Peach fruit ripening: A proteomic comparative analysis of the mesocarp of two cultivars with different flesh firmness at two ripening stages

A proteomic analysis was conducted on peach fruit mesocarp in order to better elucidate the biochemical and physiological events which characterize the transition of fruit from the “unripe” to the “ripe” phase.The first goal of the present work was to set-up a protocol suitable for improving protein extraction from peach mesocarp. The use of freeze-dried powdered tissue, together with the addition of phenol prior to the extraction with an aqueous buffer, significantly increased the protein yield and the quality of 2-DE gels. The proteomic profiles of the mesocarp from peach fruit of a non-melting flesh (NMF; ‘Oro A’) and a melting flesh (MF; ‘Bolero’) cultivar, at “unripe” and “ripe” stages as defined by some parameters typical of ripening, were then analyzed.The comparative analysis of the 2-DE gels showed that in NMF and MF peaches the relative volumes of 53 protein spots significantly changed in relation to both the ripening stage (“unripe” versus “ripe”) and/or the genetic background of the cultivar (‘Oro A’ versus ‘Bolero’).Thirty out of the 53 differently abundant spots were identified by LC-ESI-MS/MS. The analysis revealed enzymes involved in primary metabolism (e.g. C-compounds, carbohydrates, organic acids and amino acids) and in ethylene biosynthesis as well as proteins involved in secondary metabolism and responses to stress.Among these, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) appeared to be one of the proteins with the largest change in relative abundance during the fruit transition from the pre-climacteric (“unripe”) to the climacteric (“ripe”) phase. Other proteins, such as S-adenosylmethionine synthetase and β-cyanoalanine synthase involved in ethylene metabolism, were also identified. Moreover, the changes in the relative abundances of a sucrose synthase and an α-amylase suggested differences between the two cultivars in the carbohydrate import activity of ripe fruit. The different accumulation of a few typical ROS-scavenger enzymes suggested that a higher oxidative stress occurred in MF with respect to NMF fruit. This result, together with data concerning the levels of total proteins and free amino acids and those regarding proteins involved in the maintenance of tissue integrity, was consistent with the hypothesis that the last phase of ripening in MF fruit is characterized by the appearance of a senescence status.The present study appears to define well some of the biochemical and physiological events that characterize the ripening of peach and, at the same time, provides interesting indications that could be employed in future marker assisted selection (MAS) programmes aimed to obtain MF fruits with higher ability to preserve tissue functionality maintaining for a longer time their organoleptic characteristics.     

A candidate gene association study for nine economically important traits in Italian Holstein cattle

We genotyped 58 single nucleotide polymorphisms (SNPs) in 25 candidate genes in about 800 Italian Holstein sires. Fifty‐six (minor allele frequency >0.02) were used to evaluate their association with single traits: milk yield (MY), milk fat yield (FY), milk protein yield (PY), milk fat percentage (FP), milk protein percentage (PP), milk somatic cell count (MSCC); and complex indexes: longevity, fertility and productivity–functionality type (PFT), using deregressed proofs, after adjustment for familial relatedness. Thirty‐two SNPs were significantly associated (proportion of false positives <0.05) with different traits: 16 with MSCC, 15 with PY, 14 with MY, 12 with PFT, eight with longevity, eight with FY, eight with PP, five with FP and two with fertility. In particular, a SNP in the promoter region of the PRLR gene was associated with eight of nine traits. DGAT1 polymorphisms were highly associated with FP and FY. Casein gene markers were associated with several traits, confirming the role of the casein gene cluster in affecting milk yield, milk quality and health traits. Other SNPs in genes located on chromosome 6 were associated with PY, PP, PFT, MY (PPARGC1A) and MSCC (KIT). This latter association may suggest a biological link between the degree of piebaldism in Holstein and immunological functions affecting somatic cell count and mastitis resistance. Other significant SNPs were in the ACACA, CRH, CXCR1, FASN, GH1, LEP, LGB (also known as PAEP), MFGE8, SRC, TG, THRSP and TPH1 genes. These results provide information that can complement QTL mapping and genome‐wide association studies in Holstein.     

The main pollen season of Parietaria in Genoa (Italy): forecast possibilities

The authors propose a statistical model for evaluating the Beginning of the Main Pollen Season (BMPS) of Urticaceae (cf. Parietaria) in Genoa (Italy). For eight consecutive years (1981–1988) the mean daily concentrations of this pollen grain were studied in relation to the two meteorological factors which are most significant in triggering the beginning of maximal pollination, namely, mean daily air temperature and rainfall. From a multiple-regression statistical analysis, a formula has been extrapolated, which, one the basis of these meteorological data for the month of January, can be used to predict the onset of the BMPS to within nine days.     

Intrapleural liquid flow down a gravity-dependent hydraulic pressure gradient

We studied the vertical movement of 2 mg technetium-labeled albumin injected intrapleurally in 0.5 ml saline (15% of pleural liquid volume) in eight spontaneously breathing anesthetized dogs subject to a sudden change in posture (prone to supine or vice versa). The albumin movements were evaluated through a large field gamma camera placed laterally to the animal and detecting total (AT) and regional activities from two superimposed equal areas (At and Ab, top and bottom, respectively). The At/Ab ratio decreased from 2.1 to 1.3 in four animals up to 20 min from the change in posture and from 0.9 to 0.5 in four more animals studied from 50 to 90 min from turning maneuver. The rate of change in At and Ab was similar in the two groups of animals and unaffected by the acquisition posture. AT decreased by 7.7 and 3.5% for the two groups, respectively, reflecting albumin clearance from the pleural space. The opposite time course of regional activities and the independence of their rate of change of the At/Ab ratio and of the animal posture suggest a top-to-bottom albumin transfer occurring through a bulk flow of liquid estimated at 0.006 ml.kg-1.h-1. The data are consistent with a measured vertical pleural liquid pressure gradient that does not reflect a hydrostatic condition.     

Relative extended haplotype homozygosity signals across breeds reveal dairy and beef specific signatures of selection

Background

A number of methods are available to scan a genome for selection signatures by evaluating patterns of diversity within and between breeds. Among these, “extended haplotype homozygosity” (EHH) is a reliable approach to detect genome regions under recent selective pressure. The objective of this study was to use this approach to identify regions that are under recent positive selection and shared by the most representative Italian dairy and beef cattle breeds.

Results

A total of 3220 animals from Italian Holstein (2179), Italian Brown (775), Simmental (493), Marchigiana (485) and Piedmontese (379) breeds were genotyped with the Illumina BovineSNP50 BeadChip v.1. After standard quality control procedures, genotypes were phased and core haplotypes were identified. The decay of linkage disequilibrium (LD) for each core haplotype was assessed by measuring the EHH. Since accurate estimates of local recombination rates were not available, relative EHH (rEHH) was calculated for each core haplotype. Genomic regions that carry frequent core haplotypes and with significant rEHH values were considered as candidates for recent positive selection. Candidate regions were aligned across to identify signals shared by dairy or beef cattle breeds. Overall, 82 and 87 common regions were detected among dairy and beef cattle breeds, respectively. Bioinformatic analysis identified 244 and 232 genes in these common genomic regions. Gene annotation and pathway analysis showed that these genes are involved in molecular functions that are biologically related to milk or meat production.

Conclusions

Our results suggest that a multi-breed approach can lead to the identification of genomic signatures in breeds of cattle that are selected for the same production goal and thus to the localisation of genomic regions of interest in dairy and beef production.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0113-9) contains supplementary material, which is available to authorized users.     

Circulating microRNAs,miR-939, miR-595, miR-519d and miR-494, Identify Cirrhotic Patients with HCC

The performance of circulating biomarkers for the diagnosis of hepatocellular carcinoma (HCC) is sub-optimal. In this study we tested circulating microRNAs as biomarkers for HCC in cirrhotic patients by performing a two stage study: a discovery phase conducted by microarray and a validation phase performed by qRT-PCR in an independent series of 118 patients. Beside miRNAs emerged from the discovery phase, miR-21, miR-221, miR-519d were also tested in the validation setting on the basis of literary and tissue findings. Deregulated microRNAs were assayed in HCC-derived cells in the intracellular compartment, cell culture supernatant and exosomal fraction. Serum and tissue microRNA levels were compared in 14 patients surgically treated for HCC. From the discovery study, it emerged that seven circulating microRNAs were differentially expressed in cirrhotic patients with and without HCC. In the validation set, miR-939, miR-595 and miR-519d were shown to differentiate cirrhotic patients with and without HCC. MiR-939 and miR-595 are independent factors for HCC. ROC curves of miR-939, miR-595 and miR-519d displayed that AUC was higher than AFP. An exosomal secretion of miR-519d, miR-21, miR-221 and miR-1228 and a correlation between circulating and tissue levels of miR-519d, miR-494 and miR-21 were found in HCC patients. Therefore, we show that circulating microRNAs deserve attention as non-invasive biomarkers in the diagnostic setting of HCC and that exosomal secretion contributes to discharging a subset of microRNAs into the extracellular compartment.     

MicroRNA profiling reveals that miR-21, miR486 and miR-214 are upregulated and involved in cell survival in S��zary syndrome

Sézary syndrome (SS) is an incurable leukemic variant of cutaneous T-cell lymphoma and its pathogenesis is still unknown. Diagnosis/prognosis may strongly ameliorate the management of SS individuals. Here, we profiled the expression of 470 microRNAs (miRNAs) in a cohort of 22 SS patients, and we identified 45 miRNAs differentially expressed between SS and controls. Using predictive analysis, a list of 19 miRNAs, including miR-21, miR-214, miR-486, miR-18a, miR-342, miR-31 and let-7 members were also found. Moreover, we defined a signature of 14 miRNAs including again miR-21, potentially able to discriminate patients with unfavorable and favorable outcome. We validated our data for miR-21, miR-214 and miR-486 by qRT-PCR, including an additional set of array-independent SS cases. In addition, we also provide an in vitro evidence for a contribution of miR-214, miR-486 and miR-21 to apoptotic resistance of CTCL cell line.     

MicroRNA expression changes during human leukemic HL-60 cell differentiation induced by 4-hydroxynonenal,a product of lipid peroxidation

4-Hydroxynonenal (HNE) is one of several lipid oxidation products that may have an impact on human pathophysiology. It is an important second messenger involved in the regulation of various cellular processes and exhibits antiproliferative and differentiative properties in various tumor cell lines. The mechanisms by which HNE affects cell growth and differentiation are only partially clarified. Because microRNAs (miRNAs) have the ability to regulate several cellular processes, we hypothesized that HNE, in addition to other mechanisms, could affect miRNA expression. Here, we present the results of a genome-wide miRNA expression profiling of HNE-treated HL-60 leukemic cells. Among 470 human miRNAs, 10 were found to be differentially expressed between control and HNE-treated cells (at p < 0.05). Six miRNAs were down-regulated (miR-181a*, miR-199b, miR-202, miR-378, miR-454-3p, miR-575) and 4 were up-regulated (miR-125a, miR-339, miR-663, miR-660). Three of these regulated miRNAs (miR-202, miR-339, miR-378) were further assayed and validated by quantitative real-time RT-PCR. Moreover, consistent with the down-regulation of miR-378, HNE also induced the expression of the SUFU protein, a tumor suppressor recently identified as a target of miR-378. The finding that HNE could regulate the expression of miRNAs and their targets opens new perspectives on the understanding of HNE-controlled pathways. A functional analysis of 191 putative gene targets of miRNAs modulated by HNE is discussed.