First-strand cDNA Research Articles

Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, precise RNA-mediated insertion of transgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

28S rRNA-Derived Fragments Represent an Independent Molecular Predictor of Short-Term Relapse in Prostate Cancer.

Prostate cancer (PCa) is a global health concern, being a leading cause of cancer-related mortality among males. Early detection and accurate prognosis are crucial for effective management. This study delves into the diagnostic and prognostic potential of 28S rRNA-derived fragments (rRFs) in PCa. Total RNA extracted from 89 PCa and 53 benign prostate hyperplasia (BPH) tissue specimens. After 3'-end polyadenylation, we performed reverse transcription to create first-strand cDNA. Using an in-house quantitative real-time PCR (qPCR) assay, we quantified 28S rRF levels. Post-treatment biochemical relapse served as the clinical endpoint event for survival analysis, which we validated internally through bootstrap analysis. Our results revealed downregulated 28S rRF levels in PCa compared to BPH patients. Additionally, we observed a significant positive correlation between 28S rRF levels and higher Gleason scores and tumor stages. Furthermore, PCa patients with elevated 28S rRF expression had a significantly higher risk of post-treatment disease relapse independently of clinicopathological data. In conclusion, our study demonstrates, for the first time, the prognostic value of 28S rRF in prostate adenocarcinoma. Elevated 28S rRF levels independently predict short-term PCa relapse and enhance risk stratification. This establishes 28S rRF as a potential novel molecular marker for PCa prognosis.

Single-Molecule Poly(A) Tail Sequencing (SM-PATseq) Using the PacBio Platform.

The polyadenylation of the 3' ends of messenger RNAs is an important regulator of stability and translation. We developed the single-molecule poly(A) tail sequencing method, SM-PATseq, to assay tail lengths of the whole transcriptome at nucleotide resolution using long-read sequencing. This method generates cDNA using an oligo-dT 3' splint adaptor ligation to prime first-strand cDNA synthesis, followed by random hexamer priming for second-strand synthesis. By directly sequencing the cDNA on long-read platforms, we can resolve tail lengths at nucleotide resolution, identify non-A bases within the tail, and quantify transcript abundance analogous to traditional RNAseq methods. Here, we discuss the method for generating, sequencing, and primary analysis of poly(A) tail data from total RNA using the Pacific Biosciences Sequel platform.

Novel circular RNAs of the apoptosis-related BAX and BCL2L12 genes identified in a chronic lymphocytic leukemia cell line using nanopore sequencing.

Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key regulators of gene expression, with deregulated expression and established involvement in leukemia. The products of BCL2 and its homologs, including BAX and BCL2L12, are implicated in chronic lymphocytic leukemia (CLL). However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their role in CLL. We sought to further elucidate the contribution of BAX and BCL2L12 in CLL by unraveling the identity, localization, and potential role of their circRNAs. Therefore, total RNA from the EHEB cell line and peripheral blood mononuclear cells (PBMCs) of CLL patients and non-leukemic blood donors was extracted and reverse-transcribed using random hexamers. Next, nested PCRs with divergent primers were performed and the purified PCR products were subjected to 3rd generation nanopore sequencing. Nested PCRs were also applied to first-strand cDNAs synthesized from total RNA extracts of PBMCs from CLL patients and non-leukemic blood donors. Lastly, a single-molecule resolution fluorescent in situ hybridization (smFISH) method called circFISH was used to visualize the circRNA distribution in EHEB cells. We discovered several novel circRNAs produced by BAX and BCL2L12, which were characterized by great exon structure diversity. In addition, intriguing findings regarding their formation emerged. Interestingly, visualization of the most abundant circRNAs showed distinct intracellular localization. Moreover, a complex BAX and BCL2L12 circRNA expression pattern was revealed in CLL patients and non-leukemic blood donors. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.

Gene regulation of MMPs and TIMPs by somatostatin in human fibroblasts

Introduction:SST is a widespread neuropeptide with generally inhibitory function. The aim of this study is to evaluatethe effect of somatostatin in different concentrations on mRNA expression of MMPs and TIMPsin cultured, human, gingival fibroblasts. Methodology and resources: Human gingival fibroblasts were stimulated with 10-4, 10-9 or 10-12 M somatostatin DMEM without fetal calf serum; untreated fibroblasts served as controls. After incubation period, the RNA was extracted, and the first-strand cDNA was synthesized. Alterations in the expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-11, TIMP-1 and TIMP-2 mRNA were evaluated using real-time polymerase chain reaction (PCR). b-actin mRNA expression was used to normalize the data. Results: In 24 h treatment, SST, at the highest concentration induced a down-regulation on MMP-1, -2, -3 and -7 expression, and up-regulation on MMP-11 expression; while at the lowest concentration induced an up-regulation on MMP-1, -2, -3, TIMP-1 and -2 expression. At 72 hours treatment, similar effects were observed, except for the up-regulation on TIMP-2 at the higher SST concentration, an up-regulation on MMP-7 and -11 expression and a down-regulation on MMP-2 and TIMP-2 expression, at the lower SST concentration. Discussion:The modulation of inflammation by SST is still unclear. The findings of this work,suggests that it can modulate the gene expression of MMPs and TIMPs by cultured fibroblasts and its effects depend on the concentration. This may represent one of the mechanisms of inflammation suppression by SST.

Transcriptomic and Metabolomic Analyses Reveal the Roles of Flavonoids and Auxin on Peanut Nodulation.

Rhizobia form symbiotic relationships with legumes, fixing atmospheric nitrogen into a plant-accessible form within their root nodules. Nitrogen fixation is vital for sustainable soil improvements in agriculture. Peanut (Arachis hypogaea) is a leguminous crop whose nodulation mechanism requires further elucidation. In this study, comprehensive transcriptomic and metabolomic analyses were conducted to assess the differences between a non-nodulating peanut variety and a nodulating peanut variety. Total RNA was extracted from peanut roots, then first-strand and second-strand cDNA were synthesized and purified. After sequencing adaptors were added to the fragments, the cDNA libraries were sequenced. Our transcriptomic analysis identified 3362 differentially expressed genes (DEGs) between the two varieties. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that the DEGs were mainly involved in metabolic pathways, hormone signal transduction, secondary metabolic biosynthesis, phenylpropanoid biosynthesis, or ABC transport. Further analyses indicated that the biosynthesis of flavonoids, such as isoflavones, flavonols, and flavonoids, was important for peanut nodulation. A lack of flavonoid transport into the rhizosphere (soil) could prevent rhizobial chemotaxis and the activation of their nodulation genes. The downregulation of AUXIN-RESPONSE FACTOR (ARF) genes and lower auxin content could reduce rhizobia's invasion of peanut roots, ultimately reducing nodule formation. Auxin is the major hormone that influences the cell-cycle initiation and progression required for nodule initiation and accumulates during different stages of nodule development. These findings lay the foundation for subsequent research into the nitrogen-fixation efficiency of peanut nodules.

DNA methyltransferase expression (DNMT1, DNMT3a and DNMT3b) as a potential biomarker for anti-VEGF diabetic macular edema response.

DNA methylation is involved in Diabetic Retinopathy progression showing a metabolic memory mechanism. However, the association of DNA methyltransferase with diabetic macular edema is still unknown. We aimed to describe the differences in DNA methyltransferase gene expression in patients with different diabetic macular edema responses. A total of 27 diabetic patients, aged 59-90 years, were prospectively enrolled in this cross-sectional study. The participants were classified into control group (CG, n = 11), diabetic macular edema responders (rDME, n = 9) and non-responder diabetic macular edema (nrDME, n = 7) after anti-vascular endothelial growth factor (anti-VEGF) treatment. Only cases with a complete ophthalmological examination, digital 133° color fundus, and SD-OCT assessments were used. After RNA extraction and first-strand cDNA synthesis, quantitative real-time PCR was performed with specific primers on the CFX Connect™ Real-Time PCR Detection System to assess differential transcriptional expression patterns. The DNMT1 gene showed a positive correlation (r = 0.617; p = 0.043) with Best Corrected Visual Acuity (BCVA) in CG, a positive correlation (r = 0.917; p = 0.010) with HbA1c in nrDME and a negative correlation (r = -0.659; p = 0.049) with GCL-IPL thickness in rDME. DNMT3A gene showed a positive correlation (r = -0.890; p = 0.001) with Sub-foveal Choroidal thickness in rDME whereas DNMT3b gene showed a negative correlation (r = -0.815; p = 0.007) with HbA1c and RNFL (r = -0.664; p = 0.026) in CG. Patients with similar metabolic profile risk factors showed associated DNA methyltransferase transcriptional expression patterns differences fitting with the anti-VEGF diabetic macular edema response. Further studies are needed to clarify if these results (1) reflect disease evolution, (2) translate the therapeutic impact, (3) or can help to predict the therapeutic resistance profile.

Expression of the hypoxia-inducible factor as a predictor of the resistance of the organism of laboratory animals to hypoxia

BACKGROUND:One of the key transcriptional regulators that determine the bodys resistance to hypoxia is the hypoxia-inducible factorHIF-1, the study of the role of which in the bodys resistance to extreme influences can justify new directions in medical technologies for its increase.
 AIM:To evaluate the quantitative contribution of the level of expression of the hypoxia-inducible factorHIF-1in various tissues of laboratory animals to the increase in the resistance of animals to the effects of hypoxic hypoxia.
 MATERIALS AND METHODS:The study was carried out on outbred white laboratory rats obtained from the Rappolovo nursery weighing 180220 g. To conduct the study, animals were previously tested for an individual level of resistance to hypoxia, which made it possible to form experimental groups from highly resistant and low resistant animals. Biological material was taken from all animals (whole blood, plasma, tissues of the heart, liver, kidneys, brain), in which the expression of theHIF-1andTSPOgenes (housekeeping gene) was determined by the Real-Time-PCR method. Total RNA was isolated from the test material by affinity sorption,synthesis of the first strand of cDNA, amplification, followed by determination of the expression level of theHIF-1gene in rats was carried out according to the instructions and the manufacturers protocol by PCR with detection of the accumulation of reaction products in real time (Real-Time PCR) using a CFX-96 detecting amplifier (Bio-Rad, USA) and specific primers and probes for theHIF-1gene in rats (DNK-Sintez, Russia). Statistical processing of the obtained data was carried out using the ANOVA analysis of variance.
 RESULTS:It has been established that the level of resistance of animals to hypoxia is largely determined by their genetic characteristics. Even under normoxic conditions, the expression of theTSPOhousekeeping gene in animals with a high level of resistance to hypoxia differed with a high degree of reliability from low-resistance animals (in the kidneys, liver, and brain, on average, by 4060%; in the heart, by 25%). The values of the expression of this gene, determined in whole blood or plasma, make it possible to differentiate groups of animals according to the level of resistance to hypoxia. A similar ratio between animals with high and low resistance is also observed in tissues obtained immediately after hypoxic exposure. An analysis of the reaction of the genomic regulation system to extreme exposure showed that it increased the expression of the TSPO gene by 1.62 times equally in all tissues, regardless of the level of animal resistance. For theHIF-1gene, similar patterns were found, but the severity of their manifestations is more and significant.
 CONCLUSIONS:The main organ that provides a high level of resistance to hypoxia associated with the basic (under normoxic conditions) expression ofHIF-1is the brain. The expression of the hypoxia-inducible factor in it is more than 300 times higher than the expression of the housekeeping genes. The second most important organ is the liver, in whichHIF-1expression activity is more than 15 times higher than the expression of housekeeping genes. Under conditions of moderate hypoxia, a compensatory-adaptive reaction is noted, associated with the activation of hypoxic defense mechanisms in blood and liver cells, and in low-resistant animals, also in the brain tissue. In the myocardium, such a compensatory-adaptive reaction is activated only in the group of highly resistant animals. A high level of basal expression of theHIF-1transcription factor under daily (normoxic) conditions may be a predictor of a high level of resistance to hypoxia in a given animal.

Differential expression of selected microRNAs in cryopreserved sperm of Bos taurus sires with diverse fertility status

Our study aimed to evaluate the expression of four selected microRNAs (miRNA) in commercially produced cryopreserved semen of bulls with high vs. low fertility. The annual fertility index (FI) of 151 sires with ≥300 first services was recorded between November 2018 and November 2019. Sires lying at the extremes of the FI distribution (outside the range of mean FI±SD) were classified as low- (LF; n=13 bulls) or high-fertile (HF; n=7 bulls), with a non-return rate of 61.4±6.9% and 72.6±1.5%, respectively. For each bull, total RNA was isolated using a modified TRIzolTM-based protocol from two cryopreserved semen batches produced with 1-mo interval (40 sperm samples totally). Following preparation of first-strand cDNA, relative quantification of the mature sequences of hsa-miR-30d-5p, hsa-miR-92a-3p, hsa-miR-132-3p and hsa-miR-204-5p was carried out by real-time PCR. The four miRNAs were selected based on our previous studies showing their constant detection in sperm of sires with diverse age, breed and fertility. The quantification cycles (Cq) of each miRNA was normalized using hsa-miR-30d-5p and hsa-miR-132-3p combined as reference genes. The relative expression of the four miRNAs was quantified using the ΔCq and 2- ΔΔCq methods (Livak and Schmittgen, Methods, 25:402-8, 2001). In HF samples, the fluorescent signal of miR-92a, miR-204, miR-132 and miR-30d was detected after Cq 22.34±1.10, 26.19±5.58, 26.69±2.20 and 28.05±3.32, respectively; the Cq values recorded in the LF group were 22.72±1.30, 23.23±1.92, 26.97±2.50 and 27.43±2.81, respectively. Expression of miR-204 was approximately 18-fold lower in the HF than in the LF group; this difference was significant only for miR-204 when applying a mixed-effects linear model with fertility group as fixed effect and the semen batch nested within sire as random effect (b=18.36±6.48, adjusted P=0.014). The expression of miR-92a, miR-132 and miR-30d did not differ between the two groups (adjusted P>0.05 in all cases). Concluding, our study showed the differential expression of the mature sequence of hsa-miR-204, a miRNA involved in chromatin condensation during spermiogenesis, in cryopreserved sperm of sires with diverse fertility status.

RNA-seq Sample Preparation Kits Strongly Affect Transcriptome Profiles of a Gas-Fermenting Bacterium.

ABSTRACTTranscriptome analysis via RNA sequencing (RNA-seq) has become a standard technique employed across various biological fields of study. The rapid adoption of the RNA-seq approach has been mediated, in part, by the development of different commercial RNA-seq library preparation kits compatible with standard next-generation sequencing (NGS) platforms. Generally, the essential steps of library preparation, such as rRNA depletion and first-strand cDNA synthesis, are tailored to a specific group of organisms (e.g., eukaryotes versus prokaryotes) or genomic GC content. Therefore, the selection of appropriate commercial products is of crucial importance to capture the transcriptome of interest as closely to the native state as possible without introduction of technical bias. However, researchers rarely have the resources and time to test various commercial RNA-seq kits for their samples. This work reports a side-by-side comparison of RNA-seq data from Clostridium autoethanogenum obtained using three commercial rRNA removal and strand-specific library construction products of NuGEN Technologies, Qiagen, and Zymo Research and assesses their performance relative to published data. While all three vendors advertise their products as suitable for prokaryotes, we found significant differences in their performance regarding rRNA removal, strand specificity, and most importantly, transcript abundance distribution profiles. Notably, RNA-seq data obtained with Qiagen products were most similar to published data and delivered the best results in terms of library strandedness and transcript abundance distribution range. Our results highlight the importance of finding appropriate organism-specific workflows and library preparation products for RNA-seq studies.IMPORTANCE RNA-seq is a powerful technique for transcriptome profiling while involving elaborate sample processing before library sequencing. We show that RNA-seq library preparation kits can strongly affect the outcome of an RNA-seq experiment. Although library preparation benefits from the availability of various commercial kits, choosing appropriate products for the specific samples can be challenging for new users or for users working with unconventional organisms. Evaluating the performance of different commercial products requires significant financial and time investments infeasible for most researchers. Therefore, users are often guided in their choice of kits by published data involving similar input samples. We conclude that important consideration should be given to selecting sample processing workflows for any given organism.

Abstract 417: Elevation Of Serum Hdl And Apoe Level In A Novel Vps35l Deficient Mouse Model

Aim: Defect of the endosomal protein sorting factor VPS35L causes novel congenital anomalies syndrome, in which cholesterol dysmetabolism is one of the symptoms. To reveal the biochemical mechanisms, the a model mouse was developed and characterized. Methods: Loss-of-function mutation, N995del, VPS35L knock-in (KI) mice were generated. Serum lipoprotein profiles were assessed by LipoSEARCH™ (Immuno-Biological Laboratories, Japan). For biochemical assays, 70 micro-L of serum was sub-fractionated with Superose 6 column in PBS and total and free cholesterol detected using LabAssay™cholesterol (Fujifilm Wako Chemicals, Japan) and Pureauto™S F-CHO-N (SEKISUI MEDICAL, Japan) assay kits. Negative images of fractioned lipoproteins were observed by TEM (JEM-1400Plus, JEOL, Japan). ApoA-I and apoE in the fractions were determined by use of a mouse apoA1 ELISA PRO kit and a mouse apoE ELISA PRO kit (Mabtech AB, Sweden). Mouse liver total RNA was isolated by ISOGEN (Nippon Gene, Japan) and first-strand cDNA generated using the Invitrogen™ SuperScript™IV First-Strand Synthesis system with random hexamers. For quantitative real-time PCR, the StepOnePlus™ real-time PCR system (Thermo Fisher) was used, and the PCR end product of each well confirmed by the melt curve analysis. Results: Lipoprotein analysis indicated the KI mice showed 1.9-fold higher total cholesterol than WT mice. LDL-C and HDL-C were increased 2.9- and 1.9-fold compared to WT mice, respectively. ApoA-I levels in the large HDL and HDL fractions were all significantly increased 1.5-, and 1.6-fold, whereas apoE levels were elevated 25-, and 8.4-fold, respectively. Conclusions: Ablation of VPS35L in MEF(3T3) cells caused loss of endocytosis of the LDL receptor and LRP1, and perhaps even SR-BI, which may lead to increased serum LDL and HDL levels in the loss-of-function KI model mice. Surprisingly. a high level of apoE was detected in KI mice, especially in the HDL fraction. In this case, apoE catabolism became abnormal due to the lack of LRP1 at the cell surface. Abnormal metabolism of apoE may affect the central nervous system or neuron generation. Further examination of the phenotype of this this model mouse line are warranted.

Circulation of coronavirus in bats from northern and central Argentina: preliminary study

PurposeBats have proven throughout history to be the reservoir and disseminator of a wide variety of viruses in nature, where they have sometimes been linked to important infections in public health, such is the case of coronaviruses causing SARS-CoV, MERS-CoV and SARS-CoV-2. Considering the importance of these previous events and due to the lack of information on the coronavirus present in the bat population in Argentina, we decided to monitor their circulation in two provinces in which there is continuous contact of these animals with others (domestic and wild) and human beings.Methods & MaterialsSamples of pharyngeal swabs and fecal matter were collected from different species of bats from the provinces of Jujuy (northern Argentina) and La Pampa (central Argentina), and from other wild and domestic animals that cohabitated with bats. The samples were stored in RNA Shield® 1X until processing. The viral RNA extraction was performed with Quick-RNA Viral Kit (ZYMO RESEARCH®) and the cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (TRANS®). Each sample was analyzed individually by nested PCR directed to a conserved 440 bp fragment located within the viral polymerase gene (Chu et al., 2006).ResultsPreliminary results from 43 stool samples and 53 pharyngeal swabs from insectivorous and hematophagous bats demonstrated the presence of alfacoronavirus sequences in four fecal samples (3 insectivorous bats and 1 hematophagous bat) and in one pharyngeal swab sample from an insectivorous bat. Through comparison with GenBank sequences, the closest relatives identified were alphacoronaviruses of the species Myotis riparius/ nigricans, Molossus rufus and Desmodus rotundus. Interestingly, we found alphacoronavirus sequences of one species present in another bat species since the colonies were in close contact. Samples from other animal species are in the process of being analyzed.ConclusionThe identification of new coronaviruses that naturally inhabit bat populations and in animal species that share the biological niche, contributes to the knowledge of viral evolution and the biology behind the jump between animal species.

First Report of Tomato Brown Rugose Fruit Virus on Greenhouse Tomato in Syria.

Tomato (Solanum lycopersicum L.) is an important vegetable crop worldwide. In spring and autumn 2017, virus-like symptoms were observed on greenhouse grown tomato plants in the east of Akkar plain (south of coastal region, Tartous governorate, Syria). These symptoms were: mild to severe mosaic on the apical leaves, brown necrosis on sepals, receptacle and flower's cluster carrier, and severe symptoms of brown rugose and discoloration on fruit. During next growing seasons, disease spread was observed in most of Syrian coastal region with disease incidence ranged from 40% to 70% by 2020. Tomato brown rugose fruit virus (ToBRFV) was suspected as a main causal agent of the disease, especially since its first report in Jordan, a neighboring country (Salem et al. 2016), Palestine (Alkowni et al. 2019), Turkey (Fidan et al. 2019), Germany (Menzel et al. 2019), Italy (Panno et al. 2019), America (Camacho-Beltrán et al. 2019), Egypt (Amer and Mahmoud, 2020), and recently in Spain (Alfaro-Fernandez et al. 2021). In November and December 2020, seventy-one leaf samples from symptomatic plants (59 from Tartous and 12 from Lattakia governorates) and seven from asymptomatic ones (5 from Tartous and 2 from Lattakia) were collected and tested for the presence of ToBRFV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using ToBRFV-commercial kit (LOEWE® Biochemia, Germany) following the manufacturer's instructions. Results showed, forty-three of symptomatic samples reacted positively (38 in Tartous and 5 in Lattakia) and none of asymptomatic ones. On the other hand, sap mechanical inoculation of 10 tomato cv. Mandaloun F1 (Enza Zaden, the Netherlands) plants using a positive tomato isolate gave systemic mosaic symptoms in all plants identical to those observed in the original plants in the field, after 13 days of inoculation, and necrotic local lesions on 10 plants of Nicotiana tabacum after 5 days, indicating the presence of a tobamovirus in general. ToBRFV infection was confirmed in all mechanically-inoculated plants by DAS-ELISA. Further tests were necessary to investigate ToBRFV presence, because of its serological relationships with another tobamoviruses. Six representative symptomatic samples (ELISA-positive) and two asymptomatic (ELISA-negative) samples were subjected to total RNA extraction using the SV-Total RNA Extraction kit (Promega, U.S.A.) following the manufacturer's instructions. The samples were tested by two-step reverse transcription-polymerase chain reaction (RT-PCR) using species-specific primers and protocols for most common tomato-infecting viruses, including: tomato chlorosis virus and tomato infectious chlorosis virus (Dovas et al. 2002), pepino mosaic virus (PepMV) and tomato torrado virus (Wieczorek et al. 2013), alfalfa mosaic virus (Parrella et al. 2000), tomato spotted wilt virus (Salem et al. 2012) and a pair of primers: ToBRFV-F2 (5'-CATATCTCTCGACACCAGTAAAAGGACCCG-3') and ToBRFV-R2 (5'-TCCGAGTATAGGAAGACTCTGGTTGGTC-3') targeting a region of the RNA dependent RNA polymerase (RdRp), of the ToBRFV genome (KT383474; Salem et al. 2016). First-strand cDNA synthesis was carried out using Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Promega) and random primer according to the manufacturer's protocol, then followed by PCR with the seven species-specific primers. Only ToBRFV was detected among all tested viruses in symptomatic samples (ELISA-positive), and none of the tested viruses was detected in the asymptomatic plants. To confirm the presence of ToBRFV, two selected RdRp-specific PCR amplicons (872 bp) were purified and ligated into pGEM T-Easy Vector (Promega), and three clones were sequenced (GenBank accession nos. MZ447794 to 96). BLASTn analysis showed that the nucleotide sequences are 99.77-100% identical and shared around 99% identity to RdRp of ToBRFV isolate (MT118666) from Turkey available in the GenBank. Accordingly, the presence of ToBRFV was confirmed by bioassays on indicator plants, DAS-ELISA, RT-PCR, and further sequencing. To our knowledge, this is the first report of ToBRFV infecting tomato in Syria, and this requires special emphasis for further investigations because of the virus severity, easy transmission ability and absent of commercial resistance varieties till now.

First report of grapevine rupestris vein feathering virus in Vitis vinifera L. from Japan.

The production of wine grapes is gaining widespread popularity and being carried out on approximately 2,200 hectares of land in Japan. Scions grafted onto rootstocks generally have been imported from the EU, USA, New Zealand, and Australia into Japan. Unfortunately, viruses have spread in Japanese vineyards by slipping through the net of plant quarantine. Grapevine rupestris vein feathering virus (GRVFV), which was detected in a Greek grapevine accessions, is a member of genus Marafivirus in family Tymoviridae (El Beaino et al. 2001). GRVFV has been detected in many countries such as USA, Canada, Australia, New Zealand, Italy, Spain, Switzerland, Czech Republic, Uruguay, and Pakistan (Jo et al. 2015; Eichmeier et al. 2016; Xiao and Meng 2016; Blouin and MacDiarmid 2017; Reynard et al. 2017; Cho et al. 2018; Mahmood et al. 2019; Wu et al. 2020). Herein we report GRVFV infection in Vitis vinifera L. grapevines from Japan. In February 2021, dormant canes from 18 V. vinifera cv. Cabernet Sauvignon with leafroll-like disease symptoms, growing in a vineyard located in Kanagawa Prefecture, were collected. No typical vein banding symptom by GRVFV were observed in the grapevines during the growing season. Total RNA was isolated from the canes using an RNeasy Plant Mini Kit and QIAshredder (Qiagen, Valencia, CA), and subjected to cDNA synthesis using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). RT-PCR was performed with GRVFV_6156F and GRVFV_6600R primers for GRVFV detection (Reynard et al. 2017). The expected 445 nucleotides (nt) amplification product was obtained from four of 18 grapevines. Sequence analysis of the products revealed 91% identities to corresponding sequences of GRVFV isolates CHASS (KY513702) and Mauzac (KY513701) from Switzerland. Genome walking to determine the whole-genome sequence of the GRVFV isolates from the four grapevines was performed. Briefly, the upstream and downstream of the 445 nt amplification product were amplified from first-strand cDNA using gene-specific primers designed from the product and CHASS-specific primers. Each amplified fragment was Sanger sequenced. Next, gene-specific primers were designed to obtain the complete genome of GRVFV as 13 overlapping DNA fragments from each of the four grapevine samples. An identical complete genome of 6,704 bp was assembled from the overlapping DNA fragments using MEGA 10 software and named as NA1 isolate (DDBJ accession no. LC619667). Phylogenetic analysis of the NA1 genome and corresponding sequences of GRVFV from other countries showed that NA1 formed a cluster with isolate NZ ChTK0004 from New Zealand (MF000326; Supplementary Figure 1). In pairwise comparisons, the complete NA1 genome was most identical at 88% and 87%, respectively to isolates NZ ChTK0004 and Mauzac. The predicted amino acid sequences of NA1 polyprotein shared high homologies (96%) to the corresponding polyprotein sequences of NZ ChTK0004 and Mauzac, suggesting that NA1 is genetically similar to GRVFV isolates from New Zealand and Switzerland. The NA1-infected Cabernet Sauvignon was co-infected with Grapevine leafroll-associated virus 3, Grapevine virus A, and Grapevine rupestris stem pitting-associated virus according to RT-PCR assay for grapevine virus detection (Nakaune and Nakano 2006). The results underscore the importance of intensifying quarantine measures to prevent introduction of exotic viruses via contaminated wine grape vegetative cuttings.

A Cancer-Related microRNA Signature Shows Biomarker Utility in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological malignancy, arising from terminally differentiated B cells, namely plasma cells. miRNAs are small non-coding RNAs that participate in the post-transcriptional regulation of gene expression. In this study, we investigated the role of nine miRNAs in MM. CD138+ plasma cells were selected from bone marrow aspirates from MM and smoldering MM (sMM) patients. Total RNA was extracted and in vitro polyadenylated. Next, first-strand cDNA synthesis was performed using an oligo-dT–adapter primer. For the relative quantification of the investigated miRNAs, an in-house real-time quantitative PCR (qPCR) assay was developed. A functional in silico analysis of the miRNAs was also performed. miR-16-5p and miR-155-5p expression was significantly lower in the CD138+ plasma cells of MM patients than in those of sMM patients. Furthermore, lower levels of miR-15a-5p, miR-16-5p, and miR-222-3p were observed in the CD138+ plasma cells of MM patients with osteolytic bone lesions, compared to those without. miR-125b-5p was also overexpressed in the CD138+ plasma cells of MM patients with bone disease that presented with skeletal-related events (SREs). Furthermore, lower levels of miR-223-3p were associated with significantly worse overall survival in MM patients. In conclusion, we propose a miRNA signature with putative clinical utility in MM.

TRNA Derivatives in Multiple Myeloma: Investigation of the Potential Value of a tRNA-Derived Molecular Signature.

Multiple myeloma (MM) is a hematologic malignancy arising from the clonal proliferation of malignant plasma cells. tRNA-derived RNA fragments (tRFs) constitute a class of small non-coding RNAs, deriving from specific enzymatic cleavage of tRNAs. To the best of our knowledge, this is one of few studies to uncover the potential clinical significance of tRFs in MM. Total RNA was extracted from CD138+ plasma cells of MM and smoldering MM patients, and in vitro polyadenylated. First-strand cDNA synthesis was performed, priming from an oligo-dT-adaptor sequence. Next, real-time quantitative PCR (qPCR) assays were developed for the quantification of six tRFs. Biostatistical analysis was performed to assess the results and in silico analysis was conducted to predict the function of one of the tRFs. Our results showed that elevated levels of five out of six tRFs are indicators of favorable prognosis in MM, predicting prolonged overall survival (OS), while two of them constitute potential molecular biomarkers of favorable prognosis in terms of disease progression. Moreover, three tRFs could be used as surrogate prognostic biomarkers along with the R-ISS staging system to predict OS. In conclusion, tRFs show molecular biomarker utility in MM, while their mechanisms of function merit further investigation.

Detection of Viroids Using RT-qPCR.

Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has transformed the way the viroids are detected. The method involves using sequence-specific primers that anneal to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR.

Identification and in-silico characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) gene in Corylus avellana L.

Paclitaxel® (PC) is one of the most effective and profitable anti-cancer drugs. The most promising sources of this compound are natural materials such as tissue cultures of Taxus species and, more recently, hazelnut (Corylus avellana L.). A large part of the PC biosynthetic pathway in the yew tree and a few steps in the hazelnut have been identified. Since understanding the biosynthetic pathway of plant-based medicinal metabolites is an effective step toward their development and engineering, this paper aimed to identify taxadiene-5α-ol-O-acetyltransferase (TDAT) in hazelnut. TDAT is one of the key genes involved in the third step of the PC biosynthetic pathway. In this study, the TDAT gene was isolated using the nested-PCR method and then characterized. The cotyledon-derived cell mass induced with 150 μM of methyl jasmonate (MeJA) was utilized to isolate RNA and synthesize the first-strand cDNA. The full-length cDNA of TDAT is 1423 bp long and contains a 1302 bp ORF encoding 433 amino acids. The phylogenetic analysis of this gene revealed high homology with its ortholog genes in Quercus suber and Juglans regia. Bioinformatics analyses were used to predict the secondary and tertiary structures of the protein. Due to the lack of signal peptide, protein structure prediction suggested that this protein may operate at the cytoplasm. The homologous superfamily of the T5AT protein, encoded by TDAT, has two domains. The highest and lowest hydrophobicity of amino acids were found in proline 142 and lysine 56, respectively. T5AT protein fragment had 24 hydrophobic regions. The tertiary structure of this protein was designed using Modeler software (V.9.20), and its structure was verified based on the results of the Verify3D (89.46%) and ERRAT (90.3061) programs. The T5AT enzyme belongs to the superfamily of the transferase, and the amino acids histidine 164, cysteine 165, leucine 166, histidine 167, and Aspartic acid 168 resided at its active site. More characteristics of TDAT, which would aid PC engineering programs and maximize its production in hazelnut, were discussed.

A Molecular Signature of Circulating MicroRNA Can Predict Osteolytic Bone Disease in Multiple Myeloma.

Simple SummaryMultiple myeloma bone disease (MMBD) is one of the most important complications of multiple myeloma with a great impact on quality of life. Recent advances in the field of imaging techniques provided clinicians with a variety of imaging modalities with high sensitivity for the diagnosis of MMBD. However, no circulating biomarkers are available to support the diagnosis of MMBD in cases where the results are inconclusive. The aim of our study was to investigate the clinical utility of 19 miRNAs implicated in osteoporosis in MMBD. Our results suggest that the levels of circulating let-7b-5p, miR-143-3p, miR-17-5p, miR-335-5p, and miR-214-3p (standalone or combined in multi-miRNA models) can effectively predict the presence of MMBD in newly diagnosed MM patients.Background: Multiple myeloma bone disease (MMBD) constitutes a common and severe complication of multiple myeloma (MM), impacting the quality of life and survival. We evaluated the clinical value of a panel of 19 miRNAs associated with osteoporosis in MMBD. Methods: miRNAs were isolated from the plasma of 62 newly diagnosed MM patients with or without MMBD. First-strand cDNA was synthesized, and relative quantification was performed using qPCR. Lastly, we carried out extensive biostatistical analysis. Results: Circulating levels of let-7b-5p, miR-143-3p, miR-17-5p, miR-214-3p, and miR-335-5p were significantly higher in the blood plasma of MM patients with MMBD compared to those without. Receiver operating characteristic curve and logistic regression analyses showed that these miRNAs could accurately predict MMBD. Furthermore, a standalone multi-miRNA–based logistic regression model exhibited the best predictive potential regarding MMBD. Two of those miRNAs also have a prognostic role in MM since survival analysis indicated that lower circulating levels of both let-7b-5p and miR-335-5p were associated with significantly worse progression-free survival, independently of the established prognostic factors. Conclusions: Our study proposes a miRNA signature to facilitate MMBD diagnosis, especially in ambiguous cases. Moreover, we provide evidence of the prognostic role of let-7b-5p and miR-335-5p as non-invasive prognostic biomarkers in MM.

Reference Gene Selection for RT-qPCR Analysis in Maize Kernels Inoculated with Aspergillus flavus.

Resistance against infection by the fungus Aspergillus flavus Link in commercial maize (Zea mays L.) is the topic of many studies, but few studies have investigated the effects of A. flavus infection on gene expression levels in ear kernels. A crucial component of gene expression profiling by RT-qPCR is having a reliable set of reference genes that show relatively constant expression across the treatments and phenotypes under study. Currently, however, there is no published information on reference genes suitable for measuring changes in kernel gene expression levels after infection with A. flavus. Thus, in this study, six candidate reference genes (ACT1, β-Tub2, eIF4A2, TATA, EFIα, and GAPDH) were evaluated and ranked according to their expression stability. The genes were amplified from first-strand cDNA samples synthesized from kernels of two susceptible and two resistant maize lines that were either inoculated with A. flavus or water or not inoculated. Three software packages were used to calculate and rank the stability of expression for these genesgeNorm, NormFinder, and BestKeeper. The analysis revealed that the most stable genes to normalize expression levels from maize kernels responding to A. flavus inoculation and wounding were ACT1, EFIα, and eIF4A2.