Novel Circulating miRNA Signatures for Early Detection of Pancreatic Neoplasia

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) presents the lowest survival rate of all cancers because only 6% of patients reach five-year survival. Alterations in the expression of several microRNAs (miRNAs) occur in the tumor of PDAC and in preneoplastic lesions as the called intraductal papillary mucinous neoplasm (IPMN). Here, we aimed at identifying which miRNAs are significantly altered in liquid biopsies from patients with PDAC and IPMN to find new noninvasive biomarkers for early detection of PDAC. METHODS: We analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR) the expression of 17 circulating miRNAs, previously found to be significantly overexpressed in tissue pancreatic neoplasms, in a set of 182 plasma samples (94 PDAC, 19 IPMN, 18 chronic pancreatitis, and 51 disease-free controls). Then, we analyzed CA19.9 levels in the same plasma set, and we assessed the diagnostic values of differentially expressed miRNAs, CA19.9, and all possible combinations. RESULTS: Of note, 16, 14, and 9 miRNAs were significantly increased in PDAC, IPMN, and chronic pancreatitis, respectively, compared with control plasmas. miR-21-5p, miR-33a-3p, miR-320a, and miR-93-5p showed the highest discriminating capacity for pancreatic neoplasia (PDAC or IPMN) with an area under the receiver operating characteristic curve (AUC) of 0.86, 0.85, 0.85, and 0.80, respectively. 2-miRNA combinations improved these performances reaching AUC = 0.90 for "miR-33a-3p+miR-320a." Addition of CA19.9 increased the diagnostic potential of miRNA signatures even further achieving an AUC of 0.95 (93% sensitivity and 85% specificity) for the combination of "miR-33a-3p+miR-320a+CA19.9." CONCLUSIONS: Novel signatures combining miRNAs and CA19.9 could be used as noninvasive biomarkers for early detection of PDAC

Genetic variability of the coat protein gene of isolates of Citrus variegation virus from Campania (southern Italy)

Six new sequences of the coat protein (CP) gene of Citrus variegation virus (CVV) isolates, some producing crinkly leaf symptoms and some asymptomatic, and collected in Campania (southern Italy), are presented. Comparison with previously reported CP gene sequences of isolates from various locations worldwide confirmed a high degree of coat protein gene conservation in Campania (lowest similarity among all CVV sequences 92.4%). No relationship was found between amino-acid substitutions and host species or symptoms. Phylogenetic analysis proved that CP sequences from Campania isolates clustered in a new group when compared to those reported in the data bank

Viroid infection and rootstocks affect productivity and fruit quality of the Tunisian citrus cultivar Maltaise demi sanguine

In Tunisia, sweet orange citrus cultivars are usually grafted on sour orange rootstock. However, this rootstock is susceptible to Citrus tristeza virus (CTV). A trial was established in 2005 to evaluate the performance of newly introduced rootstocks compared to sour orange, using the sweet orange cultivar ‘Maltaise demi sanguine’ as the grafted scion. The effect of single or mixed viroid infections were monitored over 12 years. Once established, tree growth, cumulative yield, tree performance and fruit quality of ‘Maltaise sweet orange’ were assessed from 2008. Mixed viroid infections caused significant decreases (39 to 60%) in the canopy volume of ‘Maltaise’ grafted on Carrizo citrange, Swingle citrumelo, Cleopatra mandarin, Rangpur lime, Volkamer lemon and Trifoliate orange. The cumulative yield of trees grafted on’Alemow’ (Citrus macrophylla) and inoculated with Hop stunt viroid (HSVd) was 76% less than the healthy control. Mixed infections caused production decreases of 56% from trees grafted on Swingle citrumelo and 69% from those grafeted on Trifoliate orange. When quality parameters of fruit from trees infected with each viroid, and combinations of viroids were compared, no significant differences were recorded.

Viroid infection and rootstocks affect productivity and fruit quality of the Tunisian citrus cultivar Maltaise demi sanguine

In Tunisia, sweet orange citrus cultivars are usually grafted on sour orange rootstock. However, this rootstock is susceptible to Citrus tristeza virus (CTV). A trial was established in 2005 to evaluate the performance of newly introduced rootstocks compared to sour orange, using the sweet orange cultivar ‘Maltaise demi sanguine’ as the grafted scion. The effect of single or mixed viroid infections were monitored over 12 years. Once established, tree growth, cumulative yield, tree performance and fruit quality of ‘Maltaise sweet orange’ were assessed from 2008. Mixed viroid infections caused significant decreases (39 to 60%) in the canopy volume of ‘Maltaise’ grafted on Carrizo citrange, Swingle citrumelo, Cleopatra mandarin, Rangpur lime, Volkamer lemon and Trifoliate orange. The cumulative yield of trees grafted on’Alemow’ (Citrus macrophylla) and inoculated with Hop stunt viroid (HSVd) was 76% less than the healthy control. Mixed infections caused production decreases of 56% from trees grafted on Swingle citrumelo and 69% from those grafeted on Trifoliate orange. When quality parameters of fruit from trees infected with each viroid, and combinations of viroids were compared, no significant differences were recorded.

Biochemical–molecular–genetic biomarkers in the tear film, aqueous humor, and blood of primary open-angle glaucoma patients

Introduction: Glaucoma is a chronic neurodegenerative disease, which is the leading cause of irreversible blindness worldwide. As a response to high intraocular pressure, the clinical and molecular glaucoma biomarkers indicate the biological state of the visual system. Classical and uncovering novel biomarkers of glaucoma development and progression, follow-up, and monitoring the response to treatment are key objectives to improve vision outcomes. While the glaucoma imaging field has successfully validated biomarkers of disease progression, there is still a considerable need for developing new biomarkers of early glaucoma, that is, at the preclinical and initial glaucoma stages. Outstanding clinical trials and animal-model study designs, innovative technology, and analytical approaches in bioinformatics are essential tools to successfully uncover novel glaucoma biomarkers with a high potential for translation into clinical practice. Methods: To better understand the clinical and biochemical-molecular-genetic glaucoma pathogenesis, we conducted an analytical, observational, and case-comparative/control study in 358 primary open-angle glaucoma (POAG) patients and 226 comparative-control individuals (CG) to collect tears, aqueous humor, and blood samples to be processed for identifying POAG biomarkers by exploring several biological pathways, such as inflammation, neurotransmitter/neurotrophin alteration, oxidative stress, gene expression, miRNAs fingerprint and its biological targets, and vascular endothelial dysfunction, Statistics were done by using the IBM SPSS 25.0 program. Differences were considered statistically significant when p ≤ 0.05. Results: Mean age of the POAG patients was 70.03 ± 9.23 years, and 70.62 ± 7.89 years in the CG. Malondialdehyde (MDA), nitric oxide (NO), interleuquin (IL)-6, endothelin-1 (ET-1), and 5 hydroxyindolacetic acid (5-HIAA), displayed significantly higher levels in the POAG patients vs. the CG (p < 0.001). Total antioxidant capacity (TAC), brain derived neurotrophic factor (BDNF), 5-hydroxy tryptamine (5-HT), solute carrier family 23-nucleobase transporters-member 2 (SLC23A2) gene, and the glutathione peroxidase 4 (GPX4) gene, showed significantly lower levelsin the POAG patients than in the CG (p < 0.001). The miRNAs that differentially expressed in tear samples of the POAG patients respect to the CG were the hsa miR-26b-5p (involved in cell proliferation and apoptosis), hsa miR-152-3p (regulator of cell proliferation, and extracellular matrix expression), hsa miR-30e-5p (regulator of autophagy and apoptosis), and hsa miR-151a-3p (regulator of myoblast proliferation). Discussion: We are incredibly enthusiastic gathering as much information as possible on POAG biomarkers to learn how the above information can be used to better steer the diagnosis and therapy of glaucoma to prevent blindness in the predictable future. In fact, we may suggest that the design and development of blended biomarkers is a more appropriate solution in ophthalmological practice for early diagnosis and to predict therapeutic response in the POAG patients

Validation of miR-1228-3p as Housekeeping for MicroRNA Analysis in Liquid Biopsies from Colorectal Cancer Patients

BACKGROUND: Circulating microRNA (miRNA) analysis is a growing research field. However, it usually requires an endogenous control or housekeeping (HK) in order to normalize expression of specific miRNAs throughout different samples. Unfortunately, no adequate HK for circulating miRNA analysis is still known in the colorectal cancer (CRC) context whereas several have been suggested. Hence, our aims were to validate the previously suggested miR-1228-3p as HK for CRC studies, to compare its suitability with the widely used miR-16-5p, and to evaluate the influence of hemolysis on both miRNAs. METHODS: We analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) the expression of miR-1228-3p, miR-16-5p and the spike-in cel-miR-39 in a set of 297 plasmas (92 CRC, 101 advanced adenomas -AA-, and 100 controls) and 213 serum samples (59 CRC, 74 AA and 80 controls). We also analyzed both miRNAs depending on the hemolysis degree in 7 plasmas and 31 serums. RESULTS: Levels of miR-1228-3p and miR-16-5p did not show significant differences between groups although miR-16-5p exhibited more variability in plasma and serum samples. Importantly, the combination of cel-miR-39 and miR-1228-3p was the most stable one. Moreover, we observed that miR-16-5p was significantly influenced by hemolysis in contrast with miR-1228-3p that exhibited no correlation with this confounding factor in both biofluids. CONCLUSION: MiR-1228-3p has been validated as an adequate endogenous control for circulating miRNA analysis in CRC and AA liquid biopsies

Mature seeds for in vitro sanitation of the Grapevine leafroll associated virus (GLRaV-1 and GLRaV-3) from grape (Vitis vinifera L.)

The conservation of old grapevine varieties is important since they are adapted to specific climate conditions and may carry genes interesting to breeders. As virus infection is common in grapevine varieties, the use of virus free materials is of great importance. In this work, we used somatic embryogenesis for the sanitation of GLRaV-1 and GLRaV-3 viruses that were found after analyzing the putative presence of the five most common, economically important grape viruses by real-time multiplex RT-PCR in the old cultivar Grumet Negre . Unopened and opened inflorescences, fecundated ovaries, and, also, mature seeds were used as starting explants. Explants were cultured on plates with two embryogenesis induction media (Nitsch & McCown Woody plant medium) that contained the growth regulator thidiazuron and differed in their salt and vitamin compositions. One half of each kindof explant was cut prior to being cultured. After five months of culture, embryos had only developed from seeds that were cut previous to sowing. To the best of our knowledge, this is the first time that mature seeds have been used for inducing embryogenesis in grape. A total of 42% of the embryos transferred to tubes for germination regenerated into normal plantlets. The absence of both the GLRaV-1 and GLRaV-3 viruses in all regenerated plants was confirmed by real-time uniplex RT-PCR. So, this protocol can be used for sanitation and also for micropropagation.This work was supported by the INIA RTA2011-00067-C04-04 and RTA2011-00067-C04-01 projects co-funded with FEDER funds.Peiró Barber, RM.; Gammoudi, N.; Yuste Del Carmen, A.; Olmos Castelló, A.; Gisbert Domenech, MC. (2015). Mature seeds for in vitro sanitation of the Grapevine leafroll associated virus (GLRaV-1 and GLRaV-3) from grape (Vitis vinifera L.). 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