Total RNA Research Articles

Microarray-based analysis of differential gene expression profile in rotenone-induced Parkinson's disease zebrafish model.

Despite much clinical and laboratory research that has been performed to explore the mechanisms of Parkinson's disease (PD), its pathogenesis remains elusive to date. Therefore, this study aimed to identify possible regulators of neurodegeneration by performing microarray analysis of the zebrafish PD model's brain following rotenone exposure. A total of 36 adult zebrafish were divided into two groups: control (n = 17) and rotenone-treated (n = 19). Fish were treated with rotenone water (5 µg/L water) for 28 days and subjected to locomotor behavior analysis. Total RNA was extracted from the brain tissue after rotenone treatment. The cDNA synthesized was subjected to microarray analysis and subsequently validated by qPCR. Administration of rotenone has significantly reduced locomotor activity in zebrafish (p < 0.05), dysregulated dopamine-related gene expression (dat, th1, and th2, p < 0.001), and reduced dopamine level in the brain (p < 0.001). In the rotenone-treated group, genes involved in cytotoxic T lymphocytes (gzm3, cd8a, p < 0.001) and T cell receptor signaling (themis, lck, p < 0.001) were upregulated significantly. Additionally, gene expression involved in microgliosis regulation (tyrobp, p < 0.001), cellular response to IL-1 (ccl34b4, il2rb, p < 0.05), and regulation of apoptotic process (dedd1, p < 0.001) were also upregulated significantly. The mechanisms of T cell receptor signaling, microgliosis regulation, cellular response to IL-1, and apoptotic signaling pathways have potentially contributed to PD development in rotenone-treated zebrafish.

First report of the Horse nettle virus infecting tomato (Solanum lycopersicum L.) in the United States.

Tomato (Solanum lycopersicum L.), a member of the Solanaceae family, represents one of the most extensively cultivated vegetable species worldwide and traces its origin to western South America (Caruso et al. 2022). In a field survey conducted in 2023 in Bixby, Tulsa County, Oklahoma, distinct symptoms were noted in two plants: one exhibited mottling and cupping of leaves and brown discoloration on leaves, petioles, and stems, while the other displayed a downward curling of leaves. Leaf samples from both symptomatic tomato plants (labelled as K4 and K5) were collected, and total RNA was extracted individually via the TRI Reagent® method (Molecular Research Center Inc., Cincinnati, OH, USA). Subsequently, the RNA samples were pooled and subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 high-output kit v2.5 (Illumina, U.S.A.) at the genomic facility, Oklahoma State University (Stillwater, OK). Total read count of 8,227,020 (average length =150.5 bp) was obtained, trimmed, and de novo assembled using CLC Genomics Workbench v22.0.1 (QIAGEN) and used for BLASTn and BLASTx analysis. Two contigs: 6,375 bp (average coverage 2,915.92, read count 142,538) and 3,564 bp (average coverage 3,035.91, read count 82,370) from the pooled sample showed 88.6% and 96.7% nucleotide identities with RNA 1 (OP292294) and RNA 2 (OP292295) of Horse nettle virus A (HNA-A) isolate MD-1, respectively. Sequences of both partial contigs (RNA 1, accession no. PP063196) and RNA 2, accession no. PP063197) were submitted to GenBank. The HTS data did not reveal any other viral or viroid sequences in these two tomato samples. To further confirm the presence of HNV-A, total RNA from K4 and K5 samples was tested individually by RT-PCR using HNV specific primers (Supplementary Table 1) based on the two partial contig sequences. The expected PCR products (491 bp and 451 bp) were obtained only from the K4 sample and none from the K5 sample. PCR products were extracted from an agarose gel, cloned into the pGEM®-T Easy vector (Promega), and transformed into Escherichia coli DH5α cells (New England Bio Labs). Two clones for each PCR product were sequenced by Sanger sequencing. Nucleotide sequence comparisons and BLASTn analysis of 491 bp and 451 bp showed 86% and 97% nucleotide identity with RNA 1 and RNA 2 of HNV-A isolate MD-1 (OP292294 and OP292295), respectively. Additionally, eight more leaf samples from eight different symptomatic tomato plants were collected in the same field and tested by RT-PCR as described above. All eight samples were positive by RT-PCR, but no PCR band was obtained in the total RNA from a healthy tomato leaf used as a control. Sequences from the PCR products were identical to the obtained HTS sequences. Our results confirmed for the first time that HNV-A can infect tomatoes. Currently, HNV-A has been reported to only infect a single weed (Horse nettle, Solanum carolinense) (Zhou et al. 2023). The identification of HNV-A in tomatoes in Oklahoma suggests a potential host shift is of concern for local growers as well as tomato growers worldwide. This shift underscores the urgency for an in-depth investigation into the transmission and host specificity of HNV-A. This is the first report in the United States and the world that HNV-A could infect tomatoes naturally in a grower field.

First Report of Tobacco Streak Virus in Cannabis sativa in New York.

In 2022, virus-like symptoms were observed in a field of diverse hemp (Cannabis sativa L.) germplasm in Ontario County, New York. Less than 1% of plants exhibited stunting and curled leaves (Figure S1), consistent with tobacco streak virus (TSV) symptoms on other plants (Liu et al. 2022). Most typically, the plants were considerably reduced in overall size, with upwards, adaxial curling along the leaf margin with newer leaves appearing to be the most affected. Fifteen symptomatic plants representing nine accessions were tested for 12 viruses and viroids through Agdia Testing Services (Elkhart, IN). Of these, eight plants representing five accessions including: G 33204 21UO SD ('Cherry Wine S1'), G 33211 21UO SD ('Wife'), G 33225 22CL01 CL ('Candida #2'), G 33270 22UO SD ('Falkowski CBD Mix'), and G 33365 22UO SD ('Queen Dream'), were positive for TSV, a type of Ilarvirus in the Bromoviridae family. Presence of TSV was confirmed through enzyme-linked immunosorbent assay testing. TSV is a positive-sense, single-stranded RNA virus with a wide host range that can be transmitted by thrips, mechanical injury, seed, and pollen (Zambrana-Echevarría et al. 2021). To confirm the presence of TSV, two putatively TSV-infected samples were subjected to RNA-Seq analysis. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Aarhus, Denmark) per manufacturer's direction. Stranded RNA libraries were prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Plant kit (San Diego, California, USA). Paired-end 2x150bp sequencing was performed on an Illumina NovaSeq6000 sequencer. RNA-Seq data was trimmed using the fastp program (Chen et al. 2018) with default parameters to remove adapter sequences and low-quality bases. After filtering, 49,696,041 and 56,126,804 paired-end reads were retained from 'Wife' and 'Falkowski CBD Mix' samples, respectively. Filtered RNA-seq reads were mapped to TSV genome accession GCF_000865505.1 using the bowtie2 (Langmead & Salzberg 2012) aligner with default parameters. From 'Wife' and 'Falkowski CBD Mix' samples, 153 and 139 reads mapped to the TSV reference genome. To further validate the presence of TSV reads, RNA-Seq data was analyzed using the Kraken2 pipeline (Wood et al. 2019). Using the Kraken2 virus database, reads associated with TSV (NCBI taxonomy ID: 12317) were identified. This analysis identified 172 and 151 TSV reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Higher numbers of reads identified using the Kraken2 analysis is due to the more permissive k-mer matching approach implemented in Kraken2. Furthermore, we identified several other virus taxa in the samples. Of note, both samples had a high number of reads associated with Amazon lily mild mottle virus with 254,493 and 116,150 reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Among other virus species belonging to Ilarviruses, Cassava Ivorian bacilliform virus and Cowpea chlorotic mottle viruses were detected from both samples. To further validate infection by TSV, samples from both ELISA-positive and ELISA-negative plants were subjected to PCR using the primers and protocol described in Zambrana-Echevarría et al. 2021. Amplification of an approximately 700 base-pair product was observed in the putatively ELISA-positive samples, but not in the ELISA-negative samples. The amplicons were further cloned into the pGEM-T Easy vector (Promega, Madison, WI, U.S.A) using the manufacturer's protocol and sequenced using M13 forward and M13 reverse primers (Functional Biosciences, Madison, WI, U.S.A). Sequencing results indicated considerable similarity to TSV genomes available in GenBank, between 88% and 99%. Raw sequence data generated from this study was deposited in NCBI under the bioproject ID PRJNA1009441. Though it cannot be ruled out that the observed symptoms were caused exclusively by TSV infection due to the high number of other viral reads, the results contribute to the literature that indicates hemp can host TSV and should be considered a potential source of TSV inoculum (Chiginsky et al. 2021). This new inoculum source could cause significant crop damage and economic loss when grown with TSV susceptible row and specialty crops.

First report of cucurbit yellow stunting disorder virus infecting cucurbit crops in Jamaica.

The increasing prevalence of whitefly-transmitted viruses affecting cucurbit crops has emerged as a significant concern for global cucurbit production. Two of the most widely prevalent threats in the Americas are cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) (Crinivirus, Closteroviridae). These viruses induce similar foliar symptoms on cucurbit crops (Mondal et al., 2023) leading to loss of photosynthetic capability and decreased yields. Cantaloupe (Cucumis melo), watermelon (Citrullus lanatus), and cucumber (Cucumis sativus) are major cucurbit crops in St. Elizabeth, Jamaica, which is the principal fruit and vegetable producing region of the country. In August 2018, foliar symptoms were observed on cantaloupe, watermelon, and cucumber plants in several commercial farms in St. Elizabeth. These symptoms, mainly on the older leaves, consisted of severe yellowing or interveinal mottle and they appeared more pronounced on cantaloupe and cucumber plants compared to watermelon. Growers noticed the production of smaller than normal fruit. Disease incidence ranged from 10 to 100% and whiteflies (Bemisia tabaci Gennadius) were observed in the fields. To identify virus(es) associated with the disease, six plants (cantaloupe [n = 3], cucumber [n = 1] and watermelon [n = 2) exhibiting symptoms were sampled from four fields for preliminary screening. Total RNA was extracted from leaf tissues as described in Tamang et al. (2021) and samples tested by a multiplex reverse transcription RT-PCR method that targeted the RNA-dependent RNA polymerase (RdRp) of the whitefly transmitted viruses, CYSDV, CCYV, squash vein yellowing virus (SqVYV), and the aphid- transmitted cucurbit aphid-borne yellows virus (CABYV) (Mondal et al. 2023). RT-PCR amplified the expected 494-bp fragment of the CYSDV RdRp gene (Mondal et al., 2023) from two symptomatic plants; one cantaloupe, one cucumber, as well as from CYSDV-infected control plants but not from healthy controls. Further testing was conducted during the June-August 2020 growing season after similar symptoms were observed on additional farms in St. Elizabeth and two regions, Manchester and Clarendon, located to the east of St. Elizabeth. Twenty-one cucurbit leaf samples (11 cantaloupe, seven watermelon and two cucumber from St. Elizabeth and one cantaloupe from Clarendon) exhibiting foliar yellowing progressing from the crown outward, and mottling were collected. Whiteflies (5) from these fields in St. Elizabeth and 20 asymptomatic weed samples were also collected and sent to the USDA-ARS laboratory at Salinas, CA. Total RNA from leaf samples was extracted as described above and tested for CYSDV, CCYV, and CABYV. Total leaf DNA was also extracted (Mondal et al. 2016) and assayed with PCR (Gilbertson 2001) to detect the presence of the whitefly-transmitted cucurbit leaf crumple virus (CuLCrV), a begomovirus, commonly found in the southeastern United States (Gadhave et al., 2018; Keinath et al., 2018). Nineteen of the 21 cucurbit samples tested positive for the presence of CYSDV by RT-PCR (Mondal et al. 2023). Of the 19 CYSDV-positive samples, 13 cantaloupe, one cucumber, and five watermelon samples were singly infected with CYSDV, and one cantaloupe sample was infected with both CYSDV and CABYV. Amplicons of the Jamaica isolate from cantaloupe were sequenced (OR399555) and a 494 nt section of the RdRp gene was found to share 100% sequence identity to the Arizona 1 isolate (EF547827.1). The presence of CYSDV, was further confirmed using a second set of primers that amplified a 394-nt portion of the CYSDV coat protein gene (Polston et al., 2008). Among the weed samples, CABYV was detected in one sample from a Leonotis nepetifolia plant (Lamiaceae) and two Cleome sp. (Capparaceae) collected from St. Elizabeth. None of the crop and weed samples tested positive for CCYV or CuLCrV. DNA from whiteflies was extracted and assayed with PCR using species specific primers (Chen et al. 2016). All whiteflies were identified as B. tabaci cryptic species MEAM1, which is widely known an efficient vector of CYSDV (Berdiales, et al. 1999). This is the first report of CYSDV in Jamaica and its first known occurrence in these hosts within the country. Further monitoring of cucurbit crops and the whitefly vector is warranted to better understand the epidemiology.

First report of infections with isolates of the Asian genotype of Cucumber green mottle mosaic virus in cucumber crops in Belgium.

In July 2019, four rows of cucumber plants (Cucumis sativus) in a commercial glasshouse in the north of Belgium showed severe mosaic, blistering and distortion of the leaves, with symptoms resembling those caused by Cucumber green mottle mosaic virus (CGMMV). CGMMV is a Tobamovirus that mainly affects cucurbit crops worldwide (Dombrovsky et al., 2017). Phylogenetic analyses in previous studies have shown two major clades, one including isolates that were initially identified in Europe and Russia (European genotype) and the second one with isolates initially identified in Asia and Israel (Asian genotype) (Dombrovsky et al., 2017; Pitman et al., 2022; Mackie et al., 2023). A symptomatic leaf sample was collected and total RNA was isolated from 100 mg of leaf tissue (Spectrum™ Plant Total RNA kit, Sigma-Aldrich). CGMMV was detected using a one-step TaqMan RT-qPCR (Hongyun et al., 2008). High-throughput sequencing (HTS) confirmed the presence of CGMMV. The sample was prepared using the Novel enrichment technique of viromes protocol (NETOVIR protocol, Conceição-Neto et al., 2015). The leaf material was homogenized, enriched for virus-like particles and the RNA was extracted (QIAamp Viral RNA mini kit, QIAGEN). The extract was randomly amplified (Whole Transcriptome Amplification kit, Sigma Aldrich), used for library preparation (Nextera XT DNA library preparation kit, Illumina) and sequenced on a NovaSeq platform. HTS data analysis was performed using Geneious Prime software (Biomatters, Auckland, New Zealand, version 2023.2). After quality filtering and trimming, 26.7M reads were obtained (132 nt mean length). In total, 20.6M reads were mapped to two genomes KP772568 and GQ411361 (considered as reference for the Asian and European genotypes respectively) with Geneious. This revealed 100% coverage of the full sequences (6422 nt) with 99.4% and 90% nucleotide identities to the reference genomes of Asian and European genotypes, respectively. Phylogenetic analyses confirmed that isolate 2019-26A-BE, with GenBank ID OR724740, relates to the Asian genotype. The HTS data were additionally processed using the ViPER pipeline (De Coninck, 2021). The raw reads were quality filtered and trimmed, (Trimmomatic) and then used to perform de novo assembly (metaSPAdes). The produced contigs were classified using DIAMOND and visualized with KronaTools. The results showed that no other virus was detected in the sample. Finally, cucumber seedlings were inoculated using the original symptomatic sample and were grown in a research glasshouse. After 3 weeks, severe CGMMV symptoms, similar to the original symptoms observed in the commercial glasshouse, were observed in the inoculated plants. Infection with CGMMV was verified via RT-qPCR, and the isolate present in the inoculated plants was confirmed to belong to the Asian genotype via RT-PCR-RFLP (Crespo et al., 2017). Later samplings of symptomatic leaves confirmed the presence of isolates belonging to the Asian genotype of CGMMV at four other commercial glasshouse locations, specializing in cucumber crop, in Belgium in 2020, 2021 and 2023 by RT-PCR-RFLP. While the Asian genotype was previously found elsewhere in Europe (Pitman et al., 2022), to our knowledge, this is the first report of infections with isolates of this genotype in glasshouse cucumber crops in Belgium. Further investigation is required to determine the spread and impact of infections with isolates of the Asian genotype in cucumber crops in Belgium.

Identification and Verification of Potential Ferroptosis-Related Biomarkers in Cervical Cancer

This study screened important genes contributing to morbidity from differential ferroptosis-related genes (FRGs) in cervical cancer and to establish a risk assessment model with ferroptosis-related LncRNAs. Total RNA sequencing data were extracted from The cancer genome atlas (TCGA), Gene Expression Omnibus (GEO) and Genotype-Tissue Expression (GTEx). By differential analysis, we identified ferroptosis-related hub genes close to prevalence of cervical cancer. According to receiver operator curves (ROC) curves, hub genes have good diagnostic performance. The diagnostic potential of hub genes for occurrence of the disease was further assessed and verified. Further, a risk-assessing model based on ferroptosis-related LncRNAs was established. Finally, the differential expressions of hub genes were verified through qRT-PCR. Five hub genes were identified, and we found through GO, KEGG and immune infiltration, that the hub genes are connection with cervical cancer. The Area Under Curve (AUC) values were all greater than 0.8 in ROC curve, and the hub genes presented differences between disease and control groups in validation set GSE29570. We created a risk assessment model with 16 ferroptosis-related LncRNAs. There was a difference in survival between high-risk and low-risk groups. The AUC result for risk assessment model reached 0.792, and there were significant expression differences of Hub genes in Huvec and Hela cells. The study screened 5 hub genes and constructed the risk-assessment model based on 16 LncRNAs associated with ferroptosis genes.

Occurrence of grapevine yellow speckle viroid 2 and Australian grapevine viroid in Idaho grapevines.

Grapevine yellow speckle viroid 2 (GYSVd-2; Pospiviroidae, Apscaviroid) causes yellow speckle disease in grapevine (Koltunow et al. 1989) and was found in Australia, Iran, Italy, China, and Nigeria (Koltunow et al. 1989; Habili 2017; Zongoma et al. 2018). In the U.S., GYSVd-2 was found in the State of Washington (Vitis vinifera L. cv. Merlot; Alabi et al., 2012). Australian grapevine viroid (AGVd; Pospiviroidae, Apscaviroid) was reported in Australia, Italy, China, Tunisia, Iran, and in the U.S. wine grapes (V. vinifera) (Habili 2017). In the U.S., AGVd was reported from California (Al Rwahnih et al. 2009), from Washington State (V. vinifera cv. Syrah; GU327604), and from the State of New York (an unknown cv. of V. vinifera; KY081960). In Idaho, two other viroids, hop stunt viroid (HSVd; Pospiviroidae, Hostuviroid) and grapevine yellow speckle viroid 1 (GYSVd-1; Pospiviroidae, Apscaviroid), common in grapevines were previously found in wine grapes (Thompson et al. 2019) but neither GYSVd-2 nor AGVd were identified in the same high-throughput sequencing (HTS) outputs. In September 2020, 16 leaf and petiole samples were collected from six vineyards in Canyon and Nez Perce counties of Idaho, representing six different wine grape cultivars and an unknown table grape cultivar, and subjected to HTS analysis. One of the samples was from a table grape plant at the edge of a declining 'Chardonnay' wine grape block that was grown next to a wine tasting room deck for aesthetic, ornamental purposes; the table grape and 'Chardonnay' plants were own-rooted and planted in 1981. Ribodepleted total RNAs prepared from these samples, as described previously, were subjected to a HTS analysis on a NovaSeq platform (Dahan et al. 2023), producing 15,095,042 to 31,500,611 250-bp paired-end reads per sample. Raw reads were adapter and quality cleaned and mapped against the V. vinifera, reference genome. Unmapped paired-end reads were assembled, and contigs were analyzed using BLASTn and DIAMOND (Buchfink et al. 2021) programs. Fifteen samples were found infected with HSVd and with GYSVd-1, while one was infected with GYSVd-2 and AGVd; in particular, the table grape plant (arbitrarily designated RBTG) was found infected with all four viroid species. The HTS-derived, 490-nt GYSVd-2-specific contig from the table grape sample represented ∼1.35 genome of the Idaho isolate of GYSVd-2 (GYSVd-2-RBTG) and was 100% identical to the GYSVd-2 sequence JQ686716 from Iran. The HTS-derived, 488-nt AGVd-specific contig represented ∼1.32 genome of the Idaho isolate of AGVd (AGVd-RBTG) and was 100% identical to the AGVd sequence KF876037 from Iran. To validate the HTS data and confirm the presence of the four viroids in the original 16 samples, all of them were subjected to RT-PCR using the viroid-specific primers described by Gambino et al. (2014); all 16 samples were found positive for HSVd and GYSVd-1, and one found positive for AGVd. The RBTG sample was confirmed to be infected with HSVd, GYSVd-1, and AGVd by RT-PCR. GYSVd-2 sequence was not amplified, although primers designed by Gambino et al. (2014) matched the HTS-derived GYSVd-2-RBTG sequence; this may be related to a lower concentration of this viroid in the sample and to properties of the primers. The sampled table grape plant was asymptomatic; all four viroids were apparently not associated with any visible abnormalities in this table grape plant, consistent with the findings that viroids found in grapevines typically do not seem to be associated with visible diseases (Habili 2017).

First report of strawberry polerovirus 1 in strawberry in Italy.

Strawberry (Fragaria × ananassa Duch.) was introduced in Sicily (Italy) in the 1930s in the small town of Maletto, on the slopes of Etna volcan, where it's currently cultivated in a total area of 30 ha. The French cv. 'Madame Moutot', appreciated for its unique flavor and intense fragrance, was there propagated vegetatively and after decades, the distinctive 'Etna ecotype' originated by adaptation to the peculiar environmental conditions of the area (Milella et al., 2006). In May 2023, in a 0.5 ha "Etna ecotype" strawberry field, virus-like symptoms were observed in approximately 50% of the plants. Symptoms included severe dwarfing, leaf cupping and chlorotic spotting which lead to decline of infected plants. To investigate the etiology of the disease, leaf samples were collected from eight symptomatic plants for analysis by High-Throughput Sequencing (HTS). To this aim, total RNAs were extracted by using the RNeasy PowerPlant Kit (Qiagen, Germany). The RNAs were pooled, depleted of ribosomal RNA (QIAseq FastSelect; Qiagen), and a library was prepared according to the Illumina DNA Prep Kit. Sequencing on a NextSeq2000 instrument at Leibniz Institute DSMZ (Braunschweig, Germany) generated 31,149,784 of paired-end reads (150 nt), which were further analyzed in Geneious Prime version 2023.2 (Biomatters) using a custom workflow for virus discovery and genome assembly. Analysis of the assembled contigs by local BLASTn and BLASTp alignments against a custom plant virus database of NCBI nuclear-core (NC) reference sequences assigned a number of contigs to accession NC_025435, strawberry polerovirus 1 (SPV-1). Reconstruction of the virus genome by assembly of contigs and reads alignment resulted in a nearly complete genome sequence of SPV-1 (GenBank Acc. No. OR989958) showing by BLASTn 98.69% identity to the SPV-1 NC reference sequence, and 98.99 % identity with an isolate from the Czech Republic (GenBank Acc. OL421571). To confirm the presence of SPV-1 in each sample, RT-PCR using specific primers designed in this study SPV-1-CP-1F (5'-TCGAGATACGTCTAGAACTGCAA-3') and SPV-1-CP-1R (5'-GAGAGGCCCCTTCTACCTATTTG-3') targeting the entire 623 bp coat protein (CP) gene was performed. Amplicons of the expected size were obtained in five samples and Sanger-sequenced. The resulting sequences shared 99.85% - 100% of identity to the HTS - derived sequence (GenBank Acc. No. OR989958) through BLASTn analysis. Strawberry mottle virus (SMoV), strawberry mild yellow edge virus (SMYEV) and strawberry crinkle virus (SCV) were detected in the same library in addition to SPV-1 and then confirmed by RT-PCR using specific primers (Martin & Tzanetakis 2013). Strawberry polerovirus 1, related to the genus Polerovirus in the family Solemoviridae, was first reported in strawberries in Canada (Xiang et al. 2015) and was thereafter detected in the United States (Thekke-Veetil & Tzanetakis 2016), Argentina (Luciani et al. 2016), and Nepal (Kuwak et al. 2022). To date, the virus has been reported in Europe only in the Czech Republic (Franova et al. 2021). To our knowledge, this is the first report of SPV-1 in strawberry plants in Italy. Although the correlation between SPV-1 and strawberry decline (SD) is still uncertain (Xiang et al. 2015) transmission of the virus via aphids has recently been demonstrated (Franova et al. 2021). Our report let to hypothesize that its dissemination in Europe can be considered as increasing.

RNA isolation by two different methods in analysing adipose-biomarkers of mouse adipose tissues

The RNeasy Mini Kit and the TRI Reagent protocol are commonly employed techniques for extracting total RNA and are widely utilised in research pertaining to the expression of adipose biomarkers. This study was undertaken to establish and determine an appropriate method for isolating RNA from frozen adipose tissue, with the objective of analysing adipose biomarkers. Total RNA was extracted from frozen white adipose tissues of mice (-80oC) using both the RNeasy Mini Kit and the TRI Reagent protocol. The concentration and purity of the RNA were assessed using NanoDrop. One-step RT-qPCR was employed to quantify the mRNA expression levels of three adipose biomarkers: MEST, SFRP5, and SCD1. The results of this investigation indicated that the RNeasy Mini Kit yielded a higher RNA concentration when compared to the TRI Reagent method. Furthermore, the RNeasy Mini Kit provided RNA samples with superior purity, as evidenced by a higher 260/230 ratio. Although RNA extracted using the TRI Reagent exhibited slightly elevated mRNA expression levels for the three biomarkers, these differences did not reach statistical significance. In summary, both methods are suitable for total RNA extraction, but the RNeasy Mini Kit is recommended for obtaining higher RNA concentration and purity. The choice of method should be contingent upon the intended downstream application of the RNA.

Quantification of tRNA m1A modification by templated-ligation qPCR.

N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. M1A is generally located in the T loop of cytosolic tRNA and between acceptor and D stems of mitochondrial tRNAs, it is involved in tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template followed by qPCR using the ligation product as the template. M1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using sub-nanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

Evaluation of the Effects of Opium on the Expression of SOX2 and OCT4 in Wistar Rat Bladder

BACKGROUND: Bladder cancer is a malignancy greatly affected by behavioral habits. The aim of this study was to examine the effect of opium on changes in the expression of OCT4 and SOX2 in the bladder tissue of rats. METHOD: Thirty six rats were divided into six groups: 24 rats in the addicted group received morphine and opium for 4 months with 12 rats in the control group. Blood testing was done for the evaluation of CBC, MDA, and TAC. The bladder tissue was removed and checked by histopathological examination. All total RNA was extracted, then cDNAs were synthesized and the OCT4 and SOX2 gene expressions were evaluated by Real-time PCR. RESULTS: The OCT4 mRNA expression level in the opium group of rats was significantly increased compared to the control group (13.5 and 6.8 fold in males and females respectively). Also, in the morphine group, similar augmentation was detected (3.8 and 6.7 fold in males and females respectively). The SOX2 mRNA over-expression level was seen in the morphine group of both genders as compared to the control group (3.7 and 4.2 fold in male and female respectively) but in the opium group, enhancement of mRNA level was seen only in males (6.6 fold). Opium increases both OCT4 and SOX2 expression more than morphine in male rats, but in female rats, SOX2 is increased more by morphine. CONCLUSION: Over expression of OCT4 and SOX2 was observed in rats treated with opium and morphine. Increased OCT4 and SOX2 expression was seen in opium-treated male rats, but in female rats, SOX2 was increased more by morphine.

The RNA sequencing results revealed the expression of different genes and signaling pathways during chemotherapy resistance in peripheral T-cell lymphoma

BackgroundPeripheral T-cell lymphoma (PTCL) is a subtype of non-Hodgkin's lymphoma that occurs primarily at extranodal sites and is commonly treated using chemotherapy and radiotherapy. PTCL is more malignant than other lymphoid tumors, resulting in a poor prognosis.The 5-year recurrence rate remains high, and there is a lack of standard treatment for patients with relapse-resistant disease. However, the molecular mechanisms underlying the resistance of peripheral T-cell lymphoma cells to chemotherapeutic drugs, as well as identifying strategies to overcome drug resistance remains unclear. In this study, we aimed to identify pivotal genes and signaling pathways associated with chemotherapy resistance in PTCL.MethodsIn this study, a total of 5 healthy controls and 7 clinical patients were enrolled; 4 patients were classified as chemotherapy sensitive, and 3 patients were classified as chemotherapy resistant. Peripheral blood samples were collected from each participant, and total RNA was extracted from the white blood cells. RNA sequencing was conducted on the Illumina HiSeq platform to obtain comprehensive gene expression profiles. Subsequently, the expression patterns of the DEGs associated with the most enriched signaling pathways, with a special focus on cancer-related genes, were validated using quantitative real-time polymerase chain reaction (qRT–PCR) in peripheral TCL patients.ResultsRNA sequencing (RNA-seq) analysis revealed 4063 differentially expressed genes (DEGs) in peripheral T-cell lymphoma specimens from patients with chemotherapy resistance, of which 1128 were upregulated and 2935 were downregulated. Subsequent quantitative gene expression analysis confirmed a differential expression pattern in all the libraries, with 9 downregulated genes and 10 upregulated genes validated through quantitative real-time PCR in 6 clinical specimens from patients with chemotherapy resistance. KEGG pathway analysis revealed significant alterations in several pathways, with 6 downregulated pathways and 9 upregulated pathways enriched in the DEGs. Notably, the TNF signaling pathway, which is extensively regulated, was among the pathways that exhibited significant changes. These findings suggest that DEGs and the TNF signaling pathway may play crucial roles in chemotherapy resistance in peripheral T-cell lymphoma.ConclusionOur study revealed that the expression of specific genes, including TNFRSF1B, TRADD2, and MAP3K7, may play an important role in chemotherapy resistance in peripheral T-cell lymphoma. Moreover, we identified the downregulation of the TNF signaling pathway, a crucial pathway involved in cell survival, death, and differentiation, as a potential contributor to the development of chemotherapy resistance in peripheral T-cell lymphoma. These findings provide valuable insights into the molecular mechanisms underlying chemotherapy resistance and highlight potential targets for overcoming treatment resistance in this challenging disease.

Changes in the expression of genes encoding xanthophyl acyltransferases during the postharvest ripening of avocado (Persea americana) fruit.

Avocado fruit is rich in xanthophylls, which have been related to positive effects on human health. Xanthophyl acetyltransferases (XATs) are enzymes catalyzing the esterification of carboxylic acids to the hydroxyl group of the xanthophyll molecule. This esterification is thought to increase the lipophilic nature of the xanthophyll and its stability in a lipophilic environment. Studies on XATs in fruits are very scarce, and no studies had been carried out in avocado fruit during postharvest. The objective of this work was to investigate the changes in the expression of genes encoding XAT, during avocado fruit ripening. Avocado fruits were obtained from a local market and stored at 15 °C for 8 days. The fruit respiration rate, ethylene production, and fruit peel's color space parameters (L*, a*, b*) were measured during storage. Fruit mesocarp samples were taken after 1, 3, 5, and 7 days of storage and frozen with liquid nitrogen. Total RNA was extracted from fruit mesocarp, and the quantification of the two genes designated as COGE_ID: 936743791 and COGE_ID: 936800185 encoding XATs was performed with real-time quantitative reverse transcription polymerase chain reaction using actin as a reference gene. The presence of a climacteric peak and large changes in color were recorded during postharvest. The two genes studied showed a large expression after 3 days of fruit storage. We conclude that during the last stages of ripening in avocado fruit there was an active esterification of xanthophylls with carboxylic acids, which suggests the presence of esterified xanthophylls in the fruit mesocarp. © 2024 Society of Chemical Industry.

Glucose ingestion before and after resistance training sessions does not augment ribosome biogenesis in healthy moderately trained young adults.

Resistance training-induced skeletal muscle hypertrophy seems to depend on ribosome biogenesis and content. High glucose treatment may augment ribosome biogenesisthroughpotentiatingresistance training-induced adaptations. This was investigated with total RNA and ribosomal RNA abundances as main outcomes, with relevant transcriptional/translational regulators (c-Myc/UBF/rpS6) as a secondary outcome. Sixteen healthy, moderately trained individuals [male/female, n = 9/7; age, 24.1 (3.3)] participated in a within-participant crossover trial with unilateral resistance training (leg press and knee extension, 3 sets of 10 repetitions maximum) and pre- and post-exercise ingestion of either glucose (3 × 30g, 90g total) or placebo supplements (Stevia rebaudiana, 3 × 0.3g, 0.9g total), together with protein (2 × 25g, 50g total), on alternating days for 12days. Six morning resistance exercise sessions were conducted per condition, and the sessions were performed in an otherwise fasted state. Micro-biopsies were sampled from m. vastus lateralis before and after the intervention. Glucose ingestion did not have beneficial effects on resistance training-induced increases of ribosomal content (mean difference 7.6% [-7.2, 24.9], p = 0.34; ribosomal RNA, 47S/18S/28S/5.8S/5S, range 7.6-37.9%, p = 0.40-0.98) or levels of relevant transcriptional or translational regulators (c-MYK/UBF/rpS6, p = 0.094-0.292). Of note, both baseline and trained state data of total RNA showed a linear relationship with UBF; a ∼14% increase in total RNA corresponded to 1 SD unit increase in UBF (p = 0.003). Glucose ingestion before and after resistance training sessions did not augment ribosomal RNA accumulation during twelve days of heavy-load resistance training in moderately trained young adults.

Obesity affects brain cortex gene expression in an APOE genotype and sex dependent manner.

Obesity is the top modifiable risk factor for Alzheimer's disease. We hypothesized that high fat diet (HFD)-induced obesity alters brain transcriptomics in APOE-genotype and sex dependent manners. Here, we investigated interactions between HFD, APOE, and sex, using a knock-in mouse model of the human APOE3 and APOE4 alleles. Six-month-old APOE3-TR and APOE4-TR mice were treated with either HFD or control chow. After 4 months, total RNA was extracted from the cerebral cortices and analyzed by poly-A enriched RNA sequencing on the Illumina platform. Female mice demonstrated profound HFD-induced transcriptomic changes while there was little to no effect in males. In females, APOE3 brains demonstrated about five times more HFD-induced transcriptomic changes (399 up-regulated and 107 down-regulated genes) compared to APOE4 brains (30 up-regulated and 60 down-regulated). Unsupervised clustering analysis revealed two gene sets that responded to HFD in APOE3 mice but not in APOE4 mice. Pathway analysis demonstrated that HFD in APOE3 mice affected cortical pathways related to feeding behavior, blood circulation, circadian rhythms, extracellular matrix, and cell adhesion. Female mice and APOE3 mice have the strongest cortical transcriptomic responses to HFD related to feeding behavior and extracellular matrix remodeling. The relative lack of response of the APOE4 brain to stress associated with obesity may leave it more susceptible to additional stresses that occur with aging and in AD.

The interrelation between the high expression level of MIR34a and the trisomic abortion materials.

The underlying mechanism and constitution of spontaneous abortions are complicated and heterogeneous. Many factors, including epigenetic scenarios like micro-ribonucleic acids (miRNAs, MIRs), can additively affect the progression of pregnancy losses. This study aimed to evaluate whether the expression levels of placental inhibitor and/or activator miRNAs had a difference between the numerically abnormal and normal karyotyped spontaneous abortions. The case-control study included 100 spontaneous abortion materials consisting of trophoblastic tissues with 42 disomies (controls), 43 aneuploidies (including trisomy 16, 21, 22, and monosomy X), and 15 triploidies. Disomic abortion materials with XX normal karyotypes were omitted from the study to exclude possible maternal decidual cell contamination. Total RNA isolation was performed with TRIzol™ reagent directly from frozen trophoblastic tissues, and the mature miRNAs were obtained by reverse transcription via quantitative real-time polymerase chain reaction (qRT-PCR). Then, the expression levels of placental activators MIR378a-5p, MIR376c, MIR195, and inhibitors MIR34a and MIR210 were relatively evaluated using MIR130 as a reference. The expression level of placental inhibitor MIR34a was detected to be high in trisomic abortion materials (trisomy 16 and 21) when compared to the disomic ones (p = 0.0324). MIR195 (p = 0.0484) and MIR34a (p = 0.0346) expression levels were increased in numerically abnormal cases with advanced maternal age compared to the disomic ones within all maternal ages. It seems likely that the high expression level of MIR34a and the coexistence of trisomic abortion materials are quite interrelated with the additive effect of advanced maternal age.

A Biotinylated cpFIT-PNA Platform for the Facile Detection of Drug Resistance to Artemisinin in Plasmodium falciparum.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

Bioinformatics analysis of the microRNA genes associated with type 2 cardiorenal syndrome

BackgroundMicroRNAs (miRNAs) are important regulatory factors in the normal developmental stages of the heart and kidney. However, it is currently unclear how miRNA is expressed in type 2 cardiorenal syndrome (CRS). This study aimed to detect the differential expression of miRNAs and to clarify the main enrichment pathways of differentially expressed miRNA target genes in type 2 CRS.MethodsFive cases of healthy control (Group 1), eight of chronic heart failure (CHF, Group 2) and seven of type 2 CRS (Group 3) were enrolled, respectively. Total RNA was extracted from the peripheral blood of each group. To predict the miRNA target genes and biological signalling pathways closely related to type 2 CRS, the Agilent miRNA microarray platform was used for miRNA profiling and bioinformatics analysis of the isolated total RNA samples.ResultsAfter the microarray analysis was done to screen for differentially expressed circulating miRNAs among the three different groups of samples, the target genes and bioinformatic pathways of the differential miRNAs were predicted. A total of 38 differential miRNAs (15 up- and 23 down-regulated) were found in Group 3 compared with Group 1, and a total of 42 differential miRNAs (11 up- and 31 down-regulated) were found in Group 3 compared to Group 2. According to the Gene Ontology (GO) function and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis, the top 10 lists of molecular functions, cellular composition and biological processes, and the top 30 signalling pathways of predicted gene targets of the differentially expressed miRNAs were discriminated among the three groups.ConclusionBetween the patients with CHF and type 2 CRS, miRNAs were differentially expressed. Prediction of target genes of differentially expressed miRNAs and the use of GO function and KEGG pathway analysis may reveal the molecular mechanisms of CRS. Circulating miRNAs may contribute to the diagnosis of CRS, and further and larger studies are needed to enhance the robustness of our findings.

Blubber biopsy in common bottlenose dolphins using a novel biopsy puncher: Evaluation of the impact on living individuals and possibility of applications in cetacean research.

Biopsy has recently become a preferred protocol for sampling the skin and blubber of many cetacean species, although it is desirable to collect as minimally invasive as possible. Here, the effect of biopsy sampling on the captive common bottlenose dolphins was evaluated by analyzing the process of wound healing and changes in hematological and blood biochemical parameters after biopsy using a puncher developed to collect up to the inner layer of the dolphin blubber. Results showed that the wounds caused by biopsy were closed in as early as 1 day and completely covered with the epidermis within 5-11 days. Blood fibrinogen, which generally increases due to a wound-induced inflammatory response or activation of the coagulation system, was significantly elevated after the biopsy indicating ongoing tissue repair, while other parameters did not exhibit significant differences. Furthermore, histological observation and RNA extraction of samples were performed to investigate the versatility of this method to cetacean research. Histological examination revealed three distinct layers of the blubber in the biopsy samples. Moreover, total RNA extracted from biopsy samples exhibited sufficient quality and quantity for gene expression analyses. Overall, the puncher utilized in our study represents a valuable and minimally invasive tool for investigating various aspects of small cetacean studies.

121 Unraveling the biology of bovine conceptus elongation

Abstract The establishment of pregnancy is a well-regulated process that involves the development of a viable blastocyst, conceptus elongation, maternal recognition of pregnancy, implantation, and the onset of placentation. Pregnancy loss is a major factor limiting reproductive efficiency in livestock species. In cattle, most embryonic losses occur during the first month of gestation. The genetic and epigenetic regulation of embryonic and placental development in ruminants is still poorly understood. To investigate the transcriptome profile of the bovine conceptus during elongation, Angus heifers (n = 8) were superovulated and bred using semen from a proven high-fertility Bos indicus sire. Heifers were flushed on d 14 of gestation using a standard nonsurgical flushing technique. Recovered conceptuses were individually photographed and snap-frozen for total RNA extraction and bulk mRNA sequencing. Recovered conceptuses were classified based on size into ovoid (0.2 – 5mm; n = 6), tubular (&amp;lt;15 mm; n = 7), or filamentous (&amp;gt;16 mm; n = 7). Differentially expressed genes (DEG: FDR &amp;lt; 0.05) were determined using DESeq2 analysis and were functionally annotated using DAVID. The most pronounced transcriptomic changes were identified between the ovoid and tubular stages, with 5,937 DEGs (2,913 upregulated and 3,024 downregulated) and affected pathways were primarily involved in ribosome assembly and rRNA processing. There were 3,966 DEGs (2,069 upregulated and 1,897 downregulated) between the ovoid and filamentous stages, and the most significant pathways were involved in protein translation, embryonic development, and cell cycle. The transition between tubular to filamentous presented 1,435 DEGs (775 upregulated and 660 downregulated) and significant pathways were involved in protein degradation, cell division, cell cycle, protein stabilization, and mRNA splicing. Our findings indicate that the greatest alterations in the transcriptome occur precisely at the initiation of elongation, suggesting that the bovine conceptus undergoes major structural changes at the outset of this process. This major shift in gene expression is presumed to be necessary to support the subsequent exponential growth phase of elongation, required for maternal recognition of pregnancy.