The role of LDH serum levels in predicting global outcome in HCC patients undergoing TACE: implications for clinical management

In many tumor types serum lactate dehydrogenase (LDH) levels is an indirect marker of tumor hypoxia, neo-angiogenesis and worse prognosis. However data about hepatocellular carcinoma (HCC) are lacking in the clinical setting of patients undergoing transarterial-chemoembolization (TACE) in whom hypoxia and neo-angiogenesis may represent a molecular key to treatment failure. Aim of our analysis was to evaluate the role of LDH pre-treatment levels in determining clinical outcome for patients with HCC receiving TACE. One hundred and fourteen patients were available for our analysis. For all patients LDH values were collected within one month before the procedure. We divided our patients into two groups, according to LDH serum concentration registered before TACE (first: LDH≤450 U/l 84 patients; second: LDH>450 U/l 30 patients). Patients were classified according to the variation in LDH serum levels pre- and post-treatment (increased: 62 patients vs. decreased 52 patients). No statistically significant differences were found between the groups for all clinical characteristics analyzed (gender, median age, performance status ECOG, staging systems). In patients with LDH values below 450 U/l median time to progression (TTP) was 16.3 months, whereas it was of 10.1 months in patients above the cut-off (p = 0.0085). Accordingly median overall survival (OS) was 22.4 months and 11.7 months (p = 0.0049). In patients with decreased LDH values after treatment median TTP was 12.4 months, and median OS was 22.1 months, whereas TTP was 9.1 months and OS was 9.5 in patients with increased LDH levels (TTP: p = 0.0087; OS: p<0.0001). In our experience, LDH seemed able to predict clinical outcome for HCC patients undergoing TACE. Given the correlation between LDH levels and tumor angiogenesis we can speculate that patients with high LDH pretreatment levels may be optimal candidates for clinical trial exploring a multimodality treatment approach with TACE and anti-VEGF inhibitors in order to improve TTP and OS.     

Structural and biochemical identification of a novel bacterial oxidoreductase

By using a bioinformatics screen of the Escherichia coli genome for potential molybdenum-containing enzymes, we have identified a novel oxidoreductase conserved in the majority of Gram-negative bacteria. The identified operon encodes for a proposed heterodimer, YedYZ in Escherichia coli, consisting of a soluble catalytic subunit termed YedY, which is likely anchored to the membrane by a heme-containing trans-membrane subunit termed YedZ. YedY is uniquely characterized by the presence of one molybdenum molybdopterin not conjugated by an additional nucleotide, and it represents the only molybdoenzyme isolated from E. coli characterized by the presence of this cofactor form. We have further characterized the catalytic subunit YedY in both the molybdenum- and tungsten-substituted forms by using crystallographic analysis. YedY is very distinct in overall architecture from all known bacterial reductases but does show some similarity with the catalytic domain of the eukaryotic chicken liver sulfite oxidase. However, the strictly conserved residues involved in the metal coordination sphere and in the substrate binding pocket of YedY are strikingly different from that of chicken liver sulfite oxidase, suggesting a catalytic activity more in keeping with a reductase than that of a sulfite oxidase. Preliminary kinetic analysis of YedY with a variety of substrates supports our proposal that YedY and its many orthologues may represent a new type of membrane-associated bacterial reductase.     

Kinetics of recovery of leaf hydraulic conductance and vein functionality from cavitation-induced embolism in sunflower

The kinetics of leaf vein recovery from cavitation-induced embolism was studied in plants of sunflower cv. Margot, together with the impact of vein embolism on the overall leaf hydraulic conductance (Kleaf). During the air-dehydration of leaves to leaf water potentials (Psi L) of -1.25 MPa, Kleaf was found to decrease by about 46% with respect to values recorded in well-hydrated leaves. When leaves, previously dehydrated to Psi L= -1.1 MPa (corresponding to the turgor loss point), were put in contact with water, Kleaf recovered completely in 10 min and so did leaf water potential. Functional vein density was estimated in both dehydrating and rehydrating leaves in terms of total length of red-stained veins infiltrated with a Phloxine B solution per unit leaf surface area. Veins were found to embolize (unstained) with kinetics showing a linear relationship with Kleaf so that about a 70% loss of functional veins corresponded with a Kleaf loss of 46%. Cavitated veins recovered from embolism within 10 min from the beginning of leaf rehydration. These data indicate that: (a) leaves of sunflower underwent substantial vein embolism during dehydration; (b) vein embolism and leaf hydraulic efficiency apparently recovered from dehydration completely and rapidly upon rehydration; (c) vein refilling occurred while conduits were still at more negative xylem pressures than those required for spontaneous bubble dissolution on the basis of Henry's law. The possible consistent contribution of vital mechanisms for vein refilling is discussed.     

Methylation of ELOVL2 gene as a new epigenetic marker of age

The discovery of biomarkers able to predict biological age of individuals is a crucial goal in aging research. Recently, researchers' attention has turn toward epigenetic markers of aging. Using the Illumina Infinium HumanMethylation450 BeadChip on whole blood DNA from a small cohort of 64 subjects of different ages, we identified 3 regions, the CpG islands of ELOVL2, FHL2, and PENK genes, whose methylation level strongly correlates with age. These results were confirmed by the Sequenom's EpiTYPER assay on a larger cohort of 501 subjects from 9 to 99 years, including 7 cord blood samples. Among the 3 genes, ELOVL2 shows a progressive increase in methylation that begins since the very first stage of life (Spearman's correlation coefficient = 0.92) and appears to be a very promising biomarker of aging.     

Impact of the leaf miner<Emphasis Type="Italic"> Cameraria ohridella</Emphasis> on photosynthesis,water relations and hydraulics of<Emphasis Type="Italic"> Aesculus hippocastanum</Emphasis> leaves

The mining of leaves of Aesculus hippocastanum caused by the larvae of Cameraria ohridella leads to precocious defoliation of trees. Damage to plant productivity was estimated in terms of the photosynthetic performance as well as of leaf water relations and hydraulics of increasingly mined leaves from infested plants in comparison with the same variables measured in non-mined leaves (controls). Electron microscopy and photosynthesis measurements revealed that chloroplasts within the green portions of mined leaves were still intact and photosynthesis of these areas was close to that of non-mined leaves, i.e. damage to functional integrity of the photosynthetic system did not extend beyond the mines. Stomata below the mines were functional as they maintained their physiological kinetics but most chloroplasts in the spongy parenchyma below the mines were degraded so that a 1:1 relationship existed between photosynthesis loss and loss of leaf green areas. Leaf conductance to water vapour and transpiration rate were 60% lower in mined leaf areas but equal to controls in green portions of mined leaves. Leaf water potential was insensitive to the amount of mined leaf area and so was leaf hydraulic conductance. Anatomical observations of leaf minor veins revealed that they were structurally and functionally intact even in leaves with 90% mined surface area. Our conclusion was that the actual damage to A. hippocastanum plants in terms of loss of photosynthates and water and nutrient transport was less than that visually estimated in recent studies.     

Activation of the Double-stranded RNA-dependent Protein Kinase PKR by Small Ubiquitin-like Modifier (SUMO)

The dsRNA-dependent kinase PKR is an interferon-inducible protein with ability to phosphorylate the α subunit of the eukaryotic initiation factor (eIF)-2 complex, resulting in a shut-off of general translation, induction of apoptosis, and inhibition of virus replication. Here we analyzed the modification of PKR by the small ubiquitin-like modifiers SUMO1 and SUMO2 and evaluated the consequences of PKR SUMOylation. Our results indicate that PKR is modified by both SUMO1 and SUMO2, in vitro and in vivo. We identified lysine residues Lys-60, Lys-150, and Lys-440 as SUMOylation sites in PKR. We show that SUMO is required for efficient PKR-dsRNA binding, PKR dimerization, and eIF2α phosphorylation. Furthermore, we demonstrate that SUMO potentiates the inhibition of protein synthesis induced by PKR in response to dsRNA, whereas a PKR SUMOylation mutant is impaired in its ability to inhibit protein synthesis and shows reduced capability to control vesicular stomatitis virus replication and to induce apoptosis in response to vesicular stomatitis virus infection. In summary, our data demonstrate the important role of SUMO in processes mediated by the activation of PKR.     

Structural properties of Gerstmann-Straussler-Scheinker disease amyloid protein

Prion protein (PrP) amyloid formation is a central feature of genetic and acquired forms of prion disease such as Gerstmann-Str?ussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob disease. The major component of GSS amyloid is a PrP fragment spanning residues approximately 82-146. To investigate the determinants of the physicochemical properties of this fragment, we synthesized PrP-(82-146) and variants thereof, including entirely and partially scrambled peptides. PrP-(82-146) readily formed aggregates that were partially resistant to protease digestion. Peptide assemblies consisted of 9.8-nm-diameter fibrils having a parallel cross-beta-structure. Second derivative of infrared spectra indicated that PrP-(82-146) aggregates are primarily composed of beta-sheet (54%) and turn (24%) which is consistent with their amyloid-like properties. The peptide induced a remarkable increase in plasma membrane microviscosity of primary neurons. Modification of the amino acid sequence 106-126 caused a striking increase in aggregation rate, with formation of large amount of protease-resistant amorphous material and relatively few amyloid fibrils. Alteration of the 127-146 region had even more profound effects, with the inability to generate amyloid fibrils. These data indicate that the intrinsic properties of PrP-(82-146) are dependent upon the integrity of the C-terminal region and account for the massive deposition of PrP amyloid in GSS.     

Rhythmic,reciprocal ghrelin and leptin signaling: new insight in the development of obesity

The hypothalamus integrates metabolic, neural and hormonal signals to evoke an intermittent appetitive drive in the daily management of energy homeostasis. Three major players identified recently in the feedback communication between the periphery and hypothalamus are leptin, ghrelin and neuropeptide Y (NPY). We propose that reciprocal circadian and ultradian rhythmicities in the afferent humoral signals, anorexigenic leptin from adipocytes and orexigenic ghrelin from stomach, encode a corresponding discharge pattern in the appetite-stimulating neuropeptide Y network in the hypothalamus. An exquisitely intricate temporal relationship among these signaling modalities with varied sites of origin is paramount in sustenance of weight control on a daily basis. Our model envisages that subtle and progressive derangements in temporal communication, imposed by environmental shifts in energy intake, impel a positive energy balance culminating in excessive weight gain and obesity. This conceptual advance provides a new target for designing pharmacologic or gene transfer therapies that would normalize the rhythmic patterns of afferent hormonal and efferent neurochemical messages.