TRIzol Reagent Research Articles

LncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis

Triple-negative breast cancer (TNBC) is a highly invasive subtype of breast cancer. This study investigated the molecular mechanism and influences of MIR503HG, miR-224-5p, and homeobox A9 (HOXA9) on TNBC cell growth and migration. Dual-luciferase reporter gene and RNA immunoprecipitation were performed to examine the regulation of MIR503HG, miR-224-5p, and HOXA9. Cell proliferation, apoptosis, migration, and invasion were evaluated by colony formation, flow cytometry, and Transwell assays. Finally, nude mice were employed to investigate the influence of MIR503HG on TNBC tumor growth. HOXA9 protein levels were detected by immunohistochemical staining. MIR503HG and HOXA9 expression were reduced in TNBC, while miR-224-5p was increased. Overexpression of MIR503HG or HOXA9 reduced the cell migration ability and proliferation and promoted apoptosis, and knockdown of MIR503HG or overexpression of miR-224-5p exhibited the opposite effects. Furthermore, MIR503HG promoted HOXA9 expression by inhibiting miR-224-5p. Overexpression of miR-224-5p reversed the effects of MIR503HG overexpression on TNBC cells, while overexpression of HOXA9 reversed the effect of MIR503HG knockdown. Additionally, an in vivo study proved that MIR503HG inhibited TNBC tumor growth via the miR-224-5p/HOXA9 axis. MIR503HG inhibited cell proliferation and promoted the apoptosis of TNBC cells via the miR-224-5p/HOXA9 axis, which may function as a novel target for the treatment of TNBC.

Investigation of MRP1 and ABCG2 Gene Expression in Chronic Myeloid Leukemia (CML) Patients

Background: This study evaluated and compared the quantitative expression of multidrug resistance-associated protein 1 (MRP1) and ATP-binding cassette subfamily G member 2 (ABCG2), two Multidrug Resistance (MDR) related genes, in 30 CML patients and 27 normal subjects. Methods: Total RNA was isolated from peripheral blood mononuclear cells (MNCs) using the Trizol reagent. Then cDNAs were synthesized. Gene expression was quantified using Real-Time PCR System. The relative expression of target genes was calculated using the 2-ΔΔCt method. Results: High expression of MRP1 and ABCG2 mRNAs were detected in the patient group. Intra-group comparisons also revealed increased expression of ABCG2 in Accelerated Phase (AP)-Blastic Crisis (BC) patients compared to Chronic Phase (CP) patients. At the same time, the increased expression of MRP1 in AP-BC patients was not statistically significant. Conclusion: Considering the broad spectrum of ATP Binding Cassette (ABC) transporter superfamily substrates, they can play an essential role in cell fate determination. High expression of MRP1 and ABCG2 genes can result in the efflux of therapeutic agents and subsequent reduction in their intracellular concentration. This mechanism finally protects cells from the therapeutic effects of medications. On the other hand, these transporters can export growth factors out of the cell. Such exported molecules may have a growth-inducing effect on adjacent cells. These are the possible mechanisms for the participation of MRP1 and ABCG2 genes in conferring drug resistance to CML cells.

Expression of miR-1290 and Its Target Genes THBS1 and DKK3 in Colorectal Cancer Patients

Background: MicroRNAs (miRNAs) are small noncoding RNAs (containing approximately 22 nucleotides), which modulate and control the expression of target genes by binding them. MiRNAs play a crucial role in tumorigenesis. Thus, alterations in the expression level of miRNAs play a key role in the pathobiology of numerous cancers. In this research, the expression level of MicroRNA-1290 (miR1290) and its target genes THBS1 and DKK3 were evaluated in colorectal cancer (CRC) patients. Methods: This case-control study was carried out on 144 paraffin-embedded tissue samples of CRC and adjacent tissues from patients who referred to Imam Khomeini Hospital, Tehran, Iran. Total RNA was isolated from the tissue using Trizol reagent following the manufacturer’s instructions and then reverse transcribed to cDNA. The expression of miR-1290 and its target genes was measured by quantitative Real-Time PCR (qRT-PCR). Statistical analyses were performed using SPSS V.20 statistical software. Results: We present evidence that the miR-1290 expression in CRC tissues was significantly higher than in the normal margin, and its targets were downregulated in tumor tissue compared to the adjacent tissue. Conclusion: This study supports the essential role of miR-1290 and its contribution to CRC invasion and metastasis through targeting THBS1 and DKK3, as biomarkers for CRC diagnosis.

Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis.

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizolreagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

Frequency of two transcripts of BCR-ABL 1 fusion gene (p210 and p190) in acute lymphoblastic leukemia and chronic myeloid leukemia by reverse transcriptase polymerase chain reaction

Objective: To determine the frequency of two transcripts of BCR-ABL 1 fusion gene (P210 & P190) in acute lymphoblastic leukemia and chronic myelogenous leukemia by reverse transcriptase polymerase chain reaction. Study design: A cross-sectional study. Place and duration of study: The Armed Forces Institute of Pathology (AFIP), Rawalpindi, from December 2012 to January 2014. Methodology: 147 diagnosed patients of CML and ALL were subjected to real time reverse transcriptase polymerase chain reaction. For PCR,2-3ml of venous blood in EDTA was collected .RNA extraction was done by Trizol Reagent LS (MRC, USA) and cDNA was synthesized using reverse transcriptase and gene specific primer. Real time- PCR was done on ABI-7500.The positive samples were identified when fluorescence exceeded threshold limit. Results: Out of 147 samples, 85 were with diagnosis of CML. All of them (100%) showed P210. 58 patients had diagnosis of ALL. Out of these BCR-ABL1 is detected in 11(18.9%) patients. 9(81.8%) expressed P190 whereas P210 was present in 2(18.1%) patients. 4 patients in lymphoid blast phase of CML were identified. 3(75%) of them had PP210 and 1(25%) shows both (P190 & P210) the transcripts. Conclusion: All CML patients expressed P210 transcript whereas ALL patients mostly showed P190. These transcripts expressing specific tyrosine kinase protein have diagnostic as well prognostic significance.

Expression, Purification, and Bioactivity of a Soluble Recombinant Ovine Interferon-tau in Escherichia Coli.

IntroductionOvine interferon-tau (oIFN-τ) is a newly discovered type I interferon. This study used biochemical techniques to transform the oIFN-τ gene into Escherichia coli to obtain the mass and soluble expression of the recombinant protein.Material and MethodsFirst, total RNA was extracted from fresh sheep embryonic tissues with TRIzol reagent and then used as a template to reverse transcribe and amplify the mature oIFN-τ gene with RT-PCR. The amplified product was next digested with the HindIII and XhoI restriction enzymes and inserted into the pET-32a(+) vector to construct the prokaryotic expression plasmid. The corrected in-frame recombinant plasmid, pET-32a(+)-oIFN-τ, was transformed into E. coli Rosetta (DE3) competent cells. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was detected in bacteria. Finally, the bacteria were lysed by sonication, and the recombinant protein was purified by nickel affinity chromatography and DEAE anion exchange chromatography.ResultsThe protein was confirmed to be oIFN-τ, which mainly existed in the soluble lysate fraction, as proven by SDS-PAGE and Western blot assays.ConclusionPurified IFN-τ exists mostly in a soluble form, and its anti-vesicular stomatitis virus (VSV) activity reached 7.08×10(6)IU/mL.

200 Reduced growth performance of pigs fed methionine deficient diet may be associated with their reduced muscle cell differentiation

Abstract Methionine (Met) is the second or third limiting AA in typical swine diets and plays important roles in promoting the growth, especially, the muscle growth of pigs. This research studied the effects of dietary Met restriction on growth performance and myogenic gene expression in growing pigs. Eight genes in two families, including myogenic regulatory factor family comprising myogenic differentiation 1 (MyoD1), myogenic factor 5 (Myf5), myogenin (MyoG) and myogenic factor 6 (Myf6), and myocyte enhancer factor 2 (Mef2) family comprising Mef2A, Mef2B, Mef2C and Mef2D, were selected for analysis. Individually penned barrows (crossbred, 23.6±2.4 kg) were randomly assigned to two dietary treatments. A basal diet (Diet 1; Met-deficient) was formulated to contain 0.22% standardized ileal digestible (SID) Met and 0.52% SID Met+Cys but to meet the NRC (2012) recommendations for other nutrients. Crystalline DL-Met was added to the basal diet to generate Diet 2 adequate in Met (0.37% SID Met, 0.67% SID Met+Cys). During the four-week ad libitum feeding trial, ADG, ADFI and G:F were measured. Immediately before and after the trial, muscle samples were collected from the longissimus dorsi using a standard biopsy protocol. Total RNA was extracted from the muscle samples (TRIzol Reagent; Invitrogen) and subjected to cDNA reverse-transcription (QuantiTect Reverse Transcription Kit; Qiagen). Transcribed cDNA were used for qPCR analysis (Rotor-Gene Q System; Qiagen). Comparative ΔΔCT method was used for quantitation. Data were analyzed with Student’s T-test. Pigs fed Diet 1 (vs. Diet 2) had a lower ADG and G:F (P< 0.01). Before the feeding trial, all the tested genes had comparable mRNA levels between the two treatments (P >0.17). After the trial, Diet 1 pigs showed tendency for lower levels of Myf6 and Mef2D mRNA (P< 0.09). These results suggest that the reduced ADG and G:F is associated with the possibly-reduced muscle cell differentiation in pigs fed Met-deficient diet.

Yield and Integrity of RNA from Brain Samples are Largely Unaffected by Pre-analytical Procedures.

Gene expression studies are reported to be influenced by pre-analytical factors that can compromise RNA yield and integrity, which in turn may confound the experimental findings. Here we investigate the impact of four pre-analytical factors on brain-derived RNA: time-before-collection, tissue specimen size, tissue collection method, and RNA isolation method. We report no significant differences in RNA yield or integrity between 20mg and 60mg tissue samples collected in either liquid nitrogen or the RNAlater stabilizing solution. Isolation of RNA employing the TRIzol reagent resulted in a higher yield compared to isolation via the QIAcube kit while the latter resulted in RNA of slightly better integrity. Keeping brain tissue samples at room temperature for up to 160min prior to collection and isolation of RNA resulted in no significant difference in yield or integrity. Our findings have significant practical and financial consequences for clinical genomic departments and other laboratory settings performing large-scale routine RNA expression analysis of brain samples.

DNA Extraction with TRIzol Reagent Using a Silica Column.

TRIzol is a monophasic solution of phenol and guanidine isothiocyanate used for the extraction of RNA, DNA and proteins from tissues or cells. However, few studies have described its application to DNA extraction due to its time-consuming procedure. We present a TRIzol-modified method of extracting DNA from tissues using the TRIzol reagent and a silica column, which requires only one-third of the time required for the classic extraction procedure. Spectrophotometric analysis showed that the 260/280 and 260/230 nm optical density ratio of the DNA extracted using the TRIzol-modified method is ideal and equal to that obtained by the classic method and commercial DNAiso methods. The DNA extracted by the TRIzol-modified method had the same performance in a restriction enzyme digestion and quantitative PCR as that extracted using the classic method. Using the TRIzol-modified method saves time, simplifies the DNA extraction procedure, and facilitates various molecular biology assays.

Transcriptomic Landscape of Umbilical Cord Blood Mononuclear Cells after Genetic Modification

Introduction: Therapeutic application of umbilical cord blood mononuclear cells (UCB-MCs) has been actively studied for at least during the last 30 years. Currently, UCB-MCs are extensively being investigated in the regeneration of the central nervous system (CNS) and the treatment of human neurodegenerative disorders. Translational studies dedicated to the application of UCB-MCs have revealed their capacity for stimulation of neurogenesis in the aged brain and promising potency for being therapeutic agents for treating such diseases as Alzheimer`s disease, amyotrophic lateral sclerosis (ALS), ischemic stroke, traumatic brain injury, Parkinson`s disease etc. Still the exact mechanism providing therapeutic effect remains unclear. Several studies modulating neurodegeneration have demonstrated immunomodulating and pro-inflammatory activity of UCB-MCs. Moreover, the unique feature of UCB-MCs, which underlies in the optional concordance of HLA or immunosuppression during transplantation, has been shown. At the same time, the main challenge in the application of UCB-MCs is the limited amount of available cells from a single donor. Therefore, the development of methods increasing therapeutic potency is an important task when using UCB-MC. In this regard, in our opinion, one of the options for increasing the therapeutic potential of cells can be ex vivo genetic modification of the transplanted UCB-MC. This approach is beneficial to get cells with the desired therapeutic properties. At the same time, little is known about the effect of genetic modification and over-expression of therapeutic genes on the UCB-MC transcriptome. In this regard in our present study, we evaluated the transcriptomic landscape of gene engineered UCB-MC. Methods: UCB-MCs were separated using Ficoll density gradient and modified recombinant adenoviruses (Ad-EGFP or Ad-VEGF165, MOI 10). Total RNA from all test samples was extracted using Trizol reagent. The quality and concentration of the isolated RNA samples were evaluated using Agilent Bioanalyzer 2100. Total mRNA from genetically modified and non-treated cells was sequenced Sequencing-By-Synthesis (SBS) technology on Illumina NextSeq 500 platform in 2×75 bp mode. After quality control, reads were aligned to human reference transcriptome GRCh38 using Kallisto pseudoaligner. Differentially expressed transcripts and genes were calculated with R package "sleuth". Results: We comprehensively profiled the whole-transcriptome landscape of human genetically modified UCB-MC. Totally 12 cDNA libraries, obtained from 6 individual donors, were analyzed. Transcriptomic analysis has revealed 2,4-2,8×106 of paired reads. A total of 10164 genes in the RNA-seq data were detected and analyzed. UCB-MCs were shown to express a broad range of pro- and anti-inflammatory cytokines, chemokines, growth factors, and metalloproteinases. The principal component analysis (PCA) of the RNA-seq data showed that samples representing different biological conditions do not differ from each other and are grouped according to the source of their receipt (isolation) (Fig.1A). Genetic modification and expression of transgenes did not lead to a global shift in the transcriptome profile of UCB-MC. At the same time, the recombinant genes EGFP (log2FC = 7.15, q <0.05) and VEGF (log2FC = 4.41, q <0.05), as expected, showed increased expression compared to NTC. We performed Gene Ontology (GO) analysis of the expressed genes. The results demonstrated that most genes associated with biological processes were related to metabolism. In the category of cellular components, most detected genes were associated with cellular membrane and nucleus, while in molecular function category - protein binding (Fig.1B). Conclusion: It has been shown that genetic modification and expression of UCB-MC transgenes did not affect the global transcriptome profile. Transcriptome profiling can be useful in the creation and testing of personalized gene cell products that meet biological safety and efficacy criteria. This work was supported by the RFBR grant №18-44-160029 and subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities 0671-2020-0058. Disclosures No relevant conflicts of interest to declare.

Comprehensive Analysis of Transcriptomics and Metabolomics between the Heads and Tails of Angelica Sinensis: Genes Related to Phenylpropanoid Biosynthesis Pathway.

In Traditional Chinese Medicine (TCM), the heads and tails of Angelica sinensis (Oliv.) Diels (AS) is used in treating different diseases due to their different pharmaceutical efficacies. The underline mechanisms, however, have not been fully explored. Novel mechanisms responsible for the discrepant activities between AS heads and tails were explored by a combined strategy of transcriptomes and metabolomics. Six pairs of the heads and tails of AS roots were collected in Min County, China. Total RNA and metabolites, which were used for RNA-seq and untargeted metabolomics analysis, were respectively isolated from each AS sample (0.1 g) by Trizol and methanol reagent. Subsequently, differentially expressed genes (DEGs) and discrepant pharmaceutical metabolites were identified for comparing AS heads and tails. Key DEGs and metabolites were quantified by RT-qPCR and targeted metabolomics experiment. Comprehensive analysis of transcriptomes and metabolomics results suggested that five KEGG pathways with significant differences included 57 DEGs. Especially, fourteen DEGs and six key metabolites were related to the metabolic regulation of Phenylpropanoid biosynthesis (PB) pathway. Results of RT-qPCR and targeted metabolomics indicated that higher levels of expression of crucial genes in PB pathway, such as PAL, CAD, COMT and peroxidase in the tail of AS, were positively correlated with levels of ferulic acid-related metabolites. The average content of ferulic acid in tails (569.58±162.39 nmol/g) was higher than those in the heads (168.73 ± 67.30 nmol/g) (P.

TNF-Like Weak Inducer of Apoptosis Promotes Angiogenesis, Thereby Exacerbating Cutaneous Psoriatic Disease

TNF-Like Weak Inducer of Apoptosis Promotes Angiogenesis, Thereby Exacerbating Cutaneous Psoriatic Disease

Applicability of polyethylene glycol precipitation followed by acid guanidinium thiocyanate-phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater

The primary concentration and molecular process are critical to implement wastewater-based epidemiology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the previously developed methods were optimized for nonenveloped viruses. Few studies evaluated if the methods are applicable to the efficient recovery of enveloped viruses from various types of raw sewage. This study aims (1) to compare the whole process recovery of Pseudomonas phage φ6, a surrogate for enveloped viruses, among combinations of primary concentration [ultrafiltration (UF), electronegative membrane vortex (EMV), and polyethylene glycol precipitation (PEG)] and RNA extraction methods (spin column-based method using QIAamp Viral RNA Mini Kit and acid guanidinium thiocyanate–phenol–chloroform extraction using TRIzol reagent) for three types of raw sewage and (2) to test the applicability of the method providing the highest φ6 recovery to the detection of SARS-CoV-2 RNA. Among the tested combinations, PEG+TRIzol provided the highest φ6 recovery ratio of 29.8% to 49.8% (geometric mean). UF + QIAamp Viral RNA Mini Kit provided the second highest φ6 recovery of 6.4% to 35.8%. The comparable φ6 recovery was observed for UF + TRIzol (13.8–30.0%). PEG + QIAamp Viral RNA Mini Kit provided only 1.4% to 3.0% of φ6 recovery, while coliphage MS2, a surrogate for nonenveloped viruses, was recovered comparably with PEG + TRIzol. This indicated that the nonenveloped surrogate (MS2) did not necessarily validate the efficient recovery for enveloped viruses. EMV + QIAamp Viral RNA Mini Kit provided significantly different φ6 recovery (1.6–21%) among the types of raw sewage. Then, the applicability of modified PEG + TRIzol was examined for the raw sewage collected in Tokyo, Japan. Of the 12 grab samples, 4 were positive for SARS-CoV-2 CDC N1 and N3 assay. Consequently, PEG + TRIzol provided the highest φ6 recovery and allowed for the detection of SARS-CoV-2 RNA from raw sewage.

First Report of Mume Virus A Infection of Prunus persica in China

Mume virus A, belonging to Capillovirus in the family Betaflexiviridae, was discovered recently from Japanese apricot (Prunus mume) and exhibited diffuse chlorotic spots on leaves (Marais et al. 2018). To date, Japanese apricot is the only known host of Mume virus A (MuVA) (Maliogka et al. 2018). Peach (Prunus persica) is a delicious and healthy summer fruit grown in most temperate regions of the world. More than 20 different viruses infecting peach trees have been reported (Jo et al. 2018). In April 2019, the leaves from four peach trees showing severe yellow mosaic and leaf curling were collected from peach orchards in Xinjiang, China. The pooled total RNA was extracted from these four trees leaves with TRIzol reagent (Life Technologies Corporation, Carlsbad, CA) and subjected to high-throughput sequencing (HTS) using a PE150 sequence strategy on an Illumina HiSeq2000 (Novogene Corporation, Tianjin, China). Approximately 39.3 million of 150-bp paired-end reads were obtained and used for de novo transcriptome assembly by SPAdes Genome Assembler (Bankevich et al. 2012). A total of 89,163 contigs with N50 of 866 bp were obtained and then were annotated by BLASTX analysis against the NCBI nr virus database (Altschul et al. 1997). Six known viruses (apple chlorotic leaf spot virus, Asian prunus virus 2, cherry green ring mottle virus, nectarine stem pitting-associated virus, peach associated luteovirus, and plum bark necrosis stem pitting-associated virus) and one known viroid (hop stunt viroid) were identified. Moreover, a 7,623-bp contig with a coverage of 84.4-fold was identified as capillovirus-related sequence. To classify the capillovirus accurately, pairwise genome alignments analysis was performed between all members of the Capillovirus genus using PASC online tool (Bao et al. 2014). The closest match was the genome of MuVA (NC_040568.1), sharing 78.36% nucleotide identity, followed by those of some isolates of cherry virus A, sharing less than 58.5% nucleotide identity. This result suggests that the capillovirus from peach belongs to the same species with the MuVA from apricot but should be a different isolate. We designated it as isolate pp, and its HTS-derived viral genome sequence was deposited in GenBank (accession no. MN412555). To confirm the HTS experiment results, from the same peach orchards, 23 peach leaf samples from different trees were collected, and total RNA was extracted with TRIzol reagent with standard procedures following the instructions. The RNA was subsequently used to test for pp isolate by reverse transcription polymerase chain reaction (PCR) using specific primers DT-MVAF5716 (5′-CTTCACATCGGAGCTGTCCT-3′) and DT-MVAR6277 (5′-TGCTGATTGCTTCAGACCCT-3′), targeting a portion of the open reading frame 2 (putative movement protein). Eleven samples produced the expected 560-bp PCR fragment. Three PCR products randomly selected were cloned into pMD19-T Vector (TaKaRa) for Sanger sequencing. All sequences of amplicons shared nearly 100% sequence identity with the corresponding region of the HTS-derived viral genome sequence. However, at least the other two viruses and viroid had been detected in all 11 MuVA-positive samples, so the symptoms of the initial sample cannot be explained by MuVA infection. To our knowledge, this is the first report of MuVA infecting peach trees. This report suggests that MuVA may have a broad host range in the genus Prunus.

Development of a method for determining the levels of normalized expression of structural and functional genes in patients with knee joint arthropathy

Objective : to develop a method of molecular genetic analysis for determining the normalized expression levels of COL2A1, COL6A1, MMP-2, and MMP-9 genes in the biological material of patients with gonarthrosis. Material and methods . The biopsy samples of knee joint cartilage of 10 patients with gonarthrosis were used as a biological material for the study. Nucleic acids were isolated from the samples of the biological material using TRIZol Reagent. Reverse transcription was performed using the SuperScript III reverse transcriptase kit, dNTP and Ribonuclease inhibitor (Invitrogen, USA). Results . The usage of VectorNTI software has made it possible to select pairs of primers (forward and reverse) and TaqMan probes for COL2A1, COL6A1, MMP-2 , and MMP-9 genes, and to study the possibility of the use of the selected pairs of primers and probes for the purpose of identification of each of the studied genes. In-house test systems in the multiplex format have been designed for reference and target genes. Conclusion . High specificity (100 %) of the selected oligonucleotide primer sets was confirmed using the online application NCBI/Blast. The developed molecular genetic method can be applied to determine the normalized expression levels of COL2A1, COL6A1, MMP-2 , and MMP-9 genes in knee joint arthropathy

First Report of Hop stunt viroid Infecting Citrus Trees in Georgia, USA.

In recent years, citrus production has rapidly increased within the state of Georgia (USA), and there are now citrus plantings within at least 32 counties in residential, production, and nursery settings. Among the pathogens capable of infecting citrus are viroids, the smallest plant pathogens. Viroids are comprised of circular, single-stranded RNA ranging from 246-463 nucleotides in length (Ito et al., 2002). Hop stunt viroid (HSVd) is one of several viroids known to infect citrus. This viroid has been previously reported within Arizona, California, Florida, Texas, and Washington in the United States and in other locations throughout the world (Hadidi, 2017). HSVd is often spread mechanically on contaminated tools or through grafting. With a wide host range that includes the families Moraceae, Rosaceae, and Rutaceae (citrus), this viroid can easily move throughout a nursery and spread to other plants (Hadidi, 2017). Symptoms of HSVd include a discoloration and gumming of phloem tissues, stem pitting, bark splitting, and chlorotic and stunted growth in susceptible citrus varieties including tangerines and their hybrids (Hadidi, 2017). There are not typically symptoms on leaves or fruits; however, lime plants have shown some yellowing on leaves (Hadidi, 2017). In May and June of 2020, leaf samples were collected from 12 different citrus plants in nursery settings in Berrien and Mitchell counties in Georgia. The cultivars sampled from Citrus reticulata 'Dekopon'. The sampled trees looked relatively healthy with little or no signs of damage, but were selected for testing to ensure that they were viroid free. Reverse transcription-polymerase chain reaction (RT-PCR) was initially used to verify infection with HSVd. Genomic RNA was extracted from the leaf tissue of twelve plants using the TRIzol reagent (Thermofisher, Waltham, MA). Following cDNA synthesis, samples were tested for the presence of HSVd using the primer pair HSVd-F (5'-GGCAACTCTTCTCAGAATCCAGC-3') and HSVd-R (5'-CCGGGGCTCCTTTCTCAGGTAAGT-3') which produces a 302 bp amplicon (Sano et al., 1988). The PCR reactions for nine of the tested samples did not result in the production of any bands, however the other three samples, all Citrus reticulata 'Dekopon', produced the expected amplicon for HSVd. The amplified products were sequenced using Sanger sequencing (Retrogen Inc, San Diego, CA, USA) and the identity of the fragment sequences was confirmed using BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Partial sequences from these amplicons (deposited as accession number MT632007) shared 99% identity to corresponding HSVd sequences in Genbank (accession number MG779542). In addition to RT-PCR and sequencing, the recombinase-polymerase-amplification (RPA) technology based AmplifyRP® Acceler8™ end-point detection assay (Agdia® Inc., Elkhart, IN) was performed on previously confirmed tissue according to the manufacturer's instructions. This assay also confirmed the presence of HSVd viroid in the three samples that had been previously confirmed via RT-PCR. To the best of our knowledge, this is the first report of HSVd infecting Citrus reticulata 'Dekopon' in Georgia. If this viroid were to spread within the growing Georgia citrus industry, it could pose a significant threat to citrus plantings that contain susceptible varieties. Nursery stock infected with this viroid should be destroyed, and Georgia nursery producers and citrus growers should take appropriate precautions to prevent the spread of this viroid disease, including properly sanitizing tools used for citrus grafting and pruning. Further research is needed to determine the distribution of HSVd and its potential to impact commercial citrus production in Georgia.

EXPRESSION OF MIR-24 IN SALIVA FROM PATIENTS WITH ORAL CANCER

Objective: To evaluate the expression of miR-24 in saliva collected from patients diagnosed with oral cancer. Study Design: Plasma and saliva samples were spontaneously collected from 3 healthy donors (controls) and 8 patients with oral cancer. MicroRNA extraction was done using TRIzol reagent and quantified by spectrophotometry. cDNA was synthesized using the GoScript Reverse Transcription kit (PROMEGA). Therewith, miR-24 expression was analyzed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. β-globin was used as reference gene. Results: Seven patients were diagnosed with oral squamous cell carcinoma and 1 patient with myoepithelial carcinoma. Patients presented single and ulcerated lesions and absence of metastasis. Most of them were men (62.5%) whose average age was 64.5 years old. In plasma samples, there were no differences in expression levels between controls and patients samples; however, in saliva, miR-24 showed overexpression (4.0 Fold - P< .05) in patients with oral cancer in comparison with heathy controls. Conclusion: (1) These results suggest that the miR-24 might be a possible candidate as molecular biomarker to diagnose oral cancer through saliva, however, further studies need to be done in order to validate these results. (2) Saliva is an important source of biomolecules suitable to be validated as molecular markers to diagnose oral diseases. Objective: To evaluate the expression of miR-24 in saliva collected from patients diagnosed with oral cancer. Study Design: Plasma and saliva samples were spontaneously collected from 3 healthy donors (controls) and 8 patients with oral cancer. MicroRNA extraction was done using TRIzol reagent and quantified by spectrophotometry. cDNA was synthesized using the GoScript Reverse Transcription kit (PROMEGA). Therewith, miR-24 expression was analyzed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. β-globin was used as reference gene. Results: Seven patients were diagnosed with oral squamous cell carcinoma and 1 patient with myoepithelial carcinoma. Patients presented single and ulcerated lesions and absence of metastasis. Most of them were men (62.5%) whose average age was 64.5 years old. In plasma samples, there were no differences in expression levels between controls and patients samples; however, in saliva, miR-24 showed overexpression (4.0 Fold - P< .05) in patients with oral cancer in comparison with heathy controls. Conclusion: (1) These results suggest that the miR-24 might be a possible candidate as molecular biomarker to diagnose oral cancer through saliva, however, further studies need to be done in order to validate these results. (2) Saliva is an important source of biomolecules suitable to be validated as molecular markers to diagnose oral diseases.

EVALUATING THE LEVELS OF GENOMIC INSTABILITY IN ORAL CAVITY INJURIES: EXPRESSION OF SMYD2, SMYD3, AND EZH2 GENES

Objectives: Epigenetic alterations promote the regulation of gene expression and are related to neoplastic development. Thus, the aim of this study is to compare the expression levels of SMYD2, SMYD3, and EZH2 genes in oral cavity biopsies, according to the histopathological classification obtained. Study Design: A total of 36 samples of oral cavity biopsies were collected and classified into 4 distinct histopathological groups: Benign tissue (10), traumatic lesion (11), inflammatory lesion (7), and malignant tissue (8). To evaluate the expression levels of the SMYD2, SMYD3, and EZH2 genes, the total mRNA was obtained by extraction using the Trizol reagent, according to the established protocol. Gene expression profiles were obtained by real-time polymerase chain reaction. Results: The data obtained allowed us to identify significant differential expression of the SMYD2, SMYD3, and EZH2 genes, considering tissue classified as benign, followed by traumatic lesion, inflammatory lesion, and, finally, malignant tissue (P

DIFFERENTIAL EXPRESSION ANALYSIS OF AURKA AND AURKB GENES IN DISTINCT TISSUES OF THE ORAL CAVITY: BENIGN TO MALIGNANT

Objectives: In view of the interrelationship between neoplastic development and genomic instability, this study aimed to compare the levels of expression of aurora kinase genes (AURKA and AURKB), considered as biomarkers of neoplastic evolution, in oral biopsies, according to the histopathological classification obtained. Study Design: A total of 36 samples of oral cavity biopsies were collected and classified into 4 distinct histopathological groups: Benign tissue (10), traumatic lesion (11), inflammatory lesion (7), and malignant tissue (8). To evaluate the expression levels of the AURKA/B genes, the total mRNA was obtained by extraction using the Trizol reagent, according to the established protocol. Gene expression profiles were obtained by real-time polymerase chain reaction. Results: There was a significant diversity in AURKA/B gene expression levels, considering tissue classified as benign, followed by traumatic lesion, inflammatory lesion, and, finally, malignant tissue (P

8. Modifiable polymer promotes a pro-osteogenic, M2-like macrophage phenotype and osteoblastic differentiation of progenitor cells

8. Modifiable polymer promotes a pro-osteogenic, M2-like macrophage phenotype and osteoblastic differentiation of progenitor cells