests that this molecule, or yet unidentified metabolites, act on cellular detoxification systems supporting cell survival. Altogether, the pharmacological approach chosen here meets the necessary pre-requisites for translation into human therapy to slow down RP

Removal of radioisotopes in solution and bactericidal/bacteriostatic sterilising power in activated carbon and metal silver filters

Activated carbon filters play an important role in water filtration and purification from contaminants of different origin. Their limit consists in bacterial proliferation, which may occur only during prolonged periods of non-use and in their ability to remove radioactive contaminants present in waste water from Industry or Nuclear Medicine departments. In this work we tested a commercially available activated carbon filter for water purification enriched with silver plated parts incubating in static condition at room temperature different micro organisms (Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis, Staphylococcus aureus, Aspergillum niger), up to 78 days. The microbial growth was in general more inhibited in the presence of metal silver into the activated carbon in respect to filters with the activated carbon alone: >4 log inhibition of bacterial proliferation after 78 days of incubation the presence of silver vs. 2 log without silver. When the filters were incubated empty of carbon, the sterilizing power of silver was confirmed further. The activated carbon filters proved also their ability in removing fromwater the principal radioisotopes used for residues liquid medical and research purposes (131I, 99mTc, 201Tl, 67Ga). These results contribute useful data for the use of the silver-enriched carbon filters in water filtration both for daily use at home, and professional use in a Nuclear Medicine laboratory

Oxidative status in different settings and with different methodological approaches compared by Receiver Operating Characteristic curve analysis

Objectives: To test the performance of different analytical approaches in highlighting the occurrence of deregulated redox status in various physio-pathological situations. Design and methods: 35 light and 61 heavy smokers, 19 chronic renal failure, 59 kidney transplanted patients, and 87 healthy controls were retrospectively considered for the study. Serum oxidative stress and antioxidant status, assessed by spectrophotometric Reactive Oxygen Metabolites (d-ROMs) and Total Antioxidant Capacity (TAC) tests, respectively, were compared with plasma free (F-MDA) and total (T-MDA)malondialdehyde, both quantified by isotope-dilution-gas chromatography\u2013mass spectrometry (ID-GC\u2013MS). Sensitivity, specificity and cut-off points of T-MDA, F-MDA, d-ROMs and TAC were evaluated by both Receiver Operating Characteristic (ROC) analyses and area under the ROC curve (AUC). Results: Only T-MDA assay showed a clear absence of oxidative stress in controls and significant increase in all patients (AUC 1.00, sensitivity and specificity 100%). Accuracy was good for d-ROMs (AUC 0.87, sensitivity 72.8%, specificity 100%) and F-MDA (AUC 0.82, sensitivity 74.7%, specificity 83.9%), but not high enough for TAC to show in patients impaired antioxidant defense (AUC 0.66, sensitivity 52.0%, specificity 92.9%). Conclusions: This study reveals T-MDA as the best marker to detect oxidative stress, shows the ability of d-ROMs to identify modified oxidative status particularly in the presence of high damages, and evidences the poor TAC performance. d-ROMs and TAC assays could be useful for routine purposes; however, for an accurate clinical data evaluation, their comparison versus a \u201cgold standard method\u201d is required

Sphingolipid synthesis inhibition by myriocin administration enhances lipid consumption and ameliorates lipid response to myocardial ischemia reperfusion injury

Myocardial infarct requires prompt thrombolytic therapy or primary percutaneous coronary intervention to limit the extent of necrosis, but reperfusion creates additional damage. Along with reperfusion, a maladaptive remodeling phase might occur and it is often associated with inflammation, oxidative stress, as well as a reduced ability to recover metabolism homeostasis. Infarcted individuals can exhibit reduced lipid turnover and their accumulation in cardiomyocytes, which is linked to a deregulation of peroxisome proliferator activated receptors (PPARs), controlling fatty acids metabolism, energy production, and the anti-inflammatory response. We previously demonstrated that Myriocin can be effectively used as post-conditioning therapeutic to limit ischemia/reperfusion-induced inflammation, oxidative stress, and infarct size, in a murine model. In this follow-up study, we demonstrate that Myriocin has a critical regulatory role in cardiac remodeling and energy production, by up-regulating the transcriptional factor EB, PPARs nuclear receptors and genes involved in fatty acids metabolism, such as VLDL receptor, Fatp1, CD36, Fabp3, Cpts, and mitochondrial FA dehydrogenases. The overall effects are represented by an increased \u3b2-oxidation, together with an improved electron transport chain and energy production. The potent immunomodulatory and metabolism regulatory effects of Myriocin elicit the molecule as a promising pharmacological tool for post-conditioning therapy of myocardial ischemia/reperfusion injury

Plasma mitomycin C concentrations determined by HPLC coupled to solid-phase extraction

The aim of this study was to set up a method for quantification of plasma mitomycin C (MMC) concentrations during intravesical chemotherapy delivered in the presence of local bladder hyperthermia (HT). In comparison with existing methods, this assay, characterized by relative simplicity and efficiency, resulted in the facilitation of performance with nondedicated instrumentation or nonspecialized staff. Purification from plasma matrix was carried out by solid-phase extraction under vaccuum. The purified drug was then collected directly into the vials of the HPLC autosampler. Chromatographic analysis was performed on a reversed-phase C18 column with water:acetonitrile (85:15 by vol) as the mobile phase and the UV detector set at 365 nm. The use of porfiromycin as internal standard provided a method with good within-day precision (CV 6.0% at 5 micrograms/L, n = 6), linearity (0.5-50 micrograms/L), and specificity. The lower limit of detection (< or = 0.5 microgram/L) proved to be suitable for plasma pharmacokinetics monitoring in two tested patients treated with MMC + HT for superficial bladder cancer

Existence theorems in the geometrically non-linear 6-parametric theory of elastic plates

In this paper we show the existence of global minimizers for the geometrically exact, non-linear equations of elastic plates, in the framework of the general 6-parametric shell theory. A characteristic feature of this model for shells is the appearance of two independent kinematic fields: the translation vector field and the rotation tensor field (representing in total 6 independent scalar kinematic variables). For isotropic plates, we prove the existence theorem by applying the direct methods of the calculus of variations. Then, we generalize our existence result to the case of anisotropic plates. We also present a detailed comparison with a previously established Cosserat plate model.Comment: 19 pages, 1 figur

Long and very-long-chain ceramides correlate with a more aggressive behavior in skull base chordoma patients

Background: Skull base chordomas are rare tumors arising from notochord. Sphingolipids analysis is a promising approach in molecular oncology, and it has never been applied in chordomas. Our aim is to investigate chordoma behavior and the role of ceramides. Methods: Ceramides were extracted and evaluated by liquid chromatography and mass spectrometry in a cohort of patients with a skull base chordoma. Clinical data were also collected and correlated with ceramide levels. Linear regression and correlation analyses were conducted. Results: Analyzing the association between ceramides level and MIB-1, total ceramides and dihydroceramides showed a strong association (r = 0.7257 and r = 0.6733, respectively) with MIB-1 staining (p = 0.0033 and p = 0.0083, respectively). Among the single ceramide species, Cer C24:1 (r = 0.8814, p &lt;= 0.0001), DHCer C24:1 (r = 0.8429, p = 0.0002) and DHCer C18:0 (r = 0.9426, p &lt;= 0.0001) showed a significant correlation with MIB-1. Conclusion: Our lipid analysis showed ceramides to be promising tumoral biomarkers in skull base chordomas. Long- and very-long-chain ceramides, such as Cer C24:1 and DHCer C24:1, may be related to a prolonged tumor survival and aggressiveness, and the understanding of their effective biological role will hopefully shed light on the mechanisms of chordoma radio-resistance, tendency to recur, and use of agents targeting ceramide metabolism

Quantitative cytometry of MHC class I digestion from living cells

Digestion of crude membrane preparations with papain releases the extracellular portion of major histocompatibility complex (MHC) class I molecules. MHC class I molecules are integral membrane glycoprotein complexes formed by the noncovalent association of 2 invariant molecules, the heavy chain and the beta2-microglobulin (beta2-m), to a wide array of peptides. The cleaved soluble moiety retains the antigenic properties of the intact membrane-bound complex. Here we show that MHC class I digestion may be carried out on living cells, and we quantitate the surface expression of MHC complexes by a combined cytometric/high performance liquid chromatographic (HPLC) approach. Papain digestion results in time- and dose-dependent disappearance of membrane MHC-associated-fluorescence as detected by FACS analysis with MHC-specific monoclonal antibodies (mAbs). beta2-m and peptides became detectable by HPLC analysis and western blotting in the digestion buffer and were quantitated by comparison with purified standards. The cytometric assessment of the digestion allows one to simultaneously monitor efficacy and toxicity of the treatment. The procedure we describe allows to selectively retrieve by affinity chromatography MHC from the cell membrane, avoiding any contamination due to intracellular, "immature" MHC molecules

Relationship between fatty acids composition/antioxidant potential of breast milk and maternal diet: Comparison with infant formulas

The fatty acid composition of human breast milk is relevant for the energy, immunity and eicosanoid production in infants. Additionally, the antioxidant properties of foods are essential for human health. Therefore, in the present study we aimed to investigate the relationship between maternal diet and fatty acids composition as well as the antioxidant potential of breast milk from donors to human milk bank of Perugia\u2019s hospital, Italy. Results were compared with infant formulas. We observed increased levels of total fatty acids and, in particular, saturated and monounsaturated fatty acids in milk from mothers fed on a vegetable and fruit-rich diet compared with a Mediterranean diet. In the same milk, a reduced antioxidant potential was found. All infant formulas resulted in richer total fatty acid content than human breast milk. Only some formulas were qualitatively similar to breast milk. Of note, the antioxidant potential of the formulas was higher or lower than the human milk with the exception of one sample. The antioxidant potential of four formulas was very high. Dietary supplementation with antioxidants has been shown to have a teratogenic effect and to increase the formation of metastases in adult. There are no data on the effects of excess antioxidants in the infants, but the possibility that they can be harmful cannot be excluded