RNA Analysis Research Articles

Intercellular TIMP-1-CD63 signaling directs the evolution of immune escape and metastasis in KRAS-mutated pancreatic cancer cells

Background and aimsOncogenic KRAS mutations are present in approximately 90% of pancreatic ductal adenocarcinoma (PDAC). However, Kras mutation alone is insufficient to transform precancerous cells into metastatic PDAC. This study investigates how KRAS-mutated epithelial cells acquire the capacity to escape senescence or even immune clearance, thereby progressing to advanced PDAC.MethodsSingle-cell RNA sequencing and analysis of primary PDAC tumors were conducted. Genetically engineered pancreas-specific Kras-mutated, dual specificity phosphatase-2 (Dusp2) knockout mouse models were established. Human and mouse primary pancreatic cancer cell lines were used for in vitro assessment of cancer characteristics. Tumor progression was studied via pancreas orthotopic and portal vein injection in the immune-competent mice. Clinical relevance was validated by digital spatial transcriptomic analysis of PDAC tumors.ResultsKras mutation induces the formation of pancreatic intraepithelial neoplasia (PanIN), these lesions also exhibit significant apoptotic signals. Single-cell RNA sequencing identified a subset of ERKactiveDUSP2low cells continuing to expand from early to advanced stage PDAC. In vitro and in vivo studies reveal that early infiltrating macrophage-derived tissue inhibitor of metallopeptidase 1 (TIMP-1) is the key factor in maintaining the ERKactiveDUSP2low cell population in a CD63-dependent manner. The ERKactiveDUSP2low cancer cells further exacerbate macrophage-mediated cancer malignancy, including loss of epithelial trait, increased lymphangiogenesis, and immune escape. Digital spatial profiling analysis of PDAC samples demonstrates the colocalization of TIMP-1high macrophages and CD63high cancer cells. The presence of TIMP-1high macrophages and CD63high epithelial cells correlates with poor prognosis in PDAC.ConclusionsOur study reveals the vicious cycle between early infiltrating macrophages and pancreatic cancer cells, providing a mechanistic insight into the dynamic regulation directing pancreatic cancer progression.

The Expression Pattern and Functional Analysis of Extracellular Vesicle Long Non-Coding RNAs from Uterine Fluid During Implantation in Pig

The Expression Pattern and Functional Analysis of Extracellular Vesicle Long Non-Coding RNAs from Uterine Fluid During Implantation in Pig

TRPM4 contributes to cell death in prostate cancer tumor spheroids, and to extravasation and metastasis in a zebrafish xenograft model system.

Transient receptor potential melastatin-4 (TRPM4) ion channel expression is upregulated in prostate cancer (PCa), contributing to increased cell proliferation, migration, adhesion, epithelial-to-mesenchymal transition, cell cycle shift, and alterations of intracellular Ca2+ signaling. GEO2R platform analysis of messenger RNA (mRNA) expression of ~ 6350 genes in normal and malignant prostate tissue samples from 15 PCa patients demonstrates that TRPM4 expression is upregulated sixfold and is among the most significantly upregulated genes in PCa. We find that absence of TRPM4 reduced PCa tumor spheroid size and decreased PCa tumor spheroid outgrowth. In addition, lack of TRPM4 increased cell death in PCa tumor spheroids, a phenotype that is absent in two-dimensional (2D) cancer cell systems. Lastly, absence of TRPM4 in PCa cells reduced extravasation and metastatic burden in a preclinical zebrafish cancer model. Taken together, our findings show that TRPM4 is an attractive therapeutic target in PCa and highlights the need for future development of pharmacological tools.

Developing Orthogonal Fluorescent RNAs for Photoactive Dual-color Imaging of RNAs in Live Cells.

Fluorogenic RNA aptamers have revolutionized the visualization of RNAs within complex cellular processes. A representative category of them employs the derivatives of green fluorescent protein chromophore, 4-hydroxybenzlidene imidazolinone (HBI), as chromophores. However, the structural homogeneity of their chromophoric backbones causes severe cross-reactivity with other homologous chromophores. This limitation impairs their multiplexing capabilities, which are essential for the simultaneous visualization of multiple RNA species in live cells. Herein, we rationally designed a series of red-shifted chromophores and employed SELEX-independent engineering to develop a novel fluorogenic RNA aptamer, mSquash. mSquash displays specific and intense fluorescence upon binding with our red-shifted chromophore DFHBFPD (Ex/Em = 501/624 nm). The mSquash/DFHBFPD allows orthogonal imaging of selected RNA targets alongside the established Broccoli/DFHBI-1T (Ex/Em = 472/501 nm), facilitating multiplexed live cell imaging of various targets. Moreover, we expanded the application of fluorescent RNA to photoactive imaging by constructing two genetically encoded photoactivatable fluorescent RNAs for the first time. This innovative approach allows photoactivatable control of fluorescent RNAs via specific light wavelengths (365 nm and 450 nm), enabling spatiotemporal dual-color imaging of RNAs in live cells. Our findings represent a significant advancement in fluorescent RNA-based orthogonal imaging and spatiotemporal analysis of RNAs.

Developing Orthogonal Fluorescent RNAs for Photoactive Dual‐color Imaging of RNAs in Live Cells

Fluorogenic RNA aptamers have revolutionized the visualization of RNAs within complex cellular processes. A representative category of them employs the derivatives of green fluorescent protein chromophore, 4‐hydroxybenzlidene imidazolinone (HBI), as chromophores. However, the structural homogeneity of their chromophoric backbones causes severe cross‐reactivity with other homologous chromophores. This limitation impairs their multiplexing capabilities, which are essential for the simultaneous visualization of multiple RNA species in live cells. Herein, we rationally designed a series of red‐shifted chromophores and employed SELEX‐independent engineering to develop a novel fluorogenic RNA aptamer, mSquash. mSquash displays specific and intense fluorescence upon binding with our red‐shifted chromophore DFHBFPD (Ex/Em = 501/624 nm). The mSquash/DFHBFPD allows orthogonal imaging of selected RNA targets alongside the established Broccoli/DFHBI‐1T (Ex/Em = 472/501 nm), facilitating multiplexed live cell imaging of various targets. Moreover, we expanded the application of fluorescent RNA to photoactive imaging by constructing two genetically encoded photoactivatable fluorescent RNAs for the first time. This innovative approach allows photoactivatable control of fluorescent RNAs via specific light wavelengths (365 nm and 450 nm), enabling spatiotemporal dual‐color imaging of RNAs in live cells. Our findings represent a significant advancement in fluorescent RNA‐based orthogonal imaging and spatiotemporal analysis of RNAs.

A Comprehensive Analysis of Sex-Biased Gene Expression in the Aging Human Retina Through a Combination of Single-Cell and Bulk RNA Sequencing.

Previous studies have reported divergent sexual responses to aging; however, specific variations in gene expression between aging males and females and their potential association with age-related retinal diseases remain unclear. This study collected data from public databases and developed a comprehensive comparison of retina between aging females and males. Single-cell RNA (scRNA) and bulk RNA sequencing data of the aging retina from females and males in public databases were utilized for integrated analysis to investigate sex-biased expression in retina. Additionally, in vitro experiments were conducted on individuals with retinitis pigmentosa (RP) to validate the sex difference in degenerative retina. Bulk RNA analysis revealed sex-biased expression of specific genes in retina of aging individuals, with immune pathway-related genes exhibiting higher expression in females compared to males. The scRNA analysis demonstrated that sex-biased gene expression was cell-type specific in aging retina. Furthermore, susceptibility genes for age-related macular degeneration and RP exhibited variation across different cell types and sexes. Cell-to-cell communication unveiled an increased interaction associated with TGFB1, CCL7, and VEGFA in Müller glia, microglia, and astrocytes of female retina. Notably, we observed female-biased chemokine expression in microglia contributing to heightened susceptibility to immune inflammation in female retina. Finally, we confirmed a more pronounced inflammatory response during degeneration in female rd10 mouse retina compared to males. This study provides a comprehensive comparison of retina between females and males in healthy aging human retina and highlights the significance of sex as an influential factor in retinal diseases.

Resilience and Response of Anaerobic Digestion Systems to Short-term Hydraulic Loading Shocks: Focusing on Total and Active Microbial Community Dynamics.

Anaerobic digestion is known to be sensitive to operational changes, such as hydraulic loading shock, yet the impact on the microbiome, particularly the active RNA-based community, has not been fully understood. This study aimed to investigate the performance of anaerobic reactors and their microbial communities under short-term hydraulic loading shocks. Using synthetic wastewater, the reactor was subjected to 24-hour shocks at three-fold and seven-fold the baseline loading rate, followed by DNA and RNA analyses to assess the system's resiliency and microbial responses. The research focused on shifts in major microbial groups and their functions, paying close attention to the active RNA community during loading shock events to better reflect the system's immediate condition. Findings indicated that although the microbial community structure, particularly among the archaea, was altered, the reactor quickly regained its balance. Differences were observed between DNA and RNA profiles and between regular and shock loadings; however, the alpha diversity and functions of the overall community were sustained. This study offers important insights for the design and operation of wastewater treatment plants, with the goal of achieving stable and efficient anaerobic digestion systems.

RNA analysis of patients with benign and malignant pulmonary nodules.

Pulmonary nodules are the main manifestations of early lung cancer. Non-small cell lung cancer is the most common histological type of lung cancer, and the main histological classification of non-small cell lung cancer is lung adenocarcinoma. The present study aimed to analyze the differentially expressed genes between patients with benign and malignant pulmonary nodules, and to identify potential therapeutic targets for lung adenocarcinoma. Sequencing data for benign and malignant pulmonary nodule samples and samples with no nodules were obtained from the National Center for Biotechnology Information Gene Expression Omnibus GSE135304 dataset. Differential gene analysis showed that S100 calcium binding protein P (S100P), ribonuclease A family member 2 (RNASE2), cytochrome c oxidase subunit 7C and mast cell expressed membrane protein 1 (C19orf59) were significantly upregulated among the blood samples collected from patients with malignant pulmonary nodules. Results from Kaplan-Meier plotter datasets showed that S100P, RNASE2 and C19orf59 were associated with the prognosis of lung cancer. RNASE2 expression was positively associated with nodule size and negatively associated with lung cancer prognosis. Moreover, RNASE2 was highly expressed in lung adenocarcinoma tissues compared with that in normal tissues. CCK-8 and Transwell assays indicated that overexpressed RNASE2 promoted the proliferation, migration and invasion of lung adenocarcinoma cells. In lung adenocarcinoma cells, RNASE2 was identified as a downstream target of microRNA (miR)-185-5p and was regulated by it. Inhibited cell proliferation, migration and invasion were observed following overexpression of miR-185-5p. Overexpression of RNASE2 reversed the inhibitory effect of miR-185-5p overexpression. In conclusion, in blood samples from patients with malignant pulmonary nodules and lung adenocarcinoma tissues, RNASE2 was found to be upregulated. High RNASE2 expression was associated with poor overall survival. miR-185-5p inhibited the proliferation, migration and invasion of lung adenocarcinoma cells by downregulating RNASE2 expression. These findings have implications for guiding therapeutic strategies.

Identification and Analysis of Circular RNAs in Mammary Gland from Yaks Between Lactation and Dry Period.

Lactation is a complex physiological process regulated by numerous genes and factors. Circular RNA (circRNA), a non-coding RNA, acts as a molecular sponge that sequesters microRNAs (miRNAs) to regulate target gene expression. Although circRNA has been linked to mammary gland lactation, its specific role in yaks remains underexplored. This study employed circular RNA sequencing (circRNA-seq) to examine the differential expression of circRNAs in yak mammary tissues during lactation and the dry period. Additionally, an enrichment analysis of the differentially expressed circRNAs (DECs) was performed. A competing endogenous RNA (ceRNA) network was then constructed to explore the potential of their roles in lactation and mammary gland development. We detected 18,905 circRNAs in yak mammary tissue, among which 302 showed differential expression. The host genes of these DECs were enriched in functions and pathways associated with yak milk synthesis and composition. Through the construction of a ceRNA network and the enrichment analysis of associated mRNAs, this study identified ceRNAs potentially involved in regulating lactation and mammary gland development. In conclusion, circRNAs in yak mammary tissues were identified and analyzed across lactation and dry periods, establishing a ceRNA network related to lactation regulation. These findings provide novel insights into the regulatory mechanisms governing lactation in yaks (Bos grunniens).

Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells.

The presence of non-coding RNAs, such as microRNAs (miRNAs), in mitochondria has been reported by several studies. The biological roles and functions of these mitochondrial miRNAs ("mitomiRs") have not been sufficiently characterized, but the mitochondrial localization of miRNAs has recently gained significance due to modified mitomiR-populations in certain states of diseases. Here, we describe the isolation and analysis of mitochondrial RNAs from rat liver tissue and HepG2 cells. The principle of the analysis is to prepare mitochondria by differential centrifugation. Cytosolic RNA contamination is eliminated by RNase A treatment followed by percoll gradient-purification and RNA-extraction. Small RNA content is verified by capillary electrophoresis. Mitochondrial miRNAs are detected by Q-PCR following synthesis of cDNA. After Q-PCR based mitomiR-profiling, the Normfinder algorithm is applied to identify suitable reference miRNAs to use as normalizers for mitochondrial input and data analysis. The described procedure depicts a simple way of isolating and quantifying mitomiRs in tissue and cell culture samples.

Extraction, Purification, and Next-Generation Sequencing (NGS) Analysis of DNA and RNA from Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue.

Formalin fixed paraffin embedded (FFPE) tissues have long been used for immunohistological analyses. FFPE tissues can be stored at room temperature for several years enabling analyses to be performed later. Ease of storage and transport makes these tissues an attractive source of biological material. However, formalin fixation results in chemical modifications of proteins and nucleic acids that poses a major challenge to any type of analysis. Recovery of nucleic acids for quantitative assays is rendered difficult due to degradation resulting from fixation and long-term storage, producing low usable yields. Extensive efforts in the last 20years have led to significant improvements in use of FFPE tissues for DNA and RNA analyses and resulted in development of sensitive assays for a wide range of applications, including next-generation sequencing. In this chapter, we describe the optimization of methods for sequential extraction of DNA and RNA from FFPE tissue and subsequent preparation of DNA-seq and RNA-seq libraries for use with the Illumina platform using commercially available reagents/kits.

Inhibitory effects of circR-127aa on gastric cancer progression and tumor growth.

This study investigates the function of a newly identified 127-amino acid peptide, circR-127aa, encoded by hsa_circ_0075402 (circRACK1), in gastric cancer (GC), a condition with significant prevalence in China. Utilizing a comprehensive analysis of circular RNA (circRNA) ribosome profiling data alongside experimental validations through mass spectrometry, Western blot, and immunofluorescence, we demonstrate that circR-127aa Inhibits Malignant Phenotypes and suppresses tumor growth in nude mice models. Significantly, the interaction of circR-127aa with Vimentin, a crucial element in actin-actin-cytoskeletal remodeling, indicates that circR-127aa functions as a tumor suppressor by facilitating the ubiquitination of Vimentin. These findings advance our comprehension of gastric cancer (GC) progression and propose circR-127aa as a promising therapeutic target and biomarker in the management of GC.

Whole Genome Identification and Integrated Analysis of Long Non-Coding RNAs Responding ABA-Mediated Drought Stress in Panax ginseng C.A. Meyer

Panax ginseng C.A. Meyer is a perennial herb that is used worldwide for a number of medical purposes. Long non-coding RNAs (lncRNAs) play a crucial role in diverse biological processes but still remain poorly understood in ginseng, which has limited the application of molecular breeding in this plant. In this study, we identified 17,478 lncRNAs and 3106 novel mRNAs from ginseng by high-throughput illumine sequencing. 50 and 257 differentially expressed genes (DEGs) and DE lncRNAs (DELs) were detected under drought + ABA vs. drought conditions, respectively. The DEGs and DELs target genes main enrichment is focused on the “biosynthesis of secondary metabolites”, “starch and sucrose metabolism”, and “carbon metabolism” pathways under drought + ABA vs. drought conditions according to KEGG pathway enrichment analysis, suggesting that these secondary metabolites biosynthesis pathways might be crucial for ABA-mediated drought stress response in ginseng. Together, we identified drought stress response lncRNAs in ginseng for the first time and found that the target genes of these lncRNAs mainly regulate the biosynthesis of secondary metabolites pathway to response to drought stress. These findings also open up a new visual for molecular breeding in ginseng.

Global Analysis of microRNA-like RNAs Reveals Differential Regulation of Pathogenicity and Development in Fusarium oxysporum HS2 Causing Apple Replant Disease.

This study investigated the expression profiles of microRNA-like RNAs (milRNAs) in Fusarium oxysporum HS2 (FoHS2), a key pathogen causing Apple replant disease (ARD), across spore to mycelium formation stages. Using small RNA sequencing (sRNA-seq) and bioinformatics, we identified and analyzed milRNAs, revealing their targeting of 2364 mRNAs involved in 20 functional categories, including metabolic and cellular processes, based on gene ontology (GO) analysis. An analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that these mRNAs are related to carbohydrate and amino acid metabolism pathways. Notably, the highest number of differentially or specifically expressed milRNAs (DEmilRNAs/SEmilRNAs) was found during the spore stage, with FoHS2-milR19 targeting genes encoding histone acetyltransferases, methyltransferases, and cell wall-degrading enzymes (CWDEs), which are crucial for growth, development, and pathogenicity. We validated the reliability of our sRNA-seq data and the expression of target genes using stem-loop RT-PCR and qRT-PCR. Our results highlight the stage-specific expression of milRNAs in FoHS2, particularly in the spore stage, suggesting a key role in regulating host life activities and providing a theoretical basis for developing RNA-based pesticides to control ARD.

Prognostic feature based on androgen-responsive genes in bladder cancer and screening for potential targeted drugs

ObjectivesBladder cancer (BLCA) is a tumor that affects men more than women. The biological function and prognostic value of androgen-responsive genes (ARGs) in BLCA are currently unknown. To address this, we established an androgen signature to determine the prognosis of BLCA.MethodsSequencing data for BLCA from the TCGA and GEO datasets were used for research. The tumor microenvironment (TME) was measured using Cibersort and ssGSEA. Prognosis-related genes were identified and a risk score model was constructed using univariate Cox regression, LASSO regression, and multivariate Cox regression. Drug sensitivity analysis was performed using Genomics of drug sensitivity in cancer (GDSC). Real-time quantitative PCR was performed to assess the expression of representative genes in clinical samples.ResultsARGs (especially the CDK6, FADS1, PGM3, SCD, PTK2B, and TPD52) might regulate the progression of BLCA. The different expression patterns of ARGs may lead to different immune cell infiltration. The risk model indicates that patients with higher risk scores have a poorer prognosis, more stromal infiltration, and an enrichment of biological functions. Single-cell RNA analysis, bulk RNA data, and PCR analysis support the reliability of this risk model, and a nomogram was also established for clinical use. Drug prediction analysis showed that high-risk patients had a better response to fludarabine, AZD8186, and carmustine.ConclusionARGs played an important role in the progression, immune infiltration, and prognosis of BLCA. The ARGs model has high accuracy in predicting the prognosis of BLCA patients and provides more effective medication guidelines.

Utilizing the Fungal Bicistronic System for Multi-Gene Expression to Generate Insect-Resistant and Herbicide-Tolerant Maize.

Developing simple and efficient multi-gene expression systems is crucial for multi-trait improvement or bioproduction in transgenic plants. In previous research, an IGG6-based bicistronic system from the nonpathogenic fungus Glarea lozoyensis efficiently expressed multiple enzyme proteins in yeast and maize, and the heterologous enzymes successfully performed their catalytic activity to reconstruct the biosynthetic pathway in the host organism. Unlike enzyme proteins, some heterologous functional proteins (such as insecticidal proteins) are dose-dependent and they need to express sufficient levels to perform their biological functions. It remains unclear whether the IGG6-based bicistronic system can achieve high expression of the functional proteins for practical applications in crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, vip3Aa and cry1Ab, were linked via IGG6 to form a bicistron, while two glyphosate resistance genes, gr79epsps and gat, served as monocistronic selectable marker genes. Regenerated maize plants were produced through genetic transformation. RNA and immunoblot analyses revealed that the vip3Aa-IGG6-cry1Ab bicistron was transcribed as a single transcript, which was then translated into two separate proteins. Notably, the transcription and translation of cry1Ab were significantly positively correlated with those of vip3Aa. Through ELISA and leaf bioassay, we identified two transgenic maize lines, VICGG-15 and VICGG-20, that exhibited high insecticidal activity against fall armyworm (FAW; Spodoptera frugiperda) and Asian corn borer (ACB; Ostrinia furnacalis), both of which had high expression of Vip3Aa and Cry1Ab proteins. Subsequent evaluations, including silk, ear, and field bioassays, as well as glyphosate tolerance assessments, indicated that the VICGG-15 plants displayed high resistance to FAW and ACB, and could tolerate up to 3600 g acid equivalent (a.e.) glyphosate per hectare without adversely affecting phenotype or yield. Our finding established that the IGG6-based bicistronic system can achieve high expression of functional proteins in maize, and it is a potential candidate for multi-gene assembly and expression in plants.

MRNA vaccines as cancer therapies.

Cancer remains a major global health challenge, with conventional treatments like chemotherapy and radiotherapy often hindered by significant side effects, lack of specificity, and limited efficacy in advanced cases. Among emerging therapeutic strategies, mRNA vaccines have shown remarkable potential due to their adaptability, rapid production, and capability for personalized cancer treatment. This review provides an in-depth analysis of messenger RNA (mRNA) vaccines as a therapeutic approach for cancer immunotherapy, focusing on their molecular biology, classification, mechanisms, and clinical studies. Derived from reported literature and data on clinicaltrials.gov, it examines studies on mRNA vaccines encoding tumor-specific antigens (TSAs), tumor-associated antigens (TAAs), immunomodulators, and chimeric antigen receptors (CARs) across various cancer types. The review highlights the ability of mRNA vaccines to encode TSAs and TAAs, enabling personalized cancer treatments, and classifies these vaccines into non-replicating and self-amplifying types. It further explores their mechanisms of action, including antigen presentation and immune activation, while emphasizing findings from clinical studies that demonstrate the potential of mRNA vaccines in cancer therapy. Despite their promise, challenges remain in enhancing delivery systems, improving immunogenicity, and addressing tumor heterogeneity. Overcoming these obstacles will require further investigation to fully harness the potential of mRNA vaccines in personalized cancer treatment.

Transcriptome analyses of mRNA and circular RNAs reveal dietary supplementation with freeze-dried Lactiplantibacillus plantarum primes immune memory of White leg shrimp (Penaeus vannamei) against pathogens.

The lack of a classical adaptive immunity renders the development of disease control and prevention measures in shrimp challenging. In this study, the concept of trained immunity was exploited in the development of a feed supplement. Penaeus vannamei shrimp was fed with feed supplemented with freeze-dried whole culture of Lactiplantibacillus plantarum (FD-LAB) for 15 days. RNA sequencing using Illumina platform was performed on the gill and stomach tissues collected at specific time points during the feeding period (0th day, 8th day, 15th day). Differentially-expressed genes (DEGs) previously reported to have innate immunity- and immune memory-related functions were selected for validation. Additionally, the differential expression of putatively immune-related circular RNAs (DECs) were also explored as these noncoding regulatory RNAs may also influence host immunity. Challenge tests with either the acute hepatopancreatic necrosis disease-causing strain Vibrio parahaemolyticus D6 or White Spot Syndrome Virus (WSSV) were conducted. Transcriptome analyses showed that FD-LAB supplementation resulted to DEGs and DECs related to pathogen recognition, antimicrobial peptides, transcription regulation, and immune memory. Challenge tests performed immediately after 15 days and 8 days of feeding showed protection on P. vannamei by FD-LAB against bacterial and viral pathogens. Increase in survival rates were also observed upon challenge with both pathogens 7 days and 14 days after last intake of FD-LAB, indicating trained immunity in shrimp. Our study highlighted the effects of FD-LAB on the innate immunity and immune memory of P. vannamei against bacterial and viral pathogens. These findings emphasize the possibility of immunostimulants inducing lasting enhanced immunity against infections despite the lack of a classical adaptive immunity in shrimp.

Identification of Two Novel Recombinant Types of Potato Virus Y from Solanum tuberosum Plants in Southern Region of Russia.

Potato virus Y (PVY, genus Potyvirus, family Potyviridae) is one of the most devastating and economically important potato pathogens. Members of the Potyviridae family demonstrate high recombination rates. In nature, 5 major parental variants of PVY were identified with at least 35 recombinants. In this study we report two novel PVY recombinants discovered in the sprouts of potato tubers produced in field conditions in Astrakhan region of Russia in 2021 using high-throughput sequencing and de novo genome assembly. These recombinants, which were named Ast-A-I and Ast-A-II, have previously unknown arrangements of genome sections derived from PVYO and PVYN with a novel recombination junction at the position 7850 nt of the PVY genome, in the middle part of the RNA-dependent RNA polymerase (NIb) gene, with PVYO-type sequence at the 5'-end of this junction and PVYN-type at the 3'-end. Other recombinant junctions in the novel PVY variants were previously found in PVYNTNa and PVYNTNb. This includes those at the positions 2390 and 9200 (PVYN at the 5'-end and PVYO at the 3'-end) which were present in both novel recombinants, and at the position 500 (PVYO at the 5'-end and PVYN at the 3'-end) which present in Ast-A-II. The presence of PVY variants with novel recombinant point is verified by Sanger sequencing. Phylogenetic analysis of genomic RNA showed that the sequences of these recombinants form a separate branch which do not cluster with previously described PVY strains.

Successful Amendment of an Existing Hepatitis B Screening Programme by a Guideline Recommended Hepatitis D Screening in the Primary Care Setting.

Despite European guidelines recommending anti-hepatitis D virus (HDV) screening for all hepatitis B surface antigen (HBsAg)-positive cases, screening rates remain insufficient. We analysed anti-HDV screening rates in primary care and implemented prospective HDV screening in HBsAg-positive cases identified in the preventive medical examination from the age of 35 ("Check-Up 35+"). From 2012 to 2021, we reviewed anti-HDV and HDV RNA test rates in HBsAg-positive patients at 11 sites of a large German laboratory group. From 2022 to 2023, we prospectively screened HBsAg-positive samples from the "Check-Up 35+" for anti-HDV. Anti-HDV positive patients were then contacted again for HDV RNA testing. Retrospectively, 2792/13,905 (20%) HBsAg-positive cases were tested for anti-HDV, with 142/2792 (5.1%) being positive. HDV RNA was tested in 57/142 (40%) anti-HDV-positive cases, with 26/57 (46%) being positive. In the prospective screening, 1159/225,901 (0.51%) individuals were HBsAg-positive. Of these, 700 (60%) were tested for anti-HDV, with 18/700 (2.6%) positive test results. 4/18 (22%) were successfully contacted again for HDV RNA analysis, with one case testing positive. Neither the HBsAg nor the anti-HDV positive result was known prior to screening in these cases. Anti-HDV testing could not be performed in 459/1159 (40%) HBsAg-positive cases, primarily due to insufficient blood sample volume (310/459 cases, 68%), with others missed due to logistical errors. Retrospective data show insufficient anti-HDV screening in clinical routine. The prospective anti-HDV screening provides a blueprint for using existing hepatitis B virus screening programms for population-based HDV double reflex testing, provided that adequate logistical prerequisites are established. German Clinical Trial Register: DRKS00029180.