RNA Samples Research Articles

Quality control on oil palm RNA samples for efficient genomic downstream applications.

Quality control (QC) is primordial for determining the efficiency in any downstream genomic applications. There are several steps in the verification of the quality of RNA samples destined for genomic studies. The aim of this research was to determine whether RNA should be discarded at the level of the field lab if it fails preliminary quality control using Optical Density (OD) measurements. In this study, all samples were submitted to rigorous quality control in every stage of work. RNA samples showing poor OD values still gave excellent results in downstream QC and genomic applications. At the end of the quality control exercise, it was observed that the original samples were the same and had not undergone any deterioration along the different stages of handling and manipulation. This paper shows the different and most important stages of quality control on RNA samples (RIN) for an effective down stream application in genomic studies. RNA samples should not be discarded based on preliminary QC from our field labs.

IDENTIFICATION OF TARGET GENES INVOLVED IN RESPONSES OF Populus TO POLYCHLORINATED BIPHENYLS BASED ON RNA-SEQ

Steel slag and rice husk bokashi are the ameliorant that expected can decrease P-retention Phytoremediation represents an effective, low-cost, environmentally friendly alternative that improves soil properties, although there have been significant developments in the last decade. From the scientific point of life, the main challenge is to decipher the metabolic pathways involved in response to contaminants and to understand their regulation. This information is essential if we aspire to improve the natural capacities of some plant species to remediate contaminated soils. A comprehensive study of the molecular response to polychlorinated biphenyls (PCBs), a family of persistent organic pollutants of particular relevance on a global scale, was obtained. A transcriptomic approach based on RNA-seq technology has been used to identify the genes involved in the metabolism of in-plant compounds and to quantify their activation levels in different controlled situations. It is surprising that this response is virtually unknown at the molecular level, despite its great potential applied in the context of phytoremediation technology. We apply to hybrid poplar (Populus tremula x alba) crops with Aroclor 1221. And we took samples of RNA, thus generating a matrix of four elements with their corresponding controls. On the other hand, we perform functional analyzes with bioinformatic tools based on sequence comparisons and gene co-expression networks. The response of genes of particular interest was validated using qRT-PCR technology. This is the first comprehensive study of the response of a plant organism to the presence of PCBs. A considerable number of structural and regulatory genes have been found, defining new transcription factors whose expression is proportional to the concentration of pollutant in the medium or the time of exposure to it. Correlation analyzes allow us to state that the metabolic response to PCBs, including possible degrading pathways, is involved in at least fifteen transcription factors and approximately forty proteins or enzymes that are particularly induced.

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.

Fatty Acid Binding Protein 1 is an Independent Prognostic Biomarker for Gallbladder Cancer with Direct Hepatic Invasion.

Background: Direct liver invasion (DI) is a predominant pathway of gallbladder cancer (GBC) metastasis, but the molecular alterations associated with DI remain addressed. This study identified specific genes correlated with DI, which may offer a potential biomarker for the diagnosis and prognosis of advanced GBC. Methods: RNA samples from 3 patients with DI of GBC were used for RNA-seq analysis. Differentially expressed genes and metabolic pathways between primary tumor (T) and DI tissue was used to analyze aberrant gene expressions. Immunohistochemistry (IHC) of fatty acid binding protein 1 (FABP1) in 62 patients with DI was engaged to evaluate its association with clinicopathological characteristics and prognosis. IHC of CD3+ and CD8+ T cells was analyzed for their correlation with FABP1 expression, clinicopathological features and prognosis. Univariate and multivariate Cox hazards regression analyses were performed to identify independent prognostic factors for disease-free survival (DFS) and overall survival (OS). Results: FABP1 mRNA levels were significantly upregulated in DI region compared to T tissue. IHC results showed identical results with elevated FABP1 (p < 0.0001). Expression of FABP1 in DI region was significantly associated with lymph node metastasis (P = 0.028), reduced DFS (P = 0.013) and OS (P = 0.022); in contrast, its expression in T region was not associated with clinicopathological characteristics and prognosis (P > 0.05). The density of CD8+ T cells in DI region with higher FABP1 expression was significantly lower than that with lower FABP1 expression (p = 0.0084). Multivariate analysis unveiled those hepatic metastatic nodules (HR = 3.35, 95%CI: 1.37-8.15, P = 0.008) and FABP1 expression in DI region (HR = 2.01, 95%CI: 1.05-3.88, P = 0.036) were high risk factors for OS, and FABP1(HR = 2.05, 95%CI: 1.04-4.06, P = 0.039) was also a high risk factor for DFS. Conclusions: Elevated expression of FABP1 in DI region serves as a potential prognostic biomarker for advanced GBC with DI.

Application of Bioinformatics in Predicting the Efficacy of Digestive Tumour Immunotherapy Target TIM-3 and its Inhibitors.

Background: Our main objective is to apply bioinformatics in predicting the efficacy of digestive tumour immunotherapy target TIM-3 and its inhibitors. Methods: Our study used the gene expression omnibus (GEO) database to identify datasets associated with digestive tumours and the action of TIM-3. The GSE427729 dataset based on the GPL10192 platform. The dataset consisted of six samples of total RNA derived from TIM-3 control and knockdown RAW 264.7 cells. We used GEO2R tool to identify DEGs before performing Gene Ontology and identifying the kyoto encyclopedia of genes and genomes (KEGG) pathways. Lastly, we determined the PPI networks to identify hub genes. Results: Our study identified 57 differentially expressed genes based on an adjusted p-value of less than 0.05 and a log2 fold change of 2.0. There were 26 down-regulated genes with 31 up-regulated genes while 22, 404 genes were non-significant. The DEGs were enriched in biological pathways such as activating leukocytes, cells, and development of the immune system. Additionally, we identified four significant KEGG pathways that were implicated in digestive tumour immunotherapy and TIM-3; pathways of pancreatic cancer, NF-Kappa B signalling pathway, Toll-like receptor signalling pathway and C-type lectin receptor signalling pathway. The PPI networks identified 10 hub genes that were implicated in digestive tumour immunotherapy target TIM-3 (Myd88, Traf6, Irf7, Cdk4, Ccnd2, Mapkap1, Prr5, Mpp3, Serpinb6b and Pvrl3). Conclusion: Targeting these biological pathways, KEGG pathways, molecular functions and cellular processes can lead to novel therapeutic treatment and management in digestive tumours based on TIM-3 immunotherapy.

Purification by STA-PUT Technique of Male Germ Cells from Single Mouse and RNA-Extraction for Transcriptomic Analysis.

Transcriptomic analyses of germ cells at different stages of differentiation have shed light on the transcriptional and post-transcriptional mechanisms regulating gene expression that ensure the correct progression of spermatogenesis and male fertility. In this chapter, we describe a method to isolate meiotic and post-meiotic germ cells, based on gravimetric sedimentation, starting from a testicular germ cell suspension isolated from a single adult mouse. We also describe how to assess the purity and quality of the collected fractions of germ cells and how to optimize the extraction from these samples of RNA for subsequent RNA-sequencing experiment. In our experience, this protocol is suitable for germ cell isolation and transcriptomic analysis for mouse models with spermatogenic defects, overcoming the limits that reduced fertility poses to the obtaining of experimental animals.

Purification of Circular RNAs Using Poly(A) Tailing Followed by RNase R Digestion.

Thousands of eukaryotic protein-coding genes can be alternatively spliced to yield linear mRNAs and circular RNAs (circRNAs). Some circRNAs accumulate to higher levels than their cognate linear mRNAs, but the vast majority are expressed at low levels. Hence, for most circRNAs, only a handful of sequencing reads, if any, that span the backsplicing junction are observed in standard RNA-seq libraries. It thus has become common to use the 3'-5' exonuclease ribonuclease R (RNase R) to selectively degrade linear RNAs when aiming to prove transcript circularity or biochemically enrich circRNAs. However, RNase R fails to degrade linear RNAs with structured 3' ends or internal G-quadruplex structures. To overcome these shortcomings, we describe an improved protocol for circRNA purification from total RNA that employs a poly(A) tailing step prior to RNase R digestion, which is performed in a Li+ containing buffer (rather than K+) to destabilize G-quadruplexes. This biochemical method enables higher enrichment (two- to threefold) of circRNAs to be obtained compared to standard RNase R protocols due to more efficient removal of linear RNAs. By then performing quantitative RT-PCR (RT-qPCR) or generating RNA-seq libraries, the expression of individual circRNAs can be examined or the entire set of expressed circRNAs defined using established annotation algorithms. We describe step-by-step methods for annotating circRNAs using the CIRI2 and CIRCexplorer2 algorithms. In total, this overall approach can be used to enrich for circRNAs from any total RNA sample, thereby enabling one to quickly identify and validate circRNAs of interest for functional studies.

Comparison of RT-ddPCR and RT-qPCR platforms for SARS-CoV-2 detection: Implications for future outbreaks of infectious diseases

The increased frequency of human infectious disease outbreaks caused by RNA viruses worldwide in recent years calls for enhanced public health surveillance for better future preparedness. Wastewater-based epidemiology (WBE) is emerging as a valuable epidemiological tool for providing timely population-wide surveillance for disease prevention and response complementary to the current clinical surveillance system. Here, we compared the analytical performance and practical applications between predominant molecular detection methods of RT-qPCR and RT-ddPCR on SARS-CoV-2 detection in wastewater surveillance. When pure viral RNA was tested, RT-ddPCR exhibited superior quantification accuracy at higher concentration levels and achieved more sensitive detection with reduced variation at low concentration levels. Furthermore, RT-ddPCR consistently demonstrated more robust and accurate measurement either in the background of the wastewater matrix or with the presence of mismatches in the target regions of the consensus assay. Additionally, by detecting mock variant RNA samples, we found that RT-ddPCR outperformed RT-qPCR in virus genotyping by targeting specific loci with signature mutations in allele-specific (AS) assays, especially at low levels of allele frequencies and concentrations, which increased the possibility for sensitive low-prevalence variant detection in the population. Our study provides insights for detection method selection in the WBE applications for future infectious disease outbreaks.

DNA nanotechnology for nucleic acid analysis: sensing of nucleic acids with DNA junction-probes.

DNA nanotechnology deals with the design of non-naturally occurring DNA nanostructures that can be used in biotechnology, medicine, and diagnostics. In this study, we introduced a nucleic acid five-way junction (5WJ) structure for direct electrochemical analysis of full-length biological RNAs. To the best of our knowledge, this is the first report on the interrogation of such long nucleic acid sequences by hybridization probes attached to a solid support. A hairpin-shaped electrode-bound oligonucleotide hybridizes with three adaptor strands, one of which is labeled with methylene blue (MB). The four strands are combined into a 5WJ structure only in the presence of specific DNA or RNA analytes. Upon interrogation of a full-size 16S rRNA in the total RNA sample, the electrode-bound MB-labeled 5WJ association produces a higher signal-to-noise ratio than electrochemical nucleic acid biosensors of alternative design. This advantage was attributed to the favorable geometry on the 5WJ nanostructure formed on the electrode's surface. The 5WJ biosensor is a cost-efficient alternative to the traditional electrochemical biosensors for the analysis of nucleic acids due to the universal nature of both the electrode-bound and MB-labeled DNA components.

Parallel DNA/RNA NGS Using an Identical Target Enrichment Panel in the Analysis of Hereditary Cancer Predisposition.

Germline DNA testing using the next-gene-ration sequencing (NGS) technology has become the analytical standard for the diagnostics of hereditary diseases, including cancer. Its increasing use places high demands on correct sample identification, independent confirmation of prioritized variants, and their functional and clinical interpretation. To streamline these processes, we introduced parallel DNA and RNA capture-based NGS using identical capture panel CZECANCA, which is routinely used for DNA analysis of hereditary cancer predisposition. Here, we present the analytical workflow for RNA sample processing and its analytical and diagnostic performance. Parallel DNA/RNA analysis allowed credible sample identification by calculating the kinship coefficient. The RNA capture-based approach enriched transcriptional targets for the majority of clinically relevant cancer predisposition genes to a degree that allowed analysis of the effect of identified DNA variants on mRNA processing. By comparing the panel and whole-exome RNA enrichment, we demonstrated that the tissue-specific gene expression pattern is independent of the capture panel. Moreover, technical replicates confirmed high reproducibility of the tested RNA analysis. We concluded that parallel DNA/RNA NGS using the identical gene panel is a robust and cost-effective diagnostic strategy. In our setting, it allows routine analysis of 48 DNA/RNA pairs using NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) in a single run with sufficient coverage to analyse 226 cancer predisposition and candidate ge-nes. This approach can replace laborious Sanger confirmatory sequencing, increase testing turnaround, reduce analysis costs, and improve interpretation of the impact of variants by analysing their effect on mRNA processing.

EXPRESSION OF GENES OF BIOMOLECULES ASSOCIATED WITH THE ETIOPATHOGENESIS OF ATHEROSCLEROSIS IN ATHEROSCLEROTIC PLAQUES OF CORONARY ARTERIES

HighlightsThe study showed differences in the expression of a number of genes in atherosclerotic plaques of different types in patients with coronary atherosclerosis. The obtained data can become the basis for the development of test systems in order to determine the dynamics of the atherosclerotic process and detect signs of destabilization of the atherosclerotic plaque as early as possible. Aim. To study the differential expression of genes encoding molecules associated with the etiopathogenesis of atherosclerosis by the method of genome-wide RNA sequencing in stable atherosclerotic plaque of fibrous type and unstable atherosclerotic plaque of dystopic-necrotic type.Methods. The study was performed on samples of atherosclerotic plaques of patients with coronary atherosclerosis without acute coronary syndrome with stable angina pectoris of functional class II–IV (FC) at the age of 45-65 years. Tissue sampling of atherosclerotic plaques was performed intraoperatively in the presence of indications. Genome-wide RNA sequencing was performed using Illumina’s TruSeq RNA Sample Preparation Kit (Illumina, USA).Results. An increase in the level of gene expression in stable atherosclerotic plaques was noted for A2M, ADAMTS13, CSF3, CX3CL1, CXCL1, FGF2, GDF15, ICAM1, IL1A, IL1B, IL6, IL10, PDGFA, PTX3. There was an eightfold statistically significant increase in the level of CFD, CXCL16, FABP4, FLT3, IFNG, IL7, IL15, SELL, TGFA, THBD, TNNT1, VCAM1 and VEGFA gene expression (p&lt;0,001) in unstable atherosclerotic plaques of dystrophic-necrotic type.Conclusion. The study showed differences in the expression of a number of genes in atherosclerotic plaques of different types in patients with coronary atherosclerosis. The obtained data can become the basis for the development of test systems in order to determine the dynamics of the atherosclerotic process and detect signs of destabilization of the atherosclerotic plaque as early as possible.

Consistency of drug-resistant mutations in plasma and peripheral blood mononuclear cells of patients with treatment-naïve and treatment-experienced HIV-1 infection.

Genotypic drug resistance testing is cursrently recommended by the World Health Organization for all patients infected with human immunodeficiency virus type 1 (HIV-1) undergoing care or switching regimes due to failure with previous antiretroviral therapy (ART). Patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) who meet the criteria for free testing for genotypic drug resistance due to poor adherence in Henan Province may resume their previous regimens before resampling. Therefore, resistance testing based on plasma RNA can fail in a proportion of patients. Resistance testing based on peripheral blood mononuclear cells (PBMCs) is an alternative option. In this study, we investigated the differences in drug-resistant mutations (DRMs) between plasma HIV RNA and proviral DNA in treatment-experienced and treatment-naïve patients. Matched plasma RNA and proviral DNA samples of 66 HIV-1 infected treatment-naïve and 78 treatment-experienced patients were selected for DRM analysis and comparison. DRMs were detected in 27.3% (18/66) of treatment-naïve and 80.8% (63/78) of treatment-experienced samples. Resistance to at least one drug was detected based on analysis of plasma RNA and proviral DNA in 7.6% (5/66) and 9.1% (6/66) of treatment-naïve patients and in 79.5% (62/78) and 78.2% (61/78) of treatment-experienced patients, respectively. Furthermore, 61/66 (92.4%) of treatment-naïve patients showed concordant RNA and DNA drug resistance. When drug resistance was defined as intermediate and high, the concordance of drug resistance profiles of paired RNA and proviral DNA samples derived from treatment-naïve patients were up to 97.0% compared with only 80.8% (63/78) in treatment-experienced patients. Our data indicate that drug resistance testing based on plasma RNA or proviral DNA might be interchangeable in treatment-naïve patients, whereas plasma RNA-based testing remains the best choice for drug resistance analysis in patients with ART failure in clinical practice.

Prevalence of hepatitis D virus infection among patients with chronic hepatitis B infection in a tertiary care centre in Thailand

Knowledge about the epidemiology of hepatitis D virus (HDV) is essential for effective screening and management. Our study aimed to update the prevalence of HDV infection among patients with hepatitis B virus (HBV) infection at hepatology clinics in Thailand. We enrolled HBV-infected patients from hepatology clinics at King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between June 2022 and November 2023. Demographic, biochemical characteristics, and liver-related complications (LRC), including cirrhosis and hepatocellular carcinoma, were reviewed. The competitive enzyme and chemiluminescence immunoassays were used to detect anti-HDV antibodies. Real-time polymerase chain reaction (RT-PCR) was used to test for HDV RNA in anti-HDV-positive patients. The HDV genotype was identified in detectable HDV RNA samples. Of the 702 enrolled patients, four (0.6%) had positive and equivocal for both anti-HDV tests. Two (50.0%) of the four patients tested positive for HDV RNA and genotype 1 was identified; one had multiple risk factors. Anti-HDV seroprevalence was not significantly different between patients with and without LRC. In conclusion, HDV co-infection is less common in Thailand than globally. Additionally, our study identified genotype 1, the predominant HDV genotype worldwide, and observed co-infection even without LRC.

Comparison of RT-LAMP and RT-qPCR assays for detecting SARS-CoV-2 in the extracted RNA and direct swab samples

Rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in infected patients is critical for infection control. Loop-mediated isothermal amplification (LAMP) has been demonstrated to be a rapid, simple, reliable, cost-effective and sensitive method to detect SARS-CoV-2 in a variety of samples in considerably less time than Real-Time PCR. In this study, we developed and optimized a rapid detection method for SARS-CoV-2 based on RT-LAMP method utilizing a specific primer set targeting the ORF1a gene and then examined its sensitivity and efficiency using a serially diluted viral RNA sample with a known concentration. Furthermore, the sensitivity of the RT-LAMP to detect SARS-CoV-2 in direct swab samples with varying Ct values were compared to a commercial molecular RT-qPCR based detection kit. According to our findings the optimal incubation time for RT-LAMP assay was 45 min. There was a complete agreement between RT-LAMP and RT-qPCR in diagnosing the viral genome in the diluted extracted RNA sample. However, it had a lower sensitivity (71%) to detect the viral genome in direct swab samples compared to RT-qPCR. In conclusion, due to its simplicity, rapidness, sensitivity, and specificity, RT-LAMP has tremendous potential as a point-of-care tool; nevertheless, more research is needed to utilize it for detecting SARS-CoV-2, particularly in direct swab samples.

The alteration of apoptosis-related genes in female pelvic supportive tissues with regard to menopausal status

PurposeWe aimed to compare the expression levels of anti-apoptotic and proapoptotic genes in the parametrium, sacrouterine and round ligaments with respect to menopausal status in women presenting without any indication of pelvic organ prolapse (POP). We hypothesized that apoptosis related gene expressions in female pelvic tissues may be altered during menopause.MethodsThe study groups consisted of pre-menopausal (n = 10) and menopausal (n = 10) females who did not have POP symptoms. Three different types of tissue samples (Parametrium, Round Ligament and Sacrouterine Ligament) were obtained and RNA was isolated from these tissues. After purifying and quantifying RNA samples, qPCR was used to determine the expression levels of anti-apoptotic and pro-apoptotic genes.ResultsBCL-2 gene expression levels were significantly lower in all the tissues of menopausal patients compared to those of premenopausal patients. In comparison to premenopausal patients, the sacrouterine ligament tissue BAD expression level was significantly high (p = 0.035), and the BCL-2/BAD ratio was significantly lower in menopausal patients (p = 0.006).ConclusionApoptosis-related protein levels change during menopause; pro-apoptotic gene expressions decrease and anti-apoptotic gene expressions increase. The significant alteration of BCL-2 and BAD expression in sacrouterine ligament with respect to menopausal status was observed and this suggested that when compared to other pelvic tissues, the sacrouterine ligament, which plays a crucial role for genital organs in restoring normal pelvic anatomy and providing support, could be affected more by menopause.

Metagenomic Analyses of Viruses in the Orchid Mycorrhizal Interaction Using Improved Assemble Tools.

In nature, mycorrhizal association with soil-borne fungi is indispensable for orchid species. Compatible mycorrhizal fungi form endo-mycorrhizal structures in orchid cells, and the fungal structures are digested in orchid cells to be supplied to orchids as nutrition. Because orchid seeds lack the reserves for germination, they keep receiving nutrition through mycorrhizal formation from seed germination until nonphotosynthetic young seedlings develop leaves and become photoautotrophic. Seeds of all orchids germinate with the help of their own fungal partners, and therefore, specific partnership has been acquired in a long evolutionary history between orchids and fungi. Assuming that horizontal transmission of viruses may occur in such a close relationship, we are focusing on viruses that infect orchids and their mycorrhizal fungi. We prepared aseptically germinated orchid plants (i.e., fungi-free plants) together with pure-cultured fungal isolates and conducted transcriptome analyses (RNA-seq) by next-generation sequencing (NGS) approach. To reconstruct virus-related sequences that would have been present in the RNA sample of interest, de novo assembly process is required using short read sequences obtained from RNA-seq. In the previous version of our protocol (see Viral Metagenomics, first edition 2018), virus searches were conducted using contig sets constructed by a single assembler, but this time we devised a method to construct more reliable contigs using multiple assemblers and again reinvestigated that viruses could be detected. Because the virus detection efficiency and number of detected virus species clearly differed depending on the assembly pipeline and the number of the input data, multiple methods should be used to identify viral infection, if possible.

EVALUATION OF METHODS FOR TOTAL RNA EXTRACTION FROM THE ENDOSPERM OF COCOS NUCIFERA VAR. MAKAPUNO IN VIETNAM FOR MOLECULAR ANALYSIS

Sap coconut (Cocos nucifera L. var. makapuno) in Vietnam is a mutant coconut variant; coconut water is in the state of lotus glue, and coconut rice is like cream. For high-quality transcriptome, sequencing, quality, purity and concentration of RNA are the key factors. However, coconut endosperm tissue has higher stiffness and fatness than the leaf tissue, which complicates the extraction process. Moreover, RNA is much more difficult to preserve than DNA. In this study, various RNA extraction methods were examined in Vietnamese waxy coconut endosperm tissue samples. Optimum extraction and preservation of the RNA using the simplest possible chemicals was the objective of this study. The modified CTAB method with LiCl and the TRI reagent method were tested and evaluated. The purity, concentration and quality of RNA after storage were improved. The findings indicated that the TRIsure extraction method with the addition of NaCl and β-mercaptoethanol yielded optimum RNA quality. The RNA concentration was 159 ng/µL, with a purity ratio of 1.94 ± 0.04 for A260/A280 and 1.58 ± 0.02 for A260/230. RNA samples remained stable for up to 3 weeks when stored in absolute ethanol at 8°C–10°C, which significantly reduced their degradation during transportation. This study facilitated the use of simple chemicals for high-quality RNA extraction from coconut endosperm and its preservation for applications in high throughput sequencing.

Tracking SARS-CoV-2 variants during the 2023 flu season and beyond in Lebanon

BackgroundEarly SARS-CoV-2 variant detection relies on testing and genomic surveillance. The Omicron variant (B.1.1.529) has quickly become the dominant type among the previous circulating variants worldwide. Several subvariants have emerged exhibiting greater infectivity and immune evasion. In this study we aimed at studying the prevalence of the Omicron subvariants during the flu season and beyond in Lebanon through genomic screening and at determining the overall standing and trajectory of the pandemic in the country. MethodsA total of 155 SARS-CoV-2 RNA samples were sequenced, using Nanopore sequencing technology. ResultsNanopore sequencing of 155 genomes revealed their distribution over 39 Omicron variants. XBB.1.5 (23.29 %) was the most common, followed by XBB.1.9.1 (10.96 %) and XBB.1.42 (7.5 %). The first batch collected between September and November 2022, included the BA.2.75.2, BA.5.2, BA.5.2.20, BA.5.2.25 and BQ.1.1.5 lineages. Between December 2022 and January 2023, those lineages were replaced by BA.2.75.5, BN.1, BN.1.4, BQ.1, BQ.1.1, BQ.1.1.23, CH.1.1, CM.4 and XBK. Starting February 2023, we observed a gradual emergence and dominance of the recombinant XBB and its sub-lineages (XBB.1, XBB.1.5, XBB.1.5.2, XBB.1.5.3, XBB.1.9, XBB.1.9.1, XBB.1.9.2, XBB.1.16, XBB.1.22 and XBB.1.42). ConclusionsThe timely detection and characterization of SARS-CoV-2 variants is important to reduce transmission through established disease control measures and to avoid introductions into animal populations that could lead to serious public health implications.

Consequences of mitochondrial DNA depletion in microglial cells

AbstractBackgroundMitochondrial dysfunction is an early and prominent feature of Alzheimer’s disease (AD). We have recently published that lower brain mitochondrial DNA copy number (mtDNAcn) is associated with a greater odds of AD neuropathological change and worse cognitive performance. As it is known that microglial cells play an important role in AD, we want to address how a reduction in mtDNAcn may affect microglial function.Methodsmicroglial BV2 cells were treated with ethidium bromide (etbr) (1ug/ml) for 5 days to achieve a 95% reduction of mitochondrial‐encoded genes expression. Cells were characterized by RNAseq and Seahorse experiments. BV2 cells were challenged with latex beads and 10uM zymosan‐pHrodo for 24 hours and phagocytosis was analyzed by flow cytometry. BV2 cells were stimulated with LPS (100ng/ml) for 3 hours to measure cytokine release by qPCR and ELISA. After BV2 stimulation, fresh media (without LPS or etbr) was conditioned for 6 hours and collected to treat neurons (SH‐SY5Y cells) and human primary astrocytes for 24 hours. RNA and protein samples were collected.ResultsmtDNAcn reduction led to a dramatic reduction in oxidative phosphorylation and a mild reduction in proliferation. RNAseq data showed an increase in the hypoxia signature and cellular senescence, coupled with a reduction in biosynthetic process and response to interferon. No changes were observed in phagocytic capacity. However, after LPS stimulation, etbr‐treated cells showed a reduction in the production of IL1B and IL6 cytokines, suggesting an impairment in the response to challenge. Neurons exposed to media from etbr‐treated BV2 cells showed an increase in MAPT and APOE expression. Samples from astrocytes are currently being analyzed.ConclusionsmtDNAcn reduction impairs mitochondrial respiration, which affects the production of cytokines in response to stimulus and leads to cell senescence in microglial cells. Cytokines released by microglia affected neuronal expression of AD‐related genes.

Study of TMEM106B expression levels under neurodegenerative conditions

AbstractBackgroundTMEM106B is a transmembrane protein involved in dendrite morphogenesis and lysosome trafficking. TMEM106B was identified as a susceptibility gene for frontotemporal lobar degeneration with TDP‐43 inclusions (FTLD‐TDP). Brain tissue from patients with FTLD‐TDP showed increased mRNA levels of TMEM106B compared to normal controls (Van Deerlin et al, Nat Genet 2010). Our goal is to study TMEM106B expression levels in other neurodegenerative conditions such as progressive supranuclear palsy (PSP), Alzheimer’s disease (AD) and a well‐established mouse model of entorhinal cortex lesions (ECL).MethodUnilateral electrolytic lesions to the entorhinal cortex were conducted on two‐to‐three months old male C57BL/6j mice by stereotaxic surgery. For sham‐operated animals, the electrode was lowered only 1 mm without current and sacrifices were performed on the same day. All other lesioned mice were sacrificed 2, 7, 14, 21 or 40 days post‐lesion (DPL). Total RNA was extracted from frozen ipsilateral and contralateral hippocampi and expression profiles were measured with the Affymetrix Mouse ClariomTM D Assay. The Mayo Clinic Brain Bank collected RNA samples from temporal cortex and measured gene expression levels using next‐generation RNA sequencing (RNAseq). Results are available for 274 subjects with the following conditions: PSP, N = 84; AD, N = 82; pathological aging (PA), N = 30; and cognitively unimpaired (CU), N = 78.ResultIn the ECL mouse model, ipsilateral Tmem106b expression levels were significantly reduced during the deafferentation phase (DPL2: p = 0.046) and reached maximum levels during the reinnervation phase (DPL14: p = 0.030 and DPL21: p = 0.019), compared to sham‐operated animals. In human temporal cortices, TMEM106B expression increased progressively from CU (10.76±0.62), to PSP (11.25±0.43), PA (12.13±1.27) and finally AD (13.10±0.44) (mean CPM ±SEM). TMEM106B mRNA levels were significantly higher in AD compared to CU (p = 0.002) and PSP (p = 0.003).ConclusionElevated levels of TMEM106B in FTLD‐TDP, AD and during the reinnervation phase of the ECL mouse model suggest an active response to tissue damage that is consistent with compensatory synaptic and terminal remodeling, independently from tau‐mediated pathology. This hypothesis is consistent with TMEM106B levels found in PSP, which is characterized by abundant neurofibrillary tangles that differ in both distribution and composition from those associated with AD.