A Vibrational Circular Dichroism Approach To The Determination Of The Absolute Configuration Of Flexible And Transparent Molecules: Fluorenone Ketals Of 1,N-Diols

The infrared absorption (IR) and vibrational circular dichroism (VCD) spectra for five ketal molecules, three of which obtained from 1,2-diols and two from 1,3-diols, were recorded in the mid-IR region. The spectra have been satisfactorily reproduced by DFT calculations, even with not too large wavefunction basis sets, especially due to the low number of conformers to be considered. The mobility of some moieties provides a recognizable signature. A characteristic couplet of VCD bands attributed to normal modes involving the methine and a phenyl ring bonded to the stereogenic carbon atom is evidenced for two ketals of the series as a signature of the absolute configuration; due comparison with existing literature is made. A relation is discussed of the present VCD data with the literature VCD data of simple alcohols and diols

Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution

Recent research on the flavoenzyme D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) has revealed new, intriguing properties of this catalyst and offers novel biotechnological applications. Among them, the reaction of RgDAAO has been exploited in the analytical determination of the D-amino acid content in biological samples. However, because the enzyme does not oxidize acidic D-amino acids, it cannot be used to detect the total amount of D-amino acids. We now present the results obtained using a random mutagenesis approach to produce RgDAAO mutants with a broader substrate specificity. The libraries of RgDAAO mutants were generated by error-prone PCR, expressed in BL21(DE3)pLysS Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. Five random mutants that have a 'modified' substrate specificity, more useful for the analytical determination of the entire content of D-amino acids than wild-type RgDAAO, have been isolated. With the only exception of Y223 and G199, none of the effective amino acid substitutions lie in segments predicted to interact directly with the bound substrate. The substitutions appear to cluster on the protein surface: it would not have been possible to predict that these substitutions would enhance DAAO activity. We can only conclude that these substitutions synergistically generate small structural changes that affect the dynamics and/or stability of the protein in a way that enhances substrate binding or subsequently catalytic turnover

Stability of Transonic Shock Solutions for One-Dimensional Euler-Poisson Equations

In this paper, both structural and dynamical stabilities of steady transonic shock solutions for one-dimensional Euler-Poission system are investigated. First, a steady transonic shock solution with supersonic backgroumd charge is shown to be structurally stable with respect to small perturbations of the background charge, provided that the electric field is positive at the shock location. Second, any steady transonic shock solution with the supersonic background charge is proved to be dynamically and exponentially stable with respect to small perturbation of the initial data, provided the electric field is not too negative at the shock location. The proof of the first stability result relies on a monotonicity argument for the shock position and the downstream density, and a stability analysis for subsonic and supersonic solutions. The dynamical stability of the steady transonic shock for the Euler-Poisson equations can be transformed to the global well-posedness of a free boundary problem for a quasilinear second order equation with nonlinear boundary conditions. The analysis for the associated linearized problem plays an essential role

Epithelial ovarian carcinoma diagnosis by desorption electrospray ionization mass spectrometry imaging

Ovarian cancer is highly prevalent among European women, and is the leading cause of gynaecological cancer death. Current histopathological diagnoses of tumour severity are based on interpretation of, for example, immunohistochemical staining. Desorption electrospray mass spectrometry imaging (DESI-MSI) generates spatially resolved metabolic profiles of tissues and supports an objective investigation of tumour biology. In this study, various ovarian tissue types were analysed by DESI-MSI and co-registered with their corresponding haematoxylin and eosin (H&E) stained images. The mass spectral data reveal tissue type-dependent lipid profiles which are consistent across the n = 110 samples (n = 107 patients) used in this study. Multivariate statistical methods were used to classify samples and identify molecular features discriminating between tissue types. Three main groups of samples (epithelial ovarian carcinoma, borderline ovarian tumours, normal ovarian stroma) were compared as were the carcinoma histotypes (serous, endometrioid, clear cell). Classification rates >84% were achieved for all analyses, and variables differing statistically between groups were determined and putatively identified. The changes noted in various lipid types help to provide a context in terms of tumour biochemistry. The classification of unseen samples demonstrates the capability of DESI-MSI to characterise ovarian samples and to overcome existing limitations in classical histopathology

Eye-Light on Age-Related Macular Degeneration: Targeting Nrf2-Pathway as a Novel Therapeutic Strategy for Retinal Pigment Epithelium

open6noThis work was supported by the University of Pavia [to MA, grant number BSR1744747; 2017] and the Italian Ministry of University and Research [to MA, FFABR2017]. The University of Bologna is acknowledged by MR [Grants from RFO].Age-related macular degeneration (AMD) is a common disease with a multifactorial aetiology, still lacking effective and curative therapies. Among the early events triggering AMD is the deterioration of the retinal pigment epithelium (RPE), whose fundamental functions assure good health of the retina. RPE is physiologically exposed to high levels of oxidative stress during its lifespan; thus, the integrity and well-functioning of its antioxidant systems are crucial to maintain RPE homeostasis. Among these defensive systems, the Nrf2-pathway plays a primary role. Literature evidence suggests that, in aged and especially in AMD RPE, there is an imbalance between the increased pro-oxidant stress, and the impaired endogenous detoxifying systems, finally reverberating on RPE functions and survival. In this in vitro study on wild type (WT) and Nrf2-silenced (siNrf2) ARPE-19 cells exposed to various AMD-related noxae (H2O2, 4-HNE, MG132 + Bafilomycin), we show that the Nrf2-pathway activation is a physiological protective stress response, leading downstream to an up-regulation of the Nrf2-targets HO1 and p62, and that a Nrf2 impairment predisposes the cells to a higher vulnerability to stress. In search of new pharmacologically active compounds potentially useful for AMD, four nature-inspired hybrids (NIH) were individually characterized as Nrf2 activators, and their pharmacological activity was investigated in ARPE-19 cells. The Nrf2 activator dimethyl-fumarate (DMF; 10 μM) was used as a positive control. Three out of the four tested NIH (5 μM) display both direct and indirect antioxidant properties, in addition to cytoprotective effects in ARPE-19 cells under pro-oxidant stimuli. The observed pro-survival effects require the presence of Nrf2, with the exception of the lead compound NIH1, able to exert a still significant, albeit lower, protection even in siNrf2 cells, supporting the concept of the existence of both Nrf2-dependent and independent pathways mediating pro-survival effects. In conclusion, by using some pharmacological tools as well as a reference compound, we dissected the role of the Nrf2-pathway in ARPE-19 stress response, suggesting that the Nrf2 induction represents an efficient defensive strategy to prevent the stress-induced damage.openCatanzaro M.; Lanni C.; Basagni F.; Rosini M.; Govoni S.; Amadio M.Catanzaro M.; Lanni C.; Basagni F.; Rosini M.; Govoni S.; Amadio M

Detection of Helicobacter pylori by PCR on gastric biopsy specimens taken for CP test: comparison with histopathological analysis.

The aims of the present study were: (i) to assess whether H. pylori could be succesfully detected by PCR from the same biopsy sample used for CPtest; and ii) to evaluate CPtest comparatively to both PCR and histology for detection of H. pylori infection in dyspeptic patients. Three antral gastric biopsies were collected from each of 80 consecutive dyspeptic patients undergoing oesophagogastroduodenoscopy. Two biopsies were for histology (gold standard), one for CPtest, scored at 20min, 1h and 24h for the presence of urease activity. Gastric biopsy was then removed from CPtest and used for ureC-targeted PCR. Fifty-five (68.7%) patients were positive for H. pylori infection by histology. CPtest yielded an overall diagnostic accuracy of 93.8% (95% CI: 91–96.4%), regardless of observation period. No erroneous categorization of H. pylori status occurred using PCR, yielding sensitivity, specificity, positive and negative predictive values, and overall diagnostic accuracy of 100%. Our results suggest that H. pylori can be detected by PCR in gastric biopsies previously taken for CPtest, so reducing the workload of the endoscopist by saving additional biopsies for culture analysis and susceptibility tests

Treatment of malignant hypercalcaemia with aminohexane bisphosphonate (neridronate).

Twenty patients with hypercalcaemia due to malignancy, which persisted following rehydration, were treated with the bisphosphonate, aminohexane bisphosphonate (AHBP), which is structurally similar to pamidronate. The treatment given was a single infusion of 125 mg of AHBP in 500 ml of normal saline infused over 4 h. Serum and urine biochemistry were measured before and after treatment. Acute toxicity was evaluated with particular attention to gastrointestinal symptoms, acute-phase reaction and change in renal function, as judged by serum creatinine. The infusion of AHBP induced a rapid fall apparent by day 3 (P < 0.001), with a nadir at day 7. The serum calcium remained lower at days 14 and 28 than at day 0, but the numbers followed up were low (n = 5 and n = 4). In all 20 patients there was a fall in serum calcium after treatment, and in 13 (65%) normocalcaemia was achieved. Failure to respond completely to AHBP appeared to be associated with a renal mechanism of hypercalcaemia. Treatment was associated with a significant decrease in fasting urinary calcium excretion (P < 0.05). There was no change in white cell count or renal function following AHBP and only two cases of mild pyrexia after infusion. We conclude that aminohexane bisphosphonate is an effective agent in the treatment of tumour-induced hypercalcaemia, with rapid onset of effect and low toxicity