Long non‐coding RNAs (lncRNAs) comprise a vast repertoire of RNAs playing a wide variety of crucial roles in tissue physiology in a cell‐specific manner. Despite being engaged in myriads of regulatory mechanisms, many lncRNAs have still remained to be assigned any functions. A constellation of experimental techniques including single‐molecule RNA in situ hybridization (sm‐RNA FISH), cross‐linking and immunoprecipitation (CLIP), RNA interference (RNAi), Clustered regularly interspaced short palindromic repeats (CRISPR) and so forth has been employed to shed light on lncRNA cellular localization, structure, interaction networks and functions. Here, we review these and other experimental approaches in common use for identification and characterization of lncRNAs, particularly those involved in different types of cancer, with focus on merits and demerits of each technique.

BackgroundInfection with hepatitis E virus (HEV) is endemic in developing countries and reveals significant regional differences. Several studies have reported virus transmission via blood transfusion. To date, however, no cases of HEV RNA detection in blood donors have been reported from Iran.ObjectivesThe aim of this study was to determine the presence of HEV RNA in plasma samples of blood donors referred to a blood transfusion center in Shiraz in the southwest of Iran. The HEV genotypes were also investigated using nucleotide sequencing.Patients and MethodsBlood samples were collected from 700 blood donors who were referred to Fars blood transfusion organization from January to March 2014. Plasma samples were screened for the presence of HEV IgG and IgM antibodies by standard enzyme immunoassay. Samples seroreactive to anti-HEV were further tested for the presence of HEV RNA using nested polymerase chain reaction (PCR) with universal primers for detection of all four HEV genotypes. Positive PCR samples were then subjected to DNA sequencing for further analysis.ResultsFifty (50, 7.1%) out of 700 plasma samples tested positive for anti-HEV antibodies. HEV RNA was detected in 7/50 (12%) of the antibody-positive samples, the majority of which were IgM positive. Sequence analysis of seven isolates of the HEV RNA ORF 2 gene region revealed > 80% similarity with genotype 1.ConclusionsThe analysis indicates that the HEV isolated from blood donors in the southwest of Iran belongs to genotype 1. However, more samples from other geographic regions of Iran are needed to confirm these findings. Because transmission of HEV by administration of blood or blood components is likely to occur, it may be sensible to screen donor blood for HEV to eliminate transfusion-transmitted HEV infection when the recipient is immunocompromised.

Neurons critically rely on the functions of RNA-binding proteins to maintain their polarity and resistance to neurotoxic stress. HnRNP R has a diverse range of post-transcriptional regulatory functions and is important for neuronal development by regulating axon growth. Hnrnpr pre-mRNA undergoes alternative splicing giving rise to a full-length protein and a shorter isoform lacking its N-terminal acidic domain. To investigate functions selectively associated with the full-length hnRNP R isoform, we generated a Hnrnpr knockout mouse (Hnrnprtm1a/tm1a) in which expression of full-length hnRNP R was abolished while production of the truncated hnRNP R isoform was retained. Motoneurons cultured from Hnrnprtm1a/tm1a mice did not show any axonal growth defects but exhibited enhanced accumulation of double-strand breaks and an impaired DNA damage response upon exposure to genotoxic agents. Proteomic analysis of the hnRNP R interactome revealed the multifunctional protein Yb1 as a top interactor. Yb1-depleted motoneurons were defective in DNA damage repair. We show that Yb1 is recruited to chromatin upon DNA damage where it interacts with γ-H2AX, a mechanism that is dependent on full-length hnRNP R. Our findings thus suggest a novel role of hnRNP R in maintaining genomic integrity and highlight the function of its N-terminal acidic domain in this context.

Background:Viral load measurements are commonly used to monitor HCV infection in patients with chronic diseases or determining the number of HCV-genomes in serum samples of patients after sustained virological response. However, in some patients, HCV viral load in serum samples is too low to be detected by PCR, especially after treatment.Objectives:The aim of this study was to develop a highly specific, sensitive, and reproducible in-house quantitative PCR using specific primers and probe cited in highly conservative region of HCV genome that allows simultaneous detection of HCV genotypes 1 - 4.Materials and Methods:In this study, three sets of primer pairs and a TaqMan probe for amplification and detection of selected region within 5’-non-coding (5’NCR) of four HCV genotypes were used. Using plasmid containing 5’NCR region of HCV, standard curve, threshold, and threshold cycle (CT) values were determined. Real-time and nested PCR were performed on HCV genotypes 1 - 4 extracted from plasma and peripheral blood mononuclear cells (PBMCs) samples collected from patients with chronic HCV infection.Results:The lower limit detection of this in-house HCV real-time RT-PCR was determined as 100 RNA copies/mL. Inter- and intra-assay coefficient of variation (CV) of this in-house HCV real-time RT-PCR ranged from 0.9% to 1.8% and 1.76% to 3.94%, respectively. The viral load of the genotyped samples ranged from 2.0 × 106 ± 0.31 to 2.7 × 105 ± 0.46 copies/mL in serum samples and 5 × 102 ± 0.36 to 4.0 × 103 ± 0.51 copies/106 cells/mL of PBMCs.Conclusions:The quite sensitive in-house TaqMan real time RT-PCR assay was able to detect and quantify all four main HCV genotypes prevailing around all geographical regions of Iran.

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Background: Discovery of non-invasive methods for acute rejection in liver transplant patients would contribute to preservation of liver function in the graft. Recently, however, outcome prediction based on biostatistical models like artificial neural networks (ANNs) is increasingly becoming impressive in medicine. Objectives: The aim of this study was to obtain a predictive model based on ANN technique and to figure out the best time for early prediction of acute allograft rejection after transplantation in liver transplant recipients. Methods: Feed-forward, back-propagation neural network was developed to predict acute rejection in liver transplant recipients using clinical and biochemical data from 148 liver transplant recipients over days 3, 7, and 14 post-transplantation. Sensitivity and receiver-operating characteristic (ROC) analysis were done to reveal the importance of input variables and the performance of the neural network. Results:The results were compared with a logistic regression (LR) model using the same data. Our results showed that the data related to day 7 gave the best results in terms of ANN performance; and the most important factors in the predictive model were aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The ANN's accuracy was 90%, sensitivity was 87%, specificity was 90% in the testing set, and the performance of the ANN was better than that of the LR model. The ANN recognized correctly eight out of ten acute rejection patients and 34 out of 36 non-rejection ones in the testing set. Conclusions: This study suggests that ANN could be a valuable adjunct to conventional liver function tests for monitoring liver transplant recipients in the early postoperative period.

Deposition of neurofibrillary tangles composed of hyperphosphorylated tau in the brain is a pathological hallmark and closely correlates with onset and course of Alzheimer's disease. While tau reduction is being pursued as therapeutic strategy, prolonged lowering of total tau might lead to adverse effects, necessitating the development of more targeted approaches. We report that the RNA-binding protein hnRNP R facilitates the axonal localization of the Mapt mRNA encoding tau. Depletion of hnRNP R reduces tau in axons but not neuronal cell bodies. Brains of Alzheimer's disease mice deficient for hnRNP R contain less tau tangles and amyloid-beta plaques. Neurons treated with blocking antisense oligonucleotides to mask hnRNP R binding sites of Mapt mRNA show reduced axonal tau levels, similar to hnRNP R-deficient neurons. Lowering of tau levels selectively in axons, a major subcellular site of tangle formation and spreading, thus represents a novel therapeutic perspective for treatment of Alzheimer's disease.

The neuronal RNA-binding protein Ptbp2 regulates neuronal differentiation by modulating alternative splicing programs in the nucleus. Such programs contribute to axonogenesis by adjusting the levels of protein isoforms involved in axon growth and branching. While its functions in alternative splicing have been described in detail, cytosolic roles of Ptbp2 for axon growth have remained elusive. Here, we show that Ptbp2 is located in the cytosol including axons and growth cones of motoneurons, and that depletion of cytosolic Ptbp2 affects axon growth. We identify Ptbp2 as a major interactor of the 3’ UTR of Hnrnpr mRNA encoding the RNA-binding protein hnRNP R. Axonal localization of Hnrnpr mRNA and local synthesis of hnRNP R protein are strongly reduced when Ptbp2 is depleted, leading to defective axon growth. Ptbp2 regulates hnRNP R translation by mediating the association of Hnrnpr with ribosomes in a manner dependent on the translation factor eIF5A2. Our data thus suggest a mechanism whereby cytosolic Ptbp2 modulates axon growth by fine-tuning the mRNA transport and local synthesis of an RNA-binding protein.