RNA Preparations Research Articles

Direct RNA Nanopore Sequencing of Pseudomonas aeruginosa Clone C Transcriptomes.

ABSTRACTThe transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules, followed by similar proportions of mRNA transcripts and noncoding RNAs. The two platforms detected similar proportions of uncharged tRNAs and 29 yet-undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNAArg, and the tRNAArg gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct nonoverlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range (a few hundred to thousands of bases) and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet-undescribed RNA molecules and an unexpected composition of the pools of tRNAs and sense and antisense RNAs.IMPORTANCE Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of rRNAs, enzymatic reverse transcription, and the fragmentation, size selection, and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the Nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that—if replicated in other species—will change our view of the bacterial RNA world. The discovery of sense-antisense pairs of transfer-messenger RNA (tmRNA), tRNAs, and mRNAs indicates a further and unknown level of gene regulation in bacteria.

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-cell Multi-omics Experiments.

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

Sensitive extraction-free SARS-CoV-2 RNA virus detection using a chelating resin

SummaryCurrent conventional detection of SARS-CoV-2 involves collection of a patient’s sample with a nasopharyngeal swab, storage of the swab during transport in a viral transport medium, extraction of RNA, and quantitative reverse transcription PCR (RT-qPCR). We developed a simplified preparation method using a chelating resin, Chelex, which obviates RNA extraction during viral testing. Direct detection RT-qPCR and digital droplet PCR were compared to the current conventional method with RNA extraction for simulated samples and patient specimens. The heat treatment in the presence of Chelex markedly improved detection sensitivity as compared to heat alone, and lack of RNA extraction shortens the overall diagnostic workflow. Furthermore, the initial sample heating step inactivates SARS-CoV-2 infectivity, thus improving workflow safety. This fast RNA preparation and detection method is versatile for a variety of samples, safe for testing personnel, and suitable for standard clinical collection and testing on high-throughput platforms.

Ameliorative effects of miR-423-5p against polarization of microglial cells of the M1 phenotype by targeting a NLRP3 inflammasome signaling pathway

Spinal cord injury (SCI) causes sensation and motion dysfunction. Activation of microglial cells (MCs) in the central nervous system (CNS) is heterogeneous. Heterogeneous types of MCs can produce cytotoxic or neuroprotective effects, secrete proinflammatory or anti-inflammatory factors. The cytotoxic effect of MCs is one of the reasons for secondary damage after SCI. The NLR family pyrin domain containing 3 (NLRP3) inflammasome is a protein that can recognize pathogen-related molecular patterns or host-derived danger signal molecules, responses to microbial infection, and sterile stressors. SCI triggers activation of the NLRP3 inflammasome in the CNS. We investigated the interaction between miR-423-5p and NLRP3 in MCs polarization after SCI. A rat model of SCI was established by a modified version of Allen’s method. Spinal samples were adopted for preparation and sequencing of RNA. We screenedapromising microRNA (miR-423-5p) according to the results. Then, we found that NLRP3 was one of the prediction targets of miR-423-5p. By intervening in expression of miR-423-5p and NLRP3, we observed the different polarization of MCs. We employeda dual-luciferase reporter study, proteomics, and transcriptomicsto ascertain the direct targeting relationship between miR-423-5p and NLRP3. MiR-423-5p expression was decreased significantly after SCI in vivo and in vitro. Upregulation of miR-423-5p expression could prevent MCs from lipopolysaccharide-induced M1 polarization. Knockdown of NLRP3 expression could prevent MCs from lipopolysaccharide-induced M1 polarization. MiR-423-5p inhibited MCs polarization to the M1 phenotype by targeting NLRP3.

Watermelon crinkle leaf-associated virus 1 and watermelon crinkle leaf-associated virus 2 have a bipartite genome with molecular signatures typical of the members of the genus Coguvirus (family Phenuiviridae).

Watermelon crinkle leaf-associated virus 1 and watermelon crinkle leaf-associated virus 2 (WCLaV-1 and WCLaV-2), two unclassified members of the order Bunyavirales, are phylogenetically related to members of the genus Coguvirus (family Phenuiviridae). The genome of both viruses was reported previously to be composed of three RNA segments. However, the terminal sequences of two genomic RNA segments, namely those encoding the putative movement protein (MP) and the nucleocapsid (NP) protein, remained undetermined. High-throughput sequencing of total RNA and small RNA preparations, combined with reverse transcription PCR amplification followed by sequencing, revealed that the WCLaV-1 and WCLaV-2 possess a bipartite genome consisting of a negative-sense RNA1, encoding the RNA-dependent RNA polymerase, and an ambisense RNA2, encoding the putative movement (MP) and nucleocapsid (NP) proteins. The two open reading frames of RNA2 are in opposite orientations and are separated by a long AU-rich intergenic region (IR) that may assume a hairpin conformation. RNA1 and RNA2 of both viruses share almost identical 5' and 3' termini, which are complementary to each other up to 20 nt. This genome organization is typical of members of the genus Coguvirus, with which WCLaV-1 and WCLaV-2 also share similar terminal 5' and 3' sequences of RNA1 and RNA2. These molecular features, together with phylogenetic reconstructions support the classification of WCLaV-1 and WCLaV2 as members of two new species in the genus Coguvirus.

A method for the enrichment, isolation and validation of Mycobacterium smegmatis population surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin.

The bacterial populations surviving in the presence of antibiotics contain cells that have gained genetic resistance, phenotypic resistance and tolerance to antibiotics. Isolation of live bacterial population, surviving against antibiotics, from the milieu of high proportions of dead/damaged cells will facilitate the study of the cellular/molecular processes used by them for survival. Here we present a Percoll gradient centrifugation based method for the isolation of enriched population of Mycobacterium smegmatis surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin. From the time of harvest, throughout the enrichment and isolation processes, and up to the lysis of the cells for total RNA preparation, we maintained the cells in the presence of the antibiotic to avoid changes in their metabolic status. The total RNA extracted from the enriched population of live antibiotic-surviving population showed structural integrity and purity. We analysed the transcriptome profile of the antibiotic-surviving population and compared it with the orthologue genes of Mycobacterium tuberculosis that conferred antibiotic tolerance on tubercle bacilli isolated from the tuberculosis patients under treatment with four antitubercular antibiotics. Statistically significant comparability between the gene expression profiles of the antibiotic tolerance associated genes of M. smegmatis and M. tuberculosis validated the reliability/utility of the method.

The Tmprss6-/- Mouse Model of Iron Refractory Iron Deficiency Anemia (IRIDA) Exhibits Disrupted Phosphate Homeostasis, Elevated Circulating FGF23 Levels, and Increased Fgf23 Expression in Bone Marrow

While the effects of chronic iron deficiency on erythropoiesis are well documented, the effects of iron deficiency on metabolism remain poorly understood. Recent studies suggest that iron deficiency anemia may impact systemic phosphate regulation (Farrow et al ., PNAS, 2011; Clinkenbeard et al ., J Bone Miner Res, 2014; David et al ., Kidney Int, 2016; Hanudel et al ., Am J Physiol Renal Physiol, 2016). Here we used Tmprss6-/- mice, a model of iron refractory iron deficiency anemia (IRIDA) characterized by hepcidin elevation, to explore the relationship between systemic iron deficiency and phosphate homeostasis. By intercrossing Tmprss6+/- mice, we generated Tmprss6-/- mice and littermate controls, which were raised on a standard rodent diet (Teklad 2018S containing 200 ppm iron and 0.7% phosphorus) prior to study at 8 weeks of age. Tmprss6-/- mice displayed a trend toward lower serum phosphate concentration, as well as a significant elevation in the urinary phosphate/creatinine ratio. Because of the role of phosphate in bone mineralization, we also analyzed the tibiae of Tmprss6-/- mice and found that they showed alterations in bone histology and biomechanical properties.To determine if fibroblast growth factor 23 (FGF23), a key phosphate-regulating hormone, may contribute to these changes in phosphate balance, we compared plasma FGF23 levels of 8-week-old Tmprss6-/- mice to littermate controls using enzyme-linked immunosorbant assays (ELISAs). Tmprss6-/- mice showed significantly higher levels of the biologically-active (“intact”) form of FGF23 as well as “total” FGF23 (a measure of both the intact form and the C-terminal cleavage product of FGF23). This FGF23 elevation was also observed in 3-week-old Tmprss6-/- pups (i.e. prior to weaning). Because osteocytes in bone have been described as a major site of FGF23 production, we measured Fgf23 mRNA levels by qRT-PCR in RNA that was prepared from femur bones of Tmprss6-/- mice or littermate controls after marrow removal by flushing. We observed a four-fold increase in Fgf23 expression in the femurs of Tmprss6-/- mice as compared to littermate controls. Because Fgf23 expression has previously been reported in Ter119+ erythroid cells from wild type mice (Coe et al ., J Biol Chem, 2014), and because our RNA preparations from femur bones showed evidence of residual bone marrow contamination (as assessed by alpha hemoglobin expression), we also measured Fgf23 mRNA expression in RNA prepared from total bone marrow. Tmprss6-/- mice exhibited significantly higher bone marrow Fgf23 mRNA levels than littermate controls. Notably, when RNA was prepared from bones that had been more effectively depleted of marrow by mechanical disruption and hypotonic lysis, Tmprss6-/- mice no longer showed an increase in Fgf23 mRNA levels in the femur RNA as compared to littermate controls. Collectively, these results suggest that increased Fgf23 production by one or more bone marrow cell populations contributes to elevated circulating FGF23 in Tmprss6-/- mice.FGF23 has been proposed as the basis for a bone-kidney axis that coordinates bone health with renal handling of phosphate. Our findings suggest that altered phosphate homeostasis in Tmprss6-/- mice may result from increased Fgf23 expression in the bone marrow and a corresponding elevation in circulating FGF23. These results may have relevance to the skeletal health of patients with IRIDA due to TMPRSS6 mutation and those with other forms of iron-restricted anemia resulting from hepcidin elevation. Additionally, as Tmprss6-/- mice are known to exhibit growth retardation, our results raise the possibility that disrupted phosphate homeostasis may contribute to the commonly observed association of childhood iron deficiency with growth impairment. Future studies will characterize the source of Fgf23 production in the bone marrow of Tmprss6-/- mice as well as the mechanism by which iron deficiency elevates Fgf23 expression. DisclosuresNo relevant conflicts of interest to declare.

The Effect of Genomic DNA Contamination on the Detection of Circulating Long Non-Coding RNAs: The Paradigm of MALAT1.

The presence of contaminating gDNA in RNA preparations is a frequent cause of false positives in RT-PCR-based analysis. However, in some cases, this cannot be avoided, especially when there are no exons–intron junctions in the lncRNA sequences. Due to the lack of exons in few of long noncoding RNAs (lncRNAs) and the lack of DNAse treatment step in most studies reported so far, serious questions are raised about the specificity of lncRNA detection and the potential of reporting false-positive results. We hypothesized that minute amounts of gDNA usually co-extracted with RNA could give false-positive signals since primers would specifically bind to gDNA due to the lack of junction. In the current study, we evaluated the effect of gDNA and other forms of DNA like extrachromosomal circular DNAs (eccDNAs) contamination and the importance of including a DNAse treatment step on lncRNAsexpression.As a model, we have chosen as one of the most widely studied lncRNAs in cancer namely MALAT1, which lacks exons. When we tested this hypothesis in plasma and primary tissue samples from NSCLC patients, our findings clearly indicated that results on MALAT1 expression are highly affected by the presence of DNA contamination and that the DNAse treatment step is absolutely necessary to avoid false positive results.

CircFAT1 is Overexpressed in Colorectal Cancer and Suppresses Cancer Cell Proliferation, Invasion and Migration by Increasing the Maturation of miR-10a.

PurposeIt has been reported that circFAT1 plays opposite roles in gastric cancer and osteosarcoma. This study aimed to explore the involvement of circFAT1 in colorectal cancer (CRC).Patients and MethodsPaired CRC and non-tumor tissues from 64 CRC patients were subjected to RNA preparations and RT-qPCRs to determine the expression of circFAT1, premature miR-10a, and mature miR-10a. In CRC cells, the effects of circFAT1 overexpression on miR-10a maturation were analyzed by determining the expression of both premature miR-10a and mature miR-10a using RT-qPCRs. The role of circFAT1 and miR-10a in regulating the proliferation, invasion, and migration of CRC cells was analyzed by BrdU and Transwell assays, respectively.ResultsWe found that circFAT1 was upregulated in CRC. Mature miR-10a, but not premature miR-10a, was overexpressed in CRC and positively correlated with circFAT1. CircFAT1 overexpression increased the expression of mature miR-10a, but not premature miR-10a. Overexpression of circFAT1 and miR-10a increased cell proliferation, invasion and migration, and miR-10a inhibitor suppressed cell behaviors and reversed the effects of circFAT1 overexpression.ConclusioncircFAT1 is overexpressed in CRC and may suppress CRC cell proliferation, invasion and migration by increasing miR-10a maturation.

Chronic Dexamethasone Treatment Leads to Less Weight Gain and Ameliorated Glucose Tolerance in Mice; Role of the Cytoprotective Nrf2 Pathway

Introduction: Chronic glucocorticoid administration is necessary in a variety of conditions including but not limited to autoimmune, inflammatory and cancer-related diseases in order to relieve symptoms and sustain disease progression. However, there are adverse effects that include increase in glucose levels and others whose severity depends on the dose and duration of glucocorticoid exposure. It has been described that dexamethasone induces oxidative stress in cells by increasing reactive oxygen species (ROS) and this is one of the causes of insulin resistance at the cellular level. Nrf2 is a transcription factor which co-ordinates the antioxidant response and its activation has been shown to ameliorate insulin resistance in murine models. Hypothesis: We hypothesized that deletion of Nrf2 will lead to a more glucose intolerant insulin resistant phenotype in mice chronically treated with dexamethasone as cells would be exposed to higher ROS levels. Methods: To this end, 3-months old wild-type (WT) and Nrf2 knockout (KO) C57BL6J mice were treated intraperitoneally with 2 mg/kg dexamethasone or saline 3 times per week for 3 months. 5-10 mice were included per genotype per treatment and both male and female mice were used. Weekly measurements of body weights were performed and intraperitoneal glucose tolerance tests were done on the second and third month of treatment. Mice were sacrificed 24 hours after the last dose of dexamethasone and blood, white adipose tissue, soleus muscle and liver were collected for RNA preparation and quantitative RT-PCR analysis. Quantitative analysis of trabecular bone parameters was performed by micro-CT. Results: Both male and female mice treated with dexamethasone gained less weight over time and surprisingly were more glucose tolerant than the control group. Absence of Nrf2 did not seem to considerably affect the body weight but KO mice tended to have lower body weights after dexamethasone treatment in both genders with the effect on male mice being statistically significant (25% lower, p<0.05). Surprisingly, both WT and KO mice of both genders showed lower fasting blood glucose levels after 3 months of treatment and better glucose tolerance. Livers of KO mice showed lower levels (~50%) of the cytoprotective genes Nqo1 and Gclc as expected but no difference was observed after dexamethasone treatment. Sarcopenia muscle markers Mafbx1 and Murf1 showed no significant changes. Male mice showed increased expression of Pnpla3 in white adipose tissue indicating increased lipolysis upon dexamethasone exposure. Micro-CT showed minor changes in the bone parameters without difference between male WT and Nrf2KO mice. Conclusions: Dexamethasone unexpectedly led to better glucose tolerance and lower body weight which is uncommon in humans but it has been described previously in mouse models. More analyses are in progress to fully elucidate this phenotype.

High-Throughput NanoString Analysis of Oncogenic Human Papillomavirus and Tumor Microenvironment Transcription in Head and Neck Squamous Cell Carcinoma.

Human papillomaviruses (HPVs), specifically high-risk HPVs, are responsible for up to 3% of all cancers in women and up to 2% of all cancers in men. They have been identified as the etiological agent of cervical cancer and have been increasingly found to be the driver behind head and neck cancers of the oropharynx. A system in which we can simultaneously observe transcriptional changes to both a host's tumor microenvironment and its associated oncogenic driver (e.g., HPV) would be highly valuable for understanding HPV's role in tumorigenesis. This article describes a detailed methodology for utilizing high-throughput RNA analysis to study viral transcription in formalin-fixed, paraffin-embedded clinical tumor samples. Although our lab utilizes these methods for the study of head and neck cancer, the principles contained within are widely applicable to all fields of HPV study. © 2021 Wiley Periodicals LLC. Basic Protocol: HPV16 transcript analysis using NanoString Support Protocol 1: Preparation of RNA from formalin-fixed, paraffin-embedded slides Support Protocol 2: Preparation of RNA from cell lysates Support Protocol 3: Fluorometric RNA concentration and RNA integrity analysis Support Protocol 4: Determination of input RNA based on DV300 calculation.

Effect of Myd88 deficiency on gene expression profiling in salivary glands of female non-obese diabetic (NOD) mice

ObjectivesSjögren's syndrome (SS) is a chronic autoimmune disease characterized by inflammatory lesions in the salivary and lacrimal glands, which are caused by distinct lymphocytic infiltrates. Female non-obese diabetic (NOD) mice spontaneously develop inflammatory lesions of the salivary glands with SS-like pathological features. Previous studies have shown that MyD88, a crucial adaptor protein that activates innate immune signaling, affects lymphocytic infiltration, but its detailed role remains unclear. In this study, we investigated the role of MyD88 through gene expression profiling in the early phase of pathogenesis in the salivary glands of female NOD mice. MethodsSubmandibular glands collected from 10-week-old female wild-type and Myd88-deficient NOD mice were used for RNA preparation, followed by microarray analysis. The microarray dataset was analyzed to identify Myd88-dependent differentially expressed genes (DEGs). Data generated were used for GO enrichment, KEGG pathway, STRING database, and INTERFEROME database analyses. ResultsMyd88 deficiency was found to affect 230 DEGs, including SS-associated genes, such as Cxcl9 and Bpifa2. Most of the DEGs were identified as being involved in immunological processes. KEGG pathway analysis indicated that the DEGs were putatively involved in autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Furthermore, the DEGs included 149 interferon (IFN)-regulated genes. ConclusionsMyD88 is involved in the expression of specific genes associated with IFN-associated immunopathological processes in the salivary glands of NOD mice. Our findings are important for understanding the role of MyD88-dependent innate immune signaling in SS manifestation.

Л И М Ф О Ц И Т А Р Н А Я Р Н К К А К В О З М О Ж Н Ы Й А Д Ъ Ю В А Н Т П Р И С О З Д А Н И И П Р О Т И В О В И Р У С Н

Previously, we have demonstrated, using several experimental models, the presence of a pronounced morphogenetic activity of total RNA preparations isolated from lymphoid cells of healthy young animals, as well as lymphocytes of human peripheral blood, and the adequacy of this activity to that of the lymphocytes themselves was proved. Based on the revealed properties of such preparations, we propose to use the total RNA obtained from lymphocytes at the earliest stage of the recovery process in vaccinology as a universal adjuvant that will accelerate the induction of the reactivity of the infected person’s lymphocytes and increase the level of their activity, and therefore, enhance the body’s defense function and increase the duration of the immunity produced.

A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients

Plasmodium vivax is a world-threatening human malaria parasite, whose biology remains elusive. The unavailability of in vitro culture, and the difficulties in getting a high number of pure parasites makes RNA isolation in quantity and quality a challenge. Here, a methodological outline for RNA-seq from P. vivax isolates with low parasitemia is presented, combining parasite maturation and enrichment with efficient RNA extraction, yielding ~ 100 pg.µL−1 of RNA, suitable for SMART-Seq Ultra-Low Input RNA library and Illumina sequencing. Unbiased coding transcriptome of ~ 4 M reads was achieved for four patient isolates with ~ 51% of transcripts mapped to the P. vivax P01 reference genome, presenting heterogeneous profiles of expression among individual isolates. Amongst the most transcribed genes in all isolates, a parasite-staged mixed repertoire of conserved parasite metabolic, membrane and exported proteins was observed. Still, a quarter of transcribed genes remain functionally uncharacterized. In parallel, a P. falciparum Brazilian isolate was also analyzed and 57% of its transcripts mapped against IT genome. Comparison of transcriptomes of the two species revealed a common trophozoite-staged expression profile, with several homologous genes being expressed. Collectively, these results will positively impact vivax research improving knowledge of P. vivax biology.

Sensitive Extraction-Free SARS-CoV-2 RNA Virus Detection Using a Novel RNA Preparation Method

Current conventional detection of SARS-CoV-2 involves collection of a patient sample with a nasopharyngeal swab, storage of the swab during transport in a viral transport medium, extraction of RNA, and quantitative reverse transcription PCR (RT-qPCR). We developed a simplified and novel preparation method using a Chelex resin that obviates RNA extraction during viral testing. Direct detection RT-qPCR and digital-droplet PCR was compared to the current conventional method with RNA extraction for simulated samples and patient specimens. The heat-treatment in the presence of Chelex markedly improved detection sensitivity as compared to heat alone, and lack of RNA extraction shortens the overall diagnostic workflow. Furthermore, the initial sample heating step inactivates SARS-CoV-2 infectivity, thus improving workflow safety. This fast RNA preparation and detection method is versatile for a variety of samples, safe for testing personnel, and suitable for standard clinical collection and testing on high throughput platforms.Funding: This work was supported by the Intramural Research Programs of the National Institutes of Health (Clinical Center, NEI, NIDCR).Conflict of Interest: NEI (B.G. and R.B.H.) filed an invention disclosure. NEI has protected the intellectual property around this technology which is available for licensing and co-development. The remaining authors have no competing interests to declare.Ethical Approval: Samples were collected from healthy volunteers and subjects who provided informed consent to National Institutes of Health (NIH) Institutional Review Board (IRB)-approved protocols (20-D-0094, NCT04348240; 20-CC-0128, NCT04424446) at the NIH Clinical Center.

PURE mRNA display and cDNA display provide rapid detection of core epitope motif via high-throughput sequencing.

The reconstructed in vitro translation system known as the PURE system has been used in a variety of cell-free experiments such as the expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE-based display method for the preparation of stable messenger RNA (mRNA) and complementary DNA (cDNA)-peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry-over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. We chose the commercially available anti-FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 × 1012 random sequences, a round-by-round high-throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG epitope motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr (Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, the comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. We also show that gel purification steps in the refined PURE-based display method improve conjugate formation efficiency and enhance the enrichment rate of FLAG epitope motifs in later rounds of selection especially for mRNA display. Overall, the generalized procedure and consistent performance of two different display methods achieved by the commercially available PURE system will be useful for future studies to explore the sequence and functional space of diverse polypeptides.

Environmentally friendly method of RNA isolation

The diversity of organisms, tissues and cells is so great that, to date, no universal method for RNA extraction from these biological materials exist. The RNA isolation technique with a mix of guanidine thiocyanate, phenol, and chloroform is most widely used. Extraction and purification of RNA methods using selling guanidinium-phenol (TRIzol)-based and silica-based column kits have limitations on toxicity, or RNA isolation, particularly for plants, and scaling. The agents' toxicity is particularly relevant when employing for mass analysis in practice while gaining RNA preparations during the pandemics, epizootics, and epiphytotic. In modern diagnostics of infections at the molecular level, powerful RT-PCR technology is used, which amplifies the detection of RNA pathogens by hundreds of millions of times. We proposed obtaining RNA samples from viruses, bacteria, and plants for the reverse transcription reactions with a subsequent amplification of cDNAs by the polymerase chain reaction using potent and nontoxic chaotropic agent ammonium trichloroacetate. The method works in the analytical and preparative range and can be useful in the case of extraordinary circumstances during mass infections. Potentially this method can be adapted for obtaining RNA samples ready for the RT-isothermal PCR in the field.

Next-Generation Sequencing Reveals a Novel Emaravirus in Diseased Maple Trees From a German Urban Forest.

While the focus of plant virology has been mainly on horticultural and field crops as well as fruit trees, little information is available on viruses that infect forest trees. Utilization of next-generation sequencing (NGS) methodologies has revealed a significant number of viruses in forest trees and urban parks. In the present study, the full-length genome of a novel Emaravirus has been identified and characterized from sycamore maple (Acer pseudoplatanus) – a tree species of significant importance in urban and forest areas – showing leaf mottle symptoms. RNA-Seq was performed on the Illumina HiSeq2500 system using RNA preparations from a symptomatic and a symptomless maple tree. The sequence assembly and analysis revealed the presence of six genomic RNA segments in the symptomatic sample (RNA1: 7,074 nt-long encoding the viral replicase; RNA2: 2,289 nt-long encoding the glycoprotein precursor; RNA3: 1,525 nt-long encoding the nucleocapsid protein; RNA4: 1,533 nt-long encoding the putative movement protein; RNA5: 1,825 nt-long encoding a hypothetical protein P5; RNA6: 1,179 nt-long encoding a hypothetical protein P6). Two independent NGS sequencing runs from the same symptomatic maple tree detected the same genome segments. For one of these sequencing runs the cDNA library was prepared using a primer targeting the conserved genome terminal region, known to be shared between emaraviruses genome segments. We suggest, therefore, that the six identified genome segments represent the complete genome of a novel emaravirus from maple, which we tentatively name maple mottle-associated virus (MaMaV). Phylogenetic and sequence homology analyses place this virus on the distinct “subgroup a” clade within the Emaravirus genus along with – among others – rose rosette virus, Actinidia emaravirus 2, and fig mosaic virus. Validation RT-PCR assays performed on symptomatic and non-symptomatic trees suggest that MaMaV may be the symptom-inducing virus in the diseased trees. To our knowledge, this is the first time an Emaravirus is described from maple and is fully genetically characterized. With the discovery of MaMaV, the genus Emaravirus comprising negative-sense single-stranded viruses with very divergent genomes – that were until recently overlooked – has substantially increased counting 22 established and putative members.

Identification and Functional Characterization of Viroid Circular RNAs.

Viroids are relatively small, noncoding, plant circular RNAs. In contrast to other plant circular RNAs of endogenous origin, viroids are infectious agents able to replicate autonomously in the appropriate host. Because of their highly base-paired structures, they can be purified from infected tissue extracts using nonionic CF11 chromatography. Depending on the host plant species, viroid RNA preparation may also require polysaccharide removal by an extraction with 2-methoxyethanol followed by precipitation with cetyltrimethylammonium bromide. Electrophoretic analyses of this kind of preparations frequently show differential bands corresponding to the viroid circular molecules, which are absent in those from healthy plants. These RNA preparations can also be used for viroid transmission to new plants by mechanical inoculation.

The response of zinc transporter gene expression of selected tissues in a pig model of subclinical zinc deficiency

This study compared the relative mRNA expression of all mammal zinc (Zn) transporter genes in selected tissues of weaned piglets challenged with short-term subclinical Zn deficiency (SZD). The dietary model involved restrictive feeding (450 g/animal*day−1) of a high-phytate diet (9 g/kg) supplemented with varying amounts of zinc from ZnSO4*7H2O ranging from deficient to sufficient supply levels (total diet Zn: 28.1, 33.6, 38.8, 42.7, 47.5, 58.2, 67.8, 88.0 mg Zn/kg). Total RNA preparations comprised jejunal and colonic mucosa as well as hepatic and nephric tissue. Statistical modelling involved broken-line regression (P≤.05). ZIP10 and ZIP12 mRNAs were not detected in any tissue and ZnT3 mRNA was only identified in the kidney. All other genes were expressed in all tissues but only a few gene expression patterns allowed a significant (P<.0001) fitting of broken-line regression models, indicating homeostatic regulation under the present experimental conditions. Interestingly, these genes could be subcategorized by showing significant turnarounds in their response patterns, either at ~40 or ~60 mg Zn/kg diet (P<.0001). In conclusion, the present study showed clear differences in Zn transporter gene expression in response to SZD compared to the present literature on clinical models. We recognized that certain Zn transporter genes were regulated under the present experimental conditions by two distinct homeostatic networks. For the best of our knowledge, this represents the first comprehensive screening of Zn transporter gene expression in a highly translational model to human physiology.