PRospective Imaging of CErvical cancer and neoadjuvant treatment (PRICE) study: role of ultrasound to assess residual tumor in locally advanced cervical cancer patients undergoing chemoradiation and radical surgery.

OBJECTIVE: To determine the diagnostic performance of two-dimensional (2D) ultrasound parameters, three-dimensional (3D) power Doppler and contrast-enhanced indices in detecting residual disease in locally advanced cervical cancer patients triaged to neoadjuvant treatment followed by radical surgery. METHODS: Between October 2010 and June 2014, we screened 108 women with histologically documented locally advanced cervical cancer Stage IB2-IVA, of whom 88 were included in the final analysis. 2D ultrasound parameters, 3D power Doppler and contrast-ultrasound parameters were assessed 5 weeks after the end of neoadjuvant chemoradiation therapy. The pathological response was defined as complete (absence of any residual tumor after treatment) or partial (including microscopic and/or macroscopic residual tumor at pathology examination). The two response groups were compared and receiver-operating characteristics (ROC) curves generated to determine the best cut-off value of sonographic tumor diameter to predict residual disease. Histology was considered as reference. RESULTS: Complete pathological response to chemoradiation was observed in 40 (45.5%) patients and partial response in 48 (54.5%). The presence of residual disease, as confirmed at pathology examination, was detected by 2D grayscale ultrasound with a sensitivity of 64.6% and specificity of 65%. Color Doppler examination in the cases with lesions visualized on grayscale imaging detected the presence of residual disease, confirmed at pathology, with a sensitivity of 87.1% and specificity of 21.4%. The best area under the ROC curve (0.817) was for the detection of pathological residual disease of at least 6 mm in diameter, using a cut-off value of 12 mm for the largest tumor diameter assessed using 2D grayscale ultrasound (sensitivity, 95%; specificity, 70.6%). Neither 3D vascular indices nor contrast-ultrasound parameters obtained for lesions suspected at ultrasound following chemoradiation differed significantly between patients with histological complete and those with partial response. CONCLUSIONS: Our results show that grayscale and color Doppler ultrasound have a low level of diagnostic performance in detecting residual disease after neoadjuvant chemoradiation in patients with locally advanced cervical cancer. The best performance was achieved in detection of macroscopic (≥ 6 mm) residual disease. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Lung recruitment strategies and surfactant in neonatal intensive care unit.

Several studies in the lamb model have shown that hyperinflation of the lungs early in life may cause a blunted response to surfactant with signs of lung injury and any attempt to recruit lung volume in the surfactant deficient preterm infant by large lung inflations at birth should be potentially dangerous. As regards the situation when surfactant is given later, as rescue treatment for established RDS, the evidence for a clinically beneficial effect of a recruitment maneuver is yet insufficient and, hopefully, future studies will gather more data on this aspect.

Molecular packing parameters of bipolar lipids.

The bipolar lipid fractions extracted from the thermophilic archaeobacterium Sulfolobus solfataricus have different chemical structures and geometrical shapes. The conditions which lead to the formation of vesicles were investigated in order to study the self-assembly of these molecules. Such conditions are fulfilled when an appropriate mixture of two different molecular species (both bipolar or bipolar and monopolar) is used. According to the theory introduced by Israelachvili and co-workers, lipid self-assembly results from the balance of interaction free energy, entropy and molecular geometry. We have shown that this theory can be extended to bipolar lipids, in spite of their more complex nature, and the experimental results obtained combining 1H-NMR, light scattering and entrapped volume techniques closely match theoretical expectations. To carry out calculations, it was necessary to introduce hypotheses about the disposition of bipolar molecules in the vesicle membrane. These hypotheses have been tested indirectly by measuring the transport properties mediated by carriers or channels, whose transport mechanism can be considered to be a probe of the membrane structure.

Valinomycin acts as a channel in ultrathin lipid membranes.

When the thickness of monolayer membranes formed by bolaform archaeal lipids is reduced to the approximate length of two valinomycin molecules, the zero-current conductance does not show any more a linear dependence on valinomycin concentration; instead, a quadratic behaviour is observed. This suggests that a dimer permeation pore is formed and therefore the conduction mechanism changes from carrier to channel.

Stability against temperature and external agents of vesicles composed of archael bolaform lipids and egg PC.

The bolaform lipid PLE extracted from the thermophilic archaeon Sulfolobus solfataricus and its mixtures with egg phosphatidyl-choline (egg PC) have been used to prepare sonicated vesicles. The leakage of entrapped calcein was continuously monitored by fluorescence dequenching. The half times of leakage have been used to compare vesicle stability under different conditions of temperature, lipid composition and presence of destabilizing agents like Ca2+ ions and poly(ethylene glycol) (PEG). It has been found that leakage is primarily modulated by the monopolar/bipolar lipid ratio. In particular, the half time of leakage for vesicles formed from a mixture of the polar lipid extract (PLE) and egg PC is characterized by a maximum at about 1:2 molar ratio. The free energy of mixing has been evaluated from pressure-area isotherms on monolayers at the air/water interface. The results indicate a non monotonous behaviour of the excess free energy of mixing as a function of the molar ratio and the occurrence of a minimum at a fixed molar ratio. The possible formation of a complex is discussed and compared with previous calorimetric measurements on similar compounds.

Calcium-induced interaction and fusion of archaeobacterial lipid vesicles: a fluorescence study.

The lipids extracted from the membrane of the thermophilic archaeobacterium Sulfolobus solfataricus have an unusual bipolar structure. Each molecule is formed by two isoprenoid chains (with up to four cyclopentane groups per chain) ether-linked at both ends to glycerol or nonitol groups. These groups can be variably substituted, mainly with complex sugars. Fluorescence resonance energy transfer, aqueous contents mixing and calcein release assays were employed to assess whether bipolar lipid vesicles were able to undergo a calcium-induced fusion process. The possibility of getting fusion depends strongly on the phase behaviour of the lipids. With vesicles formed by the natural polar lipid extract (PLE), a mixture showing a complex polymorphic behaviour, the fusion process was observed above the temperature T congruent to 60 degrees C at 15 mM Ca2+. By contrast, no fusion was observed in vesicles of P2, a fraction displaying only the lamellar phase. A dramatic change of the fusion process was observed when egg PC or P2 was added to PLE. In this case only lipid mixing, but not a real fusion process occurred at T > or = 60 degrees C. The dependence of such a process on ionic conditions has also been studied. Additional experiments involving surface tension measurements on monolayers have been performed to assess the importance of a surface tension increase to get fusion. In contrast to other monopolar lipid systems, no detectable change in surface tension has been observed in our bipolar lipids even in cases in which the fusion process is present.

S-adenosylhomocysteine hydrolase from the thermophilic archaeon Sulfolobus solfataricus: purification, physico-chemical and immunological properties.

S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus, a thermoacidophilic archaeon optimally growing at 87 degrees C, has been purified to homogeneity. The specific activity of the homogeneous enzyme is 161 nmol of S-adenosylhomocysteine formed per min per mg of protein, and the overall yield, by immunoaffinity purification, is 51%. The enzyme has a molecular mass of 190 kDa, is composed of four identical subunits (subunit mass 47 kDa), and contains four molecules of tightly-bound NAD+ per tetramer of which about 40% is in the reduced form. Physico-chemical features, including amino-acid composition and secondary structure, are reported. The pure protein, used to raise specific rabbit antisera, shows immunological properties different from other S-adenosylhomocysteine-metabolizing enzymes. The enzyme is thermophilic with an optimum temperature of 75 degrees C, and shows an apparent melting temperature of 95 degrees C by measuring its residual activity after 10 min incubation at increasing temperatures.

The glycolipid of Halobacterium trapanicum.

The structural elucidation of the polar lipids in Halobacterium trapanicum is reported with particular emphasis on a new sulfated disaccharide derivative of 2,3-di-O-phytanyl-sn-glycerol. The full structural designation of this glycolipid is 2,3-di-O-phytanyl-1-O- (mannopyranosyl-(2-sulfate)-alpha-D-1-2-glucopyranosyl-alpha-D)-sn-glyce rol. The value of glycolipid structures in the taxonomy of halophilic Archaea is also discussed.

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria.

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70 degrees C) and Escherichia coli (max. growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. Subunits of C. acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77 degrees C) and E. coli counterparts (Tm = 72 degrees C). Based on the "melting' hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species. The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B. acidocaldarius and 55% G . C for E. coli. The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. Compared to E. coli the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.

ADP-ribosylation reactions in Sulfolobus solfataricus, a thermoacidophilic archaeon.

An ADP-ribosylating system was detected in a crude homogenate from Sulfolobus solfataricus, a thermophilic archaeon, optimally growing at 87 degrees C. The archaeal ADP-ribosylation reaction was time-, temperature- and NAD-dependent. It proved to be highly thermostable, with about 30% decrease of 14C incorporation from [14C]NAD on incubation at 80 degrees C for up to 24 h. The main reaction product was found to be mono-ADP-ribose. Testing both [adenine-14C(U)]NAD and [adenine-14C(U)]ADPR as substrates, it was found that acceptor proteins were modified by ADP-ribose both enzymatically, via ADP-ribosylating enzymes, and via chemical attachment of free ADP-ribose, likely produced by NAD glycohydrolase activity. The synthesis of ADP-ribose-protein complexes was shown to involve mainly acceptors with molecular masses in the 40-100 kDa range, as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate.

Glycolipids from Thermotoga maritima, a hyperthermophilic microorganism belonging to Bacteria domain.

Two novel glycolipids with a very rare alpha(1-->4) diglucosyl structure have been isolated from the thermophilic bacterium Thermotoga maritima. The structures of these compounds, on the basis of chemical procedures and spectroscopic studies (FAB-MS and NMR), were shown to be: 1(3),2-dipalmitoyl-3(1)-[glucopyranosyl-(6-decanoyl)-alpha-D-(1-->4)- glucopyranosyl-alpha-D]-glycerol (Glycolipid 1) and 1(3),2-dipalmitoyl-3(1)-[glucopyranosyl-alpha-D-(1-->4)-glucopyranosyl- alpha-D]-glycerol (Glycolipid 2).

Tetraether lipid components from a thermoacidophilic archaebacterium. Chemical structure and physical polymorphism.

As a continuation of an X-ray scattering study of the tetraether lipids extracted from the thermophilic archaebacterium Sulfolobus solfataricus, the phase behaviour of four fractions of the complex polar lipid extract (PLE) is described. Each molecule of two of these fractions (P1 and GL) carries an unsubstituted glycerol headgroup, those of another (P2) no such group; the fourth fraction (WPLE) is obtained by water-washing PLE, thus reducing its P2 content from approximately 48% to approximately 24% and increasing the average number of molecules bearing an unsubstituted glycerol headgroup from approximately 0.4 to approximately 0.6. The main result is a striking correlation between the phase behaviour and the average ratio of unsubstituted glycerol headgroups to the total number of headgroups: the fractions P1, GL and WPLE, in which that number is respectively 0.5, 0.5 and 0.3, form rod-containing phases; the fraction P2, in which that number is zero, yields a lamellar phase throughout the phase diagram. An analysis of the dimensions of the structure elements confirms our previous conclusion that, in the presence of a sufficient amount of water, the unsubstituted glycerol headgroups partition preferentially in the hydrocarbon regions rather than at the polar/apolar interfaces. These results, moreover, corroborate our previous conjectures regarding the correlations between the structure of the plasma membrane, the phase behaviour of the lipid extract and life at high temperature.

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria.

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 degrees C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C40 omega-omega' biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to "physiological" conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type alpha) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type beta'). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type alpha. (3) In all the phases with chains in the alpha conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 A. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the alpha conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunochemical detection of ADP-ribosylating enzymes in the archaeon Sulfolobus solfataricus.

Polyclonal antibodies raised against eukaryotic mono-(ADPribose)transferase and poly(ADPribose)polymerase were used to test the presence of antigenic determinants in a crude extract of Sulfolobus solfataricus, a thermophilic archaeon. Samples from eukaryotic (bull testis) and bacterial (E. coli) sources were analysed for comparison. All tested antibodies reacted with the sulfolobal sample with a specificity comparable to that of the eukaryotic preparation, as revealed by ELISA test, activity assays in the presence of antibodies and immunoblot experiments. After electrophoresis and western blot of sulfolobal proteins, a band at a mass around 50 kDa was detected by immunostaining.

Archaebacterial elongation factor Tu insensitive to pulvomycin and kirromycin.

A spermine-dependent, polyphenylalanine-synthesizing cell-free system having an optimum activity at 75-85 degrees C, has been developed from the extremely thermoacidophilic archaebacterium Caldariella acidophila. The C. acidophila system is totally insensitive to the EF-Tu targeted antibiotics pulvomycin (at 40 degrees C) and kirromycin (at 47-72 degrees C) contrary to control systems derived from both mesophilic (Escherichia coli) and thermoacidophilic (Bacillus acidocaldarius) eubacteria. The archaebacterial EF-Tu-equivalent factor is also immunologically unrelated to eubacterial EF-Tu and does not cross react with antibodies against Escherichia coli EF-Tu. The pulvomycin and kirromycin reactions thus provide new phyletic markers for archaebacterial ancestry.

Comparison of the ADP-ribosylating thermozyme from Sulfolobus solfataricus and the mesophilic poly(ADP-ribose) polymerases.

The poly(ADP-ribose) polymerase-like thermozyme purified from Sulfolobus solfataricus was characterised with respect to some physico-chemical properties. The archaeal protein exhibited a scarce electrophoretic mobility at both pH 2.9 and pH 7.5. Determination of the isoelectric point (pI=7.0-7.2) allowed us to understand the reason for the limited migration at pH 7.5, while amino acid composition analysis showed a moderate content of basic residues, which reduced mobility at pH 2.9. With respect to the charge, the archaeal enzyme behaved differently from the eukaryotic thermolabile poly(ADP-ribose) polymerase, described as a basic protein (pI=9.5). Well known inhibitors of the mesophilic polymerase like Zn(2+), nicotinamide and 3-aminobenzamide exerted a smaller effect on the enzyme from S. solfataricus, reducing the activity by at most 50%. Mg(2+) was a positive effector, although in a dose-dependent manner. It influenced the fluorescence spectrum of the archaeal protein, whereas NaCl had no effect.

Lipid profile: a useful chemotaxonomic marker for classification of a new cyanobacterium in Spirulina genus.

The morphological, physiological and genetic characteristics of an isolate cyanobacterium from hard sand of the lake Venere in the Pantelleria island (Italy) were described. The isolate with a small-size coiled helix shape, growing optimally at pH 9.2-9.5 at 30 degrees C under continuous illumination and aeration, possessed a 61.5 mol% of Guanine + Cytosine content of DNA. The lipid profile showed the presence of mono-, di-glycosyl, sulphoquinovolosyl and phosphatidyl (MGDG, DGDG, SQDG and PG). The fatty acid profile was also studied, characterized by the absence of gamma-linolenic acid and the presence of saturated and monounsaturated C16 and C18. The latter was also present as a dienoic component. The fatty acid composition was affected by growth temperature by increasing the degree of desaturation at a lower temperature and the biosynthesis of shorter acyl chains. The effects of growth conditions other than temperature, physical, nutritional and chemical on lipid composition were also studied. The overall features of the cyanobacterium isolated from Pantelleria clustered it into Spirulina genus.

Thermotropic properties of bipolar lipids of Sulfolobus solfataricus and of their mixtures with dipalmitoylphosphatidylcholine.

The thermotropic properties of the bipolar lipids, glycerol dialkylglycerol tetraether (GDGT) and glycerol dialkylnonitol tetraether (GDNT), were determined at different degrees of hydration and in mixtures with dipalmitoylphosphatidylcholine (DPPC). The number of water molecules rendered unfreezable by the GDNT molecule is 10+/-1.5 and that by the GDGT molecule 2.8+/-0.7 or about 1.1-1.5 H2O molecules per OH group. Binding of water molecules causes randomization of the two polar heads from the oriented form prevailing in the dry state. The hydration seems to be a cooperative process extending over a whole lipid domain. DPPC added in small amounts to GDNT interacts preferentially with the nonitol halves of the molecules separating them from the glycerol half molecules. In the cooperative interaction domain each DPPC molecule is surrounded by up to six GDNT molecules. Cooperative domains formed during the interaction of DPPC with GDGT are less pronounced. In both cases they affect the thermotropic properties of the system.

Monopolar-bipolar lipid interactions in model membrane systems.

1H-NMR, dynamic light scattering and negative staining electron microscopy have been used to study the formation and physico-chemical properties of aqueous dispersions of mixtures of monopolar lipids extracted from Sulfolobus solfataricus. This microorganism is a thermophilic archaeobacterium growing optimally at about 85 degrees C and pH 3. The two hydrolytic fractions of the membrane complex lipids that have been studied are: the symmetric lipid glycerol dialkyl glycerol tetraether (GDGT) and the asymmetric lipid glycerol dialkyl nonitol tetraether (GDNT). Electron micrographs of pure and mixed GDNT and GDGT dispersions show the formation of complex structures. Only above a critical monopolar/bipolar lipid ratio, typical of the bipolar lipid, could closed structures be formed and good agreement was obtained in sizing with NMR, electron microscopy and dynamic light scattering. NMR spectra have been carried out at several temperatures from 25 degrees to 85 degrees C, to obtain information on the temperature-dependent structural, dynamic and permeability properties of the co-dispersed vesicles. The results are discussed in terms of the steric constraints and the chemico-physical interactions occurring among the different parts of the molecules and compared with previous studies performed with different physical techniques.

Accumulation of osmoprotectants and lipid pattern modulation in response to growth conditions by Halomonas pantelleriense.

The effects of salinity, growth temperature, pH and composition of the medium on the accumulation of intracellular organic solutes, by nuclear magnetic resonance spectroscopy (NMR) in Halomonas pantelleriense were examined. The modulation of lipid pattern in different growth conditions was also reported. H. pantelleriense accumulated glycine betaine, ectoine, hydroxyectoine and glutamate. The type of osmoprotectant and the relative proportion depended on growth conditions. The main lipids identified by NMR studies were 1,2 diacylglycero-3-phosphorylethanolamine (PEA), 1,2 diacylglycero-3-phosphoryl-glycerol (PG) and cardiolipin, (DPG). The predominant fatty acids were C16:0 and C18:1, minor fatty acids were C16:1 and C18:0. The relative percentage of polar lipids and fatty acids were affected by growth conditions.