RNA extraction from human cumulus cells (CCs) is considered challenging due to low cell’s pop-ulation and small cell. The successful gene expression analysis from cumulus cells ultimately de-pends on an excellent concentration of Ribonucleic acid (RNA) yield by an appropriate technique. Our study aims to consolidate appropriate cumulus cell collection method, preparation and ex-traction techniques to ensure a good yield of RNA for better gene expression analysis. From the results obtained, the optimal strategy found involved mechanical denudation without an enzy-matic process, followed by the use of RNA stabilizer prior to RNA extraction. Subsequently, uti-lizing a smaller homogenizer, preferably manual pressure control with filter (Bio-Masher III®) is paramount for a pure and homogenize sample. Subsequently, during RNA extraction, the use of RNeasy micro kit and RNA carriers is recommended. These sample collection, preparation, and extraction techniques yielded an excellent-quality RNA concentration and successful gene expres-sion analysis. We also conducted a systematic review to consolidate our findings with current evidence of RNA extraction technique. Our results added a value as a current strategy for opti-mizing RNA extraction in CCs for experimental studies. Thus, our findings can contribute to for-mulating a better strategy for optimizing the RNA extraction in CCs for transcriptomic studies.

The pandemic respiratory disease COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan in December 2019 and then spread throughout the world; Italy was the most affected European country. Despite the close pet-human contact, little is known about the predisposition of pets to SARS-CoV-2. Among these, felines are the most susceptible. In this study, a domestic cat with clear symptoms of pneumonia, confirmed by Rx imaging, was found to be infected by SARS-CoV-2 using quantitative RT–qPCR from a nasal swab. This is the first Italian study reporting on the request of the scientific community to focus attention on the possible role of pets as a SARS-CoV-2 reservoir. An important question remains unanswered: did the cat die from SARS-CoV-2 infection?

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®). rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80µl of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Cq between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.80% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.

Background: the devastating outbreak of COVID-19 poses serious challenges for the diagnostics laboratories, which are often facing global shortage of reagents and equipment. With the aim of increasing the diagnostic throughput for SARS-CoV-2 molecular test, the purpose of this study was to validate an additional RNA extraction method respect to those already recommended by WHO and the US Centers for Disease Control and Prevention (CDC). Methods: a new protocol for RNA extraction from nasopharyngeal swab was set up, adapting the Qiagen RNeasy 96 plate and validated on a set of 100 clinical samples analyzed in parallel by Roche-Magnapure method (already recommended by CDC guidelines). Results: the internal control and target genes analysis showed a good agreement between the two extraction methods indicating that the two methods can be considered equivalent and that the RNeasy-adapted method can be applied for the SARS-CoV-2 diagnostics. The addition of this new extraction method resulted in a throughput increase for SARS-CoV-2 molecular test of about 2000 samples/month during the initial months of the pandemic emergency in which the lack of reagents for the extraction led to an insufficient sample processing throughput of the analysis of the swabs.

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.

Marine invertebrates are model organisms in several areas of biological sciences, being a source of massive biological information. Although, the scientific relevance of marine invertebrates, the research with them can be limited for their tissue characteristics and troubles for the replication of physical and chemical properties of seawater. Thence, the main goal of this laboratory workflow is to provide a useful methodological approach to reduce the experimental limitations during the study of marine invertebrates. The present study describes experimental methodologies for the collection, transport, and maintenance of sessile tunicates. Also, an approach to observe and characterize, a diverse population of blood cells in marine invertebrates, by several cytological stains and electron microscopy. Lastly, suggestions and protocols to extract quality RNA from samples with high concentrations of salts, pigments, secondary metabolites, and polysaccharides. This methodological approach can be easily adapted to other marine invertebrates, moreover uses low-cost reagents and widely available laboratory equipment. Making possible the study of different types of marine animals in diverse locations.

Myelodysplastic syndromes or neoplasms (MDS) are a heterogeneous group of myeloid clonal disorders characterized by peripheral blood cytopenias, blood and marrow cell dysplasia and an increased risk for evolution to acute myeloid leukemia (AML). Non-coding RNAs, especially microRNAs and long non-coding RNAs serve as regulators of normal and malignant hematopoiesis and have been implicated in carcinogenesis. This review will present a comprehensive summary of the biology and role of non-coding RNAs, including the less studied circRNA, siRNA, piRNA, and snoRNA as potential prognostic and/or predictive biomarkers or therapeutic targets in MDS.

The complexity of RNA cannot be fully expressed with the canonical A, C, G, and U alphabet. To date, over 140 distinct chemical modifications to RNA have been discovered. RNA modifications can profoundly impact the cellular outcomes of messenger RNAs (mRNAs), transfer and ribosomal RNAs, and noncoding RNAs. Additionally, aberrant RNA modifications are associated with human disease. RNA modifications are a rising topic within the fields of biochemistry and molecular biology. This review aims to provide budding scientists with an appreciation for the significance of RNA modifications, alongside the skills required to identify and fluently discuss fundamental RNA-protein interactions. The Pumilio RNA-binding protein and YT521-B homology (YTH) family of modified RNA-binding proteins serve as examples to highlight the fundamental biochemical interactions that underlie the specific recognition of both unmodified and modified ribonucleotides, respectively.

A defining feature of a productive infection is the co-opting of host cell resources for viral replication. Despite their repertoire of molecular functions, viruses subvert host defenses to take control of cellular factors such as RNA binding proteins (RBPs). RBPs are involved in virtually all steps of mRNA life forming ribonucleoprotein complexes (mRNPs) in a highly ordered and regulated process to control RNA fate and stability in the cell. Thus, the hallmark of this viral takeover is to reshape RNA fate in the cell to modulate host gene expression and evade immune responses. Here we provide an extensive review of work in this area, particularly how the host-viral interplay influences RBP functions to modulate the host cell. Overall, in this review, we highlight the myriad of ways RBPs can regulate RNA stability in either a pro-viral or antiviral manner by gathering novel insights gained from research studies in this field.

Current knowledge of the RNA world indicates two different genetic codes being present throughout the living world. In contrast to non-coding RNAs that are built of repetitive nucleotide syntax, the sequences that serve as templates for proteins share – as main characteristics – a non-repetitive syntax. The differences in their syntax structure is coherent with the difference of the functions they represent. Whereas non-coding RNAs build groups that serve as regulatory tools in nearly all genetic processes, the coding sections represent the evolutionarily successful function of the genetic information storage medium. The DNA genomes themselves are rather inactive, whereas the non-coding RNA domain is highly active, even as non-random genetic innovation operators. This indicates that repetitive syntax is the essential pre-requisite for RNA interactions to install variable RNA-group-identities, whereas the non-repetitive syntax serves as a stable conservation tool for successful selection processes out of RNA-groups cooperation and competition. The interaction opportunities of RNA loops with repetitive syntax are higher than with non-repetitive ones. Interestingly, these two genetic codes resemble the function of all natural languages, i.e., (a) everyday language use for organization and coordination of biotic group behavior, and (b) artificial (instrumental) language use for conservation of blueprints for complex protein-body constructions.

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.

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.

(1) Background: Tooth extraction is one of the oldest and most well-known surgical procedures in dental medicine. It is still routinely performed to date by both general practitioners, oral surgeons, and dental undergraduates. The Benex extraction system allows for the extraction of teeth in vertical direction, which avoids most trauma against the surrounding alveolar bone and soft tissues; (2) Methods: The study included 56 patients who were recruited from the department of Oral Surgery at the Faculty of Dental Medicine, Medical University – Plovdiv. Patients were divided into two groups of 28 patients – Group I (control group) and Group II (study group). For each group the success of the extraction, buccal cortical plate preservation, pain experience and early wound healing were assessed; (3) Results: There was no statistical significance between the success of the extractions in both groups. The Benex extractions preserved the buccal cortical plate in 95% of the cases, whereas the forceps extractions only in 71.8%, which shows a statistical significance. On the seventh day patients in group II reported less pain, without a significant difference. There was a significantly bigger number of completely healed extraction wounds on the 10th day; (4) Conclusions: Atraumatic extractions allow for more hard and soft tissues to be preserved in the extraction site. This is all crucial for the successful outcome and aesthetically pleasing results of a following prosthetic or implant restoration.

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.

Extraction techniques of important bioactive molecules from the major concern of Seafood byproducts are getting emphasis for better valorization. Employing green extraction technologies for efficient and quality production of these bioactive molecules is also strictly required. Hence, understanding the extraction process parameters to effectively design an applicable optimization strategy could make this real. In this review, statistical optimization strategies applied for the extraction process parameters of bioactive molecules from Seafood byproducts are focused. The type of experimental designs, and techniques applied to criticize and validate the effects of independent variables on the extraction output was addressed. Dominant parameters studied were enzyme/substrate ratio, pH, time, temperature and power of extraction instruments. The yield of bioactive compounds, chitin and chitosan, proteins and peptides, enzymes and carotenoids (Astaxanthins) were the most studied responses. Efficiency and/or economic and quality considerations and their selected optimization strategies that favour the production of potential bioactive molecules were also reviewed.

The groundbreaking Advanced Forest-Based Tagging Framework (AFTF) represents a paradigm shift in the domain of information extraction from medical texts. AFTF introduces a novel dual-binary tree structure that redefines how entity-relation triples are extracted. This innovative approach directly tackles the limitations of traditional linear and graph-based methods, effectively addressing challenges related to overlapping triples and computational efficiency. The AFTF model stands as a beacon of excellence, surpassing established baselines by significant margins in comprehensive evaluations on two pivotal medical datasets. Notably, AFTF achieves remarkable improvements in F1 scores, showcasing its prowess in accurate information extraction from complex medical narratives. Beyond its exceptional performance in the medical domain, AFTF exhibits remarkable versatility. This adaptability is vividly demonstrated through its robust performance across three diverse public datasets, further affirming its position as a versatile and reliable solution for information extraction tasks. This paper provides a comprehensive exposition of the AFTF architecture, shedding light on its innovative design principles and its efficient handling of intricate medical texts. AFTF represents a groundbreaking step forward in the realm of information extraction, promising enhanced accuracy, efficiency, and adaptability for a wide range of applications.

This study investigates uranium solvent extraction under AMEX process conditions. The use of pure extractants without diluents or phase modifiers allows not only to reduce the use of volatile organic compounds, but also to provide higher extraction yields without third phase formation. Pure extractants are protonated amines or quaternary ammoniums with suitable counter ion, which act at the interface between ion pairs and protic ionic liquids. The mixture of sulfates anion (SO42-) and bis(trifluoromethanesulfonyl)imide anion (NTf2-) revealed unexpected nonlinear extraction behaviors, which appear highly important to rationalize for optimized application. Spectroscopic analysis (NMR, UV-vis, FT-IR, and EXAFS) showed that uranium extraction occurs via protonated amine and three sulphates. Nonlinear extraction could further be interpreted by considering water and acid transfer between the two phases: at lower sulphate ratios, the release of acid from the organic phase into the aqueous phase was shown to influence the number of protonated amines in the organic phase, affecting uranium extraction before its enhancement. Furthermore, the extraction loss at higher sulphate ratios, was assigned to the destabilization of bidentate uranium-sulphate complexes due to a competition between water and sulphates.

Onion is not just an edible food but its seeds can also be an important source of valuable oil with various sensitive applications mainly in pharmaceutical, cosmetic and food fields. This has encouraged the use of a supercritical fluid extraction in the present study to obtain oil from local onion seeds, hence avoiding even traces of organic solvents. For several advantages carbon dioxide at supercritical conditions was used as the fluid. The effects of four key operating parameters like pressure, temperature, particle size and CO2 mass flowrate were investigated in ranges of [150, 250 bars], [35, 65°C], [0.25, 0.85mm] and [50, 100g/min], respectively, using a Box-Behnken experimental design, for a constant extraction time of 3 hours. This led to optimal operating conditions of 250 bar, 57°C, 0.25mm and 50g CO2/min for the highest oil extraction yield value of 24.36%. Furthermore, gas chromatography-mass spectrometry (GC-MS) was used to identify qualitatively and quantitatively the chemical composition of the onion seed oils. About ten components were identified in the extracts, but with four major ones, namely 9,12-Octadecadienoic acid ,methyl ester, 9,12-Octadecadienoic acid (Z,Z)-,methyl ester, 9-Octadecadienoic acid (Z)-,methyl ester and Hexadecanoic acid, methyl ester, present with significant percentages, depending on the operating parameters values. These fatty acids are well known for their benefits for human health. Finally the study shows the importance and the merits of fluids when used under particular conditions of pressure and temperature, in very sensitive industrial fields.

Structured extraction of emergency event information can effectively enhance the ability to re-spond to emergency events. This article focuses on the extraction of Chinese document-level emergency events, which mainly faces two key issues in this field: first, related datasets; Secondly, existing DEE (document-level event extraction) studies mostly use sequence annotation to extract candidate entities in the subtask of candidate entity extraction without considering the problem of role overlapping between candidate entities. On the one hand, this article constructs a Chinese document-level emergency extraction dataset, CDEEE, which first annotates the issues of argu-ment scattering, multiple events, and role overlapping. On the other hand, this article proposes a model RODEE for the problem of role overlapping in DEE tasks. This model first uses two in-dependent modules to represent the head and tail positions of candidate entities, then uses a multiplication attention mechanism to interact with the two to obtain a scoring matrix. Finally, role-overlapping candidate entities are predicted to assist in completing DEE tasks. Experiments were conducted on our manually annotated dataset, CDEEE, and the results showed that RODEE can effectively solve the problem of role overlapping in candidate entities and improve the performance of the DEE model.

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.

RNA silencing serves key roles in a multitude of cellular processes, including development, stress responses, metabolism, and maintenance of genome integrity. Dicer, Argonaute (AGO), double-stranded RNA binding (DRB), RNA-dependent RNA polymerase (RDR) and DNA-dependent RNA polymerases known as Pol IV and Pol V form core components to trigger RNA silencing. Common bean (Phaseolus vulgaris) is an important staple crop worldwide. In this study, we aimed to unravel the components of the RNA-guided silencing pathway in this non-model plant taking advantage of the availability of two genome assemblies of Andean and Meso-American origin. We identified six PvDCLs, thirteen PvAGOs, 10 PvDRB, 5 PvRDR, in both genotypes, suggesting no recent gene amplification or deletion after the gene pool separation. In addition, we identified one PvNRPD1 and one PvNRPE1 encoding the largest subunits of Pol IV and Pol V, respectively. These genes were categorized into subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different organs using online data and quantitative RT-PCR after pathogen infection. Several of the candidate genes were up-regulated after infection with the fungus Colletotrichum lindemuthianum.

After a long limbo, RNA has gained its credibility as a druggable target, fully earning its de-served role in the next-generation area of pharmaceutical R&D. We have recently probed the Trans-Activation Response element (TAR), a RNA stem–bulge–loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabiliz-ing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analyti-cal approaches for the study of multicomponent RNA-based interactions.

This review will explore the four major pillars required for design and development of an saRNA vaccine: antigen design, vector design, non-viral delivery systems, and manufacturing (both saRNA and lipid nanoparticles (LNP)). In will report on the major innovations, preclinical and clinical data reported in the last five years and will discuss future prospects.

Long non-coding RNAs (lncRNAs) have been found to be involved in many biological processes, including the regulation of cell differentiation, but a complete characterization of lncRNA is still lacking. Additionally, there is evidence that lncRNAs interact with ribosomes, raising questions about their functions in cells. Here, we used a developmentally staged protocol to induce cardiogenic commitment of hESCs and then investigated the differential association of lncRNAs with polysomes. Our results identified lncRNAs in both the ribosome-free and polysome-bound fractions during cardiogenesis and showed a very well-defined temporal lncRNA association with polysomes. Clustering of lncRNAs was performed according to the gene expression patterns during the five timepoints analyzed. In addition, differential lncRNA recruitment to polysomes was observed when comparing the differentially expressed lncRNAs in the ribosome-free and polysome-bound fractions or when calculating the polysome-bound vs ribosome-free ratio. The association of lncRNAs with polysomes could represent an additional cytoplasmic role of lncRNAs, e.g., in translational regulation of mRNA expression.

Noncoding RNA regions have emerged as pivotal players in the regulation of various cellular processes, and their involvement in HIV/AIDS pathogenesis is an evolving field of research. This review article delves into the intricate interplay between noncoding RNA regions and AIDS, shedding light on the diverse mechanisms by which these RNA molecules influence viral replication, immune responses, and disease progression. The review highlights recent discoveries, potential therapeutic applications, and the challenges ahead, illustrating the promising prospects of noncoding RNA research in the context of HIV/AIDS.

Membrane-coupled RNA transport is an emerging theme in fungal biology. This review focuses on the RNA cargo and mechanistic details of transport via two inter-related sets of organelles: endosomes and extracellular vesicles for intra- and intercellular RNA transfer. Simultaneous transport and translation of messenger RNAs (mRNAs) on the surface of shuttling endosomes is a conserved process pertinent to highly polarised eukaryotic cells, such as hyphae or neurons. Here we detail the endosomal mRNA transport machinery components and mRNA targets of the core RNA-binding protein Rrm4. Extracellular vesicles (EVs) are newly garnering interest as mediators of intercellular communication, especially between pathogenic fungi and their hosts. Landmark studies in plant-fungus interactions indicate EVs as a means of delivering various cargos, most notably small RNAs (sRNAs), for cross-kingdom RNA interference. Recent advances and implications of the nascent field of fungal EVs are discussed and potential links between endosomal and EV-mediated RNA transport are proposed.

In the intricate domain of text analysis, syntactic dependency trees play a crucial role in unraveling the web of relations among entities embedded in the text. These trees provide a structural roadmap, guiding us through the complex syntax to pinpoint the interactions and connections between different entities. However, the challenge lies in sifting through this intricate structure to extract relevant information, a task that requires precision and discernment. Traditional approaches often rely on rule-based pruning methods to simplify these dependency structures, focusing on certain parts while discarding others. Yet, this approach has its pitfalls, as it can overlook critical nuances and connections that are vital for a comprehensive understanding of the text. Addressing this gap, our research introduces the Syntactic Dependency-Aware Neural Networks (SDANNs), a groundbreaking model designed to harness the full power of the entire dependency tree. This approach marks a significant departure from the conventional methods. Instead of the rigid rule-based pruning, SDANNs implement a more flexible and dynamic 'soft-pruning' technique. This method allows the model to adaptively focus on the sub-structures within the dependency tree that are most relevant for understanding the relationships between entities. By doing so, it ensures that no vital information is overlooked, and all potential connections are considered. The efficacy of SDANNs is not just theoretical but has been empirically validated through extensive testing and evaluations across a wide range of tasks. These tasks include the extraction of complex relations spanning multiple sentences, as well as detailed analyses at the sentence level. In each of these scenarios, SDANNs have demonstrated a remarkable ability to leverage the full structural complexity of dependency trees. This capability sets them apart from existing models, enabling a more nuanced and comprehensive analysis of textual relations. The results of these evaluations consistently show that SDANNs not only meet but significantly exceed the performance of prior models. This superiority is evident in the way SDANNs handle the multifaceted and often subtle interactions within the text, offering insights that were previously inaccessible with conventional methods. In summary, the Syntactic Dependency-Aware Neural Networks represent a significant advancement in the field of text analysis. By fully embracing the complexity of syntactic dependency trees and employing a sophisticated 'soft-pruning' approach, SDANNs open new avenues for exploring and understanding the intricate relationships that exist within written language. This model stands as a testament to the potential of combining advanced neural network architectures with a deep understanding of linguistic structures, paving the way for more accurate, nuanced, and comprehensive analyses of text.

An enormous interest in the development of efficient protocols for cellulose extraction has been demonstrated in the last years, however, they were usually based on non-sustainable chemical and thermal approaches. In this work we describe a new and more sustainable method for cellulose extraction from eucalyptus and pine tree wood waste products exclusively performed using microwave-assisted radiation. The methodology includes 3 main steps: i) alkaline treatment; ii) bleaching I, using H2O2; and iii) bleaching II, an acidic treatment. Samples obtained in each step were characterized by Fourier-transform Infrared (FTIR) spectroscopy, powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The results were compared with those of starting materials, with the commercially available microcrystalline cellulose and with an industrial paper pulp. Results confirmed that for both type of wood wastes, cellulose was retained during the extractions procedures and that the removal of hemicellulose was achieved at the last step as seen by the FTIR spectra and TGA curves. The developed protocol is innovative since it constitutes an easy and quick approach to extract cellulose, from eucalyptus and pine tree wood waste using mild chemical and thermal conditions, microwave irradiation, and environmentally friendly purification steps. This approach offers the possibility of a future scale-up study to potentially apply the develop protocol to the extraction of cellulose in an industrial scale.

RNA interference (RNAi) is one of the main mechanisms for disease resistance and small RNA production in plants. The main proteins involved in RNAi include Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), double-stranded RNA-binding (DRB), and Argonaute (AGO). Juglandaceae contains a variety of important woody plants, and walnuts are one of the four major woody plant groups and one of the four major dried fruits in the world. To clarify the evolution and functional differentiation of RNAi-related proteins in the walnut (Juglans regia) genome, this study integrated various web resources from gene family acquisition to structural analysis and transcriptome data to correlate walnuts and their congeners. The walnut genome has 5 DCL, 13 RDR, 15 DRB and 15 AGO genes, similar genes encoding conserved protein structural domains and conserved motifs with similar subcellular localization. Walnut AGO proteins are classified into three classes and seven subclasses. The DCL is divided into four categories, while RDR is mainly divided into four categories, and DRB can be divided into six categories. The exception is that the copy number of walnut RDR1 is 9, in which seven RDR1 are distributed in clusters on chromosome 16. Purifying selection drove the formation of walnut genes, but protein classes were subjected to varying degrees of purifying selection. Additionally, these results showed some similarity in other plants of the walnut family. Moreover, different RNAi-related genes of walnut produced abundant selective expression in response to different tissues and stresses. In this study, DCL, RDR, DRB and AGO gene families were identified and analysed in the genome of the walnut family for the first time and preliminarily examined the evolution, structure and expression characteristics of these families to provide a preliminary basis for the evolution of the walnut RNAi pathway and breeding research.

Study of RNA-protein interactions and identification of RNA targets are among the key aspects of understanding the RNA biology. Currently, various methods are available to investigate these interactions, in particular, RNA pulldown assay. In the present paper, a method based on the HaloTag technology is presented that is called Halo-RPD (HaloTag RNA PullDown). The proposed protocol uses plants with stable fusion protein expression and the MagneBeads magnetic beads to capture RNA-protein complexes directly from the cytoplasmic lysate of transgenic A. thaliana plants. The key stages described in the paper are as follows: 1) preparation of the magnetic beads 2) tissue homogenization and collection of control samples 3) precipitation and wash of RNA-protein complexes; 4) evaluation of protein binding efficacy; 5) RNA isolation; 6) analysis of the obtained RNA. Recommendations for better NGS assay designs are provided.

A definitive transcriptome atlas for the non-coding expressed elements of pathogenic mycobacteria does not exist. Incomplete lists of non-coding transcripts can be obtained for some of the reference genomes (e.g. Mycobacterium tuberculosis H37Rv) but to what extent these transcripts have homologues in closely related species or even strains is not clear. This has implications for the analysis of transcriptomic data; non-coding parts of the transcriptome are often ignored in the absence of formal, reliable annotation. Here, we review the state of our knowledge of non-coding RNAs in pathogenic mycobacteria, emphasising the disparities in the information included in commonly used databases. We then proceed to review ways of combining computational solutions for predicting the non- coding transcriptome with experiments that can help refine and confirm these predictions.

Plant pathogenic fungi are the largest group of disease-causing agents on crop plants and represent a persistent and significant threat to agriculture worldwide. Conventional approaches based on the use of pesticides raise social concern for the impact on the environment and human health and alternative control methods are urgently needed. The rapid improvement and extensive implementation of RNAi technology for various model and non-model organisms has provided the initial framework to adapt this post-transcriptional gene silencing technology for the management of fungal pathogens. In this review, we describe exogenous RNAi involved in plant pathogenic fungi and discuss small RNA production, formulation, and RNAi delivery methods. We explore some challenges with possible solutions. Furthermore, exogenous RNAi holds great potential for RNAi-mediated plant pathogenic fungal disease control.

(200 words) Imidazolium ionic liquids containing acetylacetone, thenoyltrifluoroacetone, or 8-hydroxyquinoline, respectively, were used as the extracting agents for the separation of traces of iron (III) from its aqueous solutions with or without citric and oxalic acids. The results show that 8-hydroxyquinoline in imidazolium ionic liquids extract iron quantitatively from all the tested solutions including complexing ones, regardless indications of unexpected iron behavior/speciation.

Noncoding RNAs (ncRNAs) including circular RNAs (circRNAs) and N6-methyladenosine (m6A) have been shown to play a critical role in the development of various diseases including obesity and metabolic disorder-associated fatty liver disease (MAFLD). Obesity is a chronic disease considered by excessive fat accumulation in the body, which has been increased lately and is the foremost risk factor for the MAFLD. Causes of obesity may involve the interaction of genetic, behavioral, and social factors. The m6A RNA methylation might add a novel inspiration for understanding the development of obesity and MAFLD with post-transcriptional regulation of gene expression. In particular, circRNAs, microRNAs (miRNAs) and m6A might be implicated in the progression of MAFLD. Interestingly, m6A modification could modulate the translation, degradation, and other functions of ncRNAs. miRNAs/circRNAs could also modulate m6A modifications by affecting writers, erasers, and readers. In turn, ncRNAs could modulate the expression of m6A regulators in different ways. However, there is limited evidence on how these ncRNAs and m6A interact to affect the promotion of liver diseases. M6A has been appeared to occur in DNA, RNA and proteins, which may be associated with several biological properties. This study would provide a mechanistic understanding of the m6A modification and ncRNAs in the association on liver diseases, especially for MAFLD. Comprehension of the association between m6A modification and ncRNAs may contribute to the development of treatment tactics for MAFLD.

RNA interference is a highly conserved process in which non-coding small RNAs (sRNA) modulate gene expression at the post-transcriptional level influencing plant growth and development. Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) are the core elements involved in gene silencing and their gene families have been explored in many plants. However, these genes and their response to abiotic stresses have not yet been well in wheat. In this study, 82 AGO, 31 DCL, and 31 RDR genes were identified and phylogenetic analysis of these proteins showed that clustered into ten, four and four clades respectively. RNA-seq analysis revealed constitutive expression of AGO1, AGO9, and DCL2 family expression analysis in tissues under normal and stress conditions, whereas RDR1 which is known to engage in siRNA biogenesis showed higher expression levels in wheat leaf tissues. Our findings build the foundation for comparative genomics analyses of RNA silencing elements in cereal crops, as well as new insights into the functional complexity of RNA silencing in wheat stress responses, which is critical for understanding the processes underlying wheat stress responses.

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.

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.

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.

P. palmata is a viable source of nutrients with bioactive properties. The present study determined the potential role of post-extraction ultrasonication on some compositional features and antioxidant properties of resulting extracts after sequential enzymatic/alkaline treatment. No significant difference was detected in terms of protein content and recovery as well as amino acid composition of the extracts. The nitrogen-to-protein conversion factor of 5 was found to be too high for the seaweed and its enzymatic/alkaline extracts. The extracts sonicated by bath for 10 minutes and not sonicated showed the highest and lowest total phenolic contents (p&lt;0.05), respectively. The highest radical scavenging and lowest metal chelating activities were observed for the non-sonicated sample, as evidenced by IC50 values. The extract sonicated by bath for 10 minutes showed the most favorable in vitro antioxidant properties since its radical scavenging was not significantly different from that of not sonicated sample (p&gt;0.05), whereas its metal chelating activity was significantly higher (p&lt;0.05). To conclude, post-extraction ultrasonication by an ultrasonic bath for 10 minutes is recommended to increase phenolic content and improve antioxidant properties of enzymatic-alkaline extracts from P. palmata.

Based on the teaching material of "Robotics" course, this paper studied the automatic knowledge graph construction, including knowledge points extraction and knowledge point relation extraction. We proposed a new method of extracting the first-level, second-level, and third-level knowledge points as well as their prerequisite relations of knowledge points in the textbook. For the problem of insufficient knowledge of the pre-trained language model in the specific field, methods such as incremental pre-training and optimization of cost functions are employed to integrate subject knowledge into the pre-trained language model, thus improving its effectiveness. To overcome the problem that the traditional method of relationship extraction can not be applied directly to the extraction of teaching materials, a new scheme for knowledge point relationship extraction based on keyword relationship is proposed. The experimental data from textbooks shows that the F1 score of knowledge point extraction reaches 93%, considerably improved compared to the traditional model. Consequently, the knowledge point entity extraction and relationship extraction methods based on the pre-trained model can effectively extract structured information and facilitate the automatic construction of knowledge graphs.

One of the most important component of a Geographic Information Retrieval (GIR) is the geoparser, which performs toponym recognition, disambiguation, and geographic coordinate resolution from unstructured text domain. However, news articles which report several events across many place references mentioned in the document is not yet adequately modeled by regular geoparser types where the scope of resolution is either on toponym-level or document-level. The capacity to detect multiple events, geolocate its true locations and coordinates along with their numerical arguments are still missing from modern geoparsers, much less in Indonesian news corpora domain. We propose a novel type event geoparser which integrates an ACE-based event extraction model and provides precise event-level scope resolution. The geoparser casts the geotagging and event extraction as sequence labeling and uses Conditional Random Field with keywords feature obtained using Aggregated Topic Model as a semantic exploration from large corpus, which eventually increases the generalizability of the model. The geoparser also use Smallest Administrative Level feature along with Spatial Minimality-derived algorithm to improve the identification of Pseudo-location entities, resulting 19.4% increase for weighted F1 score. As a side effect of event extraction, the geoparser also extracts various numerical arguments and able to generate thematic choropleth map from a single news story.

Tobamoviruses are a group of plant viruses posing a significant threat to agricultural crops worldwide. In this review, we focus on plant immunity against tobamoviruses, including pattern-triggered immunity (PTI), effector-triggered immunity (ETI), RNA-targeting pathway, hormones and reactive oxygen species (ROS). Further, we highlight genetic resources for resistance against tobamoviruses in plant breeding and discuss future directions on plant protection against tobamoviruses.

The evolutionary origin of the genome remains elusive. Here, I hypothesize that its first iteration, the protogenome, was a multi-ribozyme RNA. It evolved, likely within liposomes (the protocells) forming in dry-wet cycling environments, through the random fusion of ribozymes by a ligase and was amplified by a polymerase. The protogenome thereby linked, in one molecule, the information required to seed the protometabolism (a combination of RNA-based autocatalytic sets) in newly forming protocells. If this combination of autocatalytic sets was evolutionarily advantageous, the protogenome would have amplified in a population of multiplying protocells. It likely was a quasispecies with redundant information, e.g., multiple copies of one ribozyme. As such, new functionalities could evolve, including a genetic code. Once one or more components of the protometabolism were templated by the protogenome (e.g., when a ribozyme was replaced by a protein enzyme), and/or addiction modules evolved, the protometabolism became dependent on the protogenome. Along with increasing fidelity of the RNA polymerase, the protogenome could grow, e.g., by incorporating additional ribozyme domains. Finally, the protogenome could have evolved into a DNA genome with increased stability and storage capacity. I will provide suggestions for experiments to test some aspects of this hypothesis.

Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5’-to-3’ direction until being stalled by well-defined RNA structures. Here we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome, hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (-) strand forms of ttsgR were produced in these cells in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins, p28 and p88, were provided in trans. Consistent with its replicational origin, ttsgR accumulation required a 5’ terminal G₃(A/U)₄ motif shown by others to be crucial for the replication of a TCV satellite RNA. More importantly, introducing a new G₃(A/U)₄ motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Collectively our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production.

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.

The telomerase RNA in yeasts is large, usually >1000 nt, and contains functional elements that have been extensively studied experimentally in several disparate species. Nevertheless, they are very difficult to detect by homology-based methods and so far have escaped annotation in the majority of the genomes of Saccharomycotina. This is a consequence of sequences that evolve rapidly at nucleotide level, are subject to large variations in size, and are highly plastic with respect to their secondary structures. Here we report on a survey that was aimed at closing this gap in RNA annotation. Despite considerable efforts and the combination of a variety of different methods, it was only partially successful. While 27 new telomerase RNAs were identified, we had to restrict our efforts to the subgroup Saccharomycetacea because even this narrow subgroup was diverse enough to require different search models for different phylogenetic subgroups. More distant branches of the Saccharomycotina still remain without annotated telomerase RNA.

Wild strawberry (Fragaria vesca L.) leaves possess diverse antioxidant properties, mainly due to the different classes of phenolics. In this study, microwave-assisted extraction (MAE) and accelerated solvent extraction (ASE) of wild strawberry phenolics were carried out to understand the impact of extraction temperature, extraction time and solvent-to-sample ratio (SSR) on the quantitative and qualitative properties of the obtained extracts. The highest total phenolic content (8027 mg GA/100 g DW), as well the highest DPPH• antiradical activity (903 mol TE/g DW) was obtained with ASE at 150 °C, static time 5 min and SSR of 40:1; while the highest ABTS•+ antiradical activity (681 mol TE/g DW,) and FRAP (2389 mol TE/g DW) was obtained with MAE after 5 min at 80 °C and a SSR of 40:1. The correlation analysis showed that phenolics largely contributed to the antioxidant properties of the studied MAE extracts, while correlation was not found in ASE extracts. A total of 54 different phenolics were identified by UPLC/MS-MS in MAE and ASE extracts obtained at optimal extraction conditions. MAE extract was shown to have a higher content of phenolic acids (40%), but lower content of proanthocyanidins (88%), flavonols (29%), flavan-3-ols (50%) and flavones (39%) than ASE extract.

Sweet cherry pomace is a by-product that can be a source of bioactive phenolic compounds. Usually, polyphenols have been extracted using conventional extraction methodologies. However, a significant fraction, called non-extractable polyphenols (NEPs), remains retained in the conventional extraction residues. Therefore, this work is aimed, for the first time, to investigate the release of NEPs from cherry pomace combining pressurized liquid extraction (PLE) and enzyme-assisted extraction (EAE) using Promod enzyme. A response surface methodology was employed to study the influence of temperature, time, and pH on the NEPs extraction. The response variables were the total phenolic content (TPC) measured by Folin-Ciocalteu method, total proanthocyanidin (PA) content evaluated by vanillin, DMAC, and butanol/HCl assays, and total antioxidant capacity determined by Trolox equivalent antioxidant capacity and inhibition of hydroxyl radical assays. The results indicated that PLE-EAE was more suitable and selective to obtain NEPs from sweet cherry pomace than PLE alone. In fact, the extracts obtained by PLE-EAE displayed higher TPC, PA content, and bioactivity than the extracts obtained by PLE under the same extraction conditions, and those obtained by conventional methods. Moreover, size-exclusion chromatography profiles showed that the combination of PLE and EAE enabled the recovery of NEPs with higher molecular weight than PLE without EAE treatment.

In the past decade, RNA fragments derived from full length small nucleolar RNAs (snoRNAs) have been shown to be specifically excised and functional. These sno-derived RNAs (sdRNAs) have been implicated as gene regulators in a multitude of cancers, controlling a variety of genes post-transcriptionally via association with the RNA-induced silencing complex (RISC). In this review, we have summarized the literature connecting sdRNAs to cancer gene regulation. SdRNAs possess miRNA-like functions, and are able to fill the role of tumor-suppressor or tumor-promoter in a tissue context-dependent manner. Indeed, there are many miRNAs that are actually derived from snoRNA transcripts, meaning that they are truly sdRNAs and as such are included in this review. As sdRNAs are frequently discarded from ncRNA analyses, we emphasize that sdRNAs are functionally relevant gene regulators and likely represent an overlooked subclass of miRNAs. Based on the evidence provided by the papers reviewed here, we propose that sdRNAs deserve more extensive study to better understand their underlying biology and to identify previously overlooked biomarkers and therapeutic targets for a multitude of human cancers.

Abstract: Background: The analysis of vitamins in baby food is a challenging task given the com-plexity of the food matrix, vitamin stability, and strict regulations of the European Union regarding permissible deviations from declared values. Vitamins in food exist in different concentrations and forms and have different stabilities, and it is not straightforward to prepare samples for reliable analysis using the same procedure. Therefore, significant attention has been devoted to optimizing sample preparation in the analysis of vitamins. Methods: This study aims to determine which of the seven applied extraction methods is the most efficient for the simultaneous extraction and deter-mination of A, D, E, and K vitamins in milk and baby food using high-performance liquid chro-matography (HPLC). Different samples of baby food were prepared in seven different ways based on four methods (saponification, enzymatic hydrolysis, solvent extraction, and solid-phase extrac-tion). Results and Conclusion: The best preparation method proved to be solid-phase extraction with a C18 stationary phase, and HPLC with a UV-VIS detector was identified as a sufficiently sensitive technique for the identification and quantification of fat-soluble vitamins in milk and baby food.

Dried blood spot (DBS) testing on confetti is one of the most common methods used to collect and extract DNA from field samples for molecular epidemiology of malaria studies. Here, we investigated whether ethanol or other solutions used to clean scissors are a source of sample contamination. DBS were prepared from 3 drops of blood from two Plasmodium falciparum cultures spotted on confetti, and 3 solutions (water, ethanol and DNase) were used to clean scissors between spots during DNA extraction. For each cleaning solution, two blank confetti were used as negative controls. Samples were analyzed by PCR-based genotyping of merozoite surface proteins 1 & 2 (msp-1 and msp-2), and P. falciparum chloroquine resistance transporter (Pfcrt). A total of 15 samples were analyzed. Based on msp-1 and msp-2 amplification, P. falciparum was detected on blank confetti when scissors were washed with ethanol. Based on Pfcrt amplification, DNA of P. falciparum was detected on blank confetti when scissors were cleaned with ethanol or water. No P. falciparum genes were detected on blank confetti when scissors were cleaned with DNase. Scissors cleaned with DNase prevented cross-contamination between samples during processing of dried blood spots, whereas ethanol (which is commonly used) fails to avert cross-contamination.

In the East, it has already become a tradition to prepare dry powdered seasonings from the fruits of the sumac plant (Rhus coriaria L.). In this work, we tested their processing by extraction. Sumac was collected in mid-September in a mountain forest near the village of Gezik (Guba, Azerbaijan). Before extraction, it was first dehydrated in a drying cabinet SH-DO-149 FG (Southern Bark) to an air-dry state; then crushed in a Tube Mill control mill (Germany) into pieces of 1-3 mm in size. One- and two-stage maceration extraction was tested together with water or acidified water at temperatures from 20 to 80 °C. Evaporation of primary extracts was carried out in a rotary evaporator Lab Rot 2 L (China). The concentration of coloring substances in the extract (in units of optical density) was used as an output parameter. It was found that the use of two-stage extraction and acidified water with a concentration of citric acid 0.3 g/cm³ contributes to the extraction of the maximum amount of coloring substances and increases the transparency of the resulting extracts, which allows the extracted extracts to be concentrated directly to obtain a new type of natural food dye. The involvement of sumac fruits in industrial processing will increase the need for these medicinal fruits, and this, in turn, will serve as an incentive for the introduction of this medicinal plant into culture.

Onions are an excellent source of a variety of essential antioxidants, polyphenols, and other phytonutrients. These onions were used to enhance the meal's flavour and aroma. Additionally, onions have long been recognised for their ability to change food texture, making it more pleasant for eating. Among the most significant phenolic compounds identified in onions was Quercetin, which has been proven to be very helpful for maintaining good health by reducing the risk of heart disease, cancer, and a number of other severe conditions. Polyphenol extraction is essential for the effective usage and research of these phytochemicals. Previously, the extraction process was complicated by a variety of traditional methods, but with the aid of contemporary technology, the procedure was simplified. Additionally, it was found that red onions had greater health benefits than yellow onions, which were the most commonly used kind at the time. Apart from their health benefits, polyphenols were well recognised for their industrial use in dyeing, polymer manufacturing, and resin synthesis.

Aim: In a human pilot case, we used a root formed temporary prosthetical device (Intra-alveolar Device - IAD), to investigate the effect of mechanical stimulation on hard tissue clinically and radiographically. Background: The extraction of a tooth determines the resorption of the alveolar bone. This determines challenge to place an implant and to integrate the prosthesis in soft tissue. Mechanical stimulation is fundamental for bone tropism but is not understood the effect of mechanical stimulation on post-extraction site. Case presentation: A 24 years old woman was enrolled. The IAD’s were used as expected by protocol. Clinical healing was uneventfully. Vertical radiographical bone resorption measured on CBCT was -0,14mm (5,6%) while horizontal bone resorption was -0,21mm (12,36%). Conclusion: This study demonstrated the feasibility of the proposed model. We need clinical prospectical studies enrolling more patients

Background: It remains unclear whether transfer RNA-derived small RNAs (tsRNAs) play a role in pathological cardiac hypertrophy (PCH). We aimed to clarify the expression profile of tsRNAs and disclose their relationship to the clinical phenotype of PCH and the putative role. Methods: Small RNA sequencing was performed in the plasma of PCH patients and healthy volunteers. In a larger sample size and angiotensin Ⅱ (Ang II)-stimulated H9c2 cells, the data were validated by real-time qPCR. The atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were examined in Ang II-stimulated H9c2 cells. The role of tsRNAs in the pathogenesis of PCH was explored by bioinformatics analysis. Results: A total of 4185 differentially expressed tsRNAs were identified, of which 4 and 5 tsRNAs were observed to be significantly differentially upregulated and downregulated expressed. Of the 5 down-regulated tsRNAs, 4 of them were verified to be significantly down-regulated in the larger sample group, among which tRF-30-3JVIJMRPFQ5D, tRF-16-R29P4PE, tRF-21-NB8PLML3E, and tRF-21-SWRYVMMV0 had areas under the curve to diagnose concentric hypertrophy. The 4 down-regulated tsRNAs were negatively correlated with left ventricular posterior wall dimensions in PCH patients (r=-0.4227; r=-0.4517; r=-0.5567; r=-0.4223). The levels of ANP and BNP as well as cell size were decreased in Ang II-stimulated H9c2 cells with 21-NB8PLML3E mimic transfection. Bioinformatics analysis revealed that the target genes of tRF-21-NB8PLML3E were mainly enriched in the metabolic pathway and involved in the regulation of ribosomes. Conclusion: The plasma tsRNAs tRF-21-NB8PLML3E might be considered biomarkers in patients with PCH with early screening potential.

Human metapneumovirus is one of major causes of common cold among children, especially infants. Its key enzyme is RNA-dependent RNA-polymerase, which performs both replication and transcription, including capping and cap methylation. The goal of the work is to find possible inhibitors of RNA-dependent RNA-polymerase across the active compounds of Rosaceae plants. The candidates were selected by molecular docking to cap-transferring domain of RNA-polymerase (PDB ID: 4UCZ) in Autodock VINA. Among all the substances tested by docking, ellagic acid derivatives showed the most promising results (affinity values below -10 kcal/mol). Hence, they could be treated as possible candidate drugs against metapneumoviral infection after experimental examination. The main advantage of using these substances should be their low toxicity, which is quite uncommon for selective RNA polymerase inhibitors used in clinical practice. Occurrence of ellagic acid derivatives among the plants from Rosaceae family like raspberry could explain their effect during common cold.

Among all economically important plant species in the world, grapevine (Vitis vinifera L.) is the most cultivated fruit plant. It has a significant impact on the economies of many countries through wine and fresh and dried fruit production. In recent years, the grape and wine industry has been facing outbreaks of known and emerging viral diseases across the world. Although high-throughput sequencing (HTS) has been used extensively in grapevine virology, the application and potential of third-generation sequencing have not been explored in understanding grapevine viruses and their impact on the grapevine. Nanopore sequencing, a third-generation technology, can be used for direct sequencing of both RNA and DNA with minimal infrastructure. Compared to other HTS methods, the MinION nanopore platform is faster and more cost-effective and allows for long-read sequencing. Due to the size of the MinION device, it can be easily carried for field viral disease surveillance. This review article discusses grapevine viruses and their diagnostic methods, the principle of nanopore sequencing technology and its application in grapevine virus detection, virus–plant interactions, as well as the characterization of viral RNA modifications.

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.

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.

Since the discovery of transposable elements (TEs) in Maize in the 1940s by Barbara McClintock [1] transposable elements have been described as junk, as selfish elements with no benefit to the host, and more recently as major determinants of genome structure and genome evolution. TEs are DNA sequences that are capable of moving to new sites in the genome and make additional copies while doing so. To limit the propagation of TEs, host silencing mechanisms are directed at transposon-encoded genes that are required for mobilization. The mutagenic properties of TEs, the potential of TEs to form new genes and affect gene expression, together with the host silencing mechanisms shape eukaryotic genomes and drive genome evolution. While TEs constitute more than half of the genome in many higher eukaryotes, transposable elements in the nematode C. ele-gans form a relatively small proportion of genome, about 15% [2]. Genetic studies of transposon silencing and the discovery of RNAi in C. elegans, propelled C. elegans to the forefront of studies of RNA-based mechanisms that silence TEs. Here, I will review the transposable elements that are present and active in the C. elegans genome, and the host defense mechanisms that silence these elements.

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.

RNA, as an important substance for regulating biological growth and development, has significant implications for visualization research. However, many spontaneously fluorescent substances in plants greatly interfere with the effectiveness of plant bioimaging. Aggregetion- induced emission luminogens (AIEgens), due to their luminescent properties, tunable molecular size, high fluorescence intensity, good photostability, and low cell toxicity, have been widely applied in the animal and medical fields. We have found that AIEgens have great potential as RNA fluorescent probes for efficient imaging in plants. In this review, we first introduce several common RNA labeling forms and point out their pros and cons. We briefly describe the development of AIEgens and the AIE mechanism, and then present various practical applications of AIEgens, including detailed examples of their use as biological markers. To further promote the application of AIE in the field of RNA, we suggest the use of AIEgens to modify target RNA via techniques such as click chemistry or CRISPR/Cas, to achieve RNA visualization in plants. highly possible to modify target RNA with AIEgens in vivo for RNA visualization.

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.

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.

This publication investigates the potential application of bark extractives as a biopolyol and extracted bark residues as a filler for producing polyurethane materials. The aim is to enhance the properties of polymeric composites while reducing the environmental impact associated with the complex utilization of bark.The comprehensive study of compositions of extractives isolated from black alder (Alnus glutinosa) and Baltic pine (Pinus silvestris) barks was performed using microwave-assisted water extraction. The yield of the extracts obtained under different extraction conditions, as well as the total phenolic and monomeric carbohydrate content in the extractives, were studied. Furthermore, UHPLC-HRMS analysis was conducted to study the detailed composition of the bark extracts.The total content of hydroxyl groups and the relative portion of aliphatic and phenolic groups in the isolated extracts were determined depending on the extraction regimes and bark origination. The dominant compounds in black alder bark extracts isolated at 70-90⁰C were identified as diarylheptanoids and their derivatives bearing sugar units, with oregonin being the main compound. Pine bark extracts isolated at the same temperature were found to be rich in oligomeric proanthocyanidins. Extracts from both black alder and pine barks obtained at different times of isothermal heating between 130-150°C were rich in carbohydrates and showed an increased proportion of aliphatic OH groups.The study also focused on the composition of residual biomass. It was indicated by Py-GC/MS/FID and wet chemistry methods that residues of both black alder and pine bark are enriched with lignin in comparison with initial barks. Therefore, it can be proposed that extracted bark residues introduced into polyurethane compositions will act as a charcoal formation promoter, thus increasing the flame resistance of materials.Overall, the study provides valuable insights into the chemical composition of bark extractives and extracted bark residues for their potential application as biopolyol and fillers of polyurethane materials, contributing to a more sustainable approach in polymer composite production.

Melanins are phenolic biopolymers synthesised by most of the living organism mainly for photoprotection or surviving in harsh conditions. Melanin is localised in different areas or complexed with different other biomolecules when observed from animals to microbes. This makes the melanin extraction procedure different in animal, plant and microbial tissues. Basically, the alkali-acid extraction is used in most protocols of which slight variations are there depending on the tissue used. This review will try to compile melanin extraction procedures from different cells and tissues ranging from animals to bacteria.

The number of documents published on the Web other languages than English grows every year. As a consequence, it increases the necessity of extracting useful information from different languages, pointing out the importance of researching Open Information Extraction (OIE) techniques. Different OIE methods have been dealing with features from a unique language. On the other hand, few approaches tackle multilingual aspects. In such approaches, multilingual is only treated as an extraction method, which results in low precision due to the use of general rules. Multilingual methods have been applied to a vast amount of problems in Natural Language Processing achieving satisfactory results and demonstrating that knowledge acquisition for a language can be transferred to other languages to improve the quality of the facts extracted. We state that a multilingual approach can enhance OIE methods, being ideal to evaluate and compare OIE systems, and as a consequence, to applying it to the collected facts. In this work, we discuss how the transfer knowledge between languages can increase the acquisition from multilingual approaches. We provide a roadmap of the Multilingual Open IE area concerning the state of the art studies. Additionally, we evaluate the transfer of knowledge to improve the quality of the facts extracted in each language. Moreover, we discuss the importance of a parallel corpus to evaluate and compare multilingual systems.

Network Embedding (NE) is an important method to learn the representations of network via a low-dimensional space. Conventional NE models focus on capturing the structure information and semantic information of vertices while neglecting such information for edges. In this work, we propose a novel NE model named BimoNet to capture both the structure and semantic information of edges. BimoNet is composed of two parts, i.e., the bi-mode embedding part and the deep neural network part. For bi-mode embedding part, the first mode named add-mode is used to express the entity-shared features of edges and the second mode named subtract-mode is employed to represent the entity-specific features of edges. These features actually reflect the semantic information. For deep neural network part, we firstly regard the edges in a network as nodes, and the vertices as links, which will not change the overall structure of the whole network. Then we take the nodes' adjacent matrix as the input of the deep neural network as it can obtain similar representations for nodes with similar structure. Afterwards, by jointly optimizing the objective function of these two parts, BimoNet could preserve both the semantic and structure information of edges. In experiments, we evaluate BimoNet on three real-world datasets and task of relation extraction, and BimoNet is demonstrated to outperform state-of-the-art baseline models consistently and significantly.

Cardamom is plant of the Zingiberaceae family. It has been used for the treatment of many diseases such as migraine, bronchitis, stomach and intestinal disorders. Cardamom contains triterpenes, resins, starch and fatty compounds. Phytosterols (stigmasterol, campesterol and β-sitosterol) are a group steroid alcohol in plants. They are used food, medicine and cosmetic industry. They are protective effects against some types of cancer too. Phytosterols are found in the vegetable oil such as the spindle, corn and soybean oil. This paper deals with the maximum oil and β-sitosterol yield were investigated by means of the supercritical CO₂ extraction of cardamom. The effect of operating parameters as temperature, pressure and CO₂ flow rate were investigated on oil yield. The amount of β-sitosterol was analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) at optimized conditions. The optimized conditions were recorded as temperature of 40^oC, pressure of 200 bar and CO₂ flow rate of 4 L/min. The maximum oil yield and amount of β-sitosterol were found as 74.83 mg oil/g seed and 4.73 mg β-sitosterol/g seed cardamom under these conditions.

Circular RNAs are a special type of RNAs which recently attracted a lot of research interest in studying its formation and function. RNA binding proteins (RBPs) that bind circRNAs are important in these processes but are relatively less studied. CLIP-Seq technology has been invented and applied to profile RBP-RNA interactions on the genome-wide scale. While mRNAs are usually the focus of CLIP-Seq experiments, RBP-circRNA interactions could also be identified through specialized analysis of CLIP-Seq datasets. However, many technical difficulties are involved in this process, such as the usually short read length of CLIP-Seq reads. In this study, we created a pipeline called Clirc specialized for profiling circRNAs in CLIP-Seq data and analyzing the characteristics of RBP- circRNAs interactions. In conclusion, this is one of the first few studies to investigate circRNAs and their binding partners through repurposing CLIP-Seq datasets to our knowledge, and we hope our work will become a valuable resource for future studies into the biogenesis and function of circRNAs. Clirc software is available at https://github.com/Minzhe/Clirc

We are currently assisting at the explosion of the epitranscriptomics, which studies the functional role of chemical modifications into RNA molecules. Among more than 100 RNA modifications, the N6-methyladenosine (m6A), in particular, has attracted the interest of researchers all around the world. m6A is the most abundant internal chemical modification in mRNA and it can control any aspect of mRNA post-transcriptional regulation. m6A is installed by “writers”, removed by “erasers”, and recognized by “readers”, thus, it can be compared to the reversible and dynamic epigenetic modifications in histones and DNA. Given its fundamental role in determining the way mRNAs are expressed, it comes as no surprise that alterations to m6A modifications have a deep impact in cell differentiation, normal development and human diseases. Here, we review the proteins involved in m6A modification in mammals, m6A role in gene expression and its contribution to cancer development. In particular, we will focus on AML that, among first, has indicated how alteration in m6A modification can disrupt normal cellular differentiation and lead to cancer.

Mushrooms have long rich history in folk medicine, traditional and functional foods due to high content of dietary myco-nutrients. Currently, there is increased interest in finding appropriate food-grade green ex-traction systems capable of extracting these bioactive compounds from dietary mushrooms for applica-tions in various food, pharmacological or nutraceutical formulations. Herein, we evaluated a modified Swiss water process (SWP) method using alkaline and acidic pH at low and high temperature under pressurized conditions as a suitable green food grade solvent to obtained extracts enriched with my-co-nutrients (dietary phenolics, total antioxidants (TAA), vitamins, and minerals) from Chaga. Ultra-high performance liquid chromatography coupled to high resolution accurate mass tandem mass spectrometry (UHPLC-HRAMS-MS/MS) was used to assess the phenolic compounds and vitamin levels in the extracts, while inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the mineral con-tents. Over twenty phenolic compounds were quantitatively evaluated in the extracts and the highest total phenolic content and antioxidant activity was observed at pH11.5 at 100°C. The most abundant phenolic compounds present in Chaga extracts included phenolic acids such as protocatechuic acid 4-glucoside (0.7-1.08µg/mL), syringic acid (0.62-1.18µg/mL), and myricetin (0.68-1.3µg/mL). Vitamins are being reported for the first time in Chaga. pH 2.5 at 100°C treatment shows superior effects in extracting the B vitamins whereas pH 2.5 at 60 and 100°C treatments were outstanding for extraction of total fat-soluble vitamins. Vitamin E content was the highest for the fat-soluble vitamins in the Chaga extract under acidic pH (2.5) and high temp. (100°C) and ranges between 50 to 175 µg/100g Chaga. Antioxidant minerals ranged from 85.94 µg/g (pH7 at 100°C) to 113.86 µg/g DW (pH2.5 at 100°C). High temperature 100°C and a pH of 2.5 or 9.5. The treatment of pH11.5 at 100°C was the most useful for recovering phenolics and antioxidants from Chaga including several phenolic compounds reported for the first time in Chaga. SWP is being proposed herein for the first time as a novel, green food-grade solvent system for the extraction of myco-nutrients from Chaga and have potential applications as a suitable approach to extract nutrients from other matrices. Chaga extracts enriched with bioactive myconutrients and antioxidants may be suitable for further use or applications in the food and nutraceutical industries.

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.

From the first isolation of the cystovirus bacteriophage Φ6 from Pseudomonas syringae 50 years ago, we have progressed to a better understanding of the structure and transformations of the many parts of the virion. The three-layered virion encapsulating the tripartite double stranded RNA (dsRNA) genome, breaches the cell envelope on infection, generates its own transcripts, and coopts the bacterial machinery to produce its proteins. The generation of new virions starts with a procapsid with a contracted shape, followed by packaging single stranded RNA segments with concurrent expansion of the capsid, and finally replication to reconstitute the dsRNA genome. The outer two layers are then added, and the fully formed virions released by cell lysis. Most of the procapsid structure composed of the proteins P1, P2, P4 and P7 is now known, as well as its transformations to the mature, packaged nucleocapsid. The outer two layers are less well studied. One additional study investigated the binding of the host protein YajQ to the infecting nucleocapsid, where it enhances the transcription of the large RNA segment that codes for the capsid proteins. Finally, we relate the structural aspects of bacteriophage Φ6 top those of other dsRNA viruses, noting the similarities and differences.

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.

Favipiravir is a broad-spectrum inhibitor of viral RNA-dependent RNA polymerase (RdRp) currently being used to manage COVID-19 in several countries. By acting as a substrate for RdRp, favipiravir gets incorporated into the nascent viral RNA and prevents strand extension. A high mutation rate of SARS-CoV-2 RdRp may facilitate antigenic drift as an answer to the host immune response, thereby generating resistance of virus to favipiravir. Therefore, it is extremely crucial to predict potential mutational sites in the RdRp and the emergence of structural modifications contributing to drug resistance. Here, we used high-throughput interface-based protein design to generate >100,000 designs and identify mutation hotspot residues in the favipiravir-binding site of RdRp. Several mutants had lower binding affinities to favipiravir, out of which hotspot residues with a high propensity to undergo positive selection were identified. The results showed that the designs retained an average of 97 to 98% sequence identity, suggesting that SARS-CoV-2 can develop favipiravir resistance with just a few mutations. Notably, we observed that out of 134 mutations predicted designs, 63 specific mutations were already present in the CoV-GLUE database, thus attaining ~47% correlation match with the clinical sequencing data. The findings improve our understanding of the potential signatures of adaptation in SARS-CoV-2 against favipiravir and management of COVID-19. Furthermore, they can help develop exhaustive strategies for robust antiviral design and discovery.

COVID-19 has emerged as deadly pandemic worldwide with no vaccine or suitable antiviral drugs to prevent or cure the disease. Because of the time-consuming process to develop new vaccines or antiviral agents, there has been a growing interest in repurposing some existing drugs to combat SARS-CoV-2. Vitamin D is known to be protective against acute respiratory distress syndrome (ARDS), pneumonia and cytokine storm. Recently it has been used as a repurposed drug for the treatment of H5N1 virus-induced lung injury. Circumstantial evidences indicate that people with low level of vitamin D are more susceptible to SARS-CoV-2. Although, vitamin D was suggested to interfere with viral replication, its interaction with any SARS-CoV-2 protein is unexplored yet. Beside this, ivermectin, a well-known anti-parasitic agent, exhibits potent anti-viral activities in vitro against viruses such as HIV-1 and dengue. Very recently, ivermectin has been found to reduce viral load of SARS-CoV-2 in vitro. We have analyzed available structures of SARS-CoV-2 proteins to identify probable binding partner(s) of vitamin D and ivermectin through knowledge-based docking studies and figured out possible implication of their binding in SARS-CoV-2 infection. Our observations suggest that the non-structural protein nsp7 possesses a potential site to house 25-hydroxyvitamin D3 (VDY) or the active form of Vitamin D, calcitrol. Binding of vitamin D with nsp7 likely to hamper the formation of nsp7-nsp8 complex which is required to bind with RNA dependent RNA polymerase (RdRP), nsp12 for optimal function. On the other hand, potential binding site of ivermectin has been identified in the S2 subunit of trimeric spike(S) glycoprotein of SARS-CoV-2. We propose that deeply inserted mode of ivermectin binding at three inter-subunit junctions may restrict large scale conformational changes of S2 helices which is necessary for efficient fusion of viral and host membrane. Our study, therefore, opens up avenues for further investigations to consider vitamin D and ivermectin as potential drugs against SARS-CoV-2.

The past decade has witnessed enormous progress, which has seen the noncoding RNAs (ncRNAs) turn from the so called dark matter RNA to critical functional molecules, influencing most physiological processes in development and disease contexts. Many ncRNAs interact with each other and are part of networks that influence the cell transcriptome and proteome and consequently the outcome of biological processes. The regulatory circuits controlled by ncRNAs have become increasingly more relevant in cancer. Further understanding of these complex network interactions and how ncRNAs are regulated, is paving the way for the identification of better therapeutic strategies in cancer.

This study explores the complexities involved in the extraction and purification of catechins, prominent bioactive compounds found in plant extracts and tea. It addresses challenges arising from the intricate molecular structure of catechins and their inherent variability across botanical sources. Despite these obstacles, catechins present significant potential, particularly in healthcare. Their potent antioxidant properties have been extensively investigated and associated with numerous health benefits, positioning them as promising therapeutic agents for combating chronic diseases. Furthermore, catechins have diverse applications across various industries, including food and beverage, cosmetics, agriculture, and materials science. This abstract emphasizes the importance of continued research and innovation in catechin extraction and utilization, which hold promise for advancing human health and technological progress across multiple domains.

: Peach processing generates significant amounts of by-products including peels, pomace, and seeds that are often discarded as waste, despite their rich content of bioactive components. Various extraction methods, such as solvent extraction, ultrasound-assisted extraction, and alkaline and acid hydrolysis, have been employed to recover valuable components from peach by-products and repurpose them in various products. These compounds have shown potential applications in the food, pharmaceutical, and cosmetic industries due to their antioxidant, antimicrobial, and anti-inflammatory properties. Furthermore, these wastes can also be used to produce functional ingredients, natural colorants, dietary supplements Alternative uses consist the use as animal feed, as composting materials and biofuels. This comprehensive review provides an overview of the valorization of peach by-products, focusing on their valorization and extraction techniques and potential applications.

Contemporary advanced models for aspect-targeted opinion word extraction (ATOWE), which predominantly utilize BERT-based encoders at a word level, have shown limited advancements when integrated with graph convolutional networks (GCNs) for syntactic tree assimilation. Recognizing the prowess of BERT subwords in encapsulating rare or context-poor words, this study pivots from syntactic trees to BERT subwords, omitting GCNs from the structural framework. Our approach, named Aspect-Enhanced Wordpiece Extraction Model (AEWEM), focuses on refining aspect representation during encoding. We propose an input format of paired sentence-aspect, diverging from traditional single-sentence inputs. AEWEM demonstrates superior performance on benchmark datasets, establishing a robust foundation for future explorations in this domain.

Demand for nutmeg essential oil in Indonesia and the world is increasing. Nutmeg essential oil is obtained from young nutmeg seeds extracted by water steam distillation (WSD). The weakness of the extraction process with WSD is the long distillation time (24 hours), wasteful of fuel, wasteful of manpower, produces polluting gases which damage the environment and requires a lot of water as a coolant. The purpose of this study was to compare the WSD and SCFE methods for extracting nutmeg essential oil. The research was conducted by extracting nutmeg essential oil using two methods are WSD and SCFE. The results showed that the chemical composition of the nutmeg essential oil produced by the two methods was almost similar. Nutmeg essential oil produced by both methods shows the same characterization. The yield between the two methods is almost the same, in the range of 10-12% using extraction time 15-60 minutes. The SCFE method has advantages over WSD are a faster extraction time, does not require heating materials and CO2 gas can be reused. In conclusion, SCFE is an alternative method to increase the production of nutmeg oil.

Keywords: Arachis quality; pod separation; plant extraction, peanut

The internal wave recognition algorithm in an ocean data buoy system can be used to realize the real-time and flexible observation of internal waves, but there is no accurate automatic recognition method. To meet the need for automatic, real-time, and reliable internal wave recognition, an automatic internal wave recognition algorithm has been proposed for a tightly profiled intelligent buoy system. The sea profile temperature data collected by the Bailong buoy system in the Andaman Sea in 2018 were used to train and test the internal wave recognition neural network model, which consists of two parts: feature extraction and feature classification. The experiment compares the long short-term memory network (LSTM), convolutional neural network (CNN) with different layers, and deep neural network (DNN) without a feature extraction network and adjusts the number of convolutional nuclei and convolutional strides to improve the feature extraction efficiency. Experiments show that the best results can be obtained when a CNN layer is used as the feature extraction network, the convolutional step length is 4, the number of convolutional kernels is 5. The recall reaches 95.31% and the precision is 97.53%. The internal wave identification delay of the algorithm is 5.0862 minutes, the number of parameters is 1593, and the number of calculations is 3024. The algorithm can be directly deployed to the ocean data buoy system to realize the demand for automatic, real-time and reliable internal wave identification at the buoy end.

In recent years, there has been a growing interest in utilizing natural antioxidants as alternatives to synthetic additives in food products. Apples and apple by-products have gained attention as a potential source of natural antioxidants due to their rich phenolic content. This review aims to provide a comprehensive overview of the antioxidant properties of apples and apple by-products, their extraction techniques as well as potential applications in food products because of their an-tioxidant or nutritional properties. The findings here reported highlights the proper utilization of apples and their by-products in food to reduce the detrimental effect on the environment and provide a positive impact on the economy.

This study compares the performance of existing studies on multimodal emotion recognition, and proposes a model that fuses two modalities with the speaker's text and voice signals as input values and detects depression. Based on the DAIC-WOZ dataset, voice features were extracted using CNN, text features were extracted using Transformers, and two modalities were fused through a tensor fusion network. We also build a model to detect whether the speaker is depressed or not using LSTM in the final layer. This study suggests the possibility of increasing access to mental illness diagnosis by enabling patients to detect depression on their own in daily conversations. If the model proposed in this study is developed and the voice conversation system is connected, it will be easier for patients who cannot visit the hospital periodically or who are reluctant to visit the hospital to check their condition and seek recovery. Furthermore, it can be expanded to multi-label classification for various mental diseases and used as a simple self-mental disease diagnosis tool.

Acanthopanax senticosus(rupr. rt. Maxim.; AS) is a medicinal plant used in the clinical treatment of cerebrovascular diseases and central nervous system disorders, significantly improving blood lipid levels and endothelial cell function in patients with acute cerebral infarction. Isofraxidin, one of the active ingredients of AS, is the core of the medical effects, and its extraction and purification depend on organic solvents. Deep eutectic solvents (DESs) is a new green solvent, synthesized by intermolecular hydrogen bonding between hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA), which has non-toxic, high separation and purification efficiency, and environmentally friendly compared with traditional organic solvents. In this paper, DES was used for the extraction of isofraxidin from AS. The primary findings demonstrate that the DESs had a viscosity higher than that of ethanol, and even adding a tiny amount of water (about 10%) would trigger solvent redistribution, leading to a considerable reduction in solvent viscosity. In comparison to ethanol, the extraction rate of isofraxidin by DESs was 2-3 times higher. Thus, this work developed a new technique for using green extraction of isofraxidin that has some practical implications.

The present study was aimed to evaluate the suitability of agro-wastes and crude vegetable oils for the cost effective production of poly-β-hydroxybutyrate (PHB), to evaluate growth kinetics and PHB production in Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1 with these carbon substrates and to study the biodegradation of PHB accumulated by these cultures. Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1 accumulate higher amounts of PHB corn (79.90% of dry cell mass) and rice straw (66.22% of dry cell mass) medium respectively. The kinetic model suggests that the Pseudomonas sp. RZS1 follows the Monod model more closely than A. faecalis RZS4. Both the cultures degrade their own PHB extract under the influence of PHB depolymerase. Corn waste and rice straw appear as the best and cost-effective substrates for the sustainable production of PHB from Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1. The biopolymer accumulated by these organisms is biodegradable in nature. The agro-wastes and crude vegetable oils are good and low cost sources of nutrients for the growth and production of PHN and other metabolites. Their use would lower the production cost of PHN and the low cost production will reduce the sailing price of PHB based products. This would promote the large scale commercialization and popularization of PHB as ecofriendly bioplastic/biopolymer.

Herein, we describe a highly efficient, non-complicated, and non-organic procedure to overcome the negative effect of chemotherapeutic drugs on the quality of the extracted DNA by applying several modulations in cell washing, suspension, and lysis of cells treated with these drugs. In this protocol, 500µl were extracted from patients who received systemic sessions of chemotherapy. The validity of this protocol for digestion with restriction endonucleases and both conventional and real-time polymerase chain reaction were tested. This protocol proved obvious purity (1.8±0.02 and 2.1±1.2, for A_260/280 and A_260/230, respectively) and adequate yields (10±2.24) µg/100 ml. The positive results of validation experiments proved the validity of the extracted DNA for downstream applications of molecular biology. In addition to the proven efficiency of this protocol to extract DNA from normal samples, its validity was also confirmed from patients who were exposed to chemotherapy. This entails a novel approach to extract a molecular biology grade DNA without having inhibitors against enzymes used in digestion, amplification, and subsequent sequencing even after the systemic sessions with several doses of chemotherapy.

Tooth bud removal called Ibyinyo is the practice of removing the developing tooth buds, usually done on an infant, typically performed by traditional healers who believe that this practice will reduce fever and diarrhea in children. This practice is most prevalent in East-African countries, including Rwanda. These procedures are mostly performed in non-sterile conditions using basic sharp instruments. We will discuss the case of a 10-year-old female patient who was presented at the dental clinic, with two malformed permanent canine teeth. Clinical examination revealed malformed enamel and elongated permanent right maxillary canine tooth left mandibular canine tooth with crown malformation. She was also presented with retained (primary) left maxillary lateral incisor tooth and missing left maxillary canine tooth. In addition, the ectopic eruption of left maxillary central incisor tooth and missing permanent right mandibular canine tooth were noted. All these complications resulted from tooth bud removal that the patient had experienced in her early childhood. Her malformed right maxillary and left lower mandibular canine teeth were reshaped, using composite filling materials, to improve her appearance aesthetically. Ibyinyo is preventable damage, done out of ignorance and superstitious practices that can be stopped by growing social awareness. Therefore, educating parents through community-based campaigns on the detrimental consequences of early childhood tooth bud removal through Ibyinyo practice might be helpful to eradicate this harmful and unnecessary practice.

Heavy metals have been implicated as Lagoon pollutants from sources such as agriculture, mining and manufacturing industries and waste water treatment works. A study was carried out in the University of Lagos lagoon to investigate the seasonal bioavailability of the heavy metal contaminants Cd, Cr, Cu, Pb and Zn. The physical parameters pH, redox potential, temperature, TDS and conductivity were measured on site. Dried sediment samples were extracted using the Community Bureau of Reference (BCR) sequential procedure and analysed by ICP-OES. A certified reference material (CRM), BCR 701 (lake sediment) was used for quality assurance with recoveries ranging between 80-120%. Statistical analysis (ANOVA) showed that there was a significant difference between metal distribution in the dry and wet seasons. Cu, Pb and Zn were in the available fractions (carbonate, Fe/Mn oxide and organic). Cu was highest in the Fe/Mn oxide and organic fractions. This indicated that an increase in organic matter and reducing agents will avail the Cu. Zn was distributed in all fractions while Pb was found in the Fe/Mn oxide fraction (3.93- 21.3 %). Results showed that the bioavailability of Cu, Pb and Zn was high. Metal bioavailability by BCR indicates a potential risk of pollution in lagoon sediments as the available metals exceeded the permissible Sediment Quality Assessment Guidelines (SQAG’s) from Environmental Protection Agency (EPA).

Chlorogenic acid or 5-Caffeoylquinic acid is a polyphenolic component present in coffee and its by-products. Chlorogenic acid has been shown to exert biological properties, particularly in relation to glucose and lipid metabolism, including antibacterial, antioxidant, anti-inflammatory activities, among others. Due to its importance, it is necessary to evaluate the reliability of the analytical method for its determination in complex matrices such as food. In this work, different methods of chlorogenic acid extraction in coffee Silverskin were studied, as well as its quantification by HPLC. The results showed that the method of extraction with a mixture of methanol:water (3:1 v/v) in an ultrasonic bath, favored the recovery of chlorogenic acid with a recovery of 77.44%. The instrument detection limit for chlorogenic acid was 3.311 µg/mL and the quantification limit was 11.037 µg/mL. For coffee Silverskin, the concentration obtained of chlorogenic acid by the three extraction methods evaluated was in the range of 57 to 224 mg/kg of coffee silverskin (dry basis).

This work is aimed at promoting a healthier means of livelihood by investigating insignificant areas of pollution. In this work, soy candles produced from soybeans were proven as healthier alternatives to paraffin candles. Soxhlet extraction method was used with hexane as solvent. The extracted oil were then solidified. The wax was moulded into candle and tests were carried out to prove its claims as a safer alternative to paraffin wax. The results supported this claims that soy candles is more economical and produced lesser soot than the paraffin candles.

In recent years, demand for consumption of marine foods, and especially fish, has substantially increased worldwide. The majority of collagen available is sourced from mammalian-derived products. Although fish derived gelatine is a viable alternative to mammalian sourced gelatine, there are some challenges related to the use of fish gelatine including odour, colour, gelling and film forming properties as well as consistency in gelatine amino acid composition. Chemicals used for pre-treatment, as well as extraction conditions such as temperature and time, can influence the length of polypeptide chains that result and the functional properties of the gelatine. Compared to mammalian sources, gelatines derived from fish show notable differences in physical and chemical properties, and great care should be paid to optimization of the production process in order to obtain a product with the best properties for intended applications. The focus of this review is to explore the feasibility of producing gelatine sourced from marine processing by-products using different pre-treatment and extraction strategies with the aim of improving the techno-functional properties of the final product and improving the clean-label status of gelatines. The bioactivities of gelatine hydrolysates are also discussed.

In recent years, demand for consumption of marine foods, and especially fish, has substantially increased worldwide. The majority of collagen available is sourced from mammalian-derived products. Although fish derived gelatine is a viable alternative to mammalian sourced gelatine, there are some challenges related to the use of fish gelatine including odour, colour, gelling and film forming properties as well as consistency in gelatine amino acid composition. Chemicals used for pre-treatment, as well as extraction conditions such as temperature and time, can influence the length of polypeptide chains that result and the functional properties of the gelatine. Compared to mammalian sources, gelatines derived from fish show notable differences in physical and chemical properties, and great care should be paid to optimization of the production process in order to obtain a product with the best properties for intended applications. The focus of this review is to explore the feasibility of producing gelatine sourced from marine processing by-products using different pre-treatment and extraction strategies with the aim of improving the techno-functional properties of the final product and improving the clean-label status of gelatines. The bioactivities of gelatine hydrolysates are also discussed.

1) Introduction: Brain parcellation is an important processing step in the analysis of structural brain MRI. Existing software implementations are optimized for fully developed adult brains, and provide inadequate results when applied to neonatal brain imaging. 2) Methods: We developed a semi-automated pipeline, NeBSS, for extracting 50 discrete brain structures from neonatal brain MRI, using an atlas registration method that leverages the existing ALBERT neonatal atlas 3) Results: We demonstrate a simple linear workflow for neonatal brain parcellation. NeBSS is robust to variation in imaging acquisition protocol and magnet field strength. 4) Conclusion: NeBSS is a robust pipeline capable of parcellating neonatal brain MRIs using a simple processing workflow. NeBSS fills a need in clinical translational research in neonatal imaging, where existing automated or semi-automated implementations are too rigid to be successfully applied to multi-center neuroprotection studies and clinically heterogeneous cohorts. The software is open source and freely available.

Chikungunya virus (CHIKV), a single-stranded RNA virus transmitted by Aedes mosquitoes, poses a significant global health threat, with severe complications observed in vulnerable populations. The only licensed vaccine, IXCHIQ, approved by the USFDA, is insufficient to address the growing disease burden, particularly in endemic regions lacking herd immunity. Monoclonal antibodies (mAbs), explicitly targeting structural proteins E1/E2, demonstrate promise in passive transfer studies, with mouse and human-derived mAbs showing protective efficacy. The article explores various vaccine candidates, including live attenuated, killed, nucleic-acid-based (DNA/RNA), virus-like particle, chimeric, subunit, and adenovirus vectored vaccines. RNA vaccines emerge as promising candidates due to their rapid response capabilities and enhanced safety profile. The review underscores the importance of E1 and E2 proteins as immunogens, emphasising their antigenic potential. Several vaccine candidates, such as CHIKV/IRES, measles vector (MV-CHIK), synthetic DNA-encoded antibodies, and mRNA-lipid nanoparticle vaccines, demonstrate encouraging preclinical and clinical study results. In addition to identifying potential molecular targets for antiviral therapy, the study looks into the roles played by Toll-like receptors, RIG-I, and NOD-like receptors in the immune response to CHIKV. It also offers insights into novel tactics and promising vaccine candidates.

The exogenous application of phenolic compounds is increasingly recognized as a valuable strategy for promoting growth and mitigating the adverse effects of abiotic stress. However, the biostimulant effect under optimal conditions have not been thoroughly explored. In this study, we investigated the impact of foliar application of flavonoids, specifically Crop Bio Life (CBL), on tomato plants grown under controlled conditions. Our study focused on determining growth parameters, such as cell size, and assessing the concentration of hormones. Principal component analysis (PCA) from all physiological variables were determined. Additionally, we utilized high-throughput mRNA sequencing technology and bioinformatic methodologies to robustly analyze the transcriptomes of tomato leaves regulated by flavonoids. The findings revealed that CBL primarily influenced cells enlargement, leading to increased growth. Furthermore, CBL-treated plants exhibited higher concentrations of the hormone zeatin but lower concentrations of IAA. Moreover, RNAseq analysis indicated that CBL-treated plants required increased mineral transport and water uptake, as evidenced by gene expression patterns. Genes related to pathways such as fatty acid degradation, phenylpropanoid biosynthesis, and ABC transporters showed regulatory mechanisms governing internal flavonoid biosynthesis, transport, and tissue concentration, ultimately resulting in higher flavonoid concentrations in tomato leaves.

The Arbas cashmere goat is a unique biological resource that plays a vital role in livestock husbandry in China. LCDM is a medium with special small molecules (consisting of human LIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride) for generation pluripotent stem cells (PSCs) with bidirectional developmental potential in mice, humans, pigs, and bovines. However, there is no report on whether LCDM can support for generation of PSCs with the same ability in Arbas cashmere goats. In this study, we applied LCDM to generation goat induced PSCs (giPSCs) from goat fetal fibroblasts (GFFs) by reprogramming. The derived giPSCs exhibited stem cell morphology, expressing pluripotent markers, and could differentiate into three germ layers. Moreover, the giPSCs differentiated into the trophectoderm lineage by spontaneous and directed differentiation in vitro. The giPSCs contributed to embryonic and extraembryonic tissue in preimplantation blastocysts and postimplantation chimeric embryos. RNA-sequencing analysis showed that the giPSCs were very close to goat embryos at the blastocyst stage and giPSCs have similar properties to typical extended PSCs (EPSCs). The establishment of giPSCs with LCDM provides a new way to generate high quality of PSCs from domestic animals and lays the foundation for basic and applied research in biology and agriculture.

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.