Total RNA Extraction Research Articles

Cloning Sheep Cytokin Genes and Preliminary Transfect to HT29 Cell Line

The study was carried out to clone sheep cytokine genes for protein expression in mammalian cell culture. Once expressed it is hypothesized that these proteins when added to a primary mast cell culture will allow the growth of these cells. Mast cells are used by sheep to combat gastro-intestinal nematode (GIN) by helping to establish the host immune response and to eliminate of GIN. Kit ligand and IL3 proteins are growth factors of mast cells that were derived from extraction of total RNA from lung and thymus tissues, respectively. The IL3 and kit ligand genes were cloned into bacterial expression pGEM – T easy vector to determine correct sequences before being sub-cloned into the mammalian expression pWPI vector. The Pac 1 enzyme was used to cut restriction sites of pGEM – T easy vector, pWPI vector, IL3 and kit ligand genes in cloning strategies. The study also established 24 different conditions for transient transfection IL3 and Kit ligand genes into HT29 cells and screen positive cells on the flow cytometry. Results showed that clones of IL3 and kit ligand with the correct sequences were created for using transient transfection. The transfection efficiency of IL3 and kit ligand DNA/ pWPI plasmid into HT29 cells were extremely low with approximately 9% for the best condition. This low transfection efficiency leads to shortage of IL3 and kit ligand for growing mast cells. Therefore, stable transfection will be required to have highest efficiencies that produce the large quantities of IL3 and kit ligand proteins required for culture of sheep mast cell lines.

Treatment with Raloxifene Induces the Expression of Kisspeptin, Insulin, and Androgen Receptors in Bones of Castrated Adult Female Rats.

Objective  To evaluate the effects of estrogen, raloxifene and genistein on the expression of KISS1 (kisspeptin), KISS1R (kisspeptin receptor), AR (androgen receptor) and INSR (insulin receptor) in the bones of ovariectomized rats. Methods  Forty-eight adult rats were randomly divided into 6 groups, containing 8 animals each: G1-nonovariectomized control; G2-ovariectomized and treated with conjugated equine estrogens (50 µg/Kg/day); G3-ovariectomized and treated with raloxifene (0.75 mg/kg/day); G4-ovariectomized animal that received soy extract with genistein (300 mg/kg/day); G5-ovariectomized animal that received estrogen and genistein; and G6-ovariectomized animal that received estrogen and raloxifene. Three months after surgery, the castrated animals received the drugs orally daily for 120 days. All animals were sacrificed after this period, by deepening the anesthesia. The left tibia was removed for total RNA extraction and analysis of gene expression of KISS1 , KISS1R , AR and INSR , by quantitative real-time polymerase chain reaction (qRT-PCR). Results   KISS1 was not detected in any of the treated groups. KISS1R , INSR and AR showed higher expression in the G3 group ( p  < 0.001), while lower levels of transcripts for these genes were observed in G4 and G5. G2 animals showed hypoexpression of the evaluated genes. Conclusion  The results indicate that raloxifene, alone or combined with estrogen, was able to induce the expression of genes associated with the recovery of bone tissue homeostasis in ovariectomized rats.

Exploring the miRNAs Profile in Dark-Cutting Beef.

In an animal production system, different stressors may cause the depletion of muscle glycogen stores, resulting in an elevated pH at 24 h post mortem (pH24), which leads to cell metabolism alterations that affect the conversion of muscle into meat, causing meat quality defects, such as dark-cutting beef, also known as dark, firm, and dry (DFD) beef. This process may involve the alteration of small non-coding RNAs (miRNAs), which play critical regulatory roles in cellular processes. Here, we determined whether differential miRNA expression in the Longissimus thoracis et lumborum muscle from the Asturiana de los Valles breed at 24 h post mortem could serve as an early indicator of beef quality defects. Following total RNA extraction, complete miRNAome sequencing revealed 12 miRNAs that were significantly upregulated (p < 0.001) in DFD beef compared to the levels in CONTROL beef. These miRNAs are mainly involved in the cellular responses to redox imbalances and apoptosis. Among these, four miRNAs known to be related to oxidative stress (bta-miR-1246, bta-miR-2332, bta-miR-23b-5p, and bta-miR-2411-3p) were validated via quantitative RT-PCR. Some of their target proteins were also analyzed using Western blotting. High 70 kDa heat shock protein and low Caspase-9 expressions (p < 0.01) were found in DFD beef, suggesting the downregulation of apoptosis. These results suggest the importance of miRNAs in regulating stress in muscle cells during early post mortem, as differences in the abundance of some of these miRNAs are still observed at 24 h post mortem. These changes lead to an inadequate conversion of muscle into meat, resulting in meats with quality defects.

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.

Characterization of rumen microbiome and immune genes expression of crossbred beef steers with divergent residual feed intake phenotypes

We investigated whole blood and hepatic mRNA expressions of immune genes and rumen microbiome of crossbred beef steers with divergent residual feed intake phenotype to identify relevant biological processes underpinning feed efficiency in beef cattle. Low-RFI beef steers (n = 20; RFI = − 1.83 kg/d) and high-RFI beef steers (n = 20; RFI = + 2.12 kg/d) were identified from a group of 108 growing crossbred beef steers (average BW = 282 ± 30.4 kg) fed a high-forage total mixed ration after a 70-d performance testing period. At the end of the 70-d testing period, liver biopsies and blood samples were collected for total RNA extraction and cDNA synthesis. Rumen fluid samples were also collected for analysis of the rumen microbial community. The mRNA expression of 84 genes related to innate and adaptive immunity was analyzed using pathway-focused PCR-based arrays. Differentially expressed genes were determined using P-value ≤ 0.05 and fold change (FC) ≥ 1.5 (in whole blood) or ≥ 2.0 (in the liver). Gene ontology analysis of the differentially expressed genes revealed that pathways related to pattern recognition receptor activity, positive regulation of phagocytosis, positive regulation of vitamin metabolic process, vascular endothelial growth factor production, positive regulation of epithelial tube formation and T-helper cell differentiation were significantly enriched (FDR < 0.05) in low-RFI steers. In the rumen, the relative abundance of PeH15, Arthrobacter, Moryella, Weissella, and Muribaculaceae was enriched in low-RFI steers, while Methanobrevibacter, Bacteroidales_BS11_gut_group, Bacteroides and Clostridium_sensu_stricto_1 were reduced. In conclusion, our study found that low-RFI beef steers exhibit increased mRNA expression of genes related to immune cell functions in whole blood and liver tissues, specifically those involved in pathogen recognition and phagocytosis regulation. Additionally, these low-RFI steers showed differences in the relative abundance of some microbial taxa which may partially account for their improved feed efficiency compared to high-RFI steers.

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.

Ctp1 and Yhm2: Two Mitochondrial Citrate Transporters to Support Metabolic Flexibility of Saccharomyces cerevisiae.

Differently from higher eukaryotic cells, in the yeast Saccharomyces cerevisiae there are two mitochondrial carrier proteins involved in the transport of citrate: Ctp1 and Yhm2. Very little is known about the physiological role of these proteins. Wild-type and mutant yeast strains deleted in CTP1 and YHM2 were grown in media supplemented with a fermentable (glucose) or a nonfermentable (ethanol) carbon source. To assess changes in Ctp1 and Yhm2 mRNA expression levels, real-time PCR was performed after total RNA extraction. In the wild-type strain, the metabolic switch from the exponential to the stationary phase is associated with an increase in the expression level of the two citrate transporters. In addition, the results obtained in the mutant strains suggest that the presence of a single citrate transporter can partially compensate for the absence of the other. Ctp1 and Yhm2 differently contribute to fermentative and respiratory metabolism. Moreover, the two mitochondrial carriers represent a link between the Krebs cycle and the glyoxylate cycle, which play a key role in the metabolic adaptation strategies of S. cerevisiae.

Effectiveness of cannabidiol (CBD) on histopathological changes and gene expression in hepatocellular carcinoma (HCC) model in male rats: the role of Hedgehog (Hh) signaling pathway.

The third most prevalent malignancy to cause mortality is hepatocellular carcinoma (HCC). The Hedgehog (Hh) signaling pathway is activated by binding to the transmembrane receptor Patched-1 (PTCH-1), which depresses the transmembrane G protein-coupled receptor Smoothened (SMO). This study was performed to examine the preventative and therapeutic effects of cannabidiol in adult rats exposed to diethyl nitrosamine (DENA)-induced HCC.A total of 50 male rats were divided into five groups of 10 rats each. Group I was the control group. Group II received intraperitoneal (IP) injections of DENA for 14 weeks. Group III included rats that received cannabidiol (CBD) orally (3-30mg/kg) for 2weeks and DENA injections for 14 weeks. Group IV rats received oral CBD for 2weeks before 14weeks of DENA injections. Group V included rats that received CBD orally for 2weeks after their last injection of DENA. Measurements were made for alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and alpha fetoprotein (AFP). Following total RNA extraction, Smo, Hhip, Ptch-1, and Gli-1 expressions were measured using quantitative real-time polymerase chain reaction (qRT-PCR). A histopathological analysis of liver tissues was performed.The liver enzymes, oxidant-antioxidant state, morphological, and molecular parameters of the adult male rat model of DENA-induced HCC showed a beneficial improvement after CBDadministration. In conclusion, by focusing on the Hh signaling system, administration of CBD showed a beneficial improvement in the liver enzymes, oxidant-antioxidant status, morphological, and molecular parameters in the DENA-induced HCC in adult male rats.

First Report of Lily Virus X Infecting Paris polyphylla var. yunnanensis in Yunnan, China

Lily virus X (LVX) is a positive-sense ssRNA virus belonging to the genus Potexvirus in the family Alphaflexiviridae. LVX is known to infect plants of the genera Lilium and Tricyrtis in the family Liliacea. LVX was first reported in an asymptomatic lily (Lilium formosanum) from England (Stone, 1980), but has been shown to infect plants in the Netherlands (Chen et al. 2005), the United States (Jordan et al. 2008) and Japan (Nijo et al. 2018). To date, the complete genomes of two LVX isolates from the Netherlands and Japan have been reported. Paris polyphylla var. yunnanensis, known as Dianchonglou in China, is a perennial plant of the family Melanthiaceae (formerly belonging to the family Trillium). In China, its rhizome is commonly used as an antispasmodic agent for stroke and cancer treatment (Chang et al. 2017). From 2019 to 2022, leaf mottle and shrinkage which are typical symptoms of viral infections were observed on the leaves of P. polyphylla var. yunnanensis plants in Dianchonglou fields in Qujing, Yunnan. Disease incidence ranged from 19% to 45% across 5 fields (90 plants per field) in Qujing. To identify the possible viral pathogen(s) associated with the disease, the mirVanaTM miRNA isolation Kit was used to extract total RNA was from a mixed sample pool of 5 symptomatic leaf samples collected from the 5 fields. RNA sequencing library was constructed using TruSeqTM RNA sample preparation kit. Sequencing on the Illumina HiSeqTM 2500 platform (Illumina, USA) with 125-bp paired-end reads yielded 23,077,786 raw reads. 22,534,100 clean reads were obtained by removing reads of low quality and poly-N using Trimmomatic software (Bolger et al. 2014). By utilizing the paired-end splicing method in Trinity software (Grabherr et al. 2011) the the raw reads were De novo assembled into 184,596 contigs, of which 303 were related to viruses, including Paris mosaic necrosis virus (PMNV), Pear alphapartitivirus (PAPV), Dahlia mosaic virus (DMV), and Lily virus X (LVX). BLASTn analysis revealed that 12 contigs (lengths ranging from 344 nt to 5,981 nt, query cover 6% to 99%) were most similar (57.32% to 91.67% nt identities) to the genome sequences of LVX, suggesting a possible infection of LVX in the plants. To confirm the result, a full-length genomic sequence of LVX was obtained by reverse transcription polymerase chain reaction (RT-PCR) using specific primers designed based on the sequence of the assembled contigs. The PCR products were cloned into pGEM-T vector (Promega Corporation, USA) and sequenced using the Sanger method (Sangon Biotech, Shanghai, China). The obtained full-length genomic sequence of the LVX isolate (LVX-PP, accession number OM100017) was 5,981 nt in length. BLASTp analysis demonstrated that the putative Rep and CP of LVX-PP shared 76.27% to 81.05% and 80.81% to 81.82% aa sequence similarities with that of other LVX isolates, respectively. Maximum-likelihood phylogenetic trees inferred from the Rep and CP aa sequences showed that LVX-PP clustered closely with LVX isolates. The leaf samples were further analyzed using a lily virus X (LVX) ELISA kit (DEIAPV181, Creative Diagnostics, U.S.A.). Healthy P. polyphylla var. yunnanensis leaves were taken as a negative control and buffer solution as a blank control. The results showed a positive reaction for all five symptomatic plants (OD = 1.259 ± 0.007) relative to the negative (OD = 0.099) and blank (OD = 0.073) controls. These results indicate that LVX can infect P. polyphylla var. yunnanensis. To our knowledge, this is the first report that LVX has been detected in P. polyphylla var. yunnannensis. This study will serve as an important reference for the study of the host range of LVX. Further studies will be required to determine how LVX spreads between P. polyphylla var. yunnannensis and other host plants.

Differential miRNA Profiling Reveals miR-4433a-5p as a Key Regulator of Chronic Obstructive Pulmonary Disease Progression via PIK3R2- mediated Phenotypic Modulation.

In this study, a high-throughput sequencing technology was used to screen the differentially expressed miRNA in the patients with "fast" and "slow" progression of chronic obstructive pulmonary disease (COPD). Moreover, the possible mechanism affecting the progression of COPD was preliminarily analyzed based on the target genes of candidate miRNAs. The "fast" progressive COPD group included 6 cases, "slow" and Normal progressive COPD groups included 5 cases each, and COPD group included 3 cases. The peripheral blood samples were taken from the participants, followed by total RNA extraction and high throughput miRNA sequencing. The differentially expressed miRNAs among the progressive COPD groups were identified using bioinformatics analysis. Then, the candidate miRNAs were externally verified. In addition, the target gene of this miRNA was identified, and its effects on cell activity, cell cycle, apoptosis, and other biological phenotypes of COPD were analyzed. Compared to the Normal group, a total of 35, 16, and 7 differentially expressed miRNAs were identified in the "fast" progressive COPD, "slow" progressive COPD group, and COPD group, respectively. The results were further confirmed using dual-luciferase reporter assay and transfection tests with phosphoinositide- 3-kinase, regulatory subunit 2 (PIK3R2) as a target gene of miR-4433a-5p; the result showed a negative regulatory correlation between the miRNA and its target gene. The phenotype detection showed that the activation of the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway might participate in the progression of COPD by promoting the proliferation of inflammatory A549 cells and inhibiting cellular apoptosis. MiR-4433a-5p can be used as a marker and potential therapeutic target for the progression of COPD. As a target gene of miR-4433a-5p, PIK3R2 can affect the progression of COPD by regulating phenotypes, such as cellular proliferation and apoptosis.

Exploring the molecular biomarker utility of circCCT3 in multiple myeloma: A favorable prognostic indicator, particularly for R-ISS II patients.

Circular RNAs (circRNAs) are associated with the pathobiology of multiple myeloma (MM). Recent findings regarding circCCT3 support its involvement in the development and progression of MM, through microRNA sponging. Thus, we aimed to examine the expression of circCCT3 in smoldering and symptomatic MM and to assess its clinical importance. Three cell lines from plasma cell neoplasms were cultured and bone marrow aspirate (BMA) samples were collected from 145 patients with MM or smoldering MM. Next, CD138+ enrichment was performed in BMA samples, followed by total RNA extraction and reverse transcription. Preamplification of circCCT3 and GAPDH cDNA was performed. Finally, a sensitive assay for the relative quantification of circCCT3 using nested real-time quantitative polymerase chain reaction was developed, optimized, and implemented in the patients' samples and cell lines. MM patients exhibited significantly higher intracellular circCCT3 expression in their CD138+ plasma cells, compared to those from SMM patients. In addition, MM patients overexpressing circCCT3 had longer progression-free and overall survival intervals. The favorable prognostic significance of high circCCT3 expression in MM was independent of disease stage (either International Staging System [ISS] or revised ISS [R-ISS]) and age of MM patients. Interestingly, circCCT3 expression could serve as a surrogate molecular biomarker of prognosis in MM patients, especially those of R-ISS stage II. In conclusion, our study sheds new light on the significance of circCCT3 as a promising molecular marker for predicting MM patients' prognosis.

First report of bean common mosaic virus naturally infecting rice bean (Vigna umbellata) in China.

Rice bean [Vigna umbellata (Thunb.) Ohwi et Ohashi] is a traditional grain legume in Asia. From 2020 and 2021, approximately 15% plants exhibiting virus-like symptoms of mosaic, crumpling, and stunting were observed in two rice bean fields in Changping district, Beijing, China. To unveil the viral agent(s) leading to the disease symptoms, leaf samples from nine symptomatic plants were collected, and used for total RNA extraction with RNAprep pure plant kit (DP432, Tiangen Biotech, Beijing, China). The RNA samples were then subjected to reverse transcription (RT)-PCR testing with degenerate primers targeting the partial NIb gene of potyvirus (Marie-Jeanne et al. 2000) as well as the respective specific primer pairs for 18 viruses that are known to infect Fabaceae crops (Suppl Table S1). While none of the 18 target viruses was detected from any of the samples, a fragment of 327 bp was obtained from each of the diseased leaf samples, but not from healthy plants, with the degenerate potyvirus primer pair. The suspected potyvirus-amplicons were Sanger sequenced at SinoGenMax (SinoGenoMax Co., Ltd. Beijing, China). BLASTn analysis of the sequences showed that they shared 100% nt identity with themselves and 99.1% with the partial NIb gene of bean common mosaic virus (BCMV; Potyvirus; Potyviridae) isolate BCMV-Az (accession no. KP903372). To confirm the BCMV identity of the virus as well as its molecular characteristics, a fragment around 3,388 bp corresponding to NIa, NIb, CP and 3'UTR of BCMV genome was amplified from 4 diseased samples with the primer pair BCMV-F (5'-AGCAAGTCAATTTACAAGGGACTTC-3') and BCMV-R (5'-GGAACAACAAACATTGCCGTAGCTAC-3') (Li et al., 2014). Five independent clones from each diseased sample (20 clones in total) were Sanger sequenced, and all showed 100% identity with each other. One representative sequence has been deposited in GenBank under the accession number OP422522. BLASTn analysis showed that the sequence shares >98% identity with a number of BCMV sequences in GenBank (e.g., 98.9% with KP903372, the isolate BCMV-Az), thus confirming the BCMV identity of the virus. Further analysis of plants (n = 88) resulting from seeds of the 9 abovementioned diseased plants revealed an infection rate of 41% based on symptoms and RT-PCR assay with the degenerate primers, demonstrating the seed transmissibility of the virus in rice bean. The virus isolate is also mechanical transmissible as mechanical inoculation of 10 healthy rice bean plants led to the successful infection of four plants, which showed mosaic symptoms and tested positive for BCMV with RT-PCR assay with BCMV-specific primers reported in Manjunatha et al. (2014). BCMV can infect many leguminous plants, such as azuki bean (Vigna angularis) (Li et al., 2014), yam bean (Pachyrhizus erosus) (Han et al., 2023), and mungbean (Vigna radiata) (Cui et al. 2014). However, to the best of our knowledge, this is the first report that BCMV naturally infects rice bean in China. Further attention should be paid to this emerging viral disease, and proper measures need to be developed and implemented to minimize the spread of the virus in bean crops in the region.

Evaluation of Potential miR-302, miR-132, miR-205, and miR-126 as Biomarkers for Laryngeal Squamous Cell Carcinoma

Background: Laryngeal tumor is the most commonly identified malignancy in head and neck cancers, containing 2.4% of all cancers, and is the 11th most prevalent and fatal cancer in the world. Previous research has revealed that microRNAs (miRNAs) have a significant role in the progression of laryngeal tumors. Additionally, research focuses on the association with the clinical function of miRNA in malignancy. Objectives: Based on previous research, miR-132, miR-302, miR-126, and miR-205 have potential roles in the tumor development of the LSCC. Then, in the current study, we pointed to study if there are any significant alters in the expression of microRNAs and whether they have any significant possibility as a diagnostic or prognostic factor for laryngeal squamous cell carcinoma. Methods: From 30 LSCC patients, we have collected tumor and marginal healthy tissues through laryngectomy. Total RNA extraction from the marginal healthy and malignancy tissues was done. Next, we control the quality of the RNAs and, then, synthesize cDNA. Lastly, the expression of the miRNAs was evaluated by qPCR. The expression level of genes and their association with the patient’s clinicopathological features were studied, using related statistical tests. Results: miR-132 (P = 0.0001, fold change: 1.28) and miR-302 (P = 0.0001, fold change: 1.o5) are significantly up-regulated in tumor tissues compared to normal peripheral tissues. Furthermore, we revealed that miR-205 (P &lt; 0.0001, fold change: 0.241) and miR-126 (P = 0.0001, fold change: 0.251) are low expressed in LSCC cancer tissue. Between these miRNAs, gust expression of the miR-205 had no relationship with clinical-pathological characteristics in malignancy tissue. Conclusions: The results of the current study showed the possibility of miR-302, miR-132, miR-126, and miR-205 as diagnostic or prognostic factors in LSCC.

Circulating exosomal microRNAs as biomarkers of lupus nephritis.

Disruption in the delicate symphony of genes, microRNA (miRNA), or protein expression can result in the dysregulation of the immune system, leading to the devastating consequences such as lupus nephritis (LN). The capacity of exosomes to transport miRNAs between cells and modify the phenotype of recipient cells implies their involvement in persistent kidney inflammation. This study unveils identifying two previously undiscovered exosomal miRNAs in the serum of LN patients, offering potential solutions to the current challenges in LN diagnosis and management. Initially, we used a reagent-based kit to isolate serum exosomes from patients with Systemic lupus erythematosus (SLE) and used Trizol method for total RNA extraction. Subsequently, we employed small RNA sequencing to screen for differential expression profiles of exosomal small RNAs. The RT-qPCR method was used to individually validate samples in both the screening and validation cohorts, enabling the identification of candidate small RNAs; specific to LN. We assessed the diagnostic potency using receiver operating characteristic (ROC) curve, and explored the biological roles of miRNAs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Compared to SLE patients without LN, SLE patients accompanied by LN exhibited significantly spiked levels of exosomal hsa-miR-4796-5p and hsa-miR-7974. The duo of miRNAs, hsa-miR-4796-5p and hsa-miR-7974, exhibited promising potential as biomarkers for diagnosing LN, with an AUC exceeding 0.8. Correlation analysis revealed a strong positive association between these miRNAs and proteinuria, as well as the SLE Disease Activity Index (SLEDAI) score. Moreover, the levels of two miRNAs in LN patients were significantly elevated in comparison to other autoimmune nephritis conditions, such as immunoglobulin A nephropathy (IgAN) and diabetic nephropathy (DN). Furthermore, the bioinformatics analysis indicated that this miRNAs duo can play a pivotal role in the regulation of immune processes by modulating signal pathways, such as the mTOR and PI3K-Akt signaling pathway. This study provides a new ground that serum exosomal miRNAs can effectively identify and predict LN in SLE patients.

Identification of hub genes to determine drug-disease correlation in breast carcinomas.

The exact molecular mechanism underlying the heterogeneous drug response against breast carcinoma remains to be fully understood. It is urgently required to identify key genes that are intricately associated with varied clinical response of standard anti-cancer drugs, clinically used to treat breast cancer patients. In the present study, the utility of transcriptomic data of breast cancer patients in discerning the clinical drug response using machine learning-based approaches were evaluated. Here, a computational framework has been developed which can be used to identify key genes that can be linked with clinical drug response and progression of cancer, offering an immense opportunity to predict potential prognostic biomarkers and therapeutic targets. The framework concerned utilizes DeSeq2, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Cytoscape, and machine learning techniques to find these crucial genes. Total RNA extraction and qRT-PCR were performed to quantify relative expression of few hub genes selected from the networks. In our study, we have experimentally checked the expression of few key hub genes like APOA2, DLX5, APOC3, CAMK2B, and PAK6 that were predicted to play an immense role in breast cancer tumorigenesis and progression in response to anti-cancer drug Paclitaxel. However, further experimental validations will be required to get mechanistic insights of these genes in regulating the drug response and cancer progression which will likely to play pivotal role in cancer treatment and precision oncology.

Potential effects of parietin on apoptosis and cell cycle related genes in SH-SY5Y neuroblastoma cells

Background: Ingredients obtained from natural products have been used in cancer treatments for years. High diversity and non-toxicity compared to chemotherapeutic agents are the main reasons for their preference. Lichens having potential for treatment of cancer consist of fungus and 1-2 species of algae. Under the name of lichen substances, many of them have also been synthesized as specific substances. The secondary metabolites in lichens are generally insoluble in water, and have many biological activities such as antiviral, antitumor, antibacterial, and antioxidant; they store in the fungal cell or on the surface of the hyphae and can only be extracted with organic solvents.&#x0D; Parietin extracted from lichen species such as xanthoria parietina is an anthraquinone pigment and a secondary metabolite. &#x0D; In our study, the effects of parietin on cytotoxicity, gene expression, migration, invasion, and colony formation in neuroblastoma cells treated with parietin were investigated. The SH-SY5Y neuroblastoma (NB), which is not treated with parietin, was administered as a control group.&#x0D; Methods: The IC50 value of the parietin was determined using XTT assay. The total RNA extractions were performed from the cells using the Tri-Reagent kit. The expressions of BCL-XL, BCL-2, MMP2, MMP9, P21, CYCLIN D1, CASAPASE-3, CASAPASE-9, BAX, P53, PUMA, and NOXA genes were investigated by Lightcycler 480 (Roche) using SYBR Green dye. Migration analysis of the control and the dose group cells were performed in accordance with the Wound-healing assay protocol. Invasion activities were determined using the “Invasion Chamber” (BD Biosciences) protocol. The colony assay was treated with crystal violet and the cells were observed under the light microscope.&#x0D; Results: The IC50 value of the parietin used for 48-hour treatment on the cells was determined as 35 µM. It was found that the expression levels of BCL-XL, BCL-2, MMP2, MMP9, P21, and CYCLIN D1 mRNA were downregulated, and it was also shown the expression levels of CASAPASE-3, CASAPASE-9, BAX, P53, PUMA, and NOXA to be upregulated. It was determined that parietin suppressed both cell invasion and migration, and colony formation in the neuroblastoma cells. &#x0D; Conclusions: Thus, it can be possible parietin to be used as an alternative, complementary, and supportive agent together with the other drugs in the treatment of neuroblastoma. However, more comprehensive studies supporting these significant effects of parietin will increase its potential in the application.

New Insights into Hop Latent Viroid Detection, Infectivity, Host Range, and Transmission.

Hop latent viroid (HLVd), a subviral pathogen from the family Pospiviroidae, is a major threat to the global cannabis industry and is the causative agent for "dudding disease". Infected plants can often be asymptomatic for a period of growth and then develop symptoms such as malformed and yellowing leaves, as well as stunted growth. During flowering, HLVd-infected plants show reduced levels of valuable metabolites. This study was undertaken to expand our basic knowledge of HLVd infectivity, transmission, and host range. HLVd-specific primers were used for RT-PCR detection in plant samples and were able to detect HLVd in as little as 5 picograms of total RNA. A survey of hemp samples obtained from a diseased production system proved sole infection of HLVd (72%) with no coexistence of hop stunt viroid. HLVd was infectious through successive passage assays using a crude sap or total RNA extract derived from infected hemp. HLVd was also highly transmissible through hemp seeds at rates of 58 to 80%. Host range assays revealed new hosts for HLVd: tomato, cucumber, chrysanthemum, Nicotiana benthamiana, and Arabidopsis thaliana (Col-0). Sequence analysis of 77 isolates revealed only 3 parsimony-informative sites, while 10 sites were detected among all HLVd isolates available in the GenBank. The phylogenetic relationship among HLVd isolates allowed for inferring two major clades based on the genetic distance. Our findings facilitate further studies on host-viroid interaction and viroid management.

Interplay between HIV and Human Pegivirus (HPgV) Load in Co-Infected Patients: Insights from Prevalence and Genotype Analysis.

Human pegivirus (HPgV) is transmitted through sexual or parenteral exposure and is common among patients receiving blood products. HPgV is associated with lower levels of human immunodeficiency virus (HIV) RNA and better survival among HIV-infected patients. This study aimed to investigate the prevalence of HPgV and determine its subtypes in HIV-infected individuals living in Istanbul, which has the highest rate of HIV infection in Türkiye. Total RNA extraction from plasma, cDNA synthesis, and nested PCR were performed for HPgV on plasma samples taken from 351 HIV-1-infected patients. The HPgV viral load was quantified on HPgV-positive samples. HPgV genotyping was performed by sequencing the corresponding amplicons. In the present study, the overall prevalence of HPgV RNA in HIV-infected patients was 27.3%. HPgV subtypes 1, 2a, and 2b were found, with subtype 2a being the most frequent (91.6%). Statistical analysis of HIV-1 viral load on HPgV viral load showed an opposing correlation between HIV-1 and HPgV loads. In conclusion, these data show that HPgV infection is common among HIV-positive individuals in Istanbul, Türkiye. Further comprehensive studies are needed to clarify both the cellular and molecular pathways of these two infections and to provide more information on the effect of HPgV on the course of the disease in HIV-infected individuals.

Determination of the Mycovirome of a Necrotrophic Fungus.

Next-generation sequencing (NGS) of total RNA has allowed the detection of novel viruses infecting different hosts, such as fungi, increasing our knowledge on virus horizontal transfer events among different hosts, virus diversity, and virus evolution. Here, we describe the detailed protocols for the isolation of the plant pathogenic fungus Botrytis cinerea, from grapevine plants showing symptoms of the mold gray disease, the culture and maintenance of the isolated B. cinerea strains, the extraction of total RNA from B. cinerea strains for NGS, the bioinformatics pipeline designed and followed to detect mycoviruses in the sequenced samples, and the validation of the in silico detected mycoviruses by different approaches.

Discovery and Identification of Viruses Infecting Oomycetes.

This chapter describes protocols suitable for the detection and identification of RNA viruses infecting oomycetes (so-called water molds of Kingdom Heterokonta, Stramenopila), focusing on species of Phytophthora and exemplified by P. fragariae. The protocol includes laboratory procedures for oomycete cultivation and total RNA extraction from harvested mycelia, followed by instructions on suitable parameters given for sequencing companies on ribosomal RNA depletion, cDNA library preparation, and total RNA-sequencing (RNA-Seq). We also describe the bioinformatics steps needed for de novo assembly of raw reads into contigs, removal of host-associated contigs, and virus identification by database searches, as well as host validation by RT-PCR. All steps are described using an exemplar RNA-Seq library containing a yet undescribed fusagravirus hosted by a P. fragariae isolate.