Further studies on the use of molecular probes to grapevine closterovirus A

Two cloned cDNA probes to genomic RNA of grapevine closterovirus A (GVA) were utilized successfully for the detection of viral sequences in infected herbaceous hosts (Nicotiana benthamiana) and grapevines. One of the probes (pGA112) was complementary to the central part of the viral genome and gave light false positive signals with healthy grapevine extracts, whereas the other (pGA240), which is presumably colinear with the 3' terminus, was virus-specific and hybridized only with infected sample extracts. The two probes recognized smaller than genome RNAs in electrophoresed N. benthamiana extracts and hybridized differentially with the bands, thus suggesting that these represent subgenomic RNAs. Probe pGA240 may be used for GVA detection, but the preparation of samples for hybridization needs further improvement for routine testing.Weitere Untersuchungen über die Verwendung von Molekülsonden beim Grapevine-Closterovirus AZwei klonierte cDNA-Sonden für genomische RNA des Grapevine-Closterovirus A (GVA} wurden mit Erfolg zum Nachweis von Virussequenzen in infizierten krautigen Wirtspflanzen (Nicotiana benthamiana) und Reben benützt. Die eine Sonde (pGA112} war komplementär zum zentralen Teil des Virusgenoms und lieferte schwache falsch-positive Nachweisreaktionen mit Extrakten aus gesunden Reben; die andere Sonde (pGA240}, die vermutlich kolinear mit dem 3'-Terminus ist, war dagegen virusspezifisch und hybridisierte nur mit Extrakten aus infizierten Proben. In elektrophoretisch aufgetrennten N. benthamiana-Extrakten "erkannten" die beiden Sonden RNAs von weniger als Genomgröße; sie hybridisierten differenziert mit den Banden, so daß diese subgenomische RNAs darstellen könnten. Die Sonde pGA240 kann für den GVA-Nachweis verwendet werden; für Routinetests muß die Vorbereitung der Proben für die Hybridisierung noch verbessert werden

Caveolin-1, breast cancer and ionizing radiation

Breast cancer (BC) recovery has increased in recent years thanks to efforts of Omics-based research in this field. However, despite the important results obtained, BC remains a complex multifactorial pathology that is difficult to treat appropriately. Caveolin-1 (CAV1), the basic constituent protein of specialized plasma membrane invaginations called caveolae, is emerging as a potential therapeutic biomarker in BC. This factor may modulate BC response to chemotherapy and radiation therapy. In addition, recent reports describe the key role of CAV1 during cell response to oxidative stress. The aim of the present review was to describe the biological roles of CAV1 in BC considering its contrasting dual functions as an oncogene and as a tumor suppressor. In addition, we report on how CAV1 may contribute to tumor cell response to ionizing radiation treatment. Finally, new roles of CAV1 in BC both on epithelium and stroma may be useful as prognostic indicators for patient treatment and help clinicians in the selection of the best personalized therapy

Production of monoclonal antibodies to Grapevine virus D and contribution to the study of its aetiological role in grapevine diseases

Six stable hybridoma cell lines secreting monoclonal antibodies (MAbs) to Grapevine virus D (GVD) were obtained by fusing spleen cells of immunized BALB/c mice with mouse myeloma cell line Sp 2/0-Ag 14, In ELISA all MAbs detected the virus in Nicotiana leaf extracts or cortical shavings from mature grapevine canes, The use of a polyclonal antiserum for coating plates and of monoclonal antibodies and antimouse-conjugated antibodies for antigen detection, gave highly efficient and reproducible results for identification of GVD in field-grown grapevines. The reliability of the ELISA kit was confirmed by GVD-transmission tests to herbaceous hosts, using in vitro explants as inoculum, 223 vines affected by one or more of the 4 syndroms of the rugose wood complex (Kober stem grooving, Corky bark, LN stem grooving and Rupestris stem pitting) were tested in ELISA for the detection of Grapevine virus A (GVA), Grapevine virus B (GVB) and GVD and by Western blot for the detection of Grapevine rupestris stem pitting associated virus (GRSPaV). The possible cause-effect relationship between GVA and KSG, GVB and Co, and GRSPaV and RSP was confirmed, but no consistent association was found between GVD and any of the 4 above syndromes, Intriguingly, a reduction in the expression of stem pitting symptoms in V. rupestris (from 90 % to 75 %) and of stem grooving symptoms in Kober 5BB (from 95 % to 70 %) was observed when vitiviruses and GRSPaV were contemporarily present in the same indicator. Preliminary data of a survey involving 676 grapevine samples showed a high incidence (31 %) of GVD, regardless of the geographical origin of samples.

Timing performance of a double layer diamond detector

In order to improve the time precision of detectors based on diamonds sensors we have built a detector with two scCVD layers connected in parallel to the same amplifier. This work describes the design and the first measurements of such a prototype performed on a particle beam at CERN. With this different configuration we have obtained an improvement larger than a factor of 1.6-1.7 for the timing precision of the measurement when compared to a one layer scCVD diamond detector.Peer reviewe

Integrated multi-omics investigations of metalloproteinases in colon cancer: Focus on MMP2 and MMP9

Colorectal cancer (CRC) develops by genetic and epigenetic alterations. However, the molecular mechanisms underlying metastatic dissemination remain unclear and could benefit from multi-omics investigations of specific protein families. Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in ECM remodeling and the processing of bioactive molecules. Increased MMP expression promotes the hallmarks of tumor progression, including angiogenesis, invasion, and metastasis, and is correlated with a shortened survival. Nevertheless, the collective role and the possible coordination of MMP members in CRC are poorly investigated. Here, we performed a multi-omics analysis of MMP expression in CRC using data mining and experimental investigations. Several databases were used to deeply mine different expressions between tumor and normal tissues, the genetic and epigenetic alterations, the prognostic value as well as the interrelationships with tumor immune-infiltrating cells (TIICs). A special focus was placed on to MMP2 and MMP9: their expression was correlated with immune markers and the interaction network of co-expressed genes disclosed their implication in epithelial to mesenchymal transition (EMT) and immune response. Finally, the activity levels of MMP2 and MMP9 in a cohort of colon cancer samples, including tissues and the corresponding sera, was also investigated by zymography. Our findings suggested that MMPs could have a high potency, as they are targeted in colon cancer, and might serve as novel biomarkers, especially for their involvement in the immune response. However, further studies are needed to explore the detailed biological functions and molecular mechanisms of MMPs in CRC, also in consideration of their expression and different regulation in several tissues

Proteomic Profiling of Colon Cancer Tissues: Discovery of New Candidate Biomarkers

Colon cancer is an aggressive tumor form with a poor prognosis. This study reports a comparative proteomic analysis performed by using two-dimensional differential in-gel electrophoresis (2D-DIGE) between 26 pooled colon cancer surgical tissues and adjacent non-tumoral tissues, to identify potential target proteins correlated with carcinogenesis. The DAVID functional classification tool revealed that most of the differentially regulated proteins, acting both intracellularly and extracellularly, concur across multiple cancer steps. The identified protein classes include proteins involved in cell proliferation, apoptosis, metabolic pathways, oxidative stress, cell motility, Ras signal transduction, and cytoskeleton. Interestingly, networks and pathways analysis showed that the identified proteins could be biologically inter-connected to the tumor-host microenvironment, including innate immune response, platelet and neutrophil degranulation, and hemostasis. Finally, transgelin (TAGL), here identified for the first time with four different protein species, collectively down-regulated in colon cancer tissues, emerged as a top-ranked biomarker for colorectal cancer (CRC). In conclusion, our findings revealed a different proteomic profiling in colon cancer tissues characterized by the deregulation of specific pathways involved in hallmarks of cancer. All of these proteins may represent promising novel colon cancer biomarkers and potential therapeutic targets, if validated in larger cohorts of patients

Proteomic profiling of 13 paired ductal infiltrating breast carcinomas and non-tumoral adjacent counterparts.

According to recent statistics, breast cancer remains one of the leading causes of death among women in Western countries. Breast cancer is a complex and heterogeneous disease, presently classified into several subtypes according to their cellular origin. Among breast cancer histotypes, infiltrating ductal carcinoma represents the most common and potentially aggressive form. Despite the current progress achieved in early cancer detection and treatment, including the new generation of molecular therapies, there is still need for identification of multiparametric biomarkers capable of discriminating between cancer subtypes and predicting cancer progression for personalized therapies. One established step in this direction is the proteomic strategy, expected to provide enough information on breast cancer profiling. To this aim, in the present study we analyzed 13 breast cancer tissues and their matched non-tumoral tissues by 2-DE. Collectively, we identified 51 protein spots, corresponding to 34 differentially expressed proteins, which may represent promising candidate biomarkers for molecular-based diagnosis of breast cancer and for pattern discovery. The relevance of these proteins as factors contributing to breast carcinogenesis is discussed

Next-generation sequencing and metagenomic analysis advances plant virus diagnosis and discovery

The advent of next generation sequencing (NGS) technologies dramatically advanced our ability to comprehensively investigate diseases of unknown etiology and expedited the entire process of virus discovery, identification, viral genome sequencing and, subsequently, the development of routine assays for new viral pathogens. Unlike traditional techniques, these novel approaches require no preliminary knowledge of the suspected virus(es). Currently, the RNA-Seq approach has been widely used to identify new viruses in infected plants, by analyzing virus-derived small interfering RNA populations, single- and double-stranded RNA (dsRNA) molecules extracted from infected plants. The method generates sequence in an unbiased fashion, likely allowing to detect all viruses that are present in a sample. We applied the Illumina NGS, coupled with metagenomic analysis, to generate large sequence dataset in different woody crops affected by diseases of unknown origin or infected with uncharacterized viruses or new strains. This approach allowed the identification of five novel viral species and, in addition, the sequencing of the whole genome of several viruses and viroids infecting Citrus spp., Prunus spp., grapes, fig, hazelnut, olive, persimmon and mulberry. Combined analysis of the datasets generated by using either siRNA fractions and dsRNA templates, enhanced the characterization of the whole virus-derived sequences in the infected tissues. Furthermore, profiling small RNAs from virus-infected plants led to a better understanding of host-plant response to virus and viroid infections in perennial plants. A general bioinformatic pipeline and an experimental validation strategy were developed and its application illustrated

Worldwide diffusion of Fig latent virus 1 in fig accessions and its detection by serological and molecular tools

A virus with filamentous particles ca. 700 nm long, denoted Fig latent virus 1 (FLV-1) is widespread in Apulian (southern Italy) fig orchards, in trees showing or not mosaic symptoms and in symptomless seedlings. The virus was transmitted by sap inoculation to a very restricted range of herbaceous hosts without inducing apparent symptoms and was transmitted through fig seeds to a very high percentage (80 to 100 %). It was successfully purified from root tissues of infected figs. A virus-specific antiserum raised in rabbits, proved useful for its detection in fig leaf dips by immunosorbent electron microscopy (ISEM), Western Blot, dot immuno-binding (DIBA), ELISA. The viral genome structure resembles that of members of the genus Trichovirus in the family Flexiviridae. Keywords: fig latent virus, Trichovirus, serology, ISEM, Western blot, DIBA, ELIS

Occurrence of grapevine virus A (GVA) and other closteroviruses in Tunisian grapevines affected by leafroll disease

Vorkommen von Grapevine-Virus A (GVA) und anderen Closteroviren in blattrollkranken tunesischen RebenReben, die aus den Hauptweinbaugebieten Tunesiens stammten, wurden auf die Anwesenheit von Closteroviren hin überprüft. Während in keiner der symptomfreien Reben Viruspartikel entdeckt wurden, enthielten alle Reben mit Blattrollsymptomen - außer zweien - Closteroviruspartikel, die durch IEM (immune electron microscopy) in konzentrierten Blattextrakten oder unmittelbar in Rohsaft durch ISEM (immunosorbent electron microscopy) identifiziert wurden. Alle vier derzeit bekannten Closteroviren (GClV-1, GClV-2, GClV-3 und GVA) waren, meistens im Gemisch, in Reben mit Blattrollsymptomen vorhanden. GClV-3 und GVA wurden in 77 bzw. 50 % der geprüften Reben entdeckt. Ein tunesisches Isolat von GVA, das durch Planococcus citri auf krautige Testpflanzen übertragen wurde, unterschied sich in biologischer Hinsicht, aber nicht in den charakteristischen physikalisch-chemischen und serologischen Eigenschaften von zwei italienischen Isolaten desselben Virus