Disease classification based on biological data is an important area in bioinformatics and biomedical research. It helps the doctors and medical practitioners for the early detection of disease and support them as a computer-aided diagnostic tool for accurate diagnosis, prognosis, and treatment of disease. Earlier Microarray gene expression data have wide application for the classification of disease, but now Next-generation sequencing (NGS) has replaced the Microarray technology. From the last few years, RNA sequence (RNA-Seq) data are widely used for the transcriptomic analysis. Hence, RNA-Seq based classification of disease is in its infancy. In this article, we present a general framework for the classification of disease constructed on RNA-Seq data. This framework will guide the researchers to process RNA-Seq, extract relevant features and apply the appropriate classifier to classify any kind of disease.

Abstract: (1) Background: Cancer immune escape is associated with the metabolic reprogramming of TME, and combining metabolic targets with immunotherapy has great potential to improve clinical outcomes. Among all metabolic processes, lipid metabolism, especially fatty acid (FA) metabolism, has a huge role in cancer cell survival, migration, and proliferation, but its mechanism and role in the tumor immune microenvironment remain to be investigated. (2) Methods: We comprehensively analyzed 309 fatty acid-related genes, screened 121 different genes, and used one-way COX regression to select 15 genes with prognostic impact. Systematically evaluated the correlation between FMGs modification patterns and Tumor Microenvironment Infiltration, prognosis, and Immunotherapy. The FMGs-Score was constructed to quantify the FMGs modification patterns using principal component analysis. (3) Results: Three clusters based on FMGs-related genes were demonstrated in breast cancer, with three patterns of distinct immune cell infiltration and biological behavior. An FMGsScore signature was constructed to reveal that patients with a low FMGsScore had higher immune checkpoint expression, higher immune checkpoint inhibitor (ICI) scores, increased immune microenvironment infiltration, better survival advantage, and were more sensitive to immunotherapy than those with a high FMGsScore. Finally, the expression and function of the signature key gene NDUFAB1 were examined by in vitro experiments. (4) Conclusions: This study significantly demonstrates the non-negligible role of FMGs in the immune microenvironment of breast cancer, and that FMGsScores can be used to guide the prediction of immunotherapy in breast cancer patients. In in vitro experiments, knockdown of the NDUFAB1 gene resulted in reduced proliferation and migration of MCF-7 cell lines.

The identification and molecular characterization of cellular hierarchies in complex tissues is key to understanding both normal cellular homeostasis and tumorigenesis. The mammary epithelium is a heterogeneous tissue consisting of two main cellular compartments, an outer basal layer containing myoepithelial cells and an inner luminal layer consisting of estrogen receptor-negative (ER⁻) ductal cells and secretory alveolar cells (in the fully functional differentiated tissue) and hormone-responsive estrogen receptor-positive (ER⁺) cells. Recent publications have used single-cell RNA-sequencing (scRNA-seq) analysis to decipher epithelial cell differentiation hierarchies in human and murine mammary glands, and reported the identification of new cell types and states based on the expression of the luminal progenitor cell marker KIT (c-Kit). These studies allow for comprehensive and unbiased analysis of the different cell types that constitute a heterogeneous tissue. Here we discuss scRNA-seq studies in the context of previous research in which mammary epithelial cell populations were molecularly and functionally characterized, and identified c-Kit⁺ progenitors and cell states analogous to those reported in the recent scRNA-seq studies.

RNA-seq faces persistent challenges due to the ongoing expanding array of data processing workflows, none of which have yet achieved standardization to date. It is imperative to determine which method most effectively preserves biological facts. Shannon entropy serves as a tool for depicting the biological status of a system. Thus, we assessed the measurement of Shannon entropy by several RNA-seq workflow approaches, employing RPKM and median normalization on paired samples of 475 TCGA RNA-seq datasets spanning eight different cancer types with 5-year overall survival rates ranging from 30% to 98%. Our analysis revealed that the RPKM normalization coupled with a threshold of log2 fold change ≥1 for indentifying differentially expressed genes, yielded the best results with a correlation coefficient of 0.91. We propose that Shannon entropy can serve as an objective metric for refining the optimization of RNA-seq workflows.

Solanesol is a noncyclic terpene alcohol composed of nine isoprene units and it mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.). Here, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase, two 1-deoxy-d-xylulose 5-phosphate reductoisomerase, two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase, four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase, two 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, four 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, two 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, six isopentenyl diphosphate isomerase, and two solanesyl diphosphate synthase (SPS) genes were identified to be involved in solanesol biosynthesis. Furthermore, the two N. tabacum SPS (NtSPS1 and NtSPS2), which had two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs, and leaves from four growing stages, corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthetic genes and the accumulation of solanesol in tobacco.

RNA sequencing techniques, like bulk RNA-seq and Single Cell (sc) RNA-seq, are critical tools for the biologist looking to analyze the genetic activity/transcriptome of a tissue or cell during an experimental procedure. Platforms like Illumina's next-generation sequencing (NGS) are used to produce the raw data for this experimental procedure. This raw FASTQ data must then be prepared via a complex series of data manipulations by bioinformaticians. This process currently takes place on an unwieldy textual user interface like a terminal/command line that requires the user to install and import multiple program packages, preventing the untrained biologist from initiating data analysis. Open-source platforms like Galaxy have produced a more user-friendly pipeline, yet the visual interface remains cluttered and highly technical, remaining uninviting for the natural scientist. To address this, SeqMate is a user-friendly tool that allows for one-click analytics by utilizing the power of a large language model (LLM) to automate both data preparation and analysis (differential expression, trajectory analysis, etc). Furthermore, by utilizing the power of generative AI, SeqMate is also capable of analyzing such findings and producing written reports of upregulated/downregulated/user-prompted genes with sources cited from known repositories like PubMed, PDB, and Uniprot.

Astrocytes perform a wide variety of essential functions defining normal operation of the nervous system, and are active contributors to the pathogenesis of neurodegenerative disorders such as Alzheimer among others. Recent data provide compelling evidence that distinct reactive astrocyte states are associated with specific stages of Alzheimer´s disease. The advent of transcriptomics technologies enables rapid progress in the characterisation of such pathological astrocyte states. In this review, we provide an overview of the origin, main functions, molecular and morphological features of astrocytes in physiological as well as pathological conditions related to Alzheimer´s disease. We will also explore the main roles of astrocytes in the pathogenesis of Alzheimer´s disease and summarize main transcriptional changes and altered molecular pathways observed in astrocytes during the course of the disease.

Аnalysis of small RNA sequencing data across a range of biofluids is a significant research area, given the diversity of RNA types that holds potential diagnostic, prognostic, and predictive value. The intricate task of segregating the complex mixture of small RNAs from both human and other species, including bacteria, fungi, and viruses, poses one of the most formidable challenges in the analysis of small RNA sequencing data, currently lacking satisfactory solutions. This study introduces sRNAflow, a user-friendly bioinformatic tool with a web interface designed for the analysis of small RNAs obtained from biological fluids. Tailored to the unique requirements of such samples, the proposed pipeline addresses various challenges, including filtering potential RNAs from reagents and environment, classifying small RNA types, managing small RNA annotation overlap, conducting differential expression assays, analysing isomiRs, and presenting an approach to identify the sources of small RNAs within samples. sRNAflow also encompasses an alternative alignment-free analysis of RNA-seq data, featuring clustering and initial RNA source identification using BLAST. This comprehensive approach facilitates meaningful comparisons of results between different analytical methods. The source code can be accessed at https://github.com/zajakin/sRNAflow under the GPL3 licence.

Expression analysis of small noncoding RNA (sRNA), including microRNA, piwi-interacting RNA, small rRNA-derived RNA, and tRNA-derived small RNA, is a novel and quickly developing field. Despite a range of proposed approaches, selecting and adapting a particular pipeline for transcriptomic analysis of sRNA remains a challenge. This paper focuses on the identification of the optimal pipeline configurations for each step of human sRNA analysis, including reads trimming, filtering, mapping, transcript abundance quantification and differential expression analysis. Based on our study, we suggest the following parameters for analysis of human sRNA in relation to categorical analyses with two groups of biosamples: (1) trimming with the lower length bound = 15 and the upper length bound = \(Read\ length - 40\% Adapter\ length\); (2) mapping on a reference genome with bowtie aligner with one mismatch allowed (-v 1 parameter); (3) filtering by mean threshold > 5; and (4) analyzing differential expression with DESeq2 with adjusted p-value < 0.05 or limma with p-value < 0.05 if there is very little signal and few transcripts.

During the last few years, advancement in the area of biochemistry, science of the material world, engineering and computer-aided testing has directed towards advancement of high-throughput tools for the sake of profiling information encoded in a gene. Single-cell RNA-sequencing (scRNA-seq) tools capable to examine the sequence data from each individual cells and it shows within population variety and permit exploring of cell conditions and transformation by extreme resolution, possibly give out cell sub-types or gene expression fluctuations that are shaded in mass sequencing processes, which shows population-averaged evaluations. Yet, the major disadvantage for this tool is the lack of success to pick out location related details of the RNA transcriptome, as this needs tissue detachment and cell isolation. Location based transcript determination is among the advancements in the area of medical biotechnology as this can find out the molecules like RNA dataset in their intact physical placement in tissue segment with spatial context at the scale of a single-cell, which is very advantageous as compare to single-cell sequencing techniques. These approaches give key observation in the areas of biomedical field sub-disciplines like neurology, embryology, carcinoma study, immune cell investigation and histological activities. Generally, this review mainly focused towards single-cell sequencing methods, technology development, its challenges observed, different expression data analysis mechanisms and their applications in different areas, such as in cancer research, microbes, central nervous system, sex organs and immune-biology, intensifying the essentiality of sequencing tools at single-cell level for the characterization of extremely dynamic individual cells.

COVID-19 was an inflammatory pneumonia caused by a respiratory infection with a coronavirus named SARS-CoV-2. Severely ill COVID-19 patients could die of acute pulmonary and systemic inflammation. Thus, there was a theory that long COVID-19 shared many similarities with systemic autoimmune diseases. There was also a theory that the SARS-CoV-2 virus could infect many cell- and tissue-types distributed throughout multiple organ systems of the body. I aim to review the above theories using unbiased high-throughput datasets cataloguing gene expression. Studies reviewed used the technology of next generation sequencing, either as bulk tissue RNA sequencing, or as single-cell RNAs sequencing; ingenious methods for signal deconvolution were then used to identify individual cell types. Datasets reviewed suggested that severe COVID-19 induced expression of genes associated with pro-inflammatory signaling, tissue development or remodeling, scarring, or cell death. The datasets also provided evidence for several inflammatory syndromes associated with COVID-19, such as neuronal COVID, acute respiratory disease syndrome, vascular inflammation, or multisystem inflammatory syndrome. Indeed, inflammatory signal during severe COVID-19 was reported in many cell types, for example in structural cells, in infiltrating immune cells, in the vascular system, or in circulating cells in the blood. There was also frequently a change in proportions of immune cell-types in the blood, epithelial cells in the lungs, or among infiltrating immune cells.

Sugarcane is an important sugar-source crop. As any other plant, it can be exposed to several abiotic stress conditions. Though some metals contribute to critical physiological processes in plants, the presence of aluminum ions (Al3+) can be very toxic. In order to develop plants that flourish in acidic soils, it is critical to gain insights into the molecular mechanisms of sugarcane response to aluminum stress. To determine the genes involved in sugarcane response to aluminum stress we generated 372 million paired-end RNA sequencing reads, from roots of CTC-2 and RB855453 two contrasting cultivars. Data normalization resulted in 162,161 contigs and 97,335 trinity genes. After the read cutoff, the differentially expressed genes were 4,858 in CTC-2 and 1,307 in the RB855453, Treatment Vs Control, respectively. The differentially expressed genes were annotated into 34 functional categories. The majority of the genes were upregulated in the CTC-2 (tolerant cultivar) and down regulated in RB855453 (sensitive cultivar). Here, we present the first root-transcriptome of sugarcane under aluminum stress. The results and conclusions of this study provide a valuable resource for future genetic and genomic studies in sugarcane. This transcriptome analysis points out that sugarcane tolerance to aluminum may be explained by an efficient detoxification mechanism combined with the lateral root formation and activation of redox enzymes. Following our results, we present here, a hypothetical model for the aluminum tolerance in CTC-2 cultivar.

One of the most frequent brain diseases, Alzheimer's is defined by poor cognitive function brought on by the build-up of Beta Amyloid plaques and the gradual death of neurons. Glucose metabolism and the development of amyloid plaques are being studied together. Under physiologically normal circumstances, glucose is the primary substrate for the adult human brain. The prodromal phases of AD are significantly influenced by glucose hypometabolism. Hypometabolism of glucose in the brain is a clear sign of mitochondrial dysfunction and bioenergetic system impairment. By regulating energy synthesis and cell death, mitochondria play a crucial role in the functioning of cells. Increased formation of reactive oxygen species (ROS) and oxidative stress are a result of mitochondrial dysfunction, which also accelerates the development of Alzheimer's disease. For the maintenance of balance, autophagy is crucial because it selectively destroys damaged mitochondria. AD affects this route for mitochondrial breakdown. Targeting specific mitochondrial ligands by interventions along this pathway might be a useful therapeutic approach. Due to a number of biological obstacles, this method has significant limitations. As a result, many nanocarriers have been created to improve drug delivery effectiveness. All potential nanotechnology-based treatments for AD have been examined in this study, with a particular emphasis on medication delivery to the mitochondria

Precision medicine emphasizes fine-grained diagnostics, taking individual variability into account to enhance treatment effectiveness. Parkinson's Disease (PD) heterogeneity among individuals is a proof that disease subtypes exist, and assigning individuals to subgroups is necessary for a better understanding of disease mechanisms and designing precise treatment approaches. The purpose of this study was to identify PD subtypes using RNA-Seq data in a combined pipeline including unsupervised machine learning, bioinformatics, and network analysis. 210 post mortem brain RNA-Seq samples from PD (n = 115) and Normal Controls (NC, n = 95) were obtained with a systematic data retrieval following PRISMA statements and a fully data-driven clustering pipeline was performed to identify PD subtypes. Bioinformatics and Network analyses were performed to characterize the disease mechanisms of the identified PD subtypes and to identify target genes for drug repurposing. Two PD clusters were identified and 42 DEGs were found (p.adjusted ≤ 0.01). PD clusters had significantly different gene network structures (p &lt; 0.0001) and phenotype-specific disease mechanisms, highlighting the differential involvement of the Wnt/β-catenin pathway regulating adult neurogenesis. NEUROD1 was identified as a key regulator of gene networks and ISX9 and PD98059 were identified as NEUROD1-interacting compounds with disease-modifying potential, reducing the effects of dopaminergic neurodegeneration. This hybrid data analysis approach could enable precision medicine applications by providing insights for the identification and characterization of pathological subtypes. This workflow has proven useful on PD brain RNA-Seq, but its application to other neurodegenerative diseases is encouraged.

The generation of different types of defective viral genomes (DVG) is an unavoidable consequence of the error-prone replication of RNA viruses. In recent years, a particular class of DVGs, those containing long deletions or genome rearrangements, has gain interest due to their potential therapeutic and biotechnological applications. Identifying such DVGs in high-throughput sequencing data has become an interesting computational problem. Up to nowadays, several algorithms have been proposed, though all incur in false positives, a problem of practical interest if such DVGs have to be synthetized and tested in the laboratory. Here we develop a novel software, DVGfinder, that wraps the two most commonly used algorithms into a pipeline that predicts DVGs. Using a gradient boosting classifier machine learning algorithm, we evaluate the performance of DVGfinder compared to previous algorithms and found that it outcompetes their precision and sensitivity in simulated datasets. DVGfinder generates user-friendly output files in HTML format that can assist users to identify DVGs based on their associated probability of being true positives.

Recently the increased cost-effectiveness of high-throughput technologies has made available a large number of RNA sequencing datasets to identify circular RNAs (circRNAs). However, despite many computational tools were developed to predict circRNAs, a limited number of workflows exists to predict and to characterize circRNAs. Moreover, to the best of our knowledge, these available workflows do not ensure computational reproducibility and require advanced bash scripting skills to be correctly installed and used. To cope with these critical aspects we present Docker4Circ, a new computational framework designed for a comprehensive analysis of circRNAs composed of: circRNAs prediction, classification and annotation using public databases, the back-splicing sequence reconstruction; the internal alternative splicing of circularizing exons; the alignment-free circRNAs quantification from RNA-Seq reads, and, finally, their differential expression analysis. Docker4Circ was specifically designed for making easier and more accessible circRNAs analysis thanks to the following features: (i) its R interface; (ii) the encapsulation of its computational tasks into a docker image; (iii) an available user-friendly Java GUI Interface. Furthermore, Docker4Circ ensures a reproducible analysis because all its tasks were embedded into a docker image following the guidelines provided by Reproducible Bioinformatics Project (RBP, http://reproducible-bioinformatics.org/). The effectiveness of Docker4Circ was demonstrated on a real case study whose goal is to characterize the circRNAs predicted in colorectal cancer cell lines and quantified in public RNA-Seq experiments performed on primary tumor tissues. In conclusion, we propose Docker4Circ as a framework for reproducible and comprehensive analyses of circRNAs to efficiently exploit their biological role.

Our recent work has focused on the application of infinite group theory and related algebraic geometric tools in the context of transcription factors and microRNAs. We were able to differentiate between “healthy" nucleotide sequences and disrupted sequences that may be associated with various diseases. In this paper, we extend our efforts to the study of messenger RNA metabolism, showcasing the power of our approach. We investigate (i) mRNA translation in prokaryotes and eukaryotes, (ii) polyadenylation in eukaryotes, which is crucial for nuclear export, translation, stability, and splicing of mRNA, (iii) miRNAs involved in RNA silencing and post-transcriptional regulation of gene expression, and (iv) the identification of disrupted sequences that could lead to potential illnesses. To achieve this, we employ (a) infinite (finitely generated) groups fp, with generators representing the r+1 distinct nucleotides and a relation between them [e.g., the consensus sequence in the mRNA translation (i), the poly(A) tail in item (ii), and the miRNA seed in item (iii)], (b) aperiodicity theory, which connects “healthy groups" fp to free groups Fr of rank r and their profinite completion F^r, and (c) the representation theory of groups fp over the space-time-spin group SL2(C), highlighting the role of surfaces with isolated singularities in the character variety. Our approach could potentially contribute to the understanding of the molecular mechanisms underlying various diseases and help develop new diagnostic or therapeutic strategies.

Estrogen receptor-positive (ER+) breast cancer is common among postmenopausal women and is frequently treated with Letrozole. Letrozole inhibits aromatase from synthesizing estrogen from androgens. Decreased estrogen slows the growth of tumors and can be an effective treatment. Letrozole resistance is increasingly being observed, which poses a unique problem for patients. We reanalyzed transcriptomic data comparing individuals who responded to Letrozole therapy (responders) to those who were resistant to treatment (non-responders). We identified SOX11 and S100A9 as two genes that significantly differed between these patients; with “PLK1 signaling events” being the most significant signaling pathway. We also identified PRDX4 and E2F8 as the top transcriptional biomarkers for ER+ treatment resistance. Many of the other significant gene products play a known role in ER+ breast cancer or other types of cancer, which partially validate our results. Several of the gene products we identified do not have a characterized role in ER+ breast cancer. Our analysis identified a set of genes that warrant further research to elucidate the molecular mechanism of Letrozole resistance in this patient population, as well as novel high-performing biomarkers. We anticipate that these findings could contribute to improved therapeutic outcomes in human patients.

Emerging infectious disease threats require rapid response tools to inform diagnostics, treatment, and outbreak control. RNA-based metagenomics offers this; however, most approaches are time-consuming and laborious. Here, we present a simple and fast protocol – the RAPIDprep assay – with the aim to provide cause agnostic laboratory diagnosis of infection within 24 hours of sample collection by sequencing ribosomal RNA-depleted total RNA. The method is based on the synthesis and amplification of double-stranded cDNA followed by short-read sequencing with minimal handling and clean-up steps to improve processing time. The approach was optimized and applied to a range of clinical respiratory samples to demonstrate diagnostic and quantitative performance. Our results showed robust depletion of both human and microbial rRNA, and library amplification across different sample types, qualities and extraction kits using a single protocol without input nucleic acid quantification or quality assessment. Furthermore, we demonstrate the genomic yield of both known and undiagnosed pathogens with complete genomes recovered in most cases to inform molecular epidemiological investigations and vaccine design. The RAPIDprep assay is a simple and effective tool, and representative of an important shift towards integration of modern genomic techniques to infectious disease investigations.

RNA sequencing (RNA-Seq) is a complicated protocol, both in the laboratory in generation of data and at the computer in analysis of results. Several decisions during RNA-Seq library construction have important implications for analysis, most notably strandedness during complementary DNA (cDNA) library construction. Here we clarify bioinformatic decisions related to strandedness in both alignment of DNA sequencing reads to reference genomes and subsequent determination of transcript abundance.

Breast cancer (BC) is the second leading cause of death in the United States in women. 1 in 8 women in their lifetime has a risk of developing breast cancer. With advances in omics technology, more data is available for diagnosis, treatment, and prognosis. The help of Machine Learning algorithms, a subdomain of Artificial Intelligence, would allow for a better clinical support tool. Machine Learning could be the key to answering a complex problem in BC. The methodology of this research question involves collecting and finding data such as RNA-Seq datasets, selecting and training appropriate machine learning algorithms, and evaluating the performance of the algorithms in predicting BC prognosis. Also, the study explores and tries to identify the most relevant genes and pathways through feature selection extraction and dimensionality reduction techniques. After applying feature extraction, the top 10 genes were extracted using the Univariate Feature Selection (UFS) method, precisely the SelectKBest technique. After employing different machine learning algorithms, such as Networks, Random Forest, Linear SVM, Logistic Regression, and Quadratic Discriminant Analysis (QDA), we found QDA was the best model in classifying the RNA-seq dataset with a 96% accuracy rate and 94% ROC AUC. This study suggests that AK2 and CD68 have a positive correlation with each other, and it could potentially be a biomarker and therapeutic target for BC.

Corynebacterium pseudotuberculosis is a Gram-positive bacterium that causes caseous lymphadenitis, a disease that predominantly affects sheep, goat, cattle, buffalo, and horses, but has also been recognized in other animals. This bacterium generates a severe economic impact on countries producing meat. Gene expression studies using RNA-seq is one of the most commonly used techniques to perform transcriptional experiments. Computational analysis on such data through reverse-engineering algorithms leads to a better understanding of the genome-wide complexity of gene interactomes, enabling the identification of genes having the most significant functions inferred by the activated stress response pathways. In this study, we identified the influential or causal genes from four RNA-seq data-sets from different stress conditions (high iron, low iron, acid, osmosis, and PH) in C. pseudotuberculosis, using a consensus-based network inference algorithm called miRsig and identified the causal genes in the network using the miRinfluence tool, which is based on the influence diffusion model. We found that over 50\% of the genes identified as influential have some essential cellular functions in the genomes. In the strains analyzed, most of the causal genes have crucial roles or participate in processes associated with response to extracellular stresses, pathogenicity, membrane components, and essential genes. This research brings new insight into the understanding of virulence and infection by C. pseudotuberculosis.

Mitochondria are semiautonomous organelles, which are pivotal for the energy metabolism of eukaryotic cells. These organelles contain their own genetic system (mtDNA, mitogenome) that encodes to some structural RNAs and organellar proteins. The biogenesis and function of mitochondria is tightly regulated by nuclear-encoded protein cofactors. The mtDNAs of land plants are characteristically large, with a complex mode of gene organization and expression, in particularly at the post-transcriptional level. To become functional, the primary organellar transcripts undergo extensive maturation steps, which include endo- and exo-nucleolytic cleavage, RNA-base editing, and both ‘cis’- and ‘trans’-splicing events. These essential processing steps rely on the activities of a large set of nuclear-encoded factors. Among these, the RNA helicases serve as key players in organellar (mt-)RNA metabolism. This review summarizes the important roles played by these factors in regulating the highly dynamic processes of transcription, RNA processing, and translation within the mitochondria of land plants. We further discuss recent advancements in understanding how dysregulation of mitochondrial RNA helicases affects the organellar gene expression, respiratory functions, and hence the growth, development and physiology of land plants.

In the 21st century, the effects of HIV-associated neurocognitive disorders (HAND) have been significantly reduced in individuals due to the development of antiretroviral therapies (ARTs). However, the growing epidemic of polysubstance use (PSU) has led to concern for the effects of PSU on HIV-seropositive individuals. To effectively treat individuals affected by HAND, it is critical to understand the biological mechanisms affected by PSU including identification of novel markers. To fill this important knowledge gap, we used an in vivo HIV-Transgenic (HIV-Tg) animal model, to investigate the effects of the combined use of chronic methamphetamine (METH) and oxycodone (oxy). RNA-Seq analysis on the striatum - a brain region that is primarily targeted by both HIV and drugs of abuse - identified key differentially expressed markers post-METH and oxy exposure. Furthermore, ClueGO analysis and Ingenuity Pathway Analysis (IPA) revealed crucial molecular and biological functions associated with ATP-activated adenosine receptor, neuropeptide hormone activity, and the oxytocin signaling pathway to be altered between the different treatment groups. The current study further reveals the harmful effects of chronic PSU and HIV infection that can subsequently impact neurological outcomes in polysubstance users with HAND.

Much evidence has accumulated over the past decade in favor of a significant association between dysbiosis, neuroinflammation and neurodegeneration. Presently, the pathogenetic mechanisms triggered by molecules produced by the altered microbiota, also responsible for the onset and evolution of Alzheimer Disease will be described. Our attention will be focused on the role of astrocytes and microglia. Numerous studies have progressively demonstrated how these glial cells are important to ensure an adequate environment for neuronal activity in healthy conditions. Furthermore, it is becoming evident how both cell types can mediate the onset of neuroinflammation and lead to neurodegeneration when subjected to pathological stimuli. Based on this information, the role of major microbiota products in shifting the activation profiles of astrocytes and microglia from a healthy to a diseased state will be discussed focussing on Alzheimer Disease pathogenesis.

The replication of species A rotaviruses (RVAs) and other RNA viruses involves recruitment of and inter-action with the cellular organelles lipid droplets (LDs), both physically and functionally. Inhibition of en-zymes involved in the cellular fatty acid biosynthesis pathway or of cellular lipases that degrade LDs re-duces the functions of ‘viral factories’ (viroplasms for rotaviruses or replication compartments of other RNA viruses) and decreases the production of infectious progeny virus. Similarly, disturbance of the cellular lipid homeostasis in various ways blocks the replication of flaviviruses (hepatitis C viruses, Zikavirus, others), HIV-1, SARS-CoV-2, picornaviruses, noroviruses, influenza viruses and other negative-strand RNA viruses.

Prostate cancer (Pca) is a highly heterogeneous disease and the second more common tumor in males. Molecular and genetic profiles have been used to identify subtypes and guide therapeutic intervention. However, roughly 26% of primary Pca are driven by unknown molecular lesions. We use Principal Component Analysis (PCA) and custom RNAseq-data normalization to identify a gene expression signature which segregates primary PRAD from normal tissues. This Core-Expression Signature (PRAD-CES) includes 33 genes and accounts for 39% of data complexity along the PC1-cancer axis. The PRAD-CES is populated by protein-coding (AMACR, TP63, HPN) and RNA-genes (PCA3, ARLN1) sparsely found in previous studies, validated/predicted biomarkers (HOXC6, TDRD1, DLX1), and/or cancer drivers (PCA3, ARLN1, PCAT-14). Of note, the PRAD-CES also comprises six over-expressed LncRNAs without previous Pca association, four of them potentially modulating driver’s genes TMPRSS2, PRUNE2 and AMACR. Overall, our PCA capture 57% of data complexity within PC1-3. GO enrichment and correlation analysis involving major clinical features (i.e., Gleason Score, AR Score, TMPRSS2-ERG fusion and Tumor Cellularity) suggest that PC2 and PC3 gene signatures might describe more aggressive and inflammation-prone transitional forms of PRAD. Of note, surfaced genes may entail novel prognostic biomarkers and molecular alterations to intervene. Particularly, our work uncovered RNA genes with appealing implications on Pca biology and progression.

Liver cancers give rise to a heavy burden of health care worldwide. Understanding the tumour microenvironment (TME) underpins the development of precision therapy. Single-cell RNA sequencing (scRNA-seq) technology has generated high-quality cell atlases of the TME, but its wider application faces enormous costs for various clinical circumstances. Fortunately, a variety of deconvolution algorithms can instead repurpose bulk RNA-seq data, alleviating the need for generating scRNA-seq datasets. In this study, we reviewed major public omics databases for relevance in this study and utilized 8 RNA-seq and 1 microarray datasets from clinical studies. To decipher the TME of liver cancer, we estimated the fractions of liver cell components by deconvoluting the samples with Cibersortx using three reference scRNA-seq atlases. We also confirmed that Cibersortx can accurately deconvolute cell types/subtypes of interest. Compared with non-tumorous liver, liver cancers showed multiple decreased cell types forming normal liver microarchitecture, as well as elevated cell types involved in fibrogenesis, abnormal angiogenesis and disturbed immune responses. Survival analysis shows that the fractions of five cell types/subtypes significantly correlated with patient outcomes, indicating potential therapeutic targets. Therefore, deconvolution of bulk RNA-seq data with scRNA-seq atlas references can be a useful tool to help understand the TME.

The transfer of protein-encoding genetic information from DNA to RNA to protein, a process formalized as the “Central Dogma of Molecular Biology”, has undergone a significant evolution since its inception. It was amended to account for the information flow from RNA to DNA, the reverse transcription, and for the information transfer from RNA to RNA, the RNA-dependent RNA synthesis. These processes, both potentially leading to protein production, were initially described only in viral systems, and although RNA-dependent RNA polymerase activity was shown to be present, and RNA-dependent RNA synthesisfound to occur, in mammalian cells, its function was presumed to be restricted to regulatory. However, recent results, obtained with multiple mRNA species in several mammalian systems, strongly indicate the occurrence of protein-encoding RNA to RNA information transfer in mammalian cells. It can result in the rapid production of the extraordinary quantities of specific proteins as was seen in cases of terminal cellular differentiation and during cellular deposition of extracellular matrix molecules. A malfunction of this process may be involved in pathologies associated either with the deficiency of a protein normally produced by this mechanism or with the abnormal abundanceof a protein or of its C-terminal fragment. It seems to be responsible for some types of familial thalassemia and may underlie the overproduction of beta amyloid in sporadic Alzheimer’s disease. The aim of the present article is to systematize the current knowledge and understanding of this pathway. The outlined framework introduces unexpected features of the mRNA amplification such as its ability to generate polypeptides non-contiguously encoded in the genome, its second Tier, a physiologically occurring intracellular polymerase chain reaction, iPCR, a Two-Tier Paradox and RNA Dark Matter. RNA-dependent mRNA amplification represents a new mode of genomic protein-encoding information transfer in mammalian cells. Its potential physiological impact is substantial, it appears relevant to multiple pathologies and its understanding opens new venues of therapeutic interference, it suggests powerful novel bioengineering approaches and its further rigorous investigations are highly warranted.

Mutations that provide environment dependent selective advantages drive adaptive divergence among species. Many phenotypic differences among related species are more likely to result from gene expression divergence rather than from non-synonymous mutations. In this regard, cis-regulatory mutations play an important part in generating functionally significant variation. Some proposed mechanisms that explore the role of cis-regulatory mutations in gene expression divergence involve microsatellites. Microsatellites exhibit high mutation rates and are abundant in both coding and non-coding regions and could influence gene function and products. Here we tested the hypothesis that microsatellites contribute to gene expression divergence among species with 50 individuals from nine closely related Helianthus species using an RNA-seq approach. Differential expression analyses of the transcriptomes revealed that genes containing microsatellites in non-coding regions (UTRs and introns) are more likely to be differentially expressed among species when compared to genes with microsatellites in the coding regions and transcripts lacking microsatellites. We detected a greater proportion of shared microsatellites in 5’UTRs and coding regions compared to 3’UTRs and non-coding transcripts among Helianthus spp. Further, allele frequency differences measured by pairwise FST at single nucleotide polymorphisms (SNPs), indicate greater genetic divergence in transcripts containing microsatellites compared to those lacking microsatellites. A gene ontology (GO) analysis revealed that microsatellite-containing differentially expressed genes are significantly enriched for GO terms associated with regulation of transcription and transcription factor activity. Collectively, our study provides compelling evidence to support the role of microsatellites in gene expression divergence.

Protamine is a natural cationic peptide mixture mostly known as a drug for the neutralization of heparin and as a compound in formulations of slow-release insulin. Protamine is also used for cellular delivery of nucleic acids due to opposite charge-driven coupling. This year marks60 years since the first use of Protamine as a transfection enhancement agent. Since then, Protamine has been broadly used as a stabilization agent for RNA delivery. It has also been involved in several compositions for RNA-based vaccinations in clinical development. Protamine stabilization of RNA shows double functionality: it not only protects RNA from degradation within biological systems, but also enhances penetration into cells. A Protamine-based RNA delivery system is a flexible and versatile platform that can be adjusted according to therapeutic goals: fused with targeting antibodies for precise delivery, digested into a cell penetrating peptide for better transfection efficiency or not-covalently mixed with functional polymers. This manuscript gives an overview of the strategies employed in protamine-based RNA delivery, including the optimization of the nucleic acid’s stability and translational efficiency, as well as the regulation of its immunostimulatory properties from early studies to recent developments.

Legume-rhizobial symbiosis leads to to the formation of root nodules, where rhizobia reside and develop into bacteroids to reduce nitrogen into ammonium for plant growth, which leaves an opening question as how plant immunity is attenuated in nodules in the presence of large number of foreign bacteria. In Medicago truncatula, mutation in NAD1 (Nodules with Activated Defense 1) only produces necrotic nodules with overactivated immunity, indicating NAD1 is an indispensable component required for suppressing immunity in nodules. In this study, a dual RNA-seq transcriptomic technology was performed to extensively analyze gene expressions in nad1-1 mutant nodules. We indenifited 89 DEGs in symbiotic nitrogen fixation and 89 DEGs in immunity in Medicago truncatula at 10 dpi. Simultaneously, we indenifited 27 DEGs in fix and nif genes in Sinorhizobium meliloti. Then we identified 56 DEGs related to stress, including ROS, NO, and NCR, most of which were upregulated in Sinorhizobium meliloti. Our analyses of nitrogen fixation-defective plant nad1-1 mutants with overactivated defense indicate that host use plant immunity to control massive bacterial colonization during early and late symbiotic stages. Our findings provide insight into the function of NAD1 in improvement or inhibition of plant immune response to maintain symbiosis during rhizobial endosymbiosis.

In China, the demand for rabbit meat is also increasing, and the production for rabbit meat is important. However, the underlying mechanisms of regulate skeletal muscle growth and development in rabbits remain unclear. The purpose of this study was to identify candidate genes related to skeletal muscle growth in rabbits and explore their potential regulatory mechanisms. RNA-seq technique was used to compare the differences of skeletal muscle transcriptome in Fujian white rabbits at different developmental stages (day 20 and 26 of embryos and birth 1, 30 and 60-day-olds) with a total of 25 individual selected. A total of 9737 DEGs that were acquired from the five groups were annotated into three ontologies of the GO database: biological process (BP), cell component (CC), and molecular function (MF). For KEGG analysis, there are 8249 genes were enriched in 1148 pathways, of which 67 pathways were significantly enriched, mainly in Hypertrophic cardiomyopathy (HCM), the PPAR signaling pathway, MAPK signaling pathway. Among all comparison groups, SE was the most abundant AS event. Real-time PCR verified that the expression patterns of differential genes were consistent with the transcriptome sequencing results. These results will provide a molecular regulation mechanism of muscle growth and development in Fujian white rabbits, and should serve an important theoretical basis for improving meat performance and growth rate of Chinese local meat rabbit breeds.

Starting from December 2019, coronavirus disease 2019 (COVID-19) has emerged as a once-in-a-century pandemic with deadly consequences, which urgently calls for new treatments, cures and supporting apparatuses. Remdesivir was reported by World Health Organization (WHO) as the most promising drug currently available for the treatment of COVID-19. Here, we use molecular dynamics simulations and free energy perturbation methods to study the inhibition mechanism of remdesivir to its target SARS-CoV-2 virus RNA-dependent RNA polymerase (RdRp). In the absence of a crystal structure of the SARS-CoV-2 RdRp, we first construct the homology model of this polymerase based on a previously available structure of SARS-CoV NSP12 RdRp (with a sequence identify of 95.8%). We then build the putative binding mode by aligning the remdesivir + RdRp complex to the ATP bound poliovirus RdRp. The putative binding structure is further optimized with molecular dynamics simulations and demonstrated to be stable, indicating a reasonable binding mode for remdesivir. The relative binding free energy of remdesivir is calculated to be -8.28 ± 0.65 kcal/mol, much stronger than the natural substrate ATP (-4.14 ± 0.89 kcal/mol) which is needed for the polymerization. The ~800-fold improvement in the K_d from remdesivir over ATP indicates an effective replacement of APT in blocking of the RdRp binding pocket. Key residues D618, S549 and R555 are found to be the contributors to the binding affinity of remdesivir. These findings demonstrate that remdesivir can potentially act as a SARS-CoV-2 RNA-chain terminator, effectively stopping its RNA reproduction, with key residues also identified for future lead optimization and/or drug resistance studies.

We developed and applied a method for characterizing bacterial promoters genome-wide by in vitro transcription coupled to transcriptome sequencing specific for native 5’-ends of transcripts. This method called ROSE (Run-Off transcription/RNA-SEquencing), only requires chromosomal DNA, ribonucleotides, RNA polymerase (RNAP) core enzyme, and a specific sigma factor, recognizing the corresponding promoters, which have to be analyzed. ROSE was performed on E. coli K-12 MG1655 genomic DNA using E. coli RNAP holoenzyme (including σ70) and yielded 3,226 transcription start sites, 2,167 of which were also identified in in vivo studies, and 598 were new. Many new promoters not yet identified by in vivo experiments might be repressed under the tested conditions. Complementary in vivo experiments with E. coli K-12 strain BW25113 and isogenic transcription factor gene knockout mutants of fis, fur, and hns were used to test this hypothesis. Comparative transcriptome analysis demonstrated that ROSE could identify bona fide promoters that were apparently repressed in vivo. In this sense, ROSE is well-suited as a bottom-up approach for characterizing transcriptional networks in bacteria and ideally complementary to top-down in vivo transcriptome studies.

The rapid expansion of transcriptomics from increased affordability of next-generation sequencing (NGS) technologies generates rocketing amounts of gene expression data across biology and medicine, and notably in cancer research. Concomitantly, many bioinformatics tools were developed to streamline gene expression analysis and quantification. We tested the concordance of NGS RNA sequencing (RNA-seq) analysis outcomes between the two predominant programs for reads alignment, HISAT2 and STAR, and the two most popular programs for quantifying gene expression in NGS experiments, edgeR and DESeq2, using RNA-seq data from a series of breast cancer progression specimens, which include histologically confirmed normal, early neoplasia, ductal carcinoma in situ and infiltrating ductal carcinoma samples microdissected from formalin fixed, paraffin embedded (FFPE) breast tissue blocks. We identified significant differences in aligners’ performance: HISAT2 was prone to misalign reads to retrogene genomic loci, STAR generated more precise alignments, especially for early neoplasia samples. edgeR and DESeq2 produced similar lists of differentially expressed genes in stage comparisons, with edgeR producing more conservative, though shorter, lists of genes. Albeit, Gene Ontology (GO) enrichment analysis revealed no skewness in significant GO categories identified among differentially expressed genes by edgeR vs DESeq2. As transcriptome analysis of archived FFPE samples becomes a vanguard of precision medicine, identification and fine-tuning of bioinformatics tools becomes critical for clinical research. Our results indicate that STAR and edgeR are well-suited tools for differential gene expression analysis from FFPE samples.

Hospital-based programs democratize mRNA therapeutics by facilitating the processes to translate a novel RNA idea from the bench to the clinic. Because mRNA is essentially biological software, therapeutic RNA constructs can be rapidly developed. The generation of small batches of clinical grade mRNA to support IND applications and first-in-man clinical trials, as well as personalized mRNA therapeutics delivered at the point-of-care, is feasible at a modest scale of cGMP manufacturing. Advances in mRNA manufacturing science and innovations in mRNA biology, are increasing the scope of mRNA clinical applications.

A novel coronavirus (SARS-CoV-2) that is initially found to trigger human severe respiratory illness in Wuhan City of China in 2019, has killed 2,718 people in China by February 26, 2020, and which has been recognized as a public health emergency of international concern as well. And the virus has spread to more than 38 countries around the world. However, the drug has not yet been officially licensed or approved to treat SARS-Cov-2 infection. NSP12-NSP7-NSP8 complex of SARS-CoV-2, essential for viral replication and transcription, is generally regarded as a potential target to fight against the virus. According to the NSP12-NSP7-NSP8 complex (PDB ID: 6NUR) structure of SARS, two homologous models were established for virtual screening in the present study, namely NSP12-NSP7 interface model and NSP12-NSP8 interface model. Seven compounds (Saquinavir, Tipranavir, Lonafarnib, Tegobuvir, Olysio, Filibuvir, and Cepharanthine) were selected for binding free energy calculations based on virtual screening and docking scores. All seven compounds can combine well with NSP12-NSP7-NSP8 in the homologous model, providing drug candidates for the treatment and prevention of SARS-CoV-2.

Ferroptosis, a regulated form of cell death characterized by iron-dependent lipid peroxide accumulation, plays a pivotal role in various pathological conditions, including neurodegenerative diseases. While reasonable evidence for ferroptosis exist e.g. in Parkinson’s disease or Alzheimer’s disease, there are only few reports on amyotrophic lateral sclerosis (ALS), a fast progressive and incurable neurodegenerative disorder characterized by progressive motor neuron degeneration. Initial studies, however, highlight that ferroptosis might be significantly involved in ALS. Key features of ferroptosis include oxidative stress, glutathione depletion, mitochondrial and dysfunction alterations in mitochondrial morphology, mediated by proteins such as GPX4, xCT, ACSL4 FSP1, Nrf2 and TfR1. Induction of ferroptosis involves small molecule compounds like erastin and RSL3, which disrupt system Xc- and GPX4 activity, respectively, yielding in lipid peroxidation and cellular demise. Mutations in Fused in Sarcoma (FUS) are associated with familial ALS. Pathophysiological hallmarks of FUS-ALS involve mitochondrial dysfunction and oxidative damage, implicating ferroptosis as putative cell death pathway in motor neuron demise. However, mechanistic understanding of ferroptosis in ALS, particularly FUS-ALS, remains limited. Here, we investigated ferroptosis vulnerability in a FUS-ALS cell model, revealing mitochondrial disturbance and increased susceptibility for ferroptosis in cells harboring the mutant FUS variant. This was accompanied by altered expression of ferroptosis-associated proteins, particularly by dramatic reduction of xCT expression, leading to cellular imbalance of the redox system and increased lipid peroxidation. Iron chelation with deferoxamine as well as inhibition of the mitochondrial calcium uniporter (MCU) significantly alleviated ferroptotic cell death and lipid peroxidation. These findings suggest a link between ferroptosis and FUS-ALS, offering potential therapeutic targets.

Infection by the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) results in the novel coronavirus disease COVID-19, which has posed a serious threat globally. Infection of SARS-CoV-2 during pregnancy is associated with complications like preterm labor and premature rupture of membranes; a proportion of neonates born to the infected mothers are also positive for the virus. During pregnancy, the placental barrier protects the fetus from pathogens and ensures healthy development. However, whether or not SARS-CoV-2 can infect the placenta is unknown. Herein, utilizing single-cell RNA-seq data, we report that the SARS-CoV-2 binding receptor ACE2 and the S protein priming protease TMPRSS2 are co-expressed by a subset of syncytiotrophoblasts (STB) in the first trimester and extra villous trophoblasts (EVT) in the second trimester human placenta. The ACE2- and TMPRSS2-positive (ACE2+TMPRSS2+) placental subsets express mRNA for proteins involved in viral budding and replication. These cells also express mRNA for proteins that interact with SARS-CoV-2 structural and non-structural proteins in the host cells. We also discovered unique signatures of genes in ACE2+TMPRSS2+ STBs and EVTs. The ACE2+TMPRSS2+ STBs are highly differentiated cells and express genes involved mitochondrial metabolism and glucose transport. The second trimester ACE2+TMPRSS2+ EVTs are enriched for markers of endovascular trophoblasts. Further, both these subtypes abundantly expressed genes in Toll like receptor pathway, the second trimester EVTs (but not first trimester STBs) are also enriched for component of the JAK-STAT pathway that drive inflammation. To conclude, herein we uncovered the cellular targets for SARS-CoV-2 entry and show that these cells can potentially drive viremia in the developing human placenta. Our results provide a basic framework towards understanding the paraphernalia involved in SARS-CoV-2 infections in pregnancy.

COVID19 is a current pandemic disease due to the novel coronavirus SARS-CoV-2. The scientific community mounted a strong response by accelerating research and innovation, and rapidly setting the basis to the understanding of molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key step of SARS-CoV2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of structural and functional data on key actors of the replicatory machinery of SARS-CoV-2, filling the gaps in the current availability of structural data using homology modelling. Moreover, learning from similar viruses, we collect literature data to reconstruct the pattern of interactions among protein actors of the SARS-CoV-2 RNA polymerase machinery. In this pattern, an important role is played by co-factors, like Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors holding the entire machinery together to enhance the efficiency of RNA replication.

The corneal epithelium (CE) is spread between two domains, the outer vascularized limbus and the avascular cornea proper. Epithelial cells undergo constant migration from the limbus to the vision-critical central cornea. Coordinated with this migration the cells undergo differentiation changes where a pool of unique stem/precursor cells at the limbus yields the mature cells that reach the corneal center. Differentiation is heralded by the expression of the corneal-specific Krt12. Processing data acquired by scRNA-Seq we found that the increase of Krt12 expression occurs in four distinct steps within the limbus plus a single continuous increase in the cornea. Differential gene analysis demonstrated that these domains reflect discreet stages of CE differentiation and yielded extensive information of the genes undergoing down or up regulation in the sequential transition from less to more differentiate conditions. The approach allowed the identification of multiple gene cohorts, including a) the genes that have maximal expression in the most primitive, Krt12-negative cell cohort, which is likely to include the stem/precursor cells; b) the sets of genes that undergo continuous increase or decrease along the whole differentiation path, and c) and the genes showing maximal positive or negative correlation with the changes in Krt12.

The current SARS- CoV-2 pandemic underscores the importance of understanding the evolution of RNA genomes. While RNA is subject to the formation of similar lesions as DNA, the evolutionary and physiological impacts RNA lesions have on viral genomes are yet to be characterized. Lesions that may drive the evolution of RNA genomes can induce breaks that are repaired by recombination or can cause base substitution mutagenesis, also known as base editing. Over the past decade or so, base editing mutagenesis of DNA genomes has been subject to many studies, revealing that exposure of ssDNA is subject to hypermutation that is involved in the etiology of cancer. However, base editing of RNA genomes has not been studied to the same extent. Recently hypermutation of single-stranded RNA viral genomes have also been documented though its role in evolution and population dynamics. Here, we will summarize the current knowledge of key mechanisms and causes of RNA genome instability covering areas from the RNA world theory to the SARS- CoV-2 pandemic of today. We will also highlight the key questions that remain as it pertains to RNA genome instability, mutations accumulation, and experimental strategies for addressing these questions.

All RNA viruses deliver their genomes into target host cells through processes distinct from normal trafficking of cellular RNA transcripts. The delivery of viral RNA into most cells hence triggers innate antiviral defenses that recognize viral RNA as foreign. In turn, viruses have evolved mechanisms to subvert these defenses, allowing them to thrive in target cells. Therefore, drugs activating defense to exogenous RNA could serve as broad-spectrum antiviral drugs. Here we show that transcriptional signatures associated with cellular responses to the delivery of a non-viral exogenous RNA sequence into human cells predict small molecules with broad-spectrum antiviral activity. In particular, transcriptional responses to the delivery of Cas9 mRNA into human hematopoietic stem and progenitor cells (HSPCs) highly matches those triggered by small molecules with broad-spectrum antiviral activity such as emetine, homoharringtonine, pyrvinium pamoate and anisomycin, indicating that these drugs are potentially active against other RNA viruses. Furthermore, these drugs have been approved for other indications and could thereby be repurposed to novel viruses. We propose that the antiviral activity of these drugs to SARS-CoV-2 should therefore be determined as they have been shown as active against other coronaviruses including SARS-CoV-1 and MERS-CoV. Indeed, two of these drugs- emetine and homoharringtonine- were independently shown to inhibit SARS-CoV-2 as this article was in preparation. These drugs could also be explored as potential adjuvants to COVID-19 vaccines in development due to their potential effect on the innate antiviral defenses that could bolster adaptive immunity when delivered alongside vaccine antigens.

The ribosome is a macromolecular complex composed of RNA and proteins that interact through an integrated and interconnected network to preserve its ancient core activities. In this review, we emphasize the pivotal role played by RNA-binding proteins as a driving force in the evolution of the current form of the ribosome, underscoring their importance in ensuring accurate protein synthesis. This category of proteins includes both ribosomal proteins and ribosome biogenesis factors. Impairment of their RNA-binding activity can also lead to ribosomopathies, a group of disorders characterized by defects in ribosome biogenesis that are detrimental to protein synthesis and cellular homeostasis. A comprehensive understanding of these intricate processes is essential for elucidating the mechanisms underlying the resulting diseases and advancing potential therapeutic interventions.

At the beginning of the 21st century, it became obvious that radical changes had taken place in the concept of living matter and, in particular, in the concept of the organization of intracellular space. The accumulated data testify to the essential importance of phase transitions of biopolymers (first of all, intrinsically disordered proteins and RNA) in the spatiotemporal organization of the intracellular space. Of particular interest is the stress-induced reorganization of the intracellular space. Examples of organelles formed in response to stress are nuclear A-bodies and nuclear stress granules. The formation of these organelles is based on LLPS of intrinsically disordered proteins and non-coding RNA. Despite the overlapping composition and similar mechanism of formation, these organelles have different functional activities and physical properties. In this review, we will focus our attention on these MLOs and describe their functions, structure, and mechanism of formation.

Cervical carcinogenesis is the leading cause of cancer related death in women and the role of high-risk human papillomavirus (HR-HPV) as a possible risk factor in the development of this cancer is well recognised. Despite the availability of multi-therapeutic approaches, there is still a major concern regarding the prevention of metastatic dissemination and excessive tissue injuries. Therefore, it is imperative to develop a safer and more efficient treatment modality. Ficus Carica, a natural plant, has shown potential therapeutic properties through its fruit latex when applied to HPV-positive cervical cancer cell lines. However, the mechanisms of action of Ficus Carica (fig) latex are not well understood. This study aims to provide a deeper insight into the biological activities of fig latex on human cervical cancer cell lines expressing high-risk HPV types 16 and 18. The data obtained from this study reveals that fig latex influences the expression of genes in-volved in "Class I MHC-mediated antigen presentation" as well as "Antigen processing: Ubiquitination and Proteasome degradation." These genes play a crucial role in host immune surveillance and the resolution of infection. Findings from this study suggest that fig latex may enhance T cell responses against oncogenic HPV, which could be beneficial for the clearance of early-stage cancer.

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell Acute Lymphoblastic Leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

Sweetpotato is an important miscellaneous grain crop in China, which is threatened by a variety of diseases and insect pests in the process of cultivation and production. sweetpotato stem rot is one of the common sweetpotato diseases, which seriously affects the yield and quality of sweetpotato. However, there are few studies on the mechanism of resistance to stem rot in sweetpotato. In this study, transcriptome sequencing analysis was carried out on the samples at different stages (T1, T2, T3) of stem rot pathogen infecting Xushu 48, and 44839 (T1-VS-T2), 81436 (T1-VS-T2) and 61932 (T2-VS-T3) differentially expressed genes (DEGs) were obtained. The DEGs were mainly concentrated in Alanine, aspartate and glutamate metabolism (ko00250), Carbon fixation in photosynthetic organisms (ko00710) and Amino sugar and nucleotide sugar metabolism (ko00520) and some candidate genes related to plant pathogen infection, such as receptor-like protein kinase (RLK5, RLK7), LRR receptor-like serine/threonine-protein kinase (SERK1), bHLH137, ERF9, MYB73 and NAC053, were screened. The results of this study provide genetic resources for the research on stem rot resistance of sweetpotato and provide theoretical basis for sweetpotato resistance breeding.

Respiratory Syncytial Virus is a common respiratory pathogen known for causing respiratory illnesses, ranging from mild symptoms to severe lower respiratory tract infections in infants and the elderly. This virus is responsible for one-third of pneumonia deaths in the pediatric population, but there are currently only a few effective vaccines and pharmacological treatments. The frac-talkine (CX3CL1) receptor (CX3CR1) is a co-receptor for RSV expressed by airway epithelial cells and a diversity of immune cells. Recent research has highlighted the role of CX3CR1 in the host immune response to RSV infection, and its potential role in the development of an efficient vaccine and novel treatments.

The blood–brain barrier (BBB) plays pivotal roles in synaptic and neuronal functioning by sealing the space between adjacent microvascular endothelial cells. BBB breakdown is present in patients with mild cognitive impairment (MCI) or Alzheimer disease (AD). Claudin-5 (CLDN-5) is a tetra-spanning protein essential for sealing the intercellular space between adjacent endothelial cells in the BBB. In this study, we developed a blood-based assay for CLDN-5 and investigated its diagnostic utility by using 100 cognitively normal (control) subjects, 100 patients with MCI, and 100 patients with AD. Plasma CLDN-5 levels were increased in patients with AD (3.08 ng/mL) compared with controls (2.77 ng/mL). Plasma levels of phosphorylated tau (pTau181), a biomarker of pathological tau, were elevated in patients with MCI or AD (2.86 and 4.20 pg/mL, respectively) compared with control subjects (1.81 pg/mL). In patients with MCI or AD, plasma levels of CLDN-5—but not pTau181—decreased with age, suggesting some age-dependent BBB changes in MCI and AD. These findings suggest that plasma CLDN-5 may a potential biochemical marker for the diagnosis of MCI and AD.

Safe sample transport is of great importance for infectious diseases diagnostics. Various treatments and buffers are used to inactivate pathogens in diagnostic samples. At the same time, adequate sample preservation, particularly of nucleic acids, is essential to allow an accurate laboratory diagnosis. For viruses with single-stranded RNA genomes of positive polarity, such as foot-and-mouth disease virus (FMDV), however, naked full-length viral RNA can itself be infectious. In order to assess the risk of infection from inactivated FMDV samples, two animal experiments were performed. In the first trial, six cattle were injected with FMDV RNA (isolate A22/IRQ/24/64) into the tongue epithelium. All animals developed clinical disease within two days and FMDV was reisolated from serum and saliva samples. In the second trial, another group of six cattle was exposed to FMDV RNA by instilling it on the tongue and spraying it into the nose. The animals were observed for 10 days after exposure. All animals remained clinically unremarkable and virus isolation as well as FMDV genome detection in serum and saliva were negative. No transfection reagent was used for any of the animal inoculations. In conclusion, cattle can be infected by injection with naked FMDV RNA, but not by non-invasive exposure to the RNA. Inactivated FMDV samples that contain full-length viral RNA carry only a negligible risk of infecting animals.

Cutaneous Melanoma (CM) is the most lethal form of skin cancer if it becomes metastatic, where treatment options and survival chances decrease dramatically. Immunotherapy treatments based on the immunologic checkpoint inhibitors (PD-1 and CTLA4) constituted a main breakthrough in the treatment of metastatic CM, particularly in the long-term benefit. However, several molecular pathways are responsible for the failure of this strategy in about 50-70% of CM patients. Some Long Non-coding RNAs (lncRNAs), and circular RNAs (circRNA) are implicated in triggering pro- and antitumorigenic responses to various cancer treatments. The relationship between lncRNA, circRNA and Immune Checkpoint Blockade (ICB) immunotherapy is not extensively explored in cutaneous metastatic melanoma (CMM). The aim of this study is to evaluate the potential role of both circRNA and lncRNA as a predictive immunotherapy biomarker in CMM. RNA-seq from 12 FFPE samples from the metastatic biopsy of metastatic melanoma patients treated with Nivolumab were analyzed. Our findings indicate that specific lncRNA and circRNA are involved in regulatory networks of the immune response against metastatic melanoma under treatment with nivolumab. Moreover, we have established a risk score that allows the prediction of Overall survival (OS) and Progression-free survival (PFS) of CMM patients with high accuracy. This proof of principle work provides a possible insight on the function of ceRNA, contributing to decipher the complex molecular mechanism of ICB cancer treatment response.

Recent emergence of novel coronavirus (SARS-CoV-2) all over the world has resulted more than 33,106 global deaths. To date well-established therapeutics modules for infected patients are unknown. In this present initiative, molecular interactions between FDA-approved antiviral drugs against the Hepatitis-C virus (HCV) have been investigated theoretically against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. HCV and SARS-CoV-2 are both +ssRNA viruses. At 25^o C beclabuvir, a non-nucleoside inhibitor of the RdRp_HCV can efficiently bind to RdRp _SARS-CoV-2 (ΔG_AutoDock = -9.95 kcal mol^-1) with an inhibition constant of 51.03 nM. Both the ΔG_London and ΔG_{GBVI / WSA} values were - 9.06 and - 6.67 kcal mol^-1, respectively for binding of beclabuvir to RdRp_SARS-CoV-2. In addition, beclabuvir has also shown better binding free energy with RdRp_SARS-CoV-2 (ΔG_vina = -8.0 kcal mol^-1) than that observed with the Thumb 1 domain of RdRp_HCV (ΔG_vina = -7.1 kcal mol^-1). InterProScan has suggested the RNA-directed 5'-3' polymerase activity exists within 549^th to 776^th amino acid residues of RdRp_SARS-CoV, where the major amino acid residues interacting being I591, Y621, C624, D625, A690, N693, L760, D762, D763, and E813-N817. Molecular interaction suggests occupancy of beclabuvir inside the active site environment of the RdRp_SARS-CoV-2, the enzyme essential for viral RNA synthesis. In conclusion, results suggest beclabuvir may serve as an anti-SARS-CoV-2 drug.

The molecular mechanisms underlying heat stress tolerance in animals to high temperatures remain unclear. This experiment was performed with Sprague-Dawley rats housed at 22 ℃ (control group; CT), and 42 ℃ for 30 min (H30), 60 min (H60), and 120 min (H120). Transcriptomic analysis using RNA-Sequencing (RNA-Seq) from blood (CT and H120), liver (CT, H30, H60, and H120), and adrenal glands (CT, H30, H60, and H120) was performed. The differentially expressed mRNA isoforms (DEIs) were identified and annotated by the CLC Genomics Workbench. Biological process and metabolic pathway analyses were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A total of 225, 5,764, and 4,988 DEIs in the blood, liver, and adrenal glands were observed. Furthermore, the number of novel differentially expressed transcript lengths with annotated genes and the novel differentially expressed transcript with non-annotated genes were 136 and 8 in blood, 3,549 and 120 in the liver, as well as 3,078 and 220 in adrenal glands, respectively. A total of 35 genes were involved in the heat stress response, out of which Dnaja1, LOC680121, Chordc1, AABR07011951.1, Hsp90aa1, Hspa1b, Cdkn1a, Hmox1, Bag3 and Dnaja4 were commonly identified in the liver and adrenal glands, suggesting that these genes may regulate heat stress response through interactions between liver and adrenal glands. The results suggest that the identified mRNA isoforms could be considered as potential candidates for selecting mammals to improve thermotolerance.

Transcription is carried out in most eukaryotes by three multimeric complexes (RNA polymerases I, II and III). However, plants contain two additional RNA polymerases (IV and V), which have evolved from RNA polymerase II. RNA polymerases II, IV and V contain both common and specific subunits that may specialize some of their functions. In this study, we conducted a search for the genes that putatively code for the specific subunits of RNA polymerases IV and V, as well as the corresponding of RNA polymerase II in olive. Based on the homology with the genes of Arabidopsis thaliana, we identified 13 genes that putatively code for the specific subunits of polymerases IV and V, and 16 genes that code for the corresponding specific subunits of polymerase II in olive. The transcriptomic analysis by RNA-Seq revealed that the expression of the RNA polymerases IV and V genes was induced during the initial stages of fruit development. Given that RNA polymerases IV and V are involved in the transcription of long non-coding RNAs, we investigated their expression and observed relevant changes in the expression of this type of RNAs. Particularly, the expression of the intergenic and intronic long non-coding RNAs tended to increase in the early steps of fruit development, suggesting their potential role in this process. The positive correlation between the expression of RNA polymerases IV and V subunits and the expression of non-coding RNAs supports the hypothesis that RNA polymerases IV and V may play a role in fruit development throughout the synthesis of this type of RNAs.

During choriogenesis in insects, chorion (eggshell) is formed by surrounding follicular epithelial cells in ovarioles. However, the regulatory endocrine factor(s) activating choriogenesis and the chemical components on eggshell deserve further exploration. In two representative Coleopterans, a Coccinellid Henosepilachna vigintioctopunctata and a Chrysomelid Leptinotarsa decemlineata, genes encoding the 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and ultraspiracle (USP), and two chitin biosynthesis enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP) and chitin synthase (ChS1), were highly expressed in ovaries of the young females. RNA interference (RNAi)-aided knockdown of either HvEcR or Hvusp in H. vigintioctopunctata inhibited oviposition, suppressed the expression of HvChS1, and lessened the positive signal of Calcofluor staining on the chorions, which suggests the reduction of a chitin-like substance (CLS) deposited on eggshells. Similarly, RNAi of LdEcR or Ldusp in L. decemlineata constrained oviposition, decreased the expression of LdUAP1 and LdChS1, and declined CLS contents in the resultant ovaries. Knockdown of LdUAP1 or LdChS1 caused similar defective phenotypes, i.e., reduced oviposition and CLS contents in the L. decemlineata ovaries. These results, for the first time, indicate that 20E signaling activates choriogenesis in two Coleopteran species. Moreover, our findings propose the deposition of a CLS on the chorions.

Background: Alzheimer's disease causes great changes, with the prefrontal cortex be-ing frequently damaged zones, affecting physical and cognitive behavioural and compromising autonomy and quality of life. Objective: To evaluate the effectiveness of feedback-based tech-nology on the improvement of physical-cognitive capacities in Alzheimer. Methods: Forty pa-tients with Alzheimer were recruited from the Santa Elena Alzheimer's Association, Seville, of which 15 ended up being pilot participants, divided randomly into controls (8) and experi-mental (7). They were evaluated with: Cognitive Mini-Examination Scale, Oddball Test and At-tention Network, Berg Scale, Tinetti, Timed up and Go and Geriatric Deterioration Scale. The experimental group was treated with feedback-based technology under the supervision of physiotherapists, during 16 thirty-minute sessions, twice a week. The control group performed their usual care. Result: A significant improvement was observed in the experimental group (Cognitive Mini-Examination and Tinetti). In the group of control, a worsening of physical and motor skills was evidenced because of the degenerative disease. Discussion/Conclusion: This treatment could be a strategy to keep the patient in early stages for longer, and improve quali-ty of life delaying dependency. It creates intergenerational bonds and use at home to promote treatment adherence.

Differential RNA editing by adenosine deaminases that act on RNA (ADARs) has been implicat-ed in several neurological disorders, including Parkinson’s disease (PD). Here, we report results of an RNAi screen of genes differentially regulated in adr-2 mutants, normally encoding the only catalytically active ADAR in Caenorhabditis elegans, ADR-2. Subsequent analysis of candidate genes that alter the misfolding of human α-synuclein (α-syn) and dopaminergic neurodegenera-tion, two PD pathologies, reveal that reduced expression of xdh-1, the ortholog of human xanthine dehydrogenase (XDH), is protective against α-synuclein-induced dopaminergic neurodegenera-tion. Further RNAi experiments show that WHT-2, the worm ortholog of the human ABCG2 transporter and a predicted interactor of XDH-1, is the rate-limiting factor in the ADR-2, XDH-1, WHT-2 system for dopaminergic neuroprotection. Proteomic analysis indicates that the editing of one nucleotide in wht-2 RNA leads to the substitution of threonine with alanine at residue 124 in the WHT-2 protein, changing its structure. Thus, we propose a model where wht-2, is edited by ADR-2 which promotes optimal export of uric acid, a known substrate of WHT-2 and a product of XDH-1 activity. In the absence of editing, uric acid export is limited provoking a reduction in xdh-1 transcription to limit uric acid production and maintain cellular homeostasis. In turn, ele-vation of uric acid is protective against dopaminergic neuronal cell death. These data indicate that modifying specific targets of RNA editing may represent a promising therapeutic strategy for PD.

It was thought until the 1990s that the eukaryotic translation machinery was unable to translate a circular RNA. However internal ribosome entry sites (IRESs) and m⁶A-induced ribosome engagement sites (MIRESs) were discovered, promoting 5’end-independent translation initiation. Today a new family of so-called “non-coding” circular RNAs (circRNAs) has emerged, revealing the pivotal role of 5’end-independent translation. CircRNAs have a strong impact on translational control via their sponge function, and form a new mRNA family as they are translated into proteins with pathophysiological roles. While there is no more doubt about translation of covalently closed circRNA, the linearity of canonical mRNA is only theoretical: it has been shown for more than thirty years that polysomes exhibit a circular form and mRNA functional circularization has been demonstrated in the 1990s by the interaction of initiation factor eIF4G with poly(A) binding protein. More recently, additional mechanisms of 3’-5’ interaction have been reported, including m⁶A modification. Functional circularization enhances translation via ribosome recycling and acceleration of the translation initiation rate. This update of covalently and non-covalently circular mRNA translation landscape shows that RNA circular shape might be the rule for translation with an important impact on disease development and biotechnological applications.

When it is in the template RNA, the naturally occurring m¹A epitranscriptomic RNA modification was recently reported to be able to stop the RNA polymerization reaction catalyzed by the RNA dependent RNA polymerase (RdRp) of SARS-CoV-2. In this report, we report that m¹A via its triphosphate form (m¹ATP) can be incorporated into RNA by the same RdRp. These two findings point a new direction for antiviral drug development based on m¹A for combatting COVID-19. More broadly, it is possible that the large pool of epigenetic RNA as well as DNA modifications could serve as a treasury for drug discovery aimed at combating various infectious and other diseases.

SARS-CoV-2 is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. Like other members of this family, the virus possesses a positive-sense single-stranded RNA genome. The genome encodes for the nsp12 protein, which houses the RNA-dependent-RNA polymerase (RdRP) activity responsible for the replication of the viral genome. A homology model of nsp12 was prepared using the structure of the SARS nsp12 (6NUR) as a model. The model was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp12. This exercise showed that vitamin B12 (methylcobalamin) may bind to the active site of the nsp12 protein. A model of the nsp12 in complex with substrate RNA and incoming NTP showed that Vitamin B12 binding site overlaps with that of the incoming nucleotide. A comparison of the calculated energies of binding for RNA plus NTP and methylcobalamin suggested that the vitamin may bind to the active site of nsp12 with significant affinity. It is, therefore, possible that methylcobalamin binding may prevent association with RNA and NTP and thus inhibit the RdRP activity of nsp12. Overall, our computational studies suggest that methylcobalamin form of vitamin B12 may serve as an effective inhibitor of the nsp12 protein.

Transcription factors (TFs) and microRNAs (miRNAs) are co-actors in genome-scale decoding and regulatory networks, often targeting common genes. In this paper, we describe the algebraic geometry of both TFs and miRNAs thanks to group theory. In TFs, the generator of the group is a DNA-binding domain while, in miRNAs, the generator is the seed of the sequence. For such a generated (infinite) group $\pi$, we compute the $SL(2,\mathbb{C})$ character variety, where $SL(2,\mathbb{C})$ is simultaneously a \lq space-time' (a Lorentz group) and a \lq quantum' (a spin) group. A noteworthy result of our approach is to recognize that optimal regulation occurs when $\pi$ looks like a free group $F_r$ ($r =1$ to $3$) in the cardinality sequence of its subgroups, a result obtained in our previous papers. A non free group structure features a potential disease. A second noteworthy result is about the structure of the Groebner basis $\mathcal{G}$ of the variety. A surface with simple singularities (like the well known Cayley cubic) within $\mathcal{G}$ is a signature of a potential disease even when $G$ looks like a free group $F_r$ in its structure of subgroups. Our methods apply to groups with a generating sequence made of two to four distinct DNA/RNA bases in $\{A,T/U,G,C\}$. Several human TFs and miRNAs are investigated in detail thanks to our approach.

RNA epigenetics is perhaps the most recent aspect of interest for translational epigeneticists. RNA modifications create such an extensive network of epigenetically driven combination whose role in physiology and pathophysiology is still far from being elucidated. Not surprisingly, some of the players determining changes into RNA structure are in common with those involved in DNA and chromatin structure regulation, while other molecules seem very specific to RNA. It is envisaged, then, that new small molecules, acting selectively on RNA epigenetic changes, will be reported soon, opening new therapeutic interventions based on the correction of the RNA epigenetic landscape. In this review, we shall summarize some aspects of RNA epigenetics limited to those in which the potential clinical translatability to cardiovascular disease is emerging.

Blackcurrant leaf chlorosis associated virus (BCLCaV) was detected recently by next-generation sequencing (NGS) and proposed as a new and distinct species in the genus Idaeovirus. Genomic components of BCLCaV that were detected and confirmed include: 1) RNA-1 that is monocistronic and encodes the replicase complex; 2) a bicistronic RNA-2 that encodes a movement protein (MP) and the coat protein (CP) of the virus, with open reading frames (ORF) that overlap by a single adenine (A) nucleotide (nt) representing the third position of an opal stop codon of the MP ORF2a and the first position of the start codon of the CP ORF2b; 3) a subgenomic form of RNA-2 (RNA-3) that contains ORF2b; and 4) a concatenated form of RNA-2 that consists of a complementary and inverted RNA-3 conjoined to the full-length RNA-2. Analysis of NGS-derived paired-end reads revealed the existence of bridge reads encompassing the 3’-terminus and 5’-terminus of RNA-2 or RNA-3 of BCLCaV. The full RNA-2 or RNA-3 could be amplified using outward facing or abutting primers; also RNA-2/RNA-3 could be detected even after three consecutive RNase R enzyme treatments with denaturation at 95 ^oC preceding each digestion. Evidence was obtained indicating that there are circular forms of BCLCaV RNA-2 and RNA-3.

The severity of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), greatly varies from patient to patient. In the present study, we explored and compared mutation profiles of SARS-CoV-2 isolated from mildly affected and severely affected COVID-19 patients in order to explore any relationship between mutation profile and disease severity. Genomic sequences of SARS-CoV-2 were downloaded from GISAID database. With the help of Genome Detective Coronavirus Typing Tool, genomic sequences were aligned with the Wuhan seafood market pneumonia virus reference sequence and all the mutations were identified. Distribution of mutant variants was then compared between mildly and severely affected groups. Among the numerous mutations detected, 14,408C>T and 23,403A>G mutations resulting in RNA-dependent RNA polymerase (RdRp) P323L and spike protein D614G mutations, respectively, were found predominantly in severely affected group (>82%) compared with mildly affected group (<46%, p<0.001). The 241C>T mutation in the non-coding region of the genome was also found predominantly in severely affected group. The 3,037C>T, a silent mutation, also appeared in relatively high frequency in severely affected group. We concluded that RdRp P323L and spike protein D614G mutations predominate in severely affected COVID-19 patients. Further studies will be required to explore whether these mutations have any impact on the severity of COVID-19.

SARS-CoV2 RNA depended RNA polymerase is an essential enzyme for the survival of the virus in hosts as it helps in the replication of viral RNA. There are no human polymerases that share either sequence or structural homology with viral RNA depended RNA polymerase. These make it a good target for inhibitor discovery, as a specific inhibitor cannot cross-react with the human polymerases. We have used virtual screening, docking, binding energy calculation and simulation to show that valproic acid Co-A, a metabolite from prodrug valproic acid, forms stable interaction with nsP12 of CoV. Our results suggest valproic acid Co-A could be a potential inhibitor of nsP12 of SARS-CoV2.

Immunosuppressed individuals, such as people living with HIV (PLWH), remain vulnerable to severe COVID-19. We analyzed the persistence of specific SARS-CoV-2 humoral and cellular immune responses in a retrospective, cross-sectional study in PLWH on antiretroviral therapy. Among 104 participants, 70.2% had anti-S IgG antibodies and 55.8% had significant neutralizing activity against the Omicron variant in a surrogate virus neutralization test. Only 38.5% were vaccinated (8.76 ± 4.1 months prior), all displaying anti-S IgG, 75% with neutralizing antibodies and anti-S IgA. 29.8% had no SARS-CoV-2 serologic markers and displayed significantly lower CD4 counts and HIV viral load. Severe immunosuppression (present in 12.5% of participants) was linked to lower levels of detectable anti-S IgG (0.0003), anti-S IgA (p &lt; 0.0001) and lack of neutralizing activity against Omicron variant (p &lt; 0.0001). T-cell responses were present in 86.7% of tested participants, even in those lacking serological markers. In PLWH without severe immunosuppression, neutralizing antibodies and T-cell responses persisted for up to 9 months post-infection or vaccination. Advanced immunosuppression led to diminished humoral immunity, but retained cellular immune.

Nuclear export of viral mRNAs, is an essential step in the HIV replication cycle. This role is played by a small regulatory protein of HIV-1 called Rev.The N-terminal region of Rev contains an arginine-rich sequence. The arginine-rich motif (ARM) is located between amino acids 38-50 and forms an alpha-helical secondary structure. Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev Response element (RRE). The major viral proteins are not produced if this transport of RNA is stopped. Therefore, knowledge of Rev structure is essential for understanding of its cooperative binding to the RRE, for understanding the mechanism of HIV infection and for the development of antiviral drugs that interfere with Rev’s essential functions and for acknowledgment of good candidate drugs for treatment of AIDS. To understand how REV interact with RRE element of HIV-RNA and its formation of oligomeric complex it is better to characterize the domain wise structure of REV with regard in function of each domain. Due to lack of structural data on Rev no single compound is reported as inhibitor of REV expect antiviral drugs. Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev Protein is much more important. The ARM is a highly specific sequence which allows for the multimerization of Rev and also binding of REV with RNA. Here we are first time exploring the structural characteristics of REV protein both in free form and in complex with RNA at domain function level especially explore the role of ARM motif in REV HIV-1 protein as RNA binding sites by molecular dynamics (MD) simulation and homology modeling studies. Results indicate that the arginine-rich motif (ARM) is crucial in stability of this complex. The residues ARG38, 39, 41, 43, 44, 48, 50, and ASN40 are most interacting with nucleobases of RRE in Crystal structure of Rev and Rev-response-element RNA complex. Our study plays a major role in elaboration of binding of RNA with REV and pave the way for further investigation for therapeutically agent for HIV.

Zika virus (ZIKV) has been characterized as one of the potential pathogens and placed under future epidemic outbreaks by the WHO. However, lack of potential therapeutics can result in uncontrolled pandemic like other human pandemic viruses; therefore, prioritized effective therapeutics development has been recommended against ZIKV. In this context, the present study adopted the strategy to explore the lead compounds from Azadirachta indica against ZIKV via concurrent inhibition of the ZIKVpro and ZIKVRdRp proteins using molecular simulations. Initially, structure-based virtual screening of 44 bioflavonoids reported in Azadirachta indica against the crystal structures of targeted ZIKV proteins resulted in the identification of top four common bioflavonoids, viz. rutin, nicotiflorin, isoquercitrin, and hyperoside. These compounds showed substantial docking energy (-7.9 to -11.01 kcal/mol) and intermolecular interactions with essential residues of ZIKVpro (His51, Asp74, and Ser135) and ZIKVRdRp (Asp540, Ile799, and Asp665) by comparison to the reference compounds, O7N inhibitor (ZIKVpro) and Sofosbuvir inhibitor (ZIKVRdRp). Long interval molecular dynamics simulation (500 ns) on the selected docked poses reveals the stability of respective docked complexes contributed by intermolecular hydrogen bonds and hydrophobic interactions. The predicted complex stability was further supported by calculated end-point binding free energy using molecular mechanics generalized born surface area (MM/GBSA) method. Consequently, the identified common bioflavonoids are recommended as promising therapeutic inhibitors of ZIKVpro and ZIKVRdRp for further experimental assessment.

Previously, we described a 19-base pair double-stranded RNA with 3'-trinucleotide overhangs, acting as immunostimulatory RNA (isRNA). This molecule demonstrated notable antiproliferative effects on cancer cells, inhibited tumor growth, and elicited immunostimulatory and antiviral responses by inducing cytokine and interferon production. Within this study, we compared the efficiency of lung fibrosis development, initiated in mice by BLM or LPS using different schemes of induction. Then we compared the effect of isRNA used in a preventive or therapeutic regimen on the development of fibrosis in selected BLM- and LPS-induced mouse models and showed that isRNA can be used in pathological conditions accompanied by the development of inflammation and the risk of fibrosis formation, without adverse side effects. Prophylactic regimen of isRNA application is beneficial for prevention of the development of pulmonary fibrosis.

We here report the de novo transcriptome assembly and functional annotation of Eusirus cf. giganteus clade g3, providing the first database of expressed sequences from this giant Antarctic amphipod. RNA-sequencing, carried out on the whole-body of a single juvenile individual likely undergoing molting, revealed the dominant expression of hemocyanins. The mRNAs encoding these oxygen-binding proteins cumulatively accounted for about 40% of the total transcriptional effort, highlighting the key biological importance of high hemocyanin production in this Antarctic amphipod species. We speculate that this observation may mirror a strategy previously described in Antarctic cephalopods, which compensate the decreased ability to release oxygen to peripheral tissues at sub-zero temperatures by massively increasing total blood hemocyanin content compared with temperate species. These preliminary results will undoubtedly require confirmation through proteomic and biochemical analyses aimed at characterizing the oxygen-binding properties of E. cf. giganteus clade g3 hemocyanins, and at investigating whether other Antarctic arthropod species exploit similar adaptations to cope with the challenges posed by the extreme conditions of the polar environment.

RNA pol II assembly occurs in the cytoplasm before the translocation of the enzyme to the nucleus. Affecting assembly influences mRNA transcription in the nucleus and mRNA decay in the cytoplasm. However, very little is known about the consequences on ncRNA synthesis. In this work, we show that impairment of RNA pol II assembly leads to a decrease in cryptic non-coding RNAs (preferentially CUTs and SUTs). This alteration is partially restored upon overcoming the assembly defect. Notably, this drop in ncRNAs is only partially dependent on the nuclear exosome, which suggests a major specific effect of enzyme assembly. Our data also point out a defect in transcription termination and lead as to proposes that CTD phosphatase Rtr1 could be involved in this process.

Circulating tumor cells (CTCs) are cells released from the primary tumor and intravasate into the blood or lymphatic vessels, where they are transported to distant sites and act as seeds that initiate cancer metastases. Recent advances in CTC research have shown their relevance as prognostic markers for early and metastatic disease detection, predictive biomarkers for relapse, and response to medical intervention or therapy. The rapidly evolving landscape of CTC biology has opened new avenues for understanding cancer progression, metastasis, and treatment response. Additionally, translating these findings into clinical applications holds promise for improving cancer diagnostics, prognosis, and personalized therapeutic strategies. This review discusses the significance of CTCs in cancer research and their associated challenges. We explore recent developments in the detection and characterization of CTCs and their implications in cancer research and clinical practice.

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood including the kinetics of formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA) and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA, immunofluorescence to detect nsp3 protein, a marker for the formation RO, and carried out a time course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 minutes post-infection (p.i.). Starting from 2 hr. p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 hr. p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-COV-2 variants of concern, and BA.1 strain exhibited slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets to early stage replication to combat COVID-19.

Abstract: Endoglin (ENG) is a causative gene of type 1 hereditary hemorrhagic telangiectasia (HHT1). HHT1 patients have arteriovenous malformations (AVMs) in multiple organs, including the brain. In mice, Eng deletion induced by R26RCreER or SM22aCre leads to AVM development in the brain and other organs. We have reported that the severity of brain AVM in mice with activin receptor-like kinase 1 (Alk1, an HHT2 causative gene) mutation is correlated with the number of Alk1 negative endothelial cells (ECs). We hypothesized that increase of Eng negative ECs will enhance AVM severity. To increase EC Eng deletion, we used a codon improved cre (icre), which is more potent in recombination of floxed alleles than the wild type (WT) cre. R26RCreER;Eng^f/f mice that have Rosa promoter driving and tamoxifen (TM) inducible WT cre expression globally and PdgfbiCreER;Eng^f/f that have Pdgfb promoter driving and TM inducible icre expression in ECs were treated with 3 intra-peritoneal injections of TM (2.5 mg/25g of body weight) to delete Eng globally or in the ECs. AAV-VEGF was stereotactically injected into the brain to induce brain focal angiogenesis and brain AVM. We found that iCre caused more Eng deletion in the brain indicated by lower level of Eng proteins (p<0.001) and fewer Eng positive ECs (p=0.01) than mice with WT cre. Mice with iCre mediated Eng deletion have more abnormal vessels (p=0.02), CD68⁺ macrophages (p=0.002), and hemorrhage (p=0.04), and less vascular pericyte and smooth muscle coverage than mice with WT cre. In addition, arteriovenous shunts were detected in the intestines of icre mice, a phenotype that have not been detected in WT cre mice before. RNAseq analysis showed that 8 out of the 10 top upregulated pathways identified by Gene Ontology (GO) analysis are related to the inflammation. Therefore, increase of Eng deletion in ECs exacerbates AVM severity, which is associated with enhanced inflammation. Strategies that can reduce Eng negative ECs could be used to develop new therapies to reduce AVM severity for HHT1 patients.

The transition from the Peptide/RNA world to the Protein/RNA world in the hydrothermal vent environment was a major event in the history of life. The advent of proteins utterly changed the conditions of emerging life, representing a watershed in its development. During subsequent translation various protein enzymes emerged driving protocells into a more complex and interconnected system. With their astonishing versatility, the protein enzymes catalyzed crucial biochemical reactions within protocells into more complex biomolecules in diverse metabolic pathways, whereas structural proteins provided strength and permeability in the cell membrane. Four major events followed after availability of various kinds of protein molecules during prebiotic synthesis. These are: (1) the modification of the phospholipid membrane into the plasma membrane; (2) the origin of primitive cytoplasm; (3) the beginnings of the virus world; and (4) the advent of DNA. The first innovation mediated by proteins was the improvement of the cell membrane. The phospholipid membrane was initially evolved in a vent environment from the gradual modification of a fatty acid membrane via an intermediate phosphatidate acid by non-enzymatic reactions. The phospholipid is then synthesized from phosphatidate acid by a series of enzymes. To make the phospholipid membrane more permeable, various protein molecules interacted with the cell membrane. Proteins not only stabilized the wall membrane, but also acted as pumps, preventing some molecules from the protocells from crossing the membrane barriers, while permitting other selected molecules and ions to enter and leave the protocell. The second modification led by proteins is the gradual conversion of the interior of the protocell from a water-like medium into a gel-like cytoplasm, which became the storehouse of a wide range of biomolecules including amino acids, proteins, nucleic acids, ribosomes, as well as salt and water. The third innovation utilizing the newly synthesized proteins was the emergence of the ancient virus world. In the milieu of different kinds of mRNAs in the prebiotic soup, jelly-roll capsid genes originated de novo within genomes of nonviral mRNAs by overprinting. These fragile capsid genes were possibly coated by proteins on the mineral substrate for stability and durability, transforming them into ancient viral particles. These protein coats were random and were not encoded by encased genes. Some protocells might have engulfed these viral particles, when the capsid genes utilized the ribosomes of the host to translate into the appropriate capsid proteins. These capsid proteins then coated the viral genes to make new copies of primordial viruses inside the protocell. Since then, viruses became capsid-encoding organisms. These primordial mRNA viruses parasitized RNA-based protocells, manipulating them to make new copies of themselves. This was the beginning of a relentless war between viruses and their protocellular hosts. The next stage in viral evolution was the emergence of a primitive retrovirus (pre-retrovirus) with a new kind of replicative strategy in a sense that it could turn its RNA into DNA using its own reverse transcriptase enzyme. This is the beginning of the Retro world that facilitated the transition from RNA to DNA genomes. The infection of RNA protocells with pre-retroviruses progressively transferred the RNA genome to a viral DNA genome by retro-transcription. The advent of DNA by the pre-retrovirus marks the fourth innovation, when a number of enzymes had already developed and were utilized by pre-retroviruses. With continued infection, DNA viruses slowly transferred not only their core replication enzymes, such as helicase, primase, and DNA polymerase, to RNA protocells, but also to their DNAs as well. Thus, began the DNA world, when DNA replaced RNA as the major genome of the protocells. With the advent of DNA, replication of information was entirely dissociated from its expression. Because DNA is much more stable than mRNA with more storage capacity, it is a superb archive for information systems in the form of base sequences. DNA progressively took over the replicative storage function of mRNA, leaving the latter for protein synthesis. The new protocell with the DNA genome will diversify into large populations of DNA protocells that will outcompete populations of RNA protocells. Genetic information began to flow from DNA to mRNA to protein in a two-step process involving transcription and translation. In the biological stage, DNA replication was central to the binary fission of the first cell, orchestrated by the duplication of genomes and then the division of the parent cell into two identical daughter cells. It was carried out by a set of enzymes that formed a Z-ring at the site of replication. With the onset of binary fission, the population of primitive cells grew rapidly in the hydrothermal vent environment, undergoing Darwinian evolution and diversification. These primordial hyperthermophiles, presumably the first life, obtained food and energy directly from the vent environment. However, such a situation was self-limiting, so the early cells evolved their own mechanisms for generating metabolic energy and synthesizing the molecules necessary for their reproduction. The earliest fossil record (≥ 3.5 Ga) of biotic activity is preserved in the Archean hydrothermal and sedimentary rocks of the Nuvvuagittuq Craton of Canada, the Isua Craton of Greenland, the Pilbara Craton of Australia, the Kaapvaal Craton of South Africa, and the Singhbhum Craton of India in the form of the carbonaceous remains of microbial cells, cellular microfossils, and stromatolites. These microscopic fossils provide crucial evidence of the origin and early evolution of prokaryotic cells, beginning with hyperthermophiles. Molecular phylogenetic analysis suggests that both domains of life ¬– Bacteria and Archaea probably split from the last universal common ancestor (LUCA), a hyperthermophilic organism. In the younger sequences of these Archean cratons, two kinds of photosynthetic bacteria, anoxygenic green sulfur bacteria, and oxygenic cyanobacteria, appeared in quick succession from the thermophilic ancestor, indicating a shift of niche from a benthic to a planktonic, with reduced thermotolerance. The development of anoxygenic and oxygenic photosynthesis would have allowed life to escape the hydrothermal setting and invade a newly evolved habitat—broad continental shelves to tap solar energy. Cyanobacteria invaded the global ocean, turned it into blue and green, produced oxygen for the first time, and left their signatures in the carbonates and stromatolites.

Chikungunya virus (CHIKV) is a mosquito borne RNA virus that poses an emerging threat to humans. As other RNA viruses, CHIKV encodes an error-prone RNA polymerase which, in addition to full-length genomes, gives rise to truncated, non-functional genomes coined defective viral genomes (DVGs). DVGs have been intensively studied in the context of therapy, as they can inhibit viral replication and dissemination in their hosts. In this work, we interrogate the influence of viral RNA secondary structures on the production of CHIKV DVGs. We experimentally map RNA secondary structures of CHIKV genome using selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), which couples chemical labelling with next-generation sequencing. We correlate the inferred secondary structure with preferred deletion sites of CHIKV DVGs. We document an increased probability of DVG generation with truncations at unpaired nucleotides within the secondary structure. We then generated a CHIKV mutant bearing synonymous changes at the nucleotide level to disrupt existing RNA secondary structure (CHIKV-D2S). We show that CHIKV-D2S presents altered DVG generation compared to wild-type virus, correlating with its change in RNA secondary structure obtained by SHAPE-MaP. Our work thus demonstrates that RNA secondary structure impacts CHIKV DVG production during replication.

With the increment of the aging population in recent years, neurodegenerative diseases exert a major global disease burden, essentially as a result of the lack of treatments that can stop the disease progression. Alzheimer’s Disease (AD) is an example of a neurodegenerative disease, that affects millions of people globally, with no effective treatment. Natural compounds have emerged as a viable therapy to fill a huge gap in AD management, and in recent years, mostly fuelled by the Covid-19 pandemic, RNA-based therapeutics have become a hot topic in the treatment of several diseases. Treatments of neurodegenerative diseases face significant limitations due to the complex and interconnected pathways that lead to their hallmarks and, also due to the necessity to cross the blood–brain barrier. Nanotechnology has contributed to surpass this bottleneck in the treatment of AD, by promoting safe and enhanced drug delivery to the brain. In particular, exosome-like nanoparticles, a hybrid delivery system combining exosomes and liposomes' advantageous features, are demonstrating great potential in the treatment of central nervous system diseases.

Abstract: Long noncoding RNAs (lncRNAs) are deregulated in human cancers and are associated with disease progression. Plasmacytoma Variant Translocation 1 (PVT1), an lncRNA, is located adjacent to MYC, linked to multiple myeloma (MM). PVT1 is expressed in MM and is associated with carcinogenesis, however, its role and regulation machinery remain uncertain. We examined PVT1/MYC expression through real time PCR in plasma cells purified from 59 MGUS and 140 MM patients. MM cell lines KMS11, KMS12PE, OPM2, and RPMI8226 were treated with JQ1, a MYC superenhancer inhibitor, or MYC inhibitor 10058-F4. The expression levels of PVT1 and MYC were significantly higher in MM than in MGUS (p &lt; 0.0001), and showed positive correlation with disease progression (r = 0.394, p &lt; 0.0001). JQ1 inhibited cell proliferation and decreased the expression levels of MYC and PVT1. However, 10054-F4 did not alter the expression level of PVT1. The positive correlation between MYC and PVT1 in patients, synchronous downregulation of MYC and PVT1 by JQ1, and no effect of MYC inhibitor on PVT1 expression suggest that the expression of these two genes is coregulated by a superenhancer. Cooperative effects between these two genes may contribute to MM pathogenesis and progression.

Computer vision is a rapidly developing field that focuses on highly sophisticated picture analysis, manipulation, and comprehension. Its objective is to analyze what is happening in front of a camera and utilize that understanding to control a computer or robotic system or to present users with fresh visuals that are more enlightening or appealing than the original camera images. Computer vision technologies make it feasible for new user interfaces, augmented reality gaming, biometrics, automobile, photography, movie creation, Web search, and many more applications. This essay seeks to explain how computer vision can be utilized to play blackjack successfully.

In the intricate dance between viruses and host cells, RNA-binding proteins (RBPs) serve as crucial orchestrators of gene expression and cellular processes. We will delve into the riveting realm of viral hijacking, where viruses deftly exploit host RBPs to manipulate cellular machinery for their replication and pathogenesis. Through a masterstroke of molecular subterfuge, viruses co-opt RBPs involved in various facets of RNA metabolism - from transcription to degradation - to promote viral gene expression and evade host immune defenses. This manipulation leads to global alterations in cellular RNA metabolism, impacting essential host genes vital for immune responses and homeostasis.Unveiling this clandestine alliance between viruses and RBPs is not just a scientific pursuit, but a critical imperative for devising innovative antiviral strategies to disrupt these interactions. By unraveling the intricate interplay between viral proteins and host RBPs throughout the viral life cycle - from entry to assembly - researchers aim to identify vulnerabilities that can be targeted for therapeutic intervention. Strategies such as disrupting viral protein-RBP interactions hold promise for inhibiting viral replication and curbing pathogenesis, offering a beacon of hope in the battle against viral infections.Our manuscript elucidates the indispensable roles played by RBPs in viral replication, translation, and pathogenesis, shedding light on the molecular mechanisms driving viral success. Delving deeper, it explores how viruses intricately entwine with host RBPs, manipulating cellular signaling pathways to create a hospitable environment for viral spread. By dissecting these manipulative tactics, researchers uncover new targets for antiviral therapy, envisioning a future where tailored interventions disrupt viral-host RBP interactions with precision and efficacy.As the narrative unfolds, the therapeutic implications of targeting RBPs or their interactions with viral proteins emerge as a promising frontier in the fight against viral infections. From small molecule inhibitors to RNA-based therapeutics, innovative approaches are on the horizon, offering new avenues for combatting viral diseases. We set the stage for future research, beckoning researchers to delve deeper into the molecular intricacies of viral hijacking of RBPs and charting a course towards novel therapeutic interventions that promise to reshape the landscape of antiviral therapy.\Ultimately, it beckons the scientific community to embark on a voyage of discovery, unraveling the secrets of viral hijacking of RBPs and paving the way for transformative advances in antiviral therapeutics. The stage is set for a new chapter in the battle against viral infections, where knowledge becomes the sword and innovation the shield against the pernicious machinations of viral pathogens.

Current cellular facts allow us to follow the link from chemical to biochemical metabolites, from the ancient to the modern world. In this context, the "RNA world" hypothesis proposes that early in the evolution of life, the ribozyme was responsible for the storage and transfer of genetic information and for the catalysis of biochemical reactions. Accordingly, the hammerhead ribozyme (HHR) and the hairpin ribozyme, belong to a family of endonucleolytic RNAs performing self-cleavage that might occur during replication. Furthermore, regarding the ultraconserved occurrence of HHR in several genomes of modern organisms (from mammals to small parasites and elsewhere), these small ribozymes have been regarded as living fossils of a primitive RNA world. They fold into 3D structures that generally require long-range intramolecular interactions to adopt the catalytically active conformation under specific physicochemical conditions. By studying viroids as plausible remains of ancient RNA, we recently demonstrated that they replicate in non-specific hosts, emphasizing their adaptability to different environments, which enhanced their survival probability over the ages. All these results exemplify ubiquitously features of life. Those are the versatility and efficiency of small RNAs, viroids and ribozymes, as well as their diversity and adaptability to various extreme conditions. All these traits must have originated in early life to generate novel RNA populations.

In March 2013, a novel avian influenza A H7N9 virus was emerged in China, which cause rapidly progressive pneumonia and with a high fatality rate. Serologic studies to evaluate neutralizing antibodies of infected patients and birds are invaluable tools for immunogenicity research of H7N9 and epidemiological investigation. Conventional neutralization assays are laborious and time-consuming which also hampered by biosafety requirement. In this study, We construct and produce pseudovirus bearing the full-length hemagglutinin (HA) of H7N9 virus in the Env-defective, luciferase-expressing HIV-1 backbone. The production of lentiviral pseudovirus was analysed by HA gene specific real-time reverse-transcription PCR, transmission electron microscopy (TEM), and Western Blot assay to prove the nucleic acid replication, the morphology of virus, and the expression of HA protein in pseudovirus. After that pseudovirus based inhibition assay was established to detect neutralizing antibodies of a panel of serum samples. Our results demonstrated that H7N9 pseudovirus which had single-cycle infection was generated. By comparing the neutralization antibody titers, pseudovirus based neutralization test could be recognized as an alternative of conventional microneutralization (MN). Hence, we conclude that it is possible to use pseudovirus inhibition assay to screen sera samples, as well as evaluate vaccine-induced neutralizing antibodies against H7N9 virus.

Despite all scientific efforts and many protracted and expensive clinical trials, no new drug has been approved by FDA for treatment of Alzheimer disease (AD) since 2003. Indeed, more than 200 investigational programs have failed or have been abandoned in the last decade. The most probable explanations for failures of disease-modifying treatments (DMTs) for AD may include late initiation of treatments during the course of AD development, inappropriate drug dosages, erroneous selection of treatment targets, and mainly an inadequate understanding of the complex pathophysiology of AD, which may necessitate combination treatments rather than monotherapy. Clinical trials’ methodological issues have also been criticized. Current drug-development research for AD is aimed to overcome these drawbacks. Preclinical and prodromal AD populations, as well as traditionally investigated populations representing all the clinical stages of AD, are included in recent trials. Systematic use of biomarkers in staging preclinical and prodromal AD and of a single primary outcome in trials of prodromal AD are regularly integrated. The application of amyloid, tau, and neurodegeneration biomarkers, including new biomarkers—such as Tau positron emission tomography, neurofilament light chain (blood and CSF biomarker of axonal degeneration) and neurogranin (CSF biomarker of synaptic functioning)—to clinical trials allows more precise staging of AD. Additionally, use of the Bayesian statistics, modifiable clinical trial designs, and clinical trial simulators enrich the trial methodology. Besides, combination therapy regimens are currently assessed in clinical trials. The abovementioned diagnostic and statistical advances, which have been recently integrated in clinical trials, are consequential to the recent failures of studies of disease-modifying treatments. Their experiential rather than theoretical origins may better equip potentially successful drug-development strategies.

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein’s host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient it induces reinforcement of antiviral silencing by AGO2, and triggers increased resistance against aphids, CMV’s insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein by sequestering sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated and red and green fluorescent protein fusions used to investigate subcellular colocalization with AGO1 and the 1a protein, and the effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein’s interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein’s ability to interact with AGO1, the 1a protein and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

Objective To investigate the functional and biological significance of telomere-related active genes in cholangiocarcinoma (CCA) using single-cell sequencing and cell communication analysis. Methods First, we identified and annotated different cell types in CCA samples using single-cell sequencing data. Pseudotime analysis revealed the transcriptional status and differentiation process of CCA cells. We then explored interactions between CCA cells and their regulatory pathways through cell communication analysis. 47 key genes related to telomere activity were identified, and GO and KEGG pathway enrichment analysis was performed. Additionally, we constructed a prognostic model and verified the model's predictive efficacy. Finally, we identified genes associated with drug sensitivity in CCA patients and identified pathways associated with CCA prognosis through tumor TMB analysis. Results We identified 452 active telomere-associated cells. Then, we assessed telomere associated gene activity, obtaining 47 differentially expressed telomere genes by intersecting with CCA differentially expressed genes. Major up-regulated signaling pathways, including MIF-(CD74+CXCR4) and HLA-A-CD8A, were identified through pseudotime analysis. Three telomere-associated prognostic genes were identified using LASSO analysis, and prognostic-related models were built. In addition, PBRM1 mutation load was found to be highest in the high-risk group, making it a potential diagnostic target. Finally, drug sensitivity analysis revealed higher sensitivity to certain therapeutic drugs in the high-risk group, providing guidance for tailored CCA treatment. Conclusion The MIF-(CD74+CXCR4) and HLA-A-CD8A signaling pathways appear to be major pathways for CCA cell proliferation. Prognostic models based on telomere-associated genes show promise for application, indicating the biological and clinical significance of telomere activity genes in CCA.

Background : Newly generated cardiomyocytes (NGC) concur to recovery of human myocarditis. However, Methods: We retrospectively assessed NGC in 213 consecutive patients with endomyocardial biopsy denoting acute myocarditis, with normal coronaries and valves. Tissue samples were processed for histology (H&E), immunohistochemistry for the evaluation of inflammatory infiltrates, immunostaining for alpha-sarcomeric-actin, junctional connexin-43, Ki-67, phosphorylated STAT3 (p-STAT3) and Western Blot (WB) for HMGB1. Frozen samples were analyzed with polymerase chain reaction (PCR) for cardiotropic viruses. Controls included 20 normal surgical biopsies. Results: NGCs were defined as small myocytes (diameter < 10 µm) with nuclear positivity to Ki-67 and p-STAT3, positive immunostaining for cytoplasmic α-sarcomeric actin and connexin 43. Their number/mm2 in relation with age and pathway of integration were evaluated. NGCs crossed the membrane and grew integrated within the empty necrotic myocytes. NGC mean diameter was 6.6 ± 3.34 vs 22.5 ± 3.11 µm adult cells; their number, in comparable LVEF, was 86.3 ± 10.3/mm2 in patients between 10 and 40 years; 50.4 ±13.8/mm2 in those between 41 and 60; 15.1 ± 5.7/ mm2 in those between 61 and 80; control NGC mean diameter was 0.2± 0.2/mm2. PCR was positive for viral genomes in 16% of cases; NGCs were not statistically different in viral and non-viral myocarditis. WB analysis revealed a higher expression of HMGB1 in myocarditis compared to myocardial controls. Conclusions: NGCs are constantly recognizable in acute human myocarditis. Their number declines with age. Their integration within necrotic myocytes consents preservation of cardiac structure and function.

The term neurodegeneration with brain iron accumulation (NBIA) brings together a broad set of progressive and disabling neurological genetic disorders in which iron is deposited preferentially in certain areas of the brain. Among NBIA disorders, the most frequent subtype is pantothenate kinase-associated neurodegeneration (PKAN) caused by pathologic variants in the PANK2 gene codifying the enzyme pantothenate kinase 2 (PANK2). Nowadays, there are no effective treatments to stop the progression of these diseases. This review discusses the utility of patient-derived cell models as a valuable tool for the identification of commercial pharmacological or natural compounds for implementing polytarget precision medicine in PKAN. In the last years, several studies have described that PKAN patient-derived fibroblasts manifest the main pathological changes associated with the disease including intracellular iron overload. Interestingly, treatment of mutant cell cultures with various supplements such as pantothenate, pantethine, thiamine, L-carnitine, vitamin E, omega 3, and -lipoic acid improved all pathophysiological alterations in PKAN fibroblasts with residual expression of the PANK2 enzyme. The information provided by pharmacological screenings in patient-derived cellular models can help to optimize therapeutic strategies in individual PKAN patients.

In the last decades, technological advances related to RNA manipulation enabled and expanded its application in vaccine development. This approach comprises synthetic single-stranded mRNA molecules that direct the translation of the antigen responsible for activating the desired immune response. The success of RNA vaccines depends on the delivery vehicle employed. Among the systems, yeasts emerge as a new approach to a natural delivery platform. The presence of β-glucans and mannans in its wall is responsible for the adjuvant action of this system. Yeasts are already employed to deliver protein antigens, with success and efficacy demonstrated through pre-clinical and clinical trials. Yeast β-glucan capsules, microparticles, and nanoparticles are capable of modulating host immune responses and have a high capacity to carry RNA and small molecules, with bioavailability upon oral immunization and with targeting to receptors present in anti-gen-presenting cells (APCs). Besides, yeasts are interesting vehicles for the protection and specific delivery of therapeutic vaccines based on shRNA or dsRNA. In this review, we present an overview of the attributes of yeast or its derivatives for the delivery of RNA-based vaccines, discussing their current challenges and prospects for using this promising strategy.

MicroRNAs (miRNAs) dysregulation contribute the cancer pathogenesis. However, the miRNA profile of canine oral melanoma (COM), one of the frequent malignant melanoma in dog is still unrevealed. The aim of this study is to reveal the miRNA profile in canine oral melanoma. MicroRNAs profile of oral tissues from normal healthy dogs and COM patients were compared by next-generation sequencing. Along with tumour suppressor microRNAs (miRNAs), we report 30 oncogenic miRNAs in COM. Expression of miRNAs were further confirmed by quantitative real-time PCR (qPCR). Pathway analysis showed that deregulated miRNAs impact on cancer and signalling pathways. Three oncogenic miRNAs targets (miR-450b, 301a, and 223) from human study also were down-regulated in COM and had significant negative co-relation with their respective miRNA. Furthermore, we found that miR-450b expression is higher in metastatic cells and regulated MMP9 expression through a PAX9-BMP4-MMP9 axis. In silico analysis indicated that miR-126, miR-20b, and miR-106a regulated the highest numbers of differentially expressed transcription factors in respect to human melanoma. Chromosomal enrichment analysis revealed the X chromosome was enriched with oncogenic miRNAs. We comprehensively analyzed the miRNA’s profile in COM which will be a useful resource for developing therapeutic interventions in both species.

Small non-coding RNAs (sRNAs) are ubiquitously found in the three domains of life playing large-scale roles in gene regulation, transposable element silencing, and defense against foreign elements. While a substantial body of experimental work has been done to uncover function of sRNAs in Bacteria and Eukarya, the functional roles of sRNAs in Archaea are still poorly understood. Recently, high throughput studies using RNA-sequencing revealed that sRNAs are broadly expressed in the Archaea, comprising thousands of transcripts within the transcriptome during non-challenged and stressed conditions. Antisense sRNAs, which overlap a portion of a gene on the opposite strand (cis-acting), are the most abundantly expressed non-coding RNAs and they can be classified based on their binding patterns to mRNAs (3’ UTR, 5’ UTR, CDS-binding). These antisense sRNAs target many genes and pathways, suggesting extensive roles in gene regulation. Intergenic sRNAs are less abundantly expressed and their targets are difficult to find because of a lack of complete overlap between sRNAs and target mRNAs (trans-acting). While many sRNAs have been validated experimentally, a regulatory role has only been reported for very few of them. Further work is needed to elucidate sRNA-RNA binding mechanisms, the molecular determinants of sRNA-mediated regulation, whether protein components are involved, and how sRNAs integrate with complex regulatory networks.

Background: Recent developments in our understanding of the interactions between long non-coding RNA (lncRNA) and cellular components have improved treatment approaches for various human diseases including cancer, vascular diseases, and neurological diseases. Although investigation of specific lncRNAs revealed their role in the metabolism of cellular RNA, our understanding of their contribution to post-transcriptional regulation is relatively limited. In this study, we explore the role of lncRNAs in modulating alternative splicing and their impact on downstream protein-RNA interaction networks. Results: Analysis of alternative splicing events across 39 lncRNA wildtype and knockout RNA-sequencing datasets from three human cell lines: HeLa (Cervical Cancer), K562 (Myeloid Leukemia), and U87 (Glioblastoma), resulted in high confidence (fdr < 0.01) identification of 4432 skipped exon events and 2474 retained intron events, implicating 759 genes to be impacted at post-transcriptional level due to the loss of lncRNAs. We observed that a majority of the alternatively spliced genes in a lncRNA knockout were specific to the cell type, in agreement with the finding that genes affected by alternative splicing also displayed enriched functions in a cell type specific manner. To understand the mechanism behind this cell-type specific alternative splicing patterns, we analyzed RNA binding protein (RBP)-RNA interaction profiles across the spliced regions. Conclusions: Despite limited RBP binding data across cell lines, alternatively spliced events detected in lncRNA perturbation experiments were associated with RBPs binding in proximal intron-exon junctions, in a cell type specific manner. The cellular functions affected by alternative splicing were also affected in a cell type specific manner. Based on the RBP binding profiles in HeLa and K562 cells, we hypothesize that several lncRNAs are likely to exhibit a sponge effect in disease contexts, resulting in the functional disruption of RBPs due to altered titration of the RBPs from their target loci. We propose that such lncRNA sponges can extensively rewire the post-transcriptional gene regulatory networks by altering the protein-RNA interaction landscape in a cell-type specific manner.

The Ro60 autoantigen is an RNA-binding protein, and a core component of nucleocytoplasmic ribonucleoprotein (RNP) complexes. Ro60 is essential in RNA metabolism, cell stress response pathways, and cellular homeostasis. It stabilises and mediates quality control and cellular dis-tribution of YRNAs, retroelement transcripts, and misfolded RNAs. Ro60 transcriptional dysregulation or loss of function can result in the generation and release of RNA fragments from YRNAs and other small RNAs. Small RNA fragments can instigate an inflammatory cascade through endosomal TLRs and cytoplasmic RNA sensors, which typically sense patho-gen-associated molecular patterns (PAMPs) and mount the first line of defence against invading pathogens. However, recognition of host-originating RNA moieties from Ro60 RNP complexes can activate inflammatory response pathways and compromise self-tolerance. Autoreactive B cells may produce antibodies targeting extracellular Ro60 RNP complexes. Ro60 autoantibodies serve as diagnostic markers for various autoimmune diseases including Sjögren's disease (SjD) and systemic lupus erythematosus (SLE), and they may also act as predictive markers for an-ti-drug antibody response among rheumatic patients. Understanding Ro60’s structure, function, and role in self-tolerance can enhance our understanding of the underlying molecular mecha-nisms of autoimmune conditions.

The COVID-19 pandemic stimulated attention to the medicinal applications of messenger RNA (mRNA). mRNA is expected to be applied not only to vaccines but also to regenerative medicines. The purity of mRNA is important for its medicinal application. However, the current mRNA synthesis techniques have problems, e.g., contamination of undesired 5’-uncapped mRNA and double-stranded RNAs. Recently, our group developed a completely capped mRNA synthesis technology that contributed to the progress of mRNA research. The introduction of chemically modified nucleosides, e.g., N1-methylpseudouridine and 5-methylcytidine, have been demonstrated by Dr. Karikó and Dr. Weissman which opened the practical application of mRNA for vaccines and regenerative medicines. Dr. Yamanaka reported the production of Induced Pluripotent Stem Cells (iPSCs) by the introduction of 4 types of genes using a retrovirus vector. iPSCs are widely used for research about regenerative medicines and the preparation of disease models to screen new drug candidates. Of the Yamanaka factors, Klf4 and c-Myc are oncogenes, and there is a tumor development risk if these are integrated into genomic DNA. Therefore, regenerative medicines using mRNA, which has no risk of genome insertion, have attracted attention. In this review, we summarized the synthesis of mRNA and its application for regenerative medicine.

X-ray diffraction (XRD) analysis of complex poly-atomic systems, especially of biomolecules, using ultra-short laser pulses (USP), is currently one of the most important fields of modern physics. The basis for interpreting and "deciphering" experimental data is the well-known theory of X-ray scattering, where the main parameter of USPs - its duration - is not taken into account. In the present work it is shown that for scattering of attosecond USPs on DNA and RNA trinucleotides the pulse length is the most important scattering parameter. In this case the diffraction pattern significantly changes with respect to the previously known scattering theory. The results obtained are extremely important in XRD when using attosecond pulses to study trinucleotides of DNA and RNA because using the previously known scattering theory which does not take into account the duration of USPs one can not correctly interpret and therefore "decode" DNA and RNA structures.

Our perspective article covers major findings concerning Feline Infectious Peritonitis (FIP) - a fatal coronaviral disease of cats. In the context of FIP pathogenesis, we outline disease signalment and focus on the challenges and promises of FIP invoking coronavirus RNA detection. In particular, we outline critical aspects of coronavirus RNA replication and biogenesis. We infer the replicative intermediates of feline coronavirus may constitute an underappreciated factor triggering the progression of the maladaptive immune response underlying FIP pathogenesis.

The role of innate immunity in neutralization of viral infections (including COVID-19) and forming long-lasting and specific immune memory is considered. It is assumed that antiviral protection is generated by the mechanism of RNA interference (RNAi) and is based on the presence of specific viral patterns in the DNA library of the host cells.

To understand the evolution of the human norovirus GII.P6-GII.6 and GII.P7-GII.6 strains, we analyzed both the RdRp region and VP1 gene in globally collected strains using authentic bioinformatics technologies. A common ancestor of the P6- and P7-type RdRp region emerged approximately 50 years ago and a common ancestor of the P6- and P7-type VP1 gene emerged approximately 110 years ago. Subsequently, the RdRp region and VP1 gene evolved. Moreover, the evolutionary rates were significantly faster for the P6-type RdRp region and VP1 gene than the P7-type RdRp region and VP1 genes. Large genetic divergence was observed in the P7-type RdRp region and VP1 gene compared with the P6-type RdRp region and VP1 gene. The phylodynamics of the RdRp region and VP1 gene fluctuated after the year 2000. Positive selection sites in VP1 proteins were located in the antigenicity-related protruding 2 domain, and these sites overlapped with conformational epitopes. These results suggest that the GII.6 VP1 gene and VP1 proteins evolved uniquely due to recombination between the P6- and P7-type RdRp regions in the HuNoV GII.P6-GII.6 and GII.P7-GII.6 virus strains.

Platelet-activating factor (PAF) is a phospholipid-derived inflammatory mediator, triggering various inflammatory conditions, including eosinophil activation and recruitment. Despite the unclear mechanism underlying the formation and maintenance of nasal polyp in chronic rhinosinusitis with nasal polyps (CRSwNP), there is strongly indication of its association with type 2 inflammation. This study aimed to quantify PAF-associated gene expression, examining enzymes involved in PAF synthesis (lysophosphatidylcholine acyltransferase 1 (LPCAT1), LPCAT2, LPCAT3, and LPCAT4), and PAF degradation (PAF acetylhydrolases 1 B2 (PAFAH1B2), PAFAH1B3, and PAF acetylhydrolases 2 (PAFAH2)), and PAF receptor (PTAFR). Transcriptomic analysis encompassed CRSwNP, including eosinophilic CRS (ECRS) (n=9), nonECRS (n=8), and a subgroup of ECRS with aspirin exacerbated respiratory disease (Asp) (n=3) based on the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC) criteria. Gene expression profiles from nasal polyps in CRSwNP patients and the uncinate process in normal subjects (controls) (n=6) were analyzed using bulk RNA barcoding and sequencing (BRB-seq). Hierarchal analysis, utilizing the average linkage method with iDEP1.1, clarified the relationship between Differentially Expressed Genes (DEG) expressions and cluster formation. Two clusters (cluster 1 (n=11) and cluster 2 (n=9)) of CRSwNP divided by hierarchal analysis showed distinct gene expression profiles in the specific pathway of “cytokine-cytokine receptor interaction” in KEGG pathway and upregulated type 2 inflammatory gene expression in cluster 2. In CRSwNP subclassification of ECRS, nonECRS, and Ctrl, PTAFR was only upregulated in ECRS and nonECRS. In cluster analysis, cluster 1 showed significant downregulation of LPCAT2 and upregulation of PTAFR expression, while cluster 2 showed significant upregulation of LPCAT1, PAFAH1B2, and PTAFR and downregulation of PAFAH2 expression. These findings suggest that cluster 2, characterized by severe type 2 inflammatory gene expression, manifest a prominent PAF-associated pathophysiology. Understanding this high PAF-associated pathophysiology in cluster 2 could provide valuable insights into the treatment and management of CRSwNP.