Search results for "Reverse"

Simultaneous determination of nine phytohormones in seaweed and algae extracts by HPLC-PDA.

2016

An RP-HPLC-PDA method for the simultaneous analysis of 9 compounds deriving from the phytohormones class was developed and optimized, namely indoleacetic acid (IAA), indolebutyric acid (IBA), phenyleacetic acid (PAA), naphtyleacetic acid (NAA), trans-zeatin (TZ), kinetin (KA), isopentenyladenine (IA), 6-benzylaminopurine (6-BA) and abscisic acid (ABA). Validation of the method was performed on the SFE-CO2 extract made out of the mixture of Baltic algae. The regression coefficients for plant hormones were in the range from 0.997 to 0.999. The LOD and LOQ were on the levels from 0.05–0.29 and 0.15–0.88 mg/L, respectively. Developed method was used for the separation and determination plant ho…

Real-time reverse transcription polymerase chain reaction development for rapid detection of Tomato brown rugose fruit virus and comparison with othe…

2019

Background Tomato brown rugose fruit virus (ToBRFV) is a highly infectious tobamovirus that causes severe disease in tomato (Solanum lycopersicum L.) crops. In Italy, the first ToBRFV outbreak occurred in 2018 in several provinces of the Sicily region. ToBRFV outbreak represents a serious threat for tomato crops in Italy and the Mediterranean Basin. Methods Molecular and biological characterisation of the Sicilian ToBRFV ToB-SIC01/19 isolate was performed, and a sensitive and specific Real-time RT-PCR TaqMan minor groove binder probe method was developed to detect ToBRFV in infected plants and seeds. Moreover, four different sample preparation procedures (immunocapture, total RNA extractio…

Consensus-Based Distributed State Estimation of Biofilm in Reverse Osmosis Membranes by WSNs

2017

The appearance of biofilm has become a serious problem in many reverse osmosis based systems such as the ones found in water treatment and desalination plants. In these systems, the use of traditional techniques such as pretreatment or dozing biocides are not effective when the biofilm reaches an irreversible attachment phase. In this work, we present a framework for the use of a WSN as an estimator of the biofilm evolution in a reverse osmosis membrane so that effective solutions can be applied before the irreversible phase is attained. This design is addressed in a complete distributed and decentralized fashion, and subject to realistic constraints where cooperation between nodes is perfo…

High-throughput sequencing for 1-methyladenosine (m1A) mapping in RNA

2016

Abstract Detection and mapping of modified nucleotides in RNAs is a difficult and laborious task. Several physico-chemical approaches based on differential properties of modified nucleotides can be used, however, most of these methods do not allow high-throughput analysis. Here we describe in details a method for mapping of rather common 1-methyladenosine (m1A) residues using high-throughput next generation sequencing (NGS). Since m1A residues block primer extension during reverse transcription (RT), the accumulation of abortive products as well as the nucleotide misincorporation can be detected in the sequencing data. The described library preparation protocol allows to capture both types …

Engineering of a DNA Polymerase for Direct m6A Sequencing

2017

Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N6-methyladenosine (m6A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m6A-containing RNA prior to sequencing, since m6A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m6A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for…

Centenarians Overexpress Pluripotency-Related Genes.

2018

Abstract Human mesenchymal cells can become pluripotent by the addition of Yamanaka factors OCT3/4, SOX2, c-MYC, KLF4. We have recently reported that centenarians overexpress BCL-xL, which has been shown to improve pluripotency; thus, we aimed to determine the expression of pluripotency-related genes in centenarians. We recruited 22 young, 32 octogenarian, and 47 centenarian individuals and determined the mRNA expression of Yamanaka factors and other stemness-related cell surface marker genes (VIM, BMP4, NCAM, BMPR2) in peripheral blood mononuclear cells by reverse transcription polymerase chain reaction. We found that centenarians overexpress OCT3/4, SOX2, c-MYC, VIM, BMP4, NCAM, and BMPR2…

Detection of RET rearrangements in papillary thyroid carcinoma using RT-PCR and FISH techniques - A molecular and clinical analysis.

2019

Abstract Introduction Oncogenic BRAF and RAS mutations as well as multiple known (and yet unknown) RET fusion oncogenes comprise the majority of causative molecular alterations in papillary thyroid carcinoma (PTC). Apparently “mutation-negative” PTCs encompass a heterogenous group impeding analysis of prognostic significance of underlying genetics. Material and methods BRAF wild type PTC tissue of 56 patients was analyzed using two established methods: hybrid-specific RT-PCR for the predominant rearrangement RET/PTC1 and fluorescent in situ hybridization (FISH). Clinical features of the cases with and without RET rearrangement were compared (patient age, gender, tumor size, focality, lymph …

Annulate Lamellae in a Desmoplastic Small Round Cell Tumor

2016

Physicochemical and Preclinical Evaluation of Spermine-Derived Surfactant Liposomes for in Vitro and in Vivo siRNA-Delivery to Liver Macrophages

2016

Herein we report on a liposomal system for siRNA delivery consisting of cholesterol (Chol), distearoylphosphatidylcholine (DSPC), and surfactant TF (1-hydroxy-50-amino-3,4,7,10,13,16,19,22-octaoxa-37,41,45-triaza-pentacontane), a novel spermine derivative (HO-EG8-C12-spermine) which has shown improved siRNA delivery to cells in vitro and in vivo. Predominantly single-walled liposomes with reproducible sizes and moderately broad size distributions were generated with an automated extrusion device. The liposomes remained stable when prepared in the presence of siRNA at N/P ratios of 17-34. However, when mixed with human serum in equal volumes, larger aggregates in the size range of several hu…

DNA demethylation caused By 5-Aza-2'-Deoxycytidine induces mitotic alterations and aneuploidy

2016

Aneuploidy, the unbalanced number of chromosomes in a cell, is considered a prevalent form of genetic instability and is largely acknowledged as a condition implicated in tumorigenesis. Epigenetic alterations like DNA hypomethylation have been correlated with cancer initiation/progression. Furthermore, a growing body of evidence suggests the involvement of epigenome-wide disruption as a cause of global DNA hypomethylation in aneuploidy generation. Here, we report that the DNA hypomethylating drug 5-aza-2′-deoxycytidine (DAC), affects the correct ploidy of nearly diploid HCT-116 human cells by altering the methylation pattern of the chromosomes. Specifically, we show that a DAC-induced reduc…